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Sample records for coiled-coil peptides inhibit

  1. Biomaterials Made from Coiled-Coil Peptides.

    Science.gov (United States)

    Conticello, Vincent; Hughes, Spencer; Modlin, Charles

    The development of biomaterials designed for specific applications is an important objective in personalized medicine. While the breadth and prominence of biomaterials have increased exponentially over the past decades, critical challenges remain to be addressed, particularly in the development of biomaterials that exhibit highly specific functions. These functional properties are often encoded within the molecular structure of the component molecules. Proteins, as a consequence of their structural specificity, represent useful substrates for the construction of functional biomaterials through rational design. This chapter provides an in-depth survey of biomaterials constructed from coiled-coils, one of the best-understood protein structural motifs. We discuss the utility of this structurally diverse and functionally tunable class of proteins for the creation of novel biomaterials. This discussion illustrates the progress that has been made in the development of coiled-coil biomaterials by showcasing studies that bridge the gap between the academic science and potential technological impact.

  2. Coiled-coil forming peptides for the induction of silver nanoparticles

    International Nuclear Information System (INIS)

    Božič Abram, Sabina; Aupič, Jana; Dražić, Goran; Gradišar, Helena; Jerala, Roman

    2016-01-01

    Biopolymers with defined sequence patterns offer an attractive alternative for the formation of silver nanoparticle (AgNP). A set of coiled-coil dimer forming peptides was tested for their AgNP formation ability. Seventeen of those peptides mediated the formation of AgNPs in aqueous solution at neutral pH, while the formation of a coiled-coil dimer inhibited the nanoparticle generation. A QSAR regression model on the relationship between sequence and function suggests that in this peptide type the patterns KXQQ and KXEE are favorable, whereas Ala residues appear to have an inhibitory effect. UV–VIS spectra of the obtained nanoparticles gave a peak at around 420 nm, typical for AgNPs in the size range around 40 nm, which was confirmed by dynamic light scattering and transmission electron microscopy. Peptide-induced AgNPs exhibited good antibacterial activity, even after a 15 min contact time, while they had low toxicity to human cells at the same concentrations. These results show that our designed peptides generate AgNPs with antibacterial activity at mild conditions and might be used for antibacterial coatings. - Highlights: • 17 of the 30 tested coiled-coil forming peptides induce AgNP formation. • Coiled-coil dimer formation suppresses AgNP generation of individual peptides. • Size of the peptide-induced silver nanoparticles is around 40 nm. • QSAR analysis points to the importance of KXQQ and KXEE motifs for AgNP generation. • Peptide-induced silver nanoparticles exhibit antibacterial activity.

  3. Coiled-coil forming peptides for the induction of silver nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Božič Abram, Sabina [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); Graduate School of Biomedicine, University of Ljubljana, Ljubljana 1000 (Slovenia); Aupič, Jana [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); Doctoral Programme in Chemical Sciences, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana 1000 (Slovenia); Dražić, Goran [Laboratory for Materials Chemistry, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); Gradišar, Helena [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); EN-FIST, Centre of Excellence, Trg Osvobodilne fronte 13, Ljubljana 1000 (Slovenia); Jerala, Roman, E-mail: roman.jerala@ki.si [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); EN-FIST, Centre of Excellence, Trg Osvobodilne fronte 13, Ljubljana 1000 (Slovenia)

    2016-04-08

    Biopolymers with defined sequence patterns offer an attractive alternative for the formation of silver nanoparticle (AgNP). A set of coiled-coil dimer forming peptides was tested for their AgNP formation ability. Seventeen of those peptides mediated the formation of AgNPs in aqueous solution at neutral pH, while the formation of a coiled-coil dimer inhibited the nanoparticle generation. A QSAR regression model on the relationship between sequence and function suggests that in this peptide type the patterns KXQQ and KXEE are favorable, whereas Ala residues appear to have an inhibitory effect. UV–VIS spectra of the obtained nanoparticles gave a peak at around 420 nm, typical for AgNPs in the size range around 40 nm, which was confirmed by dynamic light scattering and transmission electron microscopy. Peptide-induced AgNPs exhibited good antibacterial activity, even after a 15 min contact time, while they had low toxicity to human cells at the same concentrations. These results show that our designed peptides generate AgNPs with antibacterial activity at mild conditions and might be used for antibacterial coatings. - Highlights: • 17 of the 30 tested coiled-coil forming peptides induce AgNP formation. • Coiled-coil dimer formation suppresses AgNP generation of individual peptides. • Size of the peptide-induced silver nanoparticles is around 40 nm. • QSAR analysis points to the importance of KXQQ and KXEE motifs for AgNP generation. • Peptide-induced silver nanoparticles exhibit antibacterial activity.

  4. Self-assembly of coiled coil peptides into nanoparticles vs 2-d plates: effects of assembly pathway

    Science.gov (United States)

    Kim, Kyunghee; Pochan, Darrin

    Molecular solution assembly, or self-assembly, is a process by which ordered nanostructures or patterns are formed by non-covalent interactions during assembly. Biomimicry, the use of bioinspired molecules or biologically relevant materials, is an important area of self-assembly research with peptides serving a critical role as molecular tools. The morphology of peptide assemblies can be controlled by adjusting solution conditions such as the concentration of peptides, the temperature, and pH. Herein, spherical nanostructures, which have potential for creating an encapsulation system, are formed by self-assembly when coiled coil peptides are combined in solution. These peptides are homotrimeric and heterodimeric coiled-coil bundles and the homotrimer is connected with each of heterodimer through their external surfaces via disulfide bonds. The resultant covalent constructs could co-assemble into complementary trimeric hubs, respectively. The two peptide constructs are directly mixed and assembled in solution in order to produce either spherical particles or 2-d plates depending on the solution conditions and kinetic pathway of assembly. In particular, structural changes of the self-assembled peptides are explored by control of the thermal history of the assembly solution.

  5. Coiled coil peptides as universal linkers for the attachment of recombinant proteins to polymer therapeutics

    Czech Academy of Sciences Publication Activity Database

    Pechar, Michal; Pola, Robert; Laga, Richard; Ulbrich, Karel; Bednárová, Lucie; Maloň, Petr; Sieglová, Irena; Král, Vlastimil; Fábry, Milan; Vaněk, O.

    2011-01-01

    Roč. 12, č. 10 (2011), s. 3645-3655 ISSN 1525-7797 R&D Projects: GA ČR GA203/08/0543; GA MŠk 1M0505 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : coiled coil * polymer the rapeutics * drug targeting Subject RIV: CC - Organic Chemistry Impact factor: 5.479, year: 2011

  6. Coiled coil peptides and polymer-peptide conjugates: synthesis, self-assembly, characterization and potential in drug delivery systems

    Czech Academy of Sciences Publication Activity Database

    Pechar, Michal; Pola, Robert; Laga, Richard; Braunová, Alena; Filippov, Sergey K.; Bogomolova, Anna; Bednárová, Lucie; Vaněk, O.; Ulbrich, Karel

    2014-01-01

    Roč. 15, č. 7 (2014), s. 2590-2599 ISSN 1525-7797 R&D Projects: GA ČR GCP207/12/J030 Grant - others:AV ČR(CZ) AP0802 Program:Akademická prémie - Praemium Academiae Institutional support: RVO:61389013 ; RVO:61388963 Keywords : coiled coil * self-assembly * hydrophilic polymer Subject RIV: CD - Macromolecular Chemistry; FR - Pharmacology ; Medidal Chemistry (UOCHB-X) Impact factor: 5.750, year: 2014

  7. Spontaneous adsorption of coiled-coil model peptides K and E to a mixed lipid bilayer.

    Science.gov (United States)

    Pluhackova, Kristyna; Wassenaar, Tsjerk A; Kirsch, Sonja; Böckmann, Rainer A

    2015-03-26

    A molecular description of the lipid-protein interactions underlying the adsorption of proteins to membranes is crucial for understanding, for example, the specificity of adsorption or the binding strength of a protein to a bilayer, or for characterizing protein-induced changes of membrane properties. In this paper, we extend an automated in silico assay (DAFT) for binding studies and apply it to characterize the adsorption of the model fusion peptides E and K to a mixed phospholipid/cholesterol membrane using coarse-grained molecular dynamics simulations. In addition, we couple the coarse-grained protocol to reverse transformation to atomistic resolution, thereby allowing to study molecular interactions with high detail. The experimentally observed differential binding of the peptides E and K to membranes, as well as the increased binding affinity of helical over unstructered peptides, could be well reproduced using the polarizable Martini coarse-grained (CG) force field. Binding to neutral membranes is shown to be dominated by initial binding of the positively charged N-terminus to the phospholipid headgroup region, followed by membrane surface-aligned insertion of the peptide at the interface between the hydrophobic core of the membrane and its polar headgroup region. Both coarse-grained and atomistic simulations confirm a before hypothesized snorkeling of lysine side chains for the membrane-bound state of the peptide K. Cholesterol was found to be enriched in peptide vicinity, which is probably of importance for the mechanism of membrane fusion. The applied sequential multiscale method, using coarse-grained simulations for the slow adsorption process of peptides to membranes followed by backward transformation to atomistic detail and subsequent atomistic simulations of the preformed peptide-lipid complexes, is shown to be a versatile approach to study the interactions of peptides or proteins with biomembranes.

  8. Folding Topology of a Short Coiled-Coil Peptide Structure Templated by an Oligonucleotide Triplex

    DEFF Research Database (Denmark)

    Lou, Chenguang; Christensen, Niels Johan; Martos Maldonado, Manuel Cristo

    2017-01-01

    by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex......, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12nm) in solution was revealed to be independent of concentration. The topological folding...

  9. Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper).

    Science.gov (United States)

    Jelesarov, I; Dürr, E; Thomas, R M; Bosshard, H R

    1998-05-19

    The stability of a coiled coil or leucine zipper is controlled by hydrophobic interactions and electrostatic forces between the constituent helices. We have designed a 30-residue peptide with the repeating seven-residue pattern of a coiled coil, (abcdefg)n, and with Glu in positions e and g of each heptad. The glutamate side chains prevented folding at pH values above 6 because of electrostatic repulsion across the helix dimer interface as well as within the individual helices. Protonation of the carboxylates changed the conformation from a random coil monomer to a coiled coil dimer. Folding at alkaline pH where the peptide had a net charge of -7e was promoted by the addition of salts. The nature of the charge screening cation was less important than that of the anion. The high salt concentrations (>1 M) necessary to induce folding indicated that the salt-induced folding resulted from alterations in the protein-water interaction. Folding was promoted by the kosmotropic anions sulfate and fluoride and to a lesser extent by the weak kosmotrope formate, whereas chloride and the strong chaotrope perchlorate were ineffective. Kosmotropes are excluded from the protein surface, which is preferentially hydrated, and this promotes folding by strengthening hydrophobic interactions at the coiled coil interface. Although charge neutralization also contributed to folding, it was effective only when the screening cation was partnered by a good kosmotropic anion. Folding conformed to a two-state transition from random coil monomer to coiled coil dimer and was enthalpy driven and characterized by a change in the heat capacity of unfolding of 3.9 +/- 1.2 kJ mol-1 K-1. The rate of folding was analyzed by fluorescence stopped-flow measurements. Folding occurred in a biphasic reaction in which the rapid formation of an initial dimer (kf = 2 x 10(7) M-1 s-1) was followed by an equally rapid concentration-independent rearrangement to the folded dimer (k > 100 s-1).

  10. Requirement of the coiled-coil domain of PML-RARα oncoprotein for localization, sumoylation, and inhibition of monocyte differentiation

    International Nuclear Information System (INIS)

    Kim, Young-Eui; Kim, Dong-Yeon; Lee, Jang-Mi; Kim, Seong-Tae; Han, Tae-Hee; Ahn, Jin-Hyun

    2005-01-01

    Homo-oligomerization via a coiled-coil (C-C) domain has been shown to be necessary for the promyelocytic leukemia (PML)-retinoic acid receptor-α (RARα) fusion protein to acquire oncogenic potential in acute promyelocytic leukemia. We show here that PML(ΔC-C)-RARα, which contains a deletion in its C-C domain, is neither localized as characteristic microspeckles nor modified by small ubiquitin-like modifiers (SUMO). The absence of sumoylation of the ΔC-C mutant was due to the lack of binding to Ubc9, a SUMO conjugation enzyme. The integrity of RING finger domain was also needed for both sumoylation and microspeckle formation. In GAL4-DNA tethering assays, the ΔC-C mutant completely lost the inhibitory effect on retinoic acid (RA)-mediated transactivation. Furthermore, the expression of CD14 in U937 cells expressing the ΔC-C mutant in response to vitamin D3 was markedly higher than in cells expressing PML-RARα. However, the RA-mediated induction of C/EBPβ in cells expressing the ΔC-C mutant was comparable to that of control cells. Thus, our results suggest that the C-C domain-associated functions of sumoylation, localization as microspeckles, and the inhibition of monocyte differentiation all contribute to the oncogenic activity of PML-RARα

  11. Extending the self-assembly of coiled-coil hybrids

    NARCIS (Netherlands)

    Robson Marsden, Hana

    2009-01-01

    Of the various biomolecular building blocks in use in nature, coiled-coil forming peptides are amongst those with the most potential as building blocks for the synthetic self-assembly of nanostructures. Native coiled coils have the ability to function in, and influence, complex systems composed of

  12. Molecular basis of coiled-coil oligomerization-state specificity.

    Science.gov (United States)

    Ciani, Barbara; Bjelic, Saša; Honnappa, Srinivas; Jawhari, Hatim; Jaussi, Rolf; Payapilly, Aishwarya; Jowitt, Thomas; Steinmetz, Michel O; Kammerer, Richard A

    2010-11-16

    Coiled coils are extensively and successfully used nowadays to rationally design multistranded structures for applications, including basic research, biotechnology, nanotechnology, materials science, and medicine. The wide range of applications as well as the important functions these structures play in almost all biological processes highlight the need for a detailed understanding of the factors that control coiled-coil folding and oligomerization. Here, we address the important and unresolved question why the presence of particular oligomerization-state determinants within a coiled coil does frequently not correlate with its topology. We found an unexpected, general link between coiled-coil oligomerization-state specificity and trigger sequences, elements that are indispensable for coiled-coil formation. By using the archetype coiled-coil domain of the yeast transcriptional activator GCN4 as a model system, we show that well-established trimer-specific oligomerization-state determinants switch the peptide's topology from a dimer to a trimer only when inserted into the trigger sequence. We successfully confirmed our results in two other, unrelated coiled-coil dimers, ATF1 and cortexillin-1. We furthermore show that multiple topology determinants can coexist in the same trigger sequence, revealing a delicate balance of the resulting oligomerization state by position-dependent forces. Our experimental results should significantly improve the prediction of the oligomerization state of coiled coils. They therefore should have major implications for the rational design of coiled coils and consequently many applications using these popular oligomerization domains.

  13. Investigation of the network of preferred interactions in an artificial coiled-coil association using the peptide array technique

    Directory of Open Access Journals (Sweden)

    Raheleh Rezaei Araghi

    2012-04-01

    Full Text Available We screened a randomized library and identified natural peptides that bound selectively to a chimeric peptide containing α-, β- and γ-amino acids. The SPOT arrays provide a means for the systematic study of the possible interaction space accessible to the αβγ-chimera. The mutational analysis reveals the dependence of the binding affinities of α-peptides to the αβγ-chimera, on the hydrophobicity and bulkiness of the side chains at the corresponding hydrophobic interface. The stability of the resulting heteroassemblies was further confirmed in solution by CD and thermal denaturation.

  14. Coiled-coil formation of the membrane-fusion K/E peptides viewed by electron paramagnetic resonance.

    Directory of Open Access Journals (Sweden)

    Pravin Kumar

    Full Text Available The interaction of the complementary K (Ac-(KIAALKE3-GW-NH2 and E (Ac-(EIAALEK3-GY-NH2 peptides, components of the zipper of an artificial membrane fusion system (Robson Marsden H. et al. Angew Chemie Int Ed. 2009 is investigated by electron paramagnetic resonance (EPR. By frozen solution continuous-wave EPR and double electron-electron resonance (DEER, the distance between spin labels attached to the K- and to the E-peptide is measured. Three constructs of spin-labelled K- and E-peptides are used in five combinations for low temperature investigations. The K/E heterodimers are found to be parallel, in agreement with previous studies. Also, K homodimers in parallel orientation were observed, a finding that was not reported before. Comparison to room-temperature, solution EPR shows that the latter method is less specific to detect this peptide-peptide interaction. Combining frozen solution cw-EPR for short distances (1.8 nm to 2.0 nm and DEER for longer distances thus proves versatile to detect the zipper interaction in membrane fusion. As the methodology can be applied to membrane samples, the approach presented suggests itself for in-situ studies of the complete membrane fusion process, opening up new avenues for the study of membrane fusion.

  15. De novo design of peptide immunogens that mimic the coiled coil region of human T-cell leukemia virus type-1 glycoprotein 21 transmembrane subunit for induction of native protein reactive neutralizing antibodies.

    Science.gov (United States)

    Sundaram, Roshni; Lynch, Marcus P; Rawale, Sharad V; Sun, Yiping; Kazanji, Mirdad; Kaumaya, Pravin T P

    2004-06-04

    Peptide vaccines able to induce high affinity and protective neutralizing antibodies must rely in part on the design of antigenic epitopes that mimic the three-dimensional structure of the corresponding region in the native protein. We describe the design, structural characterization, immunogenicity, and neutralizing potential of antibodies elicited by conformational peptides derived from the human T-cell leukemia virus type 1 (HTLV-1) gp21 envelope glycoprotein spanning residues 347-374. We used a novel template design and a unique synthetic approach to construct two peptides (WCCR2T and CCR2T) that would each assemble into a triple helical coiled coil conformation mimicking the gp21 crystal structure. The peptide B-cell epitopes were grafted onto the epsilon side chains of three lysyl residues on a template backbone construct consisting of the sequence acetyl-XGKGKGKGCONH2 (where X represents the tetanus toxoid promiscuous T cell epitope (TT) sequence 580-599). Leucine substitutions were introduced at the a and d positions of the CCR2T sequence to maximize helical character and stability as shown by circular dichroism and guanidinium hydrochloride studies. Serum from an HTLV-1-infected patient was able to recognize the selected epitopes by enzyme-linked immunosorbent assay (ELISA). Mice immunized with the wild-type sequence (WCCR2T) and the mutant sequence (CCR2T) elicited high antibody titers that were capable of recognizing the native protein as shown by flow cytometry and whole virus ELISA. Sera and purified antibodies from immunized mice were able to reduce the formation of syncytia induced by the envelope glycoprotein of HTLV-1, suggesting that antibodies directed against the coiled coil region of gp21 are capable of disrupting cell-cell fusion. Our results indicate that these peptides represent potential candidates for use in a peptide vaccine against HTLV-1.

  16. Exploring the conserved water site and hydration of a coiled-coil trimerisation motif: a MD simulation study.

    Science.gov (United States)

    Dolenc, Jozica; Baron, Riccardo; Missimer, John H; Steinmetz, Michel O; van Gunsteren, Wilfred F

    2008-07-21

    The solvent structure and dynamics around ccbeta-p, a 17-residue peptide that forms a parallel three-stranded alpha-helical coiled coil in solution, was analysed through 10 ns explicit solvent molecular dynamics (MD) simulations at 278 and 330 K. Comparison with two corresponding simulations of the monomeric form of ccbeta-p was used to investigate the changes of hydration upon coiled-coil formation. Pronounced peaks in the solvent density distribution between residues Arg8 and Glu13 of neighbouring helices show the presence of water bridges between the helices of the ccbeta-p trimer; this is in agreement with the water sites observed in X-ray crystallography experiments. Interestingly, this water site is structurally conserved in many three-stranded coiled coils and, together with the Arg and Glu residues, forms part of a motif that determines three-stranded coiled-coil formation. Our findings show that little direct correlation exists between the solvent density distribution and the temporal ordering of water around the trimeric coiled coil. The MD-calculated effective residence times of up to 40 ps show rapid exchange of surface water molecules with the bulk phase, and indicate that the solvent distribution around biomolecules requires interpretation in terms of continuous density distributions rather than in terms of discrete molecules of water. Together, our study contributes to understanding the principles of three-stranded coiled-coil formation.

  17. A periodic table of coiled-coil protein structures.

    Science.gov (United States)

    Moutevelis, Efrosini; Woolfson, Derek N

    2009-01-23

    Coiled coils are protein structure domains with two or more alpha-helices packed together via interlacing of side chains known as knob-into-hole packing. We analysed and classified a large set of coiled-coil structures using a combination of automated and manual methods. This led to a systematic classification that we termed a "periodic table of coiled coils," which we have made available at http://coiledcoils.chm.bris.ac.uk/ccplus/search/periodic_table. In this table, coiled-coil assemblies are arranged in columns with increasing numbers of alpha-helices and in rows of increased complexity. The table provides a framework for understanding possibilities in and limits on coiled-coil structures and a basis for future prediction, engineering and design studies.

  18. Plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif.

    Directory of Open Access Journals (Sweden)

    Nora Céspedes

    Full Text Available Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous to Plasmodium falciparum were identified in the P. vivax genome. The peptides identified in silico were chemically synthesized; circular dichroism studies indicated partial or high α-helical content. Antigenicity was evaluated using human sera samples from malaria-endemic areas of Colombia and Papua New Guinea. Eight of these fragments were selected and used to assess immunogenicity in BALB/c mice. ELISA assays indicated strong reactivity of serum samples from individuals residing in malaria-endemic regions and sera of immunized mice, with the α-helical coiled coil structures. In addition, ex vivo production of IFN-γ by murine mononuclear cells confirmed the immunogenicity of these structures and the presence of T-cell epitopes in the peptide sequences. Moreover, sera of mice immunized with four of the eight antigens recognized native proteins on blood-stage P. vivax parasites, and antigenic cross-reactivity with three of the peptides was observed when reacted with both the P. falciparum orthologous fragments and whole parasites. Results here point to the α-helical coiled coil peptides as possible P. vivax malaria vaccine candidates as were observed for P. falciparum. Fragments selected here warrant further study in humans and non-human primate models to assess their protective efficacy as single components or assembled as hybrid linear epitopes.

  19. The heterotrimeric laminin coiled-coil domain exerts anti-adhesive effects and induces a pro-invasive phenotype.

    Directory of Open Access Journals (Sweden)

    Patricia Santos-Valle

    Full Text Available Laminins are large heterotrimeric cross-shaped extracellular matrix glycoproteins with terminal globular domains and a coiled-coil region through which the three chains are assembled and covalently linked. Laminins are key components of basement membranes, and they serve as attachment sites for cell adhesion, migration and proliferation. In this work, we produced a recombinant fragment comprising the entire laminin coiled-coil of the α1-, β1-, and γ1-chains that assemble into a stable heterotrimeric coiled-coil structure independently of the rest of the molecule. This domain was biologically active and not only failed to serve as a substrate for cell attachment, spreading and focal adhesion formation but also inhibited cell adhesion to laminin when added to cells in a soluble form at the time of seeding. Furthermore, gene array expression profiling in cells cultured in the presence of the laminin coiled-coil domain revealed up-regulation of genes involved in cell motility and invasion. These findings were confirmed by real-time quantitative PCR and zymography assays. In conclusion, this study shows for the first time that the laminin coiled-coil domain displays anti-adhesive functions and has potential implications for cell migration during matrix remodeling.

  20. Sub-20 nm Stable Micelles Based on a Mixture of Coiled-Coils: A Platform for Controlled Ligand Presentation.

    Science.gov (United States)

    Ang, JooChuan; Ma, Dan; Jung, Benson T; Keten, Sinan; Xu, Ting

    2017-11-13

    Ligand-functionalized, multivalent nanoparticles have been extensively studied for biomedical applications from imaging agents to drug delivery vehicles. However, the ligand cluster size is usually heterogeneous and the local valency is ill-defined. Here, we present a mixed micelle platform hierarchically self-assembled from a mixture of two amphiphilic 3-helix and 4-helix peptide-polyethylene glycol (PEG)-lipid hybrid conjugates. We demonstrate that the local multivalent ligand cluster size on the micelle surface can be controlled based on the coiled-coil oligomeric state. The oligomeric states of mixed peptide bundles were found to be in their individual native states. Similarly, mixed micelles indicate the orthogonal self-association of coiled-coil amphiphiles. Using differential scanning calorimetry, fluorescence recovery spectroscopy, and coarse-grained molecular dynamics simulation, we studied the distribution of coiled-coil bundles within the mixed micelles and observed migration of coiled-coils into nanodomains within the sub-20 nm mixed micelle. This report provides important insights into the assembly and formation of nanophase-separated micelles with precise control over the local multivalent state of ligands on the micelle surface.

  1. GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Pratibha; Savithri, H.S., E-mail: bchss@biochem.iisc.ernet.in

    2015-08-15

    Plant viruses exploit the host machinery for targeting the viral genome–movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER–GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER–GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130–138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm

  2. The coiled coil motif in polymer drug delivery systems

    Czech Academy of Sciences Publication Activity Database

    Pechar, Michal; Pola, Robert

    2013-01-01

    Roč. 31, č. 1 (2013), s. 90-96 ISSN 0734-9750 R&D Projects: GA ČR GA203/08/0543; GA MŠk 1M0505 Institutional research plan: CEZ:AV0Z40500505 Keywords : coiled coil * hydrophilic polymer * recombinant protein Subject RIV: CD - Macromolecular Chemistry Impact factor: 8.905, year: 2013

  3. CCHMM_PROF: a HMM-based coiled-coil predictor with evolutionary information

    DEFF Research Database (Denmark)

    Bartoli, Lisa; Fariselli, Piero; Krogh, Anders

    2009-01-01

    tools are available for predicting coiled-coil domains in protein sequences, including those based on position-specific score matrices and machine learning methods. RESULTS: In this article, we introduce a hidden Markov model (CCHMM_PROF) that exploits the information contained in multiple sequence...... alignments (profiles) to predict coiled-coil regions. The new method discriminates coiled-coil sequences with an accuracy of 97% and achieves a true positive rate of 79% with only 1% of false positives. Furthermore, when predicting the location of coiled-coil segments in protein sequences, the method reaches...

  4. Structural basis for cargo binding and autoinhibition of Bicaudal-D1 by a parallel coiled-coil with homotypic registry

    Energy Technology Data Exchange (ETDEWEB)

    Terawaki, Shin-ichi, E-mail: terawaki@gunma-u.ac.jp [Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); SPring-8 Center, RIKEN, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan); Yoshikane, Asuka [Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); SPring-8 Center, RIKEN, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan); Higuchi, Yoshiki [Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297 (Japan); Department of Picobiology, Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297 (Japan); SPring-8 Center, RIKEN, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan); Wakamatsu, Kaori [Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); SPring-8 Center, RIKEN, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan)

    2015-05-01

    Bicaudal-D1 (BICD1) is an α-helical coiled-coil protein mediating the attachment of specific cargo to cytoplasmic dynein. It plays an essential role in minus end-directed intracellular transport along microtubules. The third C-terminal coiled-coil region of BICD1 (BICD1 CC3) has an important role in cargo sorting, including intracellular vesicles associating with the small GTPase Rab6 and the nuclear pore complex Ran binding protein 2 (RanBP2), and inhibiting the association with cytoplasmic dynein by binding to the first N-terminal coiled-coil region (CC1). The crystal structure of BICD1 CC3 revealed a parallel homodimeric coiled-coil with asymmetry and complementary knobs-into-holes interactions, differing from Drosophila BicD CC3. Furthermore, our binding study indicated that BICD1 CC3 possesses a binding surface for two distinct cargos, Rab6 and RanBP2, and that the CC1-binding site overlaps with the Rab6-binding site. These findings suggest a molecular basis for cargo recognition and autoinhibition of BICD proteins during dynein-dependent intracellular retrograde transport. - Highlights: • BICD1 CC3 is a parallel homodimeric coiled-coil with axial asymmetry. • The coiled-coil packing of BICD1 CC3 is adapted to the equivalent heptad position. • BICD1 CC3 has distinct binding sites for two classes of cargo, Rab6 and RanBP2. • The CC1-binding site of BICD1 CC3 overlaps with the Rab6-binding site.

  5. Structural basis for cargo binding and autoinhibition of Bicaudal-D1 by a parallel coiled-coil with homotypic registry

    International Nuclear Information System (INIS)

    Terawaki, Shin-ichi; Yoshikane, Asuka; Higuchi, Yoshiki; Wakamatsu, Kaori

    2015-01-01

    Bicaudal-D1 (BICD1) is an α-helical coiled-coil protein mediating the attachment of specific cargo to cytoplasmic dynein. It plays an essential role in minus end-directed intracellular transport along microtubules. The third C-terminal coiled-coil region of BICD1 (BICD1 CC3) has an important role in cargo sorting, including intracellular vesicles associating with the small GTPase Rab6 and the nuclear pore complex Ran binding protein 2 (RanBP2), and inhibiting the association with cytoplasmic dynein by binding to the first N-terminal coiled-coil region (CC1). The crystal structure of BICD1 CC3 revealed a parallel homodimeric coiled-coil with asymmetry and complementary knobs-into-holes interactions, differing from Drosophila BicD CC3. Furthermore, our binding study indicated that BICD1 CC3 possesses a binding surface for two distinct cargos, Rab6 and RanBP2, and that the CC1-binding site overlaps with the Rab6-binding site. These findings suggest a molecular basis for cargo recognition and autoinhibition of BICD proteins during dynein-dependent intracellular retrograde transport. - Highlights: • BICD1 CC3 is a parallel homodimeric coiled-coil with axial asymmetry. • The coiled-coil packing of BICD1 CC3 is adapted to the equivalent heptad position. • BICD1 CC3 has distinct binding sites for two classes of cargo, Rab6 and RanBP2. • The CC1-binding site of BICD1 CC3 overlaps with the Rab6-binding site

  6. Plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif

    DEFF Research Database (Denmark)

    Céspedes, Nora; Habel, Catherine; Lopez-Perez, Mary

    2014-01-01

    Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous to Pla...

  7. Hypoxia activates muscle-restricted coiled-coil protein (MURC) expression via transforming growth factor-β in cardiac myocytes.

    Science.gov (United States)

    Shyu, Kou-Gi; Cheng, Wen-Pin; Wang, Bao-Wei; Chang, Hang

    2014-03-01

    The expression of MURC (muscle-restricted coiled-coil protein), a hypertrophy-regulated gene, increases during pressure overload. Hypoxia can cause myocardial hypertrophy; however, how hypoxia affects the regulation of MURC in cardiomyocytes undergoing hypertrophy is still unknown. The aim of the present study was to test the hypothesis that hypoxia induces MURC expression in cardiomyocytes during hypertrophy. The expression of MURC was evaluated in cultured rat neonatal cardiomyocytes subjected to hypoxia and in an in vivo model of AMI (acute myocardial infarction) to induce myocardial hypoxia in adult rats. MURC protein and mRNA expression were significantly enhanced by hypoxia. MURC proteins induced by hypoxia were significantly blocked after the addition of PD98059 or ERK (extracellular-signal-regulated kinase) siRNA 30 min before hypoxia. Gel-shift assay showed increased DNA-binding activity of SRF (serum response factor) after hypoxia. PD98059, ERK siRNA and an anti-TGF-β (transforming growth factor-β) antibody abolished the SRF-binding activity enhanced by hypoxia or exogenous administration of TGF-β. A luciferase promoter assay demonstrated increased transcriptional activity of SRF in cardiomyocytes by hypoxia. Increased βMHC (β-myosin heavy chain) and BNP (B-type natriuretic peptide) protein expression and increased protein synthesis was identified after hypoxia with the presence of MURC in hypertrophic cardiomyocytes. MURC siRNA inhibited the hypertrophic marker protein expression and protein synthesis induced by hypoxia. AMI in adult rats also demonstrated increased MURC protein expression in the left ventricular myocardium. In conclusion, hypoxia in cultured rat neonatal cardiomyocytes increased MURC expression via the induction of TGF-β, SRF and the ERK pathway. These findings suggest that MURC plays a role in hypoxia-induced hypertrophy in cardiomyocytes.

  8. Conformational switching in the coiled-coil domains of a proteasomal ATPase regulates substrate processing.

    Science.gov (United States)

    Snoberger, Aaron; Brettrager, Evan J; Smith, David M

    2018-06-18

    Protein degradation in all domains of life requires ATPases that unfold and inject proteins into compartmentalized proteolytic chambers. Proteasomal ATPases in eukaryotes and archaea contain poorly understood N-terminally conserved coiled-coil domains. In this study, we engineer disulfide crosslinks in the coiled-coils of the archaeal proteasomal ATPase (PAN) and report that its three identical coiled-coil domains can adopt three different conformations: (1) in-register and zipped, (2) in-register and partially unzipped, and (3) out-of-register. This conformational heterogeneity conflicts with PAN's symmetrical OB-coiled-coil crystal structure but resembles the conformational heterogeneity of the 26S proteasomal ATPases' coiled-coils. Furthermore, we find that one coiled-coil can be conformationally constrained even while unfolding substrates, and conformational changes in two of the coiled-coils regulate PAN switching between resting and active states. This switching functionally mimics similar states proposed for the 26S proteasome from cryo-EM. These findings thus build a mechanistic framework to understand regulation of proteasome activity.

  9. Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3

    International Nuclear Information System (INIS)

    Amano, Yosuke; Ishikawa, Rie; Sakatani, Toshio; Ichinose, Junji; Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori; Nakajima, Jun; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2015-01-01

    Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation. - Highlights: • TPM3-ALK fusion protein dimerizes through the coiled-coil structure of TPM3. • Longer coiled-coil structure of TPM3 leads to higher TPM3-ALK dimer formation. • Presence of TPM3-ALK dimer leads to ALK, STAT3, and ERK1/2 phosphorylation. • Presence of TPM3-ALK leads to loss of contact inhibition. • BN-PAGE is a simple technique for visualizing oncogenic dimerization

  10. Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3

    Energy Technology Data Exchange (ETDEWEB)

    Amano, Yosuke; Ishikawa, Rie; Sakatani, Toshio [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Ichinose, Junji [Department of Cardiothoracic Surgery, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Nakajima, Jun [Department of Cardiothoracic Surgery, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Nagase, Takahide; Ohishi, Nobuya [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Takai, Daiya, E-mail: dtakai-ind@umin.ac.jp [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Department of Clinical Laboratory, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan)

    2015-02-13

    Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation. - Highlights: • TPM3-ALK fusion protein dimerizes through the coiled-coil structure of TPM3. • Longer coiled-coil structure of TPM3 leads to higher TPM3-ALK dimer formation. • Presence of TPM3-ALK dimer leads to ALK, STAT3, and ERK1/2 phosphorylation. • Presence of TPM3-ALK leads to loss of contact inhibition. • BN-PAGE is a simple technique for visualizing oncogenic dimerization.

  11. Two coiled-coil domains of Chlamydia trachomatis IncA affect membrane fusion events during infection.

    Science.gov (United States)

    Ronzone, Erik; Paumet, Fabienne

    2013-01-01

    Chlamydia trachomatis replicates in a parasitophorous membrane-bound compartment called an inclusion. The inclusions corrupt host vesicle trafficking networks to avoid the degradative endolysosomal pathway but promote fusion with each other in order to sustain higher bacterial loads in a process known as homotypic fusion. The Chlamydia protein IncA (Inclusion protein A) appears to play central roles in both these processes as it participates to homotypic fusion and inhibits endocytic SNARE-mediated membrane fusion. How IncA selectively inhibits or activates membrane fusion remains poorly understood. In this study, we analyzed the spatial and molecular determinants of IncA's fusogenic and inhibitory functions. Using a cell-free membrane fusion assay, we found that inhibition of SNARE-mediated fusion requires IncA to be on the same membrane as the endocytic SNARE proteins. IncA displays two coiled-coil domains showing high homology with SNARE proteins. Domain swap and deletion experiments revealed that although both these domains are capable of independently inhibiting SNARE-mediated fusion, these two coiled-coil domains cooperate in mediating IncA multimerization and homotypic membrane interaction. Our results support the hypothesis that Chlamydia employs SNARE-like virulence factors that positively and negatively affect membrane fusion and promote infection.

  12. Two coiled-coil domains of Chlamydia trachomatis IncA affect membrane fusion events during infection.

    Directory of Open Access Journals (Sweden)

    Erik Ronzone

    Full Text Available Chlamydia trachomatis replicates in a parasitophorous membrane-bound compartment called an inclusion. The inclusions corrupt host vesicle trafficking networks to avoid the degradative endolysosomal pathway but promote fusion with each other in order to sustain higher bacterial loads in a process known as homotypic fusion. The Chlamydia protein IncA (Inclusion protein A appears to play central roles in both these processes as it participates to homotypic fusion and inhibits endocytic SNARE-mediated membrane fusion. How IncA selectively inhibits or activates membrane fusion remains poorly understood. In this study, we analyzed the spatial and molecular determinants of IncA's fusogenic and inhibitory functions. Using a cell-free membrane fusion assay, we found that inhibition of SNARE-mediated fusion requires IncA to be on the same membrane as the endocytic SNARE proteins. IncA displays two coiled-coil domains showing high homology with SNARE proteins. Domain swap and deletion experiments revealed that although both these domains are capable of independently inhibiting SNARE-mediated fusion, these two coiled-coil domains cooperate in mediating IncA multimerization and homotypic membrane interaction. Our results support the hypothesis that Chlamydia employs SNARE-like virulence factors that positively and negatively affect membrane fusion and promote infection.

  13. Multicoil2: predicting coiled coils and their oligomerization states from sequence in the twilight zone.

    Directory of Open Access Journals (Sweden)

    Jason Trigg

    Full Text Available The alpha-helical coiled coil can adopt a variety of topologies, among the most common of which are parallel and antiparallel dimers and trimers. We present Multicoil2, an algorithm that predicts both the location and oligomerization state (two versus three helices of coiled coils in protein sequences. Multicoil2 combines the pairwise correlations of the previous Multicoil method with the flexibility of Hidden Markov Models (HMMs in a Markov Random Field (MRF. The resulting algorithm integrates sequence features, including pairwise interactions, through multinomial logistic regression to devise an optimized scoring function for distinguishing dimer, trimer and non-coiled-coil oligomerization states; this scoring function is used to produce Markov Random Field potentials that incorporate pairwise correlations localized in sequence. Multicoil2 significantly improves both coiled-coil detection and dimer versus trimer state prediction over the original Multicoil algorithm retrained on a newly-constructed database of coiled-coil sequences. The new database, comprised of 2,105 sequences containing 124,088 residues, includes reliable structural annotations based on experimental data in the literature. Notably, the enhanced performance of Multicoil2 is evident when tested in stringent leave-family-out cross-validation on the new database, reflecting expected performance on challenging new prediction targets that have minimal sequence similarity to known coiled-coil families. The Multicoil2 program and training database are available for download from http://multicoil2.csail.mit.edu.

  14. Routine phasing of coiled-coil protein crystal structures with AMPLE

    Directory of Open Access Journals (Sweden)

    Jens M. H. Thomas

    2015-03-01

    Full Text Available Coiled-coil protein folds are among the most abundant in nature. These folds consist of long wound α-helices and are architecturally simple, but paradoxically their crystallographic structures are notoriously difficult to solve with molecular-replacement techniques. The program AMPLE can solve crystal structures by molecular replacement using ab initio search models in the absence of an existent homologous protein structure. AMPLE has been benchmarked on a large and diverse test set of coiled-coil crystal structures and has been found to solve 80% of all cases. Successes included structures with chain lengths of up to 253 residues and resolutions down to 2.9 Å, considerably extending the limits on size and resolution that are typically tractable by ab initio methodologies. The structures of two macromolecular complexes, one including DNA, were also successfully solved using their coiled-coil components. It is demonstrated that both the ab initio modelling and the use of ensemble search models contribute to the success of AMPLE by comparison with phasing attempts using single structures or ideal polyalanine helices. These successes suggest that molecular replacement with AMPLE should be the method of choice for the crystallographic elucidation of a coiled-coil structure. Furthermore, AMPLE may be able to exploit the presence of a coiled coil in a complex to provide a convenient route for phasing.

  15. Novel Anti-Nicotine Vaccine Using a Trimeric Coiled-Coil Hapten Carrier.

    Directory of Open Access Journals (Sweden)

    Keith D Miller

    Full Text Available Tobacco addiction represents one of the largest public health problems in the world and is the leading cause of cancer and heart disease, resulting in millions of deaths a year. Vaccines for smoking cessation have shown considerable promise in preclinical models, although functional antibody responses induced in humans are only modestly effective in preventing nicotine entry into the brain. The challenge in generating serum antibodies with a large nicotine binding capacity is made difficult by the fact that this drug is non-immunogenic and must be conjugated as a hapten to a protein carrier. To circumvent the limitations of traditional carriers like keyhole limpet hemocyanin (KLH, we have synthesized a short trimeric coiled-coil peptide (TCC that creates a series of B and T cell epitopes with uniform stoichiometry and high density. Here we compared the relative activities of a TCC-nic vaccine and two control KLH-nic vaccines using Alum as an adjuvant or GLA-SE, which contains a synthetic TLR4 agonist formulated in a stable oil-in-water emulsion. The results showed that the TCC's high hapten density correlated with a better immune response in mice as measured by anti-nicotine Ab titer, affinity, and specificity, and was responsible for a reduction in anti-carrier immunogenicity. The Ab responses achieved with this synthetic vaccine resulted in a nicotine binding capacity in serum that could prevent >90% of a nicotine dose equivalent to three smoked cigarettes (0.05 mg/kg from reaching the brain.

  16. Immunogenicity of coiled-coil based drug-free macromolecular therapeutics

    Czech Academy of Sciences Publication Activity Database

    Kverka, Miloslav; Hartley, J.M.; Chu, T.W.; Yang, J.; Heidchen, R.; Kopeček, J.

    2014-01-01

    Roč. 35, č. 2 (2014), s. 5886-5896 ISSN 1616-0177 R&D Projects: GA MŠk(CZ) EE2.3.30.0003 Grant - others:NIH(US) GM095606 Institutional support: RVO:61388971 Keywords : coiled-coil * enantiomers * HPMA copolymer Subject RIV: EC - Immunology

  17. Golgi coiled-coil proteins contain multiple binding sites for Rab family G proteins

    NARCIS (Netherlands)

    Sinka, Rita; Gillingham, Alison K.; Kondylis, Vangelis; Munro, Sean

    2008-01-01

    Vesicles and other carriers destined for the Golgi apparatus must be guided to the correct cisternae. Golgins, long coiled-coil proteins that localize to particular Golgi subdomains via their C termini, are candidate regulators of vesicle sorting. In this study, we report that the GRIP domain

  18. Automated de novo phasing and model building of coiled-coil proteins.

    Science.gov (United States)

    Rämisch, Sebastian; Lizatović, Robert; André, Ingemar

    2015-03-01

    Models generated by de novo structure prediction can be very useful starting points for molecular replacement for systems where suitable structural homologues cannot be readily identified. Protein-protein complexes and de novo-designed proteins are examples of systems that can be challenging to phase. In this study, the potential of de novo models of protein complexes for use as starting points for molecular replacement is investigated. The approach is demonstrated using homomeric coiled-coil proteins, which are excellent model systems for oligomeric systems. Despite the stereotypical fold of coiled coils, initial phase estimation can be difficult and many structures have to be solved with experimental phasing. A method was developed for automatic structure determination of homomeric coiled coils from X-ray diffraction data. In a benchmark set of 24 coiled coils, ranging from dimers to pentamers with resolutions down to 2.5 Å, 22 systems were automatically solved, 11 of which had previously been solved by experimental phasing. The generated models contained 71-103% of the residues present in the deposited structures, had the correct sequence and had free R values that deviated on average by 0.01 from those of the respective reference structures. The electron-density maps were of sufficient quality that only minor manual editing was necessary to produce final structures. The method, named CCsolve, combines methods for de novo structure prediction, initial phase estimation and automated model building into one pipeline. CCsolve is robust against errors in the initial models and can readily be modified to make use of alternative crystallographic software. The results demonstrate the feasibility of de novo phasing of protein-protein complexes, an approach that could also be employed for other small systems beyond coiled coils.

  19. Four signature motifs define the first class of structurally related large coiled-coil proteins in plants.

    Directory of Open Access Journals (Sweden)

    Meier Iris

    2002-04-01

    Full Text Available Abstract Background Animal and yeast proteins containing long coiled-coil domains are involved in attaching other proteins to the large, solid-state components of the cell. One subgroup of long coiled-coil proteins are the nuclear lamins, which are involved in attaching chromatin to the nuclear envelope and have recently been implicated in inherited human diseases. In contrast to other eukaryotes, long coiled-coil proteins have been barely investigated in plants. Results We have searched the completed Arabidopsis genome and have identified a family of structurally related long coiled-coil proteins. Filament-like plant proteins (FPP were identified by sequence similarity to a tomato cDNA that encodes a coiled-coil protein which interacts with the nuclear envelope-associated protein, MAF1. The FPP family is defined by four novel unique sequence motifs and by two clusters of long coiled-coil domains separated by a non-coiled-coil linker. All family members are expressed in a variety of Arabidopsis tissues. A homolog sharing the structural features was identified in the monocot rice, indicating conservation among angiosperms. Conclusion Except for myosins, this is the first characterization of a family of long coiled-coil proteins in plants. The tomato homolog of the FPP family binds in a yeast two-hybrid assay to a nuclear envelope-associated protein. This might suggest that FPP family members function in nuclear envelope biology. Because the full Arabidopsis genome does not appear to contain genes for lamins, it is of interest to investigate other long coiled-coil proteins, which might functionally replace lamins in the plant kingdom.

  20. CCBuilder 2.0: Powerful and accessible coiled-coil modeling.

    Science.gov (United States)

    Wood, Christopher W; Woolfson, Derek N

    2018-01-01

    The increased availability of user-friendly and accessible computational tools for biomolecular modeling would expand the reach and application of biomolecular engineering and design. For protein modeling, one key challenge is to reduce the complexities of 3D protein folds to sets of parametric equations that nonetheless capture the salient features of these structures accurately. At present, this is possible for a subset of proteins, namely, repeat proteins. The α-helical coiled coil provides one such example, which represents ≈ 3-5% of all known protein-encoding regions of DNA. Coiled coils are bundles of α helices that can be described by a small set of structural parameters. Here we describe how this parametric description can be implemented in an easy-to-use web application, called CCBuilder 2.0, for modeling and optimizing both α-helical coiled coils and polyproline-based collagen triple helices. This has many applications from providing models to aid molecular replacement for X-ray crystallography, in silico model building and engineering of natural and designed protein assemblies, and through to the creation of completely de novo "dark matter" protein structures. CCBuilder 2.0 is available as a web-based application, the code for which is open-source and can be downloaded freely. http://coiledcoils.chm.bris.ac.uk/ccbuilder2. We have created CCBuilder 2.0, an easy to use web-based application that can model structures for a whole class of proteins, the α-helical coiled coil, which is estimated to account for 3-5% of all proteins in nature. CCBuilder 2.0 will be of use to a large number of protein scientists engaged in fundamental studies, such as protein structure determination, through to more-applied research including designing and engineering novel proteins that have potential applications in biotechnology. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

  1. Strong contributions from vertical triads to helix-partner preferences in parallel coiled coils.

    Science.gov (United States)

    Steinkruger, Jay D; Bartlett, Gail J; Woolfson, Derek N; Gellman, Samuel H

    2012-09-26

    Pairing preferences in heterodimeric coiled coils are determined by complementarities among side chains that pack against one another at the helix-helix interface. However, relationships between dimer stability and interfacial residue identity are not fully understood. In the context of the "knobs-into-holes" (KIH) packing pattern, one can identify two classes of interactions between side chains from different helices: "lateral", in which a line connecting the adjacent side chains is perpendicular to the helix axes, and "vertical", in which the connecting line is parallel to the helix axes. We have previously analyzed vertical interactions in antiparallel coiled coils and found that one type of triad constellation (a'-a-a') exerts a strong effect on pairing preferences, while the other type of triad (d'-d-d') has relatively little impact on pairing tendencies. Here, we ask whether vertical interactions (d'-a-d') influence pairing in parallel coiled-coil dimers. Our results indicate that vertical interactions can exert a substantial impact on pairing specificity, and that the influence of the d'-a-d' triad depends on the lateral a' contact within the local KIH motif. Structure-informed bioinformatic analyses of protein sequences reveal trends consistent with the thermodynamic data derived from our experimental model system in suggesting that heterotriads involving Leu and Ile are preferred over homotriads involving Leu and Ile.

  2. Fine-tuning of protein domain boundary by minimizing potential coiled coil regions

    International Nuclear Information System (INIS)

    Iwaya, Naoko; Goda, Natsuko; Unzai, Satoru; Fujiwara, Kenichiro; Tanaka, Toshiki; Tomii, Kentaro; Tochio, Hidehito; Shirakawa, Masahiro; Hiroaki, Hidekazu

    2007-01-01

    Structural determination of individual protein domains isolated from multidomain proteins is a common approach in the post-genomic era. Novel and thus uncharacterized domains liberated from intact proteins often self-associate due to incorrectly defined domain boundaries. Self-association results in missing signals, poor signal dispersion and a low signal-to-noise ratio in 1 H- 15 N HSQC spectra. We have found that a putative, non-canonical coiled coil region close to a domain boundary can cause transient hydrophobic self-association and monomer-dimer equilibrium in solution. Here we propose a rational method to predict putative coiled coil regions adjacent to the globular core domain using the program COILS. Except for the amino acid sequence, no preexisting knowledge concerning the domain is required. A small number of mutant proteins with a minimized coiled coil region have been rationally designed and tested. The engineered domains exhibit decreased self-association as assessed by 1 H- 15 N HSQC spectra with improved peak dispersion and sharper cross peaks. Two successful examples of isolating novel N-terminal domains from AAA-ATPases are demonstrated. Our method is useful for the experimental determination of domain boundaries suited for structural genomics studies

  3. Fine-tuning of protein domain boundary by minimizing potential coiled coil regions.

    Science.gov (United States)

    Iwaya, Naoko; Goda, Natsuko; Unzai, Satoru; Fujiwara, Kenichiro; Tanaka, Toshiki; Tomii, Kentaro; Tochio, Hidehito; Shirakawa, Masahiro; Hiroaki, Hidekazu

    2007-01-01

    Structural determination of individual protein domains isolated from multidomain proteins is a common approach in the post-genomic era. Novel and thus uncharacterized domains liberated from intact proteins often self-associate due to incorrectly defined domain boundaries. Self-association results in missing signals, poor signal dispersion and a low signal-to-noise ratio in (1)H-(15)N HSQC spectra. We have found that a putative, non-canonical coiled coil region close to a domain boundary can cause transient hydrophobic self-association and monomer-dimer equilibrium in solution. Here we propose a rational method to predict putative coiled coil regions adjacent to the globular core domain using the program COILS. Except for the amino acid sequence, no preexisting knowledge concerning the domain is required. A small number of mutant proteins with a minimized coiled coil region have been rationally designed and tested. The engineered domains exhibit decreased self-association as assessed by (1)H-(15)N HSQC spectra with improved peak dispersion and sharper cross peaks. Two successful examples of isolating novel N-terminal domains from AAA-ATPases are demonstrated. Our method is useful for the experimental determination of domain boundaries suited for structural genomics studies.

  4. Historical review: another 50th anniversary--new periodicities in coiled coils.

    Science.gov (United States)

    Gruber, Markus; Lupas, Andrei N

    2003-12-01

    In 1953, Francis Crick and Linus Pauling both proposed models of supercoiled alpha helices ('coiled coils') for the structure of keratin. These were the first attempts at modelling the tertiary structure of a protein. Crick emphasized the packing mode of the side-chains ('knobs-into-holes'), which required a periodicity of seven residues over two helical turns (7/2) and a supercoil in the opposite sense of the constituent helices. By contrast, Pauling envisaged a broader set of periodicities (4/1, 7/2, 18/5, 15/4, 11/3) and supercoils of both senses. Crick's model became canonical and the 'heptad repeat' essentially synonymous with coiled coils, but 50 years later new crystal structures and protein sequences show that the less common periodicities envisaged by Pauling also occur in coiled coils, adding a variant packing mode ('knobs-to-knobs') to the standard model. Pauling's laboratory notebooks suggest that he searched unsuccessfully for this packing mode in 1953.

  5. Coiled-Coil Proteins Facilitated the Functional Expansion of the Centrosome

    Science.gov (United States)

    Kuhn, Michael; Hyman, Anthony A.; Beyer, Andreas

    2014-01-01

    Repurposing existing proteins for new cellular functions is recognized as a main mechanism of evolutionary innovation, but its role in organelle evolution is unclear. Here, we explore the mechanisms that led to the evolution of the centrosome, an ancestral eukaryotic organelle that expanded its functional repertoire through the course of evolution. We developed a refined sequence alignment technique that is more sensitive to coiled coil proteins, which are abundant in the centrosome. For proteins with high coiled-coil content, our algorithm identified 17% more reciprocal best hits than BLAST. Analyzing 108 eukaryotic genomes, we traced the evolutionary history of centrosome proteins. In order to assess how these proteins formed the centrosome and adopted new functions, we computationally emulated evolution by iteratively removing the most recently evolved proteins from the centrosomal protein interaction network. Coiled-coil proteins that first appeared in the animal–fungi ancestor act as scaffolds and recruit ancestral eukaryotic proteins such as kinases and phosphatases to the centrosome. This process created a signaling hub that is crucial for multicellular development. Our results demonstrate how ancient proteins can be co-opted to different cellular localizations, thereby becoming involved in novel functions. PMID:24901223

  6. Unraveling double stranded alpha-helical coiled coils: an x-ray diffraction study on hard alpha-keratin fibers.

    Science.gov (United States)

    Kreplak, L; Doucet, J; Briki, F

    2001-04-15

    Transformations of proteins secondary and tertiary structures are generally studied in globular proteins in solution. In fibrous proteins, such as hard alpha-keratin, that contain long and well-defined double stranded alpha-helical coiled coil domains, such study can be directly done on the native fibrous tissue. In order to assess the structural behavior of the coiled coil domains under an axial mechanical stress, wide angle x-ray scattering and small angle x-ray scattering experiments have been carried out on stretched horse hair fibers at relative humidity around 30%. Our observations of the three major axial spacings as a function of the applied macroscopic strain have shown two rates. Up to 4% macroscopic strain the coiled coils were slightly distorted but retained their overall conformation. Above 4% the proportion of coiled coil domains progressively decreased. The main and new result of our study is the observation of the transition from alpha-helical coiled coils to disordered chains instead of the alpha-helical coiled coil to beta-sheet transition that occurs in wet fibers.

  7. CCBuilder: an interactive web-based tool for building, designing and assessing coiled-coil protein assemblies.

    Science.gov (United States)

    Wood, Christopher W; Bruning, Marc; Ibarra, Amaurys Á; Bartlett, Gail J; Thomson, Andrew R; Sessions, Richard B; Brady, R Leo; Woolfson, Derek N

    2014-11-01

    The ability to accurately model protein structures at the atomistic level underpins efforts to understand protein folding, to engineer natural proteins predictably and to design proteins de novo. Homology-based methods are well established and produce impressive results. However, these are limited to structures presented by and resolved for natural proteins. Addressing this problem more widely and deriving truly ab initio models requires mathematical descriptions for protein folds; the means to decorate these with natural, engineered or de novo sequences; and methods to score the resulting models. We present CCBuilder, a web-based application that tackles the problem for a defined but large class of protein structure, the α-helical coiled coils. CCBuilder generates coiled-coil backbones, builds side chains onto these frameworks and provides a range of metrics to measure the quality of the models. Its straightforward graphical user interface provides broad functionality that allows users to build and assess models, in which helix geometry, coiled-coil architecture and topology and protein sequence can be varied rapidly. We demonstrate the utility of CCBuilder by assembling models for 653 coiled-coil structures from the PDB, which cover >96% of the known coiled-coil types, and by generating models for rarer and de novo coiled-coil structures. CCBuilder is freely available, without registration, at http://coiledcoils.chm.bris.ac.uk/app/cc_builder/. © The Author 2014. Published by Oxford University Press.

  8. Polymer therapeutics with a coiled coil motif targeted against murine BCL1 leukemia

    Czech Academy of Sciences Publication Activity Database

    Pola, Robert; Laga, Richard; Ulbrich, Karel; Sieglová, Irena; Král, Vlastimil; Fábry, Milan; Kabešová, Martina; Kovář, Marek; Pechar, Michal

    2013-01-01

    Roč. 14, č. 3 (2013), s. 881-889 ISSN 1525-7797 R&D Projects: GA ČR GAP301/11/0325; GA AV ČR IAAX00500803 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:61389013 ; RVO:61388971 ; RVO:68378050 Keywords : coiled coil * polymer therapeutics * scFv Subject RIV: CD - Macromolecular Chemistry; EC - Immunology (MBU-M); EB - Genetics ; Molecular Biology (UMG-J) Impact factor: 5.788, year: 2013

  9. Polymer cancerostatics with a coiled coil motif targeted against murine leukemia

    Czech Academy of Sciences Publication Activity Database

    Pechar, Michal; Pola, Robert; Janoušková, Olga; Sieglová, Irena; Král, Vlastimil; Fábry, Milan; Tomalová, Barbora; Kovář, Marek

    2017-01-01

    Roč. 6, 4 (Suppl) (2017), s. 36 ISSN 2325-9604. [International Conference and Exhibition on Nanomedicine and Drug Delivery. 29.05.2017-31.05.2017, Osaka] R&D Projects: GA ČR(CZ) GA16-17207S Institutional support: RVO:61389013 ; RVO:68378050 ; RVO:61388971 Keywords : coiled coil * polymer cancerostatics * active targeting Subject RIV: CD - Macromolecular Chemistry; EC - Immunology (MBU-M) https://www.scitechnol.com/conference-abstracts/scientific-tracks-abstracts/nanodelivery-2017-proceedings.html

  10. An analysis of 3D solvation structure in biomolecules: application to coiled coil serine and bacteriorhodopsin.

    Science.gov (United States)

    Hirano, Kenji; Yokogawa, Daisuke; Sato, Hirofumi; Sakaki, Shigeyoshi

    2010-06-17

    Three-dimensional (3D) solvation structure around coiled coil serine (Coil-Ser) and inner 3D hydration structure in bacteriorhodopsin (bR) were studied using a recently developed method named multicenter molecular Ornstein-Zernike equation (MC-MOZ) theory. In addition, a procedure for analyzing the 3D solvent distribution was proposed. The method enables us to calculate the coordination number of solvent water as well as the strength of hydrogen bonding between the water molecule and the protein. The results for Coil-Ser and bR showed very good agreement with the experimental observations.

  11. Fifty years of coiled-coils and alpha-helical bundles: a close relationship between sequence and structure.

    Science.gov (United States)

    Parry, David A D; Fraser, R D Bruce; Squire, John M

    2008-09-01

    alpha-Helical coiled coils are remarkable for the diversity of related conformations that they adopt in both fibrous and globular proteins, and for the range of functions that they exhibit. The coiled coils are based on a heptad (7-residue), hendecad (11-residue) or a related quasi-repeat of apolar residues in the sequences of the alpha-helical regions involved. Most of these, however, display one or more sequence discontinuities known as stutters or stammers. The resulting coiled coils vary in length, in the number of chains participating, in the relative polarity of the contributing alpha-helical regions (parallel or antiparallel), and in the pitch length and handedness of the supercoil (left- or right-handed). Functionally, the concept that a coiled coil can act only as a static rod is no longer valid, and the range of roles that these structures have now been shown to exhibit has expanded rapidly in recent years. An important development has been the recognition that the delightful simplicity that exists between sequence and structure, and between structure and function, allows coiled coils with specialized features to be designed de novo.

  12. Invited review the coiled coil silk of bees, ants, and hornets.

    Science.gov (United States)

    Sutherland, Tara D; Weisman, Sarah; Walker, Andrew A; Mudie, Stephen T

    2012-06-01

    In this article, we review current knowledge about the silk produced by the larvae of bees, ants, and hornets [Apoidea and Vespoidea: Hymenoptera]. Different species use the silk either alone or in composites for a variety of purposes including mechanical reinforcement, thermal regulation, or humidification. The characteristic molecular structure of this silk is α-helical proteins assembled into tetrameric coiled coils. Gene sequences from seven species are available, and each species possesses a copy of each of four related silk genes that encode proteins predicted to form coiled coils. The proteins are ordered at multiple length scales within the labial gland of the final larval instar before spinning. The insects control the morphology of the silk during spinning to produce either fibers or sheets. The silk proteins are small and non repetitive and have been produced artificially at high levels by fermentation in E. coli. The artificial silk proteins can be fabricated into materials with structural and mechanical properties similar to those of native silks. Copyright © 2011 Wiley Periodicals, Inc.

  13. d-Cysteine Ligands Control Metal Geometries within De Novo Designed Three-Stranded Coiled Coils

    DEFF Research Database (Denmark)

    Ruckthong, Leela; Peacock, Anna F.A.; Pascoe, Cherilyn E.

    2017-01-01

    Although metal ion binding to naturally occurring l-amino acid proteins is well documented, understanding the impact of the opposite chirality (d-)amino acids on the structure and stereochemistry of metals is in its infancy. We examine the effect of a d-configuration cysteine within a designed l......-amino acid three-stranded coiled coil in order to enforce a precise coordination number on a metal center. The d chirality does not alter the native fold, but the side-chain re-orientation modifies the sterics of the metal binding pocket. l-Cys side chains within the coiled-coil structure have previously...... by comparison of the structure of ZnIICl(CSL16DC)3 2- to the published structure of ZnII(H2O)(GRAND-CSL12AL16LC)3 -. Moreover, spectroscopic analysis indicates that the CdII geometry observed by using l-Cys ligands (a mixture of three- and four-coordinate CdII) is altered to a single four-coordinate species...

  14. Structure and Misfolding of the Flexible Tripartite Coiled-Coil Domain of Glaucoma-Associated Myocilin

    Energy Technology Data Exchange (ETDEWEB)

    Hill, Shannon E.; Nguyen, Elaine; Donegan, Rebecca K.; Patterson-Orazem, Athéna C.; Hazel, Anthony; Gumbart, James C.; Lieberman, Raquel L.

    2017-11-01

    Glaucoma-associated myocilin is a member of the olfactomedins, a protein family involved in neuronal development and human diseases. Molecular studies of the myocilin N-terminal coiled coil demonstrate a unique tripartite architecture: a Y-shaped parallel dimer-of-dimers with distinct tetramer and dimer regions. The structure of the dimeric C-terminal 7-heptad repeats elucidates an unexpected repeat pattern involving inter-strand stabilization by oppositely charged residues. Molecular dynamics simulations reveal an alternate accessible conformation in which the terminal inter-strand disulfide limits the extent of unfolding and results in a kinked configuration. By inference, full-length myocilin is also branched, with two pairs of C-terminal olfactomedin domains. Selected variants within the N-terminal region alter the apparent quaternary structure of myocilin but do so without compromising stability or causing aggregation. In addition to increasing our structural knowledge of naturally occurring extracellular coiled coils and biomedically important olfactomedins, this work broadens the scope of protein misfolding in the pathogenesis of myocilin-associated glaucoma.

  15. Structure and Misfolding of the Flexible Tripartite Coiled-Coil Domain of Glaucoma-Associated Myocilin.

    Science.gov (United States)

    Hill, Shannon E; Nguyen, Elaine; Donegan, Rebecca K; Patterson-Orazem, Athéna C; Hazel, Anthony; Gumbart, James C; Lieberman, Raquel L

    2017-11-07

    Glaucoma-associated myocilin is a member of the olfactomedins, a protein family involved in neuronal development and human diseases. Molecular studies of the myocilin N-terminal coiled coil demonstrate a unique tripartite architecture: a Y-shaped parallel dimer-of-dimers with distinct tetramer and dimer regions. The structure of the dimeric C-terminal 7-heptad repeats elucidates an unexpected repeat pattern involving inter-strand stabilization by oppositely charged residues. Molecular dynamics simulations reveal an alternate accessible conformation in which the terminal inter-strand disulfide limits the extent of unfolding and results in a kinked configuration. By inference, full-length myocilin is also branched, with two pairs of C-terminal olfactomedin domains. Selected variants within the N-terminal region alter the apparent quaternary structure of myocilin but do so without compromising stability or causing aggregation. In addition to increasing our structural knowledge of naturally occurring extracellular coiled coils and biomedically important olfactomedins, this work broadens the scope of protein misfolding in the pathogenesis of myocilin-associated glaucoma. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Magnetic field alignment of coil-coil diblock copolymers and blends via intrinsic chain anisotropy

    Science.gov (United States)

    Rokhlenko, Yekaterina; Majewski, Pawel; Larson, Steven; Yager, Kevin; Gopalan, Padma; Avgeropoulos, Apostolos; Chan, Edwin; Osuji, Chinedum

    Magnetic fields can control alignment of self-assembled soft materials such as block copolymers provided there is a suitably large magnetic susceptibility anisotropy present in the system. Recent results have highlighted the existence of a non-trivial intrinsic anisotropy in coil-coil diblock copolymers, specifically in lamellar-forming PS-b-P4VP, which enables alignment at field strengths of a few tesla in systems lacking mesogenic components. Alignment is predicated on correlation in the orientation of end-end vectors implied by the localization of block junctions at the microdomain interface and is observed on cooling across the order-disorder transition in the presence of the field. For appropriate combinations of field strength and grain size, we can leverage intrinsic chain anisotropy to magnetically direct self-assembly of many non-mesogenic systems, including other coil-coil BCPs like PS-b-PDMS and PS-b-PMMA, blends of BCPs of disparate morphologies and MWs, and blends of BCPs with homopolymers. This is noteworthy as blends of PS-b-P4VP with PEO provide a route to form functional materials such as nanoporous films by dissolution of PEO, or aligned ion conduction materials. We survey these various systems using TEM and in-situ X-ray scattering to study the phase behavior and temperature-, time- and field- dependent dynamics of alignment.

  17. Structure of a designed, right-handed coiled-coil tetramer containing all biological amino acids.

    Science.gov (United States)

    Sales, Mark; Plecs, Joseph J; Holton, James M; Alber, Tom

    2007-10-01

    The previous design of an unprecedented family of two-, three-, and four-helical, right-handed coiled coils utilized nonbiological amino acids to efficiently pack spaces in the oligomer cores. Here we show that a stable, right-handed parallel tetrameric coiled coil, called RH4B, can be designed entirely using biological amino acids. The X-ray crystal structure of RH4B was determined to 1.1 Angstrom resolution using a designed metal binding site to coordinate a single Yb(2+) ion per 33-amino acid polypeptide chain. The resulting experimental phases were particularly accurate, and the experimental electron density map provided an especially clear, unbiased view of the molecule. The RH4B structure closely matched the design, with equivalent core rotamers and an overall root-mean-square deviation for the N-terminal repeat of the tetramer of 0.24 Angstrom. The clarity and resolution of the electron density map, however, revealed alternate rotamers and structural differences between the three sequence repeats in the molecule. These results suggest that the RH4B structure populates an unanticipated variety of structures.

  18. Inhibition of peptide aggregation by means of enzymatic phosphorylation

    Directory of Open Access Journals (Sweden)

    Kristin Folmert

    2016-11-01

    Full Text Available As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP-dependent protein kinase that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β-sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated.

  19. Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain.

    Science.gov (United States)

    Tao, Y; Strelkov, S V; Mesyanzhinov, V V; Rossmann, M G

    1997-06-15

    Oligomeric coiled-coil motifs are found in numerous protein structures; among them is fibritin, a structural protein of bacteriophage T4, which belongs to a class of chaperones that catalyze a specific phage-assembly process. Fibritin promotes the assembly of the long tail fibers and their subsequent attachment to the tail baseplate; it is also a sensing device that controls the retraction of the long tail fibers in adverse environments and, thus, prevents infection. The structure of fibritin had been predicted from sequence and biochemical analyses to be mainly a triple-helical coiled coil. The determination of its structure at atomic resolution was expected to give insights into the assembly process and biological function of fibritin, and the properties of modified coiled-coil structures in general. The three-dimensional structure of fibritin E, a deletion mutant of wild-type fibritin, was determined to 2.2 A resolution by X-ray crystallography. Three identical subunits of 119 amino acid residues form a trimeric parallel coiled-coil domain and a small globular C-terminal domain about a crystallographic threefold axis. The coiled-coil domain is divided into three segments that are separated by insertion loops. The C-terminal domain, which consists of 30 residues from each subunit, contains a beta-propeller-like structure with a hydrophobic interior. The residues within the C-terminal domain make extensive hydrophobic and some polar intersubunit interactions. This is consistent with the C-terminal domain being important for the correct assembly of fibritin, as shown earlier by mutational studies. Tight interactions between the C-terminal residues of adjacent subunits counteract the latent instability that is suggested by the structural properties of the coiled-coil segments. Trimerization is likely to begin with the formation of the C-terminal domain which subsequently initiates the assembly of the coiled coil. The interplay between the stabilizing effect of the C

  20. Rho-associated coiled-coil kinase (ROCK) protein controls microtubule dynamics in a novel signaling pathway that regulates cell migration.

    Science.gov (United States)

    Schofield, Alice V; Steel, Rohan; Bernard, Ora

    2012-12-21

    The two members of the Rho-associated coiled-coil kinase (ROCK1 and 2) family are established regulators of actin dynamics that are involved in the regulation of the cell cycle as well as cell motility and invasion. Here, we discovered a novel signaling pathway whereby ROCK regulates microtubule (MT) acetylation via phosphorylation of the tubulin polymerization promoting protein 1 (TPPP1/p25). We show that ROCK phosphorylation of TPPP1 inhibits the interaction between TPPP1 and histone deacetylase 6 (HDAC6), which in turn results in increased HDAC6 activity followed by a decrease in MT acetylation. As a consequence, we show that TPPP1 phosphorylation by ROCK increases cell migration and invasion via modulation of cellular acetyl MT levels. We establish here that the ROCK-TPPP1-HDAC6 signaling pathway is important for the regulation of cell migration and invasion.

  1. Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment.

    Directory of Open Access Journals (Sweden)

    Xinwei Wang

    Full Text Available A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to V(H and V(L for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of V(H frameworks and V(H-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering.

  2. Low resolution crystal structure of Arenicola erythrocruorin: influence of coiled coils on the architecture of a megadalton respiratory protein.

    Science.gov (United States)

    Royer, William E; Omartian, Michael N; Knapp, James E

    2007-01-05

    Annelid erythrocruorins are extracellular respiratory complexes assembled from 180 subunits into hexagonal bilayers. Cryo-electron microscopic experiments have identified two different architectural classes. In one, designated type I, the vertices of the two hexagonal layers are partially staggered, with one hexagonal layer rotated by about 16 degrees relative to the other layer, whereas in the other class, termed type II, the vertices are essentially eclipsed. We report here the first crystal structure of a type II erythrocruorin, that from Arenicola marina, at 6.2 A resolution. The structure reveals the presence of long continuous triple-stranded coiled-coil "spokes" projecting towards the molecular center from each one-twelfth unit; interdigitation of these spokes provides the only contacts between the two hexagonal layers of the complex. This arrangement contrasts with that of a type I erythrocruorin from Lumbricus terrestris in which the spokes are broken into two triple-stranded coiled coils with a disjointed connection. The disjointed connection allows formation of a more compact structure in the type I architecture, with the two hexagonal layers closer together and additional extensive contacts between the layers. Comparison of sequences of the coiled-coil regions of various linker subunits shows that the linker subunits from type II erythrocruorins possess continuous heptad repeats, whereas a sequence gap places these repeats out of register in the type I linker subunits, consistent with a disjointed coiled-coil arrangement.

  3. Missense mutation in DISC1 C-terminal coiled-coil has GSK3β signaling and sex-dependent behavioral effects in mice

    Science.gov (United States)

    Dachtler, James; Elliott, Christina; Rodgers, R. John; Baillie, George S.; Clapcote, Steven J.

    2016-01-01

    Disrupted-in-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and affective disorders. The full-length DISC1 protein consists of an N-terminal ‘head’ domain and a C-terminal tail domain that contains several predicted coiled-coils, structural motifs involved in protein-protein interactions. To probe the in vivo effects of missense mutation of DISC1’s C-terminal tail, we tested mice carrying mutation D453G within a predicted α-helical coiled-coil region. We report that, relative to wild-type littermates, female DISC1D453G mice exhibited novelty-induced hyperlocomotion, an anxiogenic profile in the elevated plus-maze and open field tests, and reduced social exploration of unfamiliar mice. Male DISC1D453G mice displayed a deficit in passive avoidance, while neither males nor females exhibited any impairment in startle reactivity or prepulse inhibition. Whole brain homogenates showed normal levels of DISC1 protein, but decreased binding of DISC1 to GSK3β, decreased phospho-inhibition of GSK3β at serine 9, and decreased levels of β-catenin in DISC1D453G mice of either sex. Interrupted GSK3β signaling may thus be part of the mechanism underlying the behavioral phenotype associated with D453G, in common with the previously described N-terminal domain mutations Q31L and L100P in mice, and the schizophrenia risk-conferring variant R264Q in humans. PMID:26728762

  4. Peptide inhibition of human cytomegalovirus infection

    Directory of Open Access Journals (Sweden)

    Morris Cindy A

    2011-02-01

    Full Text Available Abstract Background Human cytomegalovirus (HCMV is the most prevalent congenital viral infection in the United States and Europe causing significant morbidity and mortality to both mother and child. HCMV is also an opportunistic pathogen in immunocompromised individuals, including human immunodeficiency virus (HIV- infected patients with AIDS, and solid organ and allogeneic stem cell transplantation recipients. Current treatments for HCMV-associated diseases are insufficient due to the emergence of drug-induced resistance and cytotoxicity, necessitating novel approaches to limit HCMV infection. The aim of this study was to develop therapeutic peptides targeting glycoprotein B (gB, a major glycoprotein of HCMV that is highly conserved across the Herpesviridae family, that specifically inhibit fusion of the viral envelope with the host cell membrane preventing HCMV entry and infection. Results Using the Wimley-White Interfacial Hydrophobicity Scale (WWIHS, several regions within gB were identified that display a high potential to interact with lipid bilayers of cell membranes and hydrophobic surfaces within proteins. The ability of synthetic peptides analogous to WWIHS-positive sequences of HCMV gB to inhibit viral infectivity was evaluated. Human foreskin fibroblasts (HFF were infected with the Towne-GFP strain of HCMV (0.5 MOI, preincubated with peptides at a range of concentrations (78 nm to 100 μM, and GFP-positive cells were visualized 48 hours post-infection by fluorescence microscopy and analyzed quantitatively by flow cytometry. Peptides that inhibited HCMV infection demonstrated different inhibitory concentration curves indicating that each peptide possesses distinct biophysical properties. Peptide 174-200 showed 80% inhibition of viral infection at a concentration of 100 μM, and 51% and 62% inhibition at concentrations of 5 μM and 2.5 μM, respectively. Peptide 233-263 inhibited infection by 97% and 92% at concentrations of 100

  5. Intermediate filament mechanics in vitro and in the cell: From coiled coils to filaments, fibers and networks

    OpenAIRE

    Köster, Sarah; Weitz, David; Goldman, Robert D.; Aebi, Ueli; Herrmann, Harald

    2015-01-01

    Intermediate filament proteins form filaments, fibers and networks both in the cytoplasm and the nucleus of metazoan cells. Their general structural building plan accommodates highly varying amino acid sequences to yield extended dimeric α-helical coiled coils of highly conserved design. These “rod” particles are the basic building blocks of intrinsically flexible, filamentous structures that are able to resist high mechanical stresses, i.e. bending and stretching to a considerable degree, bo...

  6. Rice Cellulose SynthaseA8 Plant-Conserved Region Is a Coiled-Coil at the Catalytic Core Entrance

    Energy Technology Data Exchange (ETDEWEB)

    Rushton, Phillip S.; Olek, Anna T.; Makowski, Lee; Badger, John; Steussy, C. Nicklaus; Carpita, Nicholas C.; Stauffacher, Cynthia V. (NEU); (Purdue)

    2016-11-22

    The crystallographic structure of a rice (Oryza sativa) cellulose synthase, OsCesA8, plant-conserved region (P-CR), one of two unique domains in the catalytic domain of plant CesAs, was solved to 2.4 Å resolution. Two antiparallel α-helices form a coiled-coil domain linked by a large extended connector loop containing a conserved trio of aromatic residues. The P-CR structure was fit into a molecular envelope for the P-CR domain derived from small-angle X-ray scattering data. The P-CR structure and molecular envelope, combined with a homology-based chain trace of the CesA8 catalytic core, were modeled into a previously determined CesA8 small-angle X-ray scattering molecular envelope to produce a detailed topological model of the CesA8 catalytic domain. The predicted position for the P-CR domain from the molecular docking models places the P-CR connector loop into a hydrophobic pocket of the catalytic core, with the coiled-coil aligned near the entrance of the substrate UDP-glucose into the active site. In this configuration, the P-CR coiled-coil alone is unlikely to regulate substrate access to the active site, but it could interact with other domains of CesA, accessory proteins, or other CesA catalytic domains to control substrate delivery.

  7. Structural mapping of the coiled-coil domain of a bacterial condensin and comparative analyses across all domains of life suggest conserved features of SMC proteins.

    Science.gov (United States)

    Waldman, Vincent M; Stanage, Tyler H; Mims, Alexandra; Norden, Ian S; Oakley, Martha G

    2015-06-01

    The structural maintenance of chromosomes (SMC) proteins form the cores of multisubunit complexes that are required for the segregation and global organization of chromosomes in all domains of life. These proteins share a common domain structure in which N- and C- terminal regions pack against one another to form a globular ATPase domain. This "head" domain is connected to a central, globular, "hinge" or dimerization domain by a long, antiparallel coiled coil. To date, most efforts for structural characterization of SMC proteins have focused on the globular domains. Recently, however, we developed a method to map interstrand interactions in the 50-nm coiled-coil domain of MukB, the divergent SMC protein found in γ-proteobacteria. Here, we apply that technique to map the structure of the Bacillus subtilis SMC (BsSMC) coiled-coil domain. We find that, in contrast to the relatively complicated coiled-coil domain of MukB, the BsSMC domain is nearly continuous, with only two detectable coiled-coil interruptions. Near the middle of the domain is a break in coiled-coil structure in which there are three more residues on the C-terminal strand than on the N-terminal strand. Close to the head domain, there is a second break with a significantly longer insertion on the same strand. These results provide an experience base that allows an informed interpretation of the output of coiled-coil prediction algorithms for this family of proteins. A comparison of such predictions suggests that these coiled-coil deviations are highly conserved across SMC types in a wide variety of organisms, including humans. © 2015 Wiley Periodicals, Inc.

  8. MURC, a muscle-restricted coiled-coil protein that modulates the Rho/ROCK pathway, induces cardiac dysfunction and conduction disturbance.

    Science.gov (United States)

    Ogata, Takehiro; Ueyama, Tomomi; Isodono, Koji; Tagawa, Masashi; Takehara, Naofumi; Kawashima, Tsuneaki; Harada, Koichiro; Takahashi, Tomosaburo; Shioi, Tetsuo; Matsubara, Hiroaki; Oh, Hidemasa

    2008-05-01

    We identified a novel muscle-restricted putative coiled-coil protein, MURC, which is evolutionarily conserved from frog to human. MURC was localized to the cytoplasm with accumulation in the Z-line of the sarcomere in the murine adult heart. MURC mRNA expression in the heart increased during the developmental process from the embryonic stage to adulthood. In response to pressure overload, MURC mRNA expression increased in the hypertrophied heart. Using the yeast two-hybrid system, we identified the serum deprivation response (SDPR) protein, a phosphatidylserine-binding protein, as a MURC-binding protein. MURC induced activation of the RhoA/ROCK pathway, which modulated serum response factor-mediated atrial natriuretic peptide (ANP) expression and myofibrillar organization. SDPR augmented MURC-induced transactivation of the ANP promoter in cardiomyocytes, and RNA interference of SDPR attenuated the action of MURC on the ANP promoter. Transgenic mice expressing cardiac-specific MURC (Tg-MURC) exhibited cardiac contractile dysfunction and atrioventricular (AV) conduction disturbances with atrial chamber enlargement, reduced thickness of the ventricular wall, and interstitial fibrosis. Spontaneous episodes of atrial fibrillation and AV block were observed in Tg-MURC mice. These findings indicate that MURC modulates RhoA signaling and that MURC plays an important role in the development of cardiac dysfunction and conduction disturbance with increased vulnerability to atrial arrhythmias.

  9. MURC, a Muscle-Restricted Coiled-Coil Protein That Modulates the Rho/ROCK Pathway, Induces Cardiac Dysfunction and Conduction Disturbance▿

    Science.gov (United States)

    Ogata, Takehiro; Ueyama, Tomomi; Isodono, Koji; Tagawa, Masashi; Takehara, Naofumi; Kawashima, Tsuneaki; Harada, Koichiro; Takahashi, Tomosaburo; Shioi, Tetsuo; Matsubara, Hiroaki; Oh, Hidemasa

    2008-01-01

    We identified a novel muscle-restricted putative coiled-coil protein, MURC, which is evolutionarily conserved from frog to human. MURC was localized to the cytoplasm with accumulation in the Z-line of the sarcomere in the murine adult heart. MURC mRNA expression in the heart increased during the developmental process from the embryonic stage to adulthood. In response to pressure overload, MURC mRNA expression increased in the hypertrophied heart. Using the yeast two-hybrid system, we identified the serum deprivation response (SDPR) protein, a phosphatidylserine-binding protein, as a MURC-binding protein. MURC induced activation of the RhoA/ROCK pathway, which modulated serum response factor-mediated atrial natriuretic peptide (ANP) expression and myofibrillar organization. SDPR augmented MURC-induced transactivation of the ANP promoter in cardiomyocytes, and RNA interference of SDPR attenuated the action of MURC on the ANP promoter. Transgenic mice expressing cardiac-specific MURC (Tg-MURC) exhibited cardiac contractile dysfunction and atrioventricular (AV) conduction disturbances with atrial chamber enlargement, reduced thickness of the ventricular wall, and interstitial fibrosis. Spontaneous episodes of atrial fibrillation and AV block were observed in Tg-MURC mice. These findings indicate that MURC modulates RhoA signaling and that MURC plays an important role in the development of cardiac dysfunction and conduction disturbance with increased vulnerability to atrial arrhythmias. PMID:18332105

  10. Alanine Zipper-Like Coiled-Coil Domains Are Necessary for Homotypic Dimerization of Plant GAGA-Factors in the Nucleus and Nucleolus

    Science.gov (United States)

    Bloss, Ulrich; Hecker, Andreas; Elgass, Kirstin; Hummel, Sabine; Hahn, Achim; Caesar, Katharina; Schleifenbaum, Frank; Harter, Klaus; Berendzen, Kenneth W.

    2011-01-01

    GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms. PMID:21347358

  11. The coiled-coil domain of MURC/cavin-4 is involved in membrane trafficking of caveolin-3 in cardiomyocytes.

    Science.gov (United States)

    Naito, Daisuke; Ogata, Takehiro; Hamaoka, Tetsuro; Nakanishi, Naohiko; Miyagawa, Kotaro; Maruyama, Naoki; Kasahara, Takeru; Taniguchi, Takuya; Nishi, Masahiro; Matoba, Satoaki; Ueyama, Tomomi

    2015-12-15

    Muscle-restricted coiled-coil protein (MURC), also referred to as cavin-4, is a member of the cavin family that works cooperatively with caveolins in caveola formation and function. Cavins are cytoplasmic proteins with coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, and its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/cavin-4 gene have been identified in patients with dilated cardiomyopathy. In the present study, we show the role of MURC/cavin-4 as a caveolar component in the heart. In H9c2 cells, MURC/cavin-4 was localized at the plasma membrane, whereas a MURC/cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. Additionally, although ΔCC did not alter Cav3 mRNA expression, ΔCC decreased the Cav3 protein level. MURC/cavin-4 and ΔCC similarly induced cardiomyocyte hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression than MURC/cavin-4. ΔCC induced ERK activation in cardiomyocytes. Transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) showed impaired cardiac function accompanied by cardiomyocyte hypertrophy and marked interstitial fibrosis. Hearts from ΔCC-Tg mice showed a reduction of the Cav3 protein level and activation of ERK. These results suggest that MURC/cavin-4 requires its coiled-coil domain to target the plasma membrane and to stabilize Cav3 at the plasma membrane of cardiomyocytes and that MURC/cavin-4 functions as a crucial caveolar component to regulate cardiac function. Copyright © 2015 the American Physiological Society.

  12. Design considerations in coiled-coil fusion constructs for the structural determination of a problematic region of the human cardiac myosin rod

    Energy Technology Data Exchange (ETDEWEB)

    Andreas, Michael P.; Ajay, Gautam; Gellings, Jaclyn A.; Rayment, Ivan (UW)

    2017-12-01

    X-ray structural determination of segments of the myosin rod has proved difficult because of the strong salt-dependent aggregation properties and repeating pattern of charges on the surface of the coiled-coil that lead to the formation of paracrystals. This problem has been resolved in part through the use of globular assembly domains that improve protein folding and prevent aggregation. The primary consideration now in designing coiled-coil fusion constructs for myosin is deciding where to truncate the coiled-coil and which amino acid residues to include from the folding domain. This is especially important for myosin that contains numerous regions of low predicted coiled-coil propensity. Here we describe the strategy adopted to determine the structure of the region that extends from Arg1677 – Leu1797 that included two areas that do not show a strong sequence signature of a conventional left-handed coiled coil or canonical heptad repeat. This demonstrates again that, with careful choice of fusion constructs, overlapping structures exhibit very similar conformations for the myosin rod fragments in the canonical regions. However, conformational variability is seen around Leu1706 which is a hot spot for cardiomyopathy mutations suggesting that this might be important for function.

  13. Regulatory coiled-coil domains promote head-to-head assemblies of AAA+ chaperones essential for tunable activity control.

    Science.gov (United States)

    Carroni, Marta; Franke, Kamila B; Maurer, Michael; Jäger, Jasmin; Hantke, Ingo; Gloge, Felix; Linder, Daniela; Gremer, Sebastian; Turgay, Kürşad; Bukau, Bernd; Mogk, Axel

    2017-11-22

    Ring-forming AAA+ chaperones exert ATP-fueled substrate unfolding by threading through a central pore. This activity is potentially harmful requiring mechanisms for tight repression and substrate-specific activation. The AAA+ chaperone ClpC with the peptidase ClpP forms a bacterial protease essential to virulence and stress resistance. The adaptor MecA activates ClpC by targeting substrates and stimulating ClpC ATPase activity. We show how ClpC is repressed in its ground state by determining ClpC cryo-EM structures with and without MecA. ClpC forms large two-helical assemblies that associate via head-to-head contacts between coiled-coil middle domains (MDs). MecA converts this resting state to an active planar ring structure by binding to MD interaction sites. Loss of ClpC repression in MD mutants causes constitutive activation and severe cellular toxicity. These findings unravel an unexpected regulatory concept executed by coiled-coil MDs to tightly control AAA+ chaperone activity.

  14. Thermodynamic analysis of cavity creating mutations in an engineered leucine zipper and energetics of glycerol-induced coiled coil stabilization.

    Science.gov (United States)

    Dürr, E; Jelesarov, I

    2000-04-18

    Protein stability in vitro can be influenced either by introduction of mutations or by changes in the chemical composition of the solvent. Recently, we have characterized the thermodynamic stability and the rate of folding of the engineered dimeric leucine zipper A(2), which has a strengthened hydrophobic core [Dürr, E., Jelesarov, I., and Bosshard, H. R. (1999) Biochemistry 38, 870-880]. Here we report on the energetic consequences of a cavity introduced by Leu/Ala substitution at the tightly packed dimeric interface and how addition of 30% glycerol affects the folding thermodynamics of A(2) and the cavity mutants. Folding could be described by a two-state transition from two unfolded monomers to a coiled coil dimer. Removal of six methylene groups by Leu/Ala substitutions destabilized the dimeric coiled coil by 25 kJ mol(-1) at pH 3.5 and 25 degrees C in aqueous buffer. Destabilization was purely entropic at around room temperature and became increasingly enthalpic at elevated temperatures. Mutations were accompanied by a decrease of the unfolding heat capacity by 0.5 kJ K(-1) mol(-1). Addition of 30% glycerol increased the free energy of folding of A(2) and the cavity mutants by 5-10 kJ mol(-1) and lowered the unfolding heat capacity by 25% for A(2) and by 50% for the Leu/Ala mutants. The origin of the stabilizing effect of glycerol varied with temperature. Stabilization of the parent leucine zipper A(2) was enthalpic with an unfavorable entropic component between 0 and 100 degrees C. In the case of cavity mutants, glycerol induced enthalpic stabilization below 50 degrees C and entropic stabilization above 50 degrees C. The effect of glycerol could not be accounted for solely by the enthalpy and entropy of transfer or protein surface from water to glycerol/water mixture. We propose that in the presence of glycerol the folded coiled coil dimer is better packed and displays less intramolecular fluctuations, leading to enhanced enthalpic interactions and to an

  15. Insights on the structure and stability of Licanantase: a trimeric acid-stable coiled-coil lipoprotein from Acidithiobacillus thiooxidans

    Directory of Open Access Journals (Sweden)

    Fernando Abarca

    2014-08-01

    Full Text Available Licanantase (Lic is the major component of the secretome of Acidithiobacillus thiooxidans when grown in elemental sulphur. When used as an additive, Lic improves copper recovery from bioleaching processes. However, this recovery enhancement is not fully understood. In this context, our aim is to predict the 3D structure of Lic, to shed light on its structure-function relationships. Bioinformatics analyses on the amino acid sequence of Lic showed a great similarity with Lpp, an Escherichia coli Lipoprotein that can form stable trimers in solution. Lic and Lpp share the secretion motif, intracellular processing and alpha helix structure, as well as the distribution of hydrophobic residues in heptads forming a hydrophobic core, typical of coiled-coil structures. Cross-linking experiments showed the presence of Lic trimers, supporting our predictions. Taking the in vitro and in silico evidence as a whole, we propose that the most probable structure for Lic is a trimeric coiled-coil. According to this prediction, a suitable model for Lic was produced using the de novo algorithm “Rosetta Fold-and-Dock”. To assess the structural stability of our model, Molecular Dynamics (MD and Replica Exchange MD simulations were performed using the structure of Lpp and a 14-alanine Lpp mutant as controls, at both acidic and neutral pH. Our results suggest that Lic was the most stable structure among the studied proteins in both pH conditions. This increased stability can be explained by a higher number of both intermonomer hydrophobic contacts and hydrogen bonds, key elements for the stability of Lic’s secondary and tertiary structure.

  16. The Structures of Coiled-Coil Domains from Type III Secretion System Translocators Reveal Homology to Pore-Forming Toxins

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; Dickenson, Nicholas E.; Patil, Mrinalini; Keightley, Andrew; Wyckoff, Gerald J.; Picking, William D.; Picking, Wendy L.; Geisbrecht, Brian V. (UMKC); (OKLU)

    2012-03-26

    Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SSs) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) that is responsible for over one million deaths per year. The Shigella type III secretion apparatus is composed of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC. While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 {angstrom} and 2.8 {angstrom} limiting resolution, respectively. These newly identified domains are composed of extended-length (114 {angstrom} in IpaB and 71 {angstrom} in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably, colicin Ia. This suggests that these mechanistically separate and functionally distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events.

  17. Coiled-coil domain-dependent homodimerization of intracellular barley immune receptors defines a minimal functional module for triggering cell death

    NARCIS (Netherlands)

    Maekawa, T.; Cheng, W.; Spiridon, L.N.; Töller, A.; Lukasik, E.; Saijo, Y.; Liu, P.; Shen, Q.H.; Micluta, M.A.; Somssich, I.E.; Takken, F.L.W.; Petrescu, A.J.; Chai, J.; Schulze-Lefert, P.

    2011-01-01

    Plants and animals have evolved structurally related innate immune sensors, designated NLRs, to detect intracellular nonself molecules. NLRs are modular, consisting of N-terminal coiled-coil (CC) or TOLL/interleukin-1 receptor (TIR) domains, a central nucleotide-binding (NB) domain, and C-terminal

  18. Conserved residues in the coiled-coil pocket of human immunodeficiency virus type 1 gp41 are essential for viral replication and interhelical interaction

    International Nuclear Information System (INIS)

    Mo Hongmei; Konstantinidis, Alex K.; Stewart, Kent D.; Dekhtyar, Tatyana; Ng, Teresa; Swift, Kerry; Matayoshi, Edmund D.; Kati, Warren; Kohlbrenner, William; Molla, Akhteruzzaman

    2004-01-01

    The human immunodeficiency virus type 1 (HIV-1) gp41 plays an important role in mediating the fusion of HIV with host cells. During the fusion process, three N-terminal helices and three C-terminal helices pack in an anti-parallel direction to form a six-helix bundle. X-ray crystallographic analysis of the gp41 core demonstrated that within each coiled-coil interface, there is a deep and large pocket, formed by a cluster of residues in the N-helix coiled-coil. In this report, we systematically analyzed the role of seven conserved residues that are either lining or packing this pocket on the infectivity and interhelical interaction using novel approaches. Our results show that residues L568, V570, W571, and K574 of the N-helix that are lining the side chain and right wall of the pocket are important for establishing a productive infection. Mutations V570A and W571A completely abolished replication, while replication of the L568A and K574A mutants was significantly attenuated relative to wild type. Similarly, residues W628, W631, and I635 of the C-helix that insert into the pocket are essential for infectivity. The impaired infectivity of these seven mutants is in part attributed to the loss in binding affinity of the interhelical interaction. Molecular modeling of the crystal structure of the coiled-coil further shows that alanine substitution of those residues disrupts the hydrophobic interaction between the N- and C-helix. These results suggest that the conserved residues in the coiled-coil domain play a key role in HIV infection and this coiled-coil pocket is a good target for development of inhibitors against HIV. In addition, our data indicate that the novel fluorescence polarization assay described in this study could be valuable in screening for inhibitors that block the interhelical interaction and HIV entry

  19. Inhibition of Chondrocyte Hypertrophy of Osteoarthritis by Disruptor Peptide

    Science.gov (United States)

    2017-07-01

    with PTHR and inhibits the pathogenic beta-catenin- mediated PTHR signaling switch. In Aim 2, we will define the role of disruptor peptide in...confirmed that disruptor peptide conjugated to penetratin can enter cells. Importantly, disruptor peptide can reverse the beta-catenin- mediated PTH... mediated PTHR signaling switch in chondrocytes. Mouse primary chondrocytes express both β-catenin and PTHR. Our data showed that Pen-dis- pep

  20. Complexes of neutralizing and non-neutralizing affinity matured Fabs with a mimetic of the internal trimeric coiled-coil of HIV-1 gp41.

    Directory of Open Access Journals (Sweden)

    Elena Gustchina

    Full Text Available A series of mini-antibodies (monovalent and bivalent Fabs targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066 broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062 non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN363 or 3-H has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen

  1. Exploring the trigger sequence of the GCN4 coiled-coil: Biased molecular dynamics resolves apparent inconsistencies in NMR measurements

    Science.gov (United States)

    Missimer, John H; Dolenc, Jožica; Steinmetz, Michel O; van Gunsteren, Wilfred F

    2010-01-01

    Trigger sequences are indispensable elements for coiled-coil formation. The monomeric helical trigger sequence of the yeast transcriptional activator GCN4 has been investigated recently using several solution NMR observables including nuclear Overhauser enhancement (NOE) intensities and 3J(HN,HCα)-coupling constants, and a set of 20 model structures was proposed. Constrained to satisfy the NOE-derived distance bounds, the NMR model structures do not appear to reproduce all the measured 3J(HN-HCα)-coupling constant values, indicating that the α-helical propensity is not uniform along the GCN4 trigger sequence. A recent methodological study of unrestrained and restrained molecular dynamics (MD) simulations of the GCN4 trigger sequence in solution showed that only MD simulations incorporating time-averaged NOE distance restraints and instantaneous or local-elevation 3J-coupling restraints could satisfy the entire set of the experimental data. In this report, we assess by means of cluster analyses the model structures characteristic of the two simulations that are compatible with the measured data and compare them with the proposed 20 NMR model structures. Striking characteristics of the MD model structures are the variability of the simulated configurations and the indication of entropic stability mediated by the aromatic N-terminal residues 17Tyr and 18His, which are absent in the set of NMR model structures. PMID:20954244

  2. Sme4 coiled-coil protein mediates synaptonemal complex assembly, recombinosome relocalization, and spindle pole body morphogenesis.

    Science.gov (United States)

    Espagne, Eric; Vasnier, Christelle; Storlazzi, Aurora; Kleckner, Nancy E; Silar, Philippe; Zickler, Denise; Malagnac, Fabienne

    2011-06-28

    We identify a large coiled-coil protein, Sme4/PaMe4, that is highly conserved among the large group of Sordariales and plays central roles in two temporally and functionally distinct aspects of the fungal sexual cycle: first as a component of the meiotic synaptonemal complex (SC) and then, after disappearing and reappearing, as a component of the spindle pole body (SPB). In both cases, the protein mediates spatial juxtaposition of two major structures: linkage of homolog axes through the SC and a change in the SPB from a planar to a bent conformation. Corresponding mutants exhibit defects, respectively, in SC and SPB morphogenesis, with downstream consequences for recombination and astral-microtubule nucleation plus postmeiotic nuclear migration. Sme4 is also required for reorganization of recombination complexes in which Rad51, Mer3, and Msh4 foci relocalize from an on-axis position to a between-axis (on-SC) position concomitant with SC installation. Because involved recombinosome foci represent total recombinational interactions, these dynamics are irrespective of their designation for maturation into cross-overs or noncross-overs. The defined dual roles for Sme4 in two different structures that function at distinct phases of the sexual cycle also provide more functional links and evolutionary dynamics among the nuclear envelope, SPB, and SC.

  3. MURC, a muscle-restricted coiled-coil protein, is involved in the regulation of skeletal myogenesis.

    Science.gov (United States)

    Tagawa, Masashi; Ueyama, Tomomi; Ogata, Takehiro; Takehara, Naofumi; Nakajima, Norio; Isodono, Koji; Asada, Satoshi; Takahashi, Tomosaburo; Matsubara, Hiroaki; Oh, Hidemasa

    2008-08-01

    Skeletal myogenesis is a multistep process by which multinucleated mature muscle fibers are formed from undifferentiated, mononucleated myoblasts. However, the molecular mechanisms of skeletal myogenesis have not been fully elucidated. Here, we identified muscle-restricted coiled-coil (MURC) protein as a positive regulator of myogenesis. In skeletal muscle, MURC was localized to the cytoplasm with accumulation in the Z-disc of the sarcomere. In C2C12 myoblasts, MURC expression occurred coincidentally with myogenin expression and preceded sarcomeric myosin expression during differentiation into myotubes. RNA interference (RNAi)-mediated knockdown of MURC impaired differentiation in C2C12 myoblasts, which was accompanied by impaired myogenin expression and ERK activation. Overexpression of MURC in C2C12 myoblasts resulted in the promotion of differentiation with enhanced myogenin expression and ERK activation during differentiation. During injury-induced muscle regeneration, MURC expression increased, and a higher abundance of MURC was observed in immature myofibers compared with mature myofibers. In addition, ERK was activated in regenerating tissue, and ERK activation was detected in MURC-expressing immature myofibers. These findings suggest that MURC is involved in the skeletal myogenesis that results from modulation of myogenin expression and ERK activation. MURC may play pivotal roles in the molecular mechanisms of skeletal myogenic differentiation.

  4. Magnetic Field Alignment of PS-P4VP: a Non-Liquid Crystalline Coil-Coil Block Copolymer

    Science.gov (United States)

    Rokhlenko, Yekaterina; Zhang, Kai; Larson, Steven; Gopalan, Padma; O'Hern, Corey; Osuji, Chinedum

    2015-03-01

    Magnetic fields provide the ability to control alignment of self-assembled soft materials such as block copolymers. Most prior work in this area has relied on the presence of ordered assemblies of anisotropic liquid crystalline species to ensure sufficient magnetic anisotropy to drive alignment. Recent experiments with poly(styrene-b-4-vinylpyridine), a non-liquid crystalline BCP, however, show field-induced alignment of a lamellar microstructure during cooling across the order-disorder transition. Using in situ x-ray scattering, we examine the roles of field strength and cooling rate on the alignment response of this low MW coil-coil BCP. Alignment is first observed at field strengths as low as 1 Tesla and improves markedly with both increasing field strength and slower cooling. We present a geometric argument to illustrate the origin of a finite, non-trivial magnetic susceptibility anisotropy for highly stretched surface-tethered polymer chains and corroborate this using coarse-grained molecular dynamics simulations. We rationalize the magnetic field response of the system in terms of the mobility afforded by the absence of entanglements, the intrinsic anisotropy resulting from the stretched polymer chains and sterically constrained conjugated rings, and the large grain size in these low molecular weight materials.

  5. Novel Antimicrobial Peptides That Inhibit Gram Positive Bacterial Exotoxin Synthesis

    Science.gov (United States)

    Merriman, Joseph A.; Nemeth, Kimberly A.; Schlievert, Patrick M.

    2014-01-01

    Gram-positive bacteria, such as Staphylococcus aureus, cause serious human illnesses through combinations of surface virulence factors and secretion of exotoxins. Our prior studies using the protein synthesis inhibitor clindamycin and signal transduction inhibitors glycerol monolaurate and α-globin and β-globin chains of hemoglobin indicate that their abilities to inhibit exotoxin production by S. aureus are separable from abilities to inhibit growth of the organism. Additionally, our previous studies suggest that inhibition of exotoxin production, in absence of ability to kill S. aureus and normal flora lactobacilli, will prevent colonization by pathogenic S. aureus, while not interfering with lactobacilli colonization. These disparate activities may be important in development of novel anti-infective agents that do not alter normal flora. We initiated studies to explore the exotoxin-synthesis-inhibition activity of hemoglobin peptides further to develop potential agents to prevent S. aureus infections. We tested synthesized α-globin chain peptides, synthetic variants of α-globin chain peptides, and two human defensins for ability to inhibit exotoxin production without significantly inhibiting S. aureus growth. All of these peptides were weakly or not inhibitory to bacterial growth. However, the peptides were inhibitory to exotoxin production with increasing activity dependent on increasing numbers of positively-charged amino acids. Additionally, the peptides could be immobilized on agarose beads or have amino acid sequences scrambled and still retain exotoxin-synthesis-inhibition. The peptides are not toxic to human vaginal epithelial cells and do not inhibit growth of normal flora L. crispatus. These peptides may interfere with plasma membrane signal transduction in S. aureus due to their positive charges. PMID:24748386

  6. An alternative conformation of the gp41 heptad repeat 1 region coiled coil exists in the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor

    International Nuclear Information System (INIS)

    Mische, Claudia C.; Yuan Wen; Strack, Bettina; Craig, Stewart; Farzan, Michael; Sodroski, Joseph

    2005-01-01

    The human immunodeficiency virus (HIV-1) transmembrane envelope glycoprotein, gp41, which mediates virus-cell fusion, exists in at least three different conformations within the trimeric envelope glycoprotein complex. The structures of the prefusogenic and intermediate states are unknown; structures representing the postfusion state have been solved. In the postfusion conformation, three helical heptad repeat 2 (HR2) regions pack in an antiparallel fashion into the hydrophobic grooves on the surface of a triple-helical coiled coil formed by the heptad repeat 1 (HR1) regions. We studied the prefusogenic conformation of gp41 by mutagenic alteration of membrane-anchored and soluble forms of the HIV-1 envelope glycoproteins. Our results indicate that, in the HIV-1 envelope glycoprotein precursor, the gp41 HR1 region is in a conformation distinct from that of a trimeric coiled coil. Thus, the central gp41 coiled coil is formed during the transition of the HIV-1 envelope glycoproteins from the precursor state to the receptor-bound intermediate

  7. HIPdb: a database of experimentally validated HIV inhibiting peptides.

    Science.gov (United States)

    Qureshi, Abid; Thakur, Nishant; Kumar, Manoj

    2013-01-01

    Besides antiretroviral drugs, peptides have also demonstrated potential to inhibit the Human immunodeficiency virus (HIV). For example, T20 has been discovered to effectively block the HIV entry and was approved by the FDA as a novel anti-HIV peptide (AHP). We have collated all experimental information on AHPs at a single platform. HIPdb is a manually curated database of experimentally verified HIV inhibiting peptides targeting various steps or proteins involved in the life cycle of HIV e.g. fusion, integration, reverse transcription etc. This database provides experimental information of 981 peptides. These are of varying length obtained from natural as well as synthetic sources and tested on different cell lines. Important fields included are peptide sequence, length, source, target, cell line, inhibition/IC(50), assay and reference. The database provides user friendly browse, search, sort and filter options. It also contains useful services like BLAST and 'Map' for alignment with user provided sequences. In addition, predicted structure and physicochemical properties of the peptides are also included. HIPdb database is freely available at http://crdd.osdd.net/servers/hipdb. Comprehensive information of this database will be helpful in selecting/designing effective anti-HIV peptides. Thus it may prove a useful resource to researchers for peptide based therapeutics development.

  8. Impact of peptide clustering on unbinding forces in the context of fusion mimetics

    International Nuclear Information System (INIS)

    Pähler, Gesa; Lorenz, Bärbel; Janshoff, Andreas

    2013-01-01

    Highlights: ► Coiled-coil peptides as SNARE mimetics for membrane fusion. ► Interaction forces assessed by colloidal probe microscopy. ► Lateral organization of lipopeptides visualized by atomic force microscopy. -- Abstract: Coiled-coil zipping and unzipping is a pivotal process in SNARE-regulated membrane fusion. In this study we examine this process mediated by a minimal model for coiled-coil formation employing force spectroscopy in the context of membrane-coated surfaces and probes. The interaction forces of several hundred pN are surprisingly low considering the proposed amount of molecular bonds in the contact zone. However, by means of high-resolution imaging employing atomic force microscopy and studying the lateral mobility of lipids and peptides as a function of coiled-coil formation, we are able to supply a detailed view on processes occurring on the membrane surfaces during force measurements. The interaction forces determined here are not only dependent on the peptide concentration on the surface, but also on the regional organization of lateral peptide clusters found prior to coiled-coil formation

  9. Peptide Extracts from Cultures of Certain Lactobacilli Inhibit Helicobacter pylori.

    Science.gov (United States)

    De Vuyst, Luc; Vincent, Pascal; Makras, Eleftherios; Leroy, Frédéric; Pot, Bruno

    2010-03-01

    Helicobacter pylori inhibition by probiotic lactobacilli has been observed in vitro and in vivo. Carefully selected probiotic Lactobacillus strains could therefore play an important role in the treatment of H. pylori infection and eradication. However, the underlying mechanism for this inhibition is not clear. The aim of this study was to examine if peptide extracts, containing bacteriocins or other antibacterial peptides, from six Lactobacillus cultures (Lactobacillus acidophilus La1, Lactobacillus amylovorus DCE 471, Lactobacillus casei YIT 9029, Lactobacillus gasseri K7, Lactobacillus johnsonii La1, and Lactobacillus rhamnosus GG) contribute to the inhibition of H. pylori. Peptide extracts from cultures of Lact. amylovorus DCE 471 and Lact. johnsonii La1 were most active, reducing the viability of H. pylori ATCC 43504 with more than 2 log units within 4 h of incubation (P < 0.001). The four other extracts were less or not active. When six clinical isolates of H. pylori were tested for their susceptibility towards five inhibitory peptide extracts, similar observations were made. Again, the peptide extracts from Lact. amylovorus DCE 471 and Lact. johnsonii La1 were the most inhibitory, while the three other extracts resulted in a much lower inhibition of H. pylori. Protease-treated extracts were inactive towards H. pylori, confirming the proteinaceous nature of the inhibitory substance.

  10. Anti-angiogenic SPARC peptides inhibit progression of neuroblastoma tumors

    Directory of Open Access Journals (Sweden)

    Tian Yufeng

    2010-06-01

    Full Text Available Abstract Background New, more effective strategies are needed to treat highly aggressive neuroblastoma. Our laboratory has previously shown that full-length Secreted Protein Acidic and Rich in Cysteine (SPARC and a SPARC peptide corresponding to the follistatin domain of the protein (FS-E potently block angiogenesis and inhibit the growth of neuroblastoma tumors in preclinical models. Peptide FS-E is structurally complex and difficult to produce, limiting its potential as a therapeutic in the clinic. Results In this study, we synthesized two smaller and structurally more simple SPARC peptides, FSEN and FSEC, that respectively correspond to the N-and C-terminal loops of peptide FS-E. We show that both peptides FSEN and FSEC have anti-angiogenic activity in vitro and in vivo, although FSEC is more potent. Peptide FSEC also significantly inhibited the growth of neuroblastoma xenografts. Histologic examination demonstrated characteristic features of tumor angiogenesis with structurally abnormal, tortuous blood vessels in control neuroblastoma xenografts. In contrast, the blood vessels observed in tumors, treated with SPARC peptides, were thin walled and structurally more normal. Using a novel method to quantitatively assess blood vessel abnormality we demonstrated that both SPARC peptides induced changes in blood vessel architecture that are consistent with blood vessel normalization. Conclusion Our results demonstrate that SPARC peptide FSEC has potent anti-angiogenic and anti-tumorigenic effects in neuroblastoma. Its simple structure and ease of production indicate that it may have clinical utility in the treatment of high-risk neuroblastoma and other types of pediatric and adult cancers, which depend on angiogenesis.

  11. The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation

    DEFF Research Database (Denmark)

    Becker-Heck, Anita; Zohn, Irene E; Okabe, Noriko

    2011-01-01

    Primary ciliary dyskinesia (PCD) is a genetically heterogeneous autosomal recessive disorder characterized by recurrent infections of the respiratory tract associated with the abnormal function of motile cilia. Approximately half of individuals with PCD also have alterations in the left-right...... organization of their internal organ positioning, including situs inversus and situs ambiguous (Kartagener's syndrome). Here, we identify an uncharacterized coiled-coil domain containing a protein, CCDC40, essential for correct left-right patterning in mouse, zebrafish and human. In mouse and zebrafish, Ccdc40...

  12. cDNA cloning of the basement membrane chondroitin sulfate proteoglycan core protein, bamacan: a five domain structure including coiled-coil motifs

    DEFF Research Database (Denmark)

    Wu, R R; Couchman, J R

    1997-01-01

    Basement membranes contain several proteoglycans, and those bearing heparan sulfate glycosaminoglycans such as perlecan and agrin usually predominate. Most mammalian basement membranes also contain chondroitin sulfate, and a core protein, bamacan, has been partially characterized. We have now....... The protein sequence has low overall homology, apart from very small NH2- and COOH-terminal motifs. At the junctions between the distal globular domains and the coiled-coil regions lie glycosylation sites, with up to three N-linked oligosaccharides and probably three chondroitin chains. Three other Ser...

  13. N-terminal aliphatic residues dictate the structure, stability, assembly, and small molecule binding of the coiled-coil region of cartilage oligomeric matrix protein.

    Science.gov (United States)

    Gunasekar, Susheel K; Asnani, Mukta; Limbad, Chandani; Haghpanah, Jennifer S; Hom, Wendy; Barra, Hanna; Nanda, Soumya; Lu, Min; Montclare, Jin Kim

    2009-09-15

    The coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) assembles into a homopentamer that naturally recognizes the small molecule 1,25-dihydroxyvitamin D(3) (vit D). To identify the residues critical for the structure, stability, oligomerization, and binding to vit D as well as two other small molecules, all-trans-retinol (ATR) and curcumin (CCM), here we perform an alanine scanning mutagenesis study. Ten residues lining the hydrophobic pocket of COMPcc were mutated into alanine; of the mutated residues, the N-terminal aliphatic residues L37, L44, V47, and L51 are responsible for maintaining the structure and function. Furthermore, two polar residues, T40 and Q54, within the N-terminal region when converted into alanine improve the alpha-helical structure, stability, and self-assembly behavior. Helical stability, oligomerization, and binding appear to be linked in a manner in which mutations that abolish helical structure and assembly bind poorly to vit D, ATR, and CCM. These results provide not only insight into COMPcc and its functional role but also useful guidelines for the design of stable, pentameric coiled-coils capable of selectively storing and delivering various small molecules.

  14. A fusion-inhibiting peptide against Rift Valley fever virus inhibits multiple, diverse viruses.

    Directory of Open Access Journals (Sweden)

    Jeffrey W Koehler

    Full Text Available For enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III based on the protein sequence and structure. For Rift Valley fever virus (RVFV, the glycoprotein Gc (Class II fusion protein mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. Here, we show that peptides analogous to the RVFV Gc stem region inhibited RVFV infectivity in cell culture by inhibiting the fusion process. Further, we show that infectivity can be inhibited for diverse, unrelated RNA viruses that have Class I (Ebola virus, Class II (Andes virus, or Class III (vesicular stomatitis virus fusion proteins using this single peptide. Our findings are consistent with an inhibition mechanism similar to that proposed for stem peptide fusion inhibitors of dengue virus in which the RVFV inhibitory peptide first binds to both the virion and cell membranes, allowing it to traffic with the virus into the endocytic pathway. Upon acidification and rearrangement of Gc, the peptide is then able to specifically bind to Gc and prevent fusion of the viral and endocytic membranes, thus inhibiting viral infection. These results could provide novel insights into conserved features among the three classes of viral fusion proteins and offer direction for the future development of broadly active fusion inhibitors.

  15. Peptide-oligonucleotide conjugates as nanoscale building blocks for assembly of an artificial three-helix protein mimic

    DEFF Research Database (Denmark)

    Lou, Chenguang; Martos-Maldonado, Manuel C.; Madsen, Charlotte Stahl

    2016-01-01

    Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain ...

  16. Polymer cancerostatics targeted with an antibody fragment bound via a coiled coil motif: in vivo therapeutic efficacy against murine BCL1 leukemia

    Czech Academy of Sciences Publication Activity Database

    Pechar, Michal; Pola, Robert; Janoušková, Olga; Sieglová, Irena; Král, Vlastimil; Fábry, Milan; Tomalová, Barbora; Kovář, Marek

    2018-01-01

    Roč. 18, č. 1 (2018), s. 1-11, č. článku 1700173. ISSN 1616-5187 R&D Projects: GA MŠk(CZ) LO1507; GA MŠk(CZ) LQ1604; GA MZd(CZ) NV16-28594A; GA ČR(CZ) GA16-17207S; GA ČR GA13-12885S Institutional support: RVO:61389013 ; RVO:68378050 ; RVO:61388971 Keywords : cancer therapy * coiled coil * drug targeting Subject RIV: CD - Macromolecular Chemistry; CD - Macromolecular Chemistry (UMG-J); EE - Microbiology, Virology (MBU-M) OBOR OECD: Polymer science; Polymer science (UMG-J); Microbiology (MBU-M) Impact factor: 3.238, year: 2016

  17. An IP-10 (CXCL10)-Derived Peptide Inhibits Angiogenesis

    Science.gov (United States)

    Yates-Binder, Cecelia C.; Rodgers, Margaret; Jaynes, Jesse; Wells, Alan; Bodnar, Richard J.; Turner, Timothy

    2012-01-01

    Angiogenesis plays a critical role in processes such as organ development, wound healing, and tumor growth. It requires well-orchestrated integration of soluble and matrix factors and timely recognition of such signals to regulate this process. Previous work has shown that newly forming vessels express the chemokine receptor CXC receptor 3 (CXCR3) and, activation by its ligand IP-10 (CXCL10), both inhibits development of new vasculature and causes regression of newly formed vessels. To identify and develop new therapeutic agents to limit or reverse pathological angiogenesis, we identified a 21 amino acid fragment of IP-10, spanning the α-helical domain residues 77–98, that mimic the actions of the whole IP-10 molecule on endothelial cells. Treatment of the endothelial cells with the 22 amino acid fragment referred to as IP-10p significantly inhibited VEGF-induced endothelial motility and tube formation in vitro, properties critical for angiogenesis. Using a Matrigel plug assay in vivo, we demonstrate that IP-10p both prevented vessel formation and induced involution of nascent vessels. CXCR3 neutralizing antibody was able to block the inhibitory effects of the IP-10p, demonstrating specificity of the peptide. Inhibition of endothelial function by IP-10p was similar to that described for IP-10, secondary to CXCR3-mediated increase in cAMP production, activation of PKA inhibiting cell migration, and inhibition of VEGF-mediated m-calpain activation. IP-10p provides a novel therapeutic agent that inhibits endothelial cell function thus, allowing for the modulation of angiogenesis. PMID:22815829

  18. Nuclear Magnetic Resonance Structures of GCN4p Are Largely Conserved When Ion Pairs Are Disrupted at Acidic pH but Show a Relaxation of the Coiled Coil Superhelix.

    Science.gov (United States)

    Kaplan, Anne R; Brady, Megan R; Maciejewski, Mark W; Kammerer, Richard A; Alexandrescu, Andrei T

    2017-03-21

    To understand the roles ion pairs play in stabilizing coiled coils, we determined nuclear magnetic resonance structures of GCN4p at three pH values. At pH 6.6, all acidic residues are fully charged; at pH 4.4, they are half-charged, and at pH 1.5, they are protonated and uncharged. The α-helix monomer and coiled coil structures of GCN4p are largely conserved, except for a loosening of the coiled coil quaternary structure with a decrease in pH. Differences going from neutral to acidic pH include (i) an unwinding of the coiled coil superhelix caused by the loss of interchain ion pair contacts, (ii) a small increase in the separation of the monomers in the dimer, (iii) a loosening of the knobs-into-holes packing motifs, and (iv) an increased separation between oppositely charged residues that participate in ion pairs at neutral pH. Chemical shifts (HN, N, C', Cα, and Cβ) of GCN4p display a seven-residue periodicity that is consistent with α-helical structure and is invariant with pH. By contrast, periodicity in hydrogen exchange rates at neutral pH is lost at acidic pH as the exchange mechanism moves into the EX1 regime. On the basis of 1 H- 15 N nuclear Overhauser effect relaxation measurements, the α-helix monomers experience only small increases in picosecond to nanosecond backbone dynamics at acidic pH. By contrast, 13 C rotating frame T 1 relaxation (T 1ρ ) data evince an increase in picosecond to nanosecond side-chain dynamics at lower pH, particularly for residues that stabilize the coiled coil dimerization interface through ion pairs. The results on the structure and dynamics of GCNp4 over a range of pH values help rationalize why a single structure at neutral pH poorly predicts the pH dependence of the unfolding stability of the coiled coil.

  19. Conformational plasticity of the coiled-coil domain of BmrR is required for bmr operator binding: the structure of unliganded BmrR.

    Science.gov (United States)

    Kumaraswami, Muthiah; Newberry, Kate J; Brennan, Richard G

    2010-04-30

    The multidrug-binding transcription regulator BmrR from Bacillus subtilis is a MerR family member that binds to a wide array of cationic lipophilic toxins to activate the transcription of the multidrug efflux pump gene bmr. Transcription activation from the sigma(A)-dependent bmr operator requires BmrR to remodel the nonoptimal 19-bp spacer between the -10 promoter element and the -35 promoter element in order to facilitate productive RNA polymerase binding. Despite the availability of several structures of BmrR bound to DNA and drugs, the lack of a BmrR structure in its unliganded or apo (DNA free and drug free) state hinders our full understanding of the structural transitions required for DNA binding and transcription activation. Here, we report the crystal structure of the constitutively active, unliganded BmrR mutant BmrR(E253Q/R275E). Superposition of the ligand-free (apo BmrR(E253Q/R275E)) and DNA-bound BmrR structures reveals that apo BmrR must undergo significant rearrangement in order to assume the DNA-bound conformation, including an outward rotation of minor groove binding wings, an inward movement of helix-turn-helix motifs, and a downward relocation of pliable coiled-coil helices. Computational analysis of the DNA-free and DNA-bound structures reveals a flexible joint that is located at the center of the coiled-coil helices. This region, which is composed of residues 94 through 98, overlaps the helical bulge that is observed only in the apo BmrR structure. This conformational hinge is likely common to other MerR family members with large effector-binding domains, but appears to be missing from the smaller metal-binding MerR family members. Interestingly, the center-to-center distance of the recognition helices of apo BmrR is 34 A and suggests that the conformational change from the apo BmrR structure to the bmr operator-bound BmrR structure is initiated by the binding of this transcription activator to a more B-DNA-like conformation. (c) 2010 Elsevier

  20. Structural Basis of Rap Phosphatase Inhibition by Phr Peptides

    Science.gov (United States)

    Gallego del Sol, Francisca; Marina, Alberto

    2013-01-01

    Two-component systems, composed of a sensor histidine kinase and an effector response regulator (RR), are the main signal transduction devices in bacteria. In Bacillus, the Rap protein family modulates complex signaling processes mediated by two-component systems, such as competence, sporulation, or biofilm formation, by inhibiting the RR components involved in these pathways. Despite the high degree of sequence homology, Rap proteins exert their activity by two completely different mechanisms of action: inducing RR dephosphorylation or blocking RR binding to its target promoter. However the regulatory mechanism involving Rap proteins is even more complex since Rap activity is antagonized by specific signaling peptides (Phr) through a mechanism that remains unknown at the molecular level. Using X-ray analyses, we determined the structure of RapF, the anti-activator of competence RR ComA, alone and in complex with its regulatory peptide PhrF. The structural and functional data presented herein reveal that peptide PhrF blocks the RapF-ComA interaction through an allosteric mechanism. PhrF accommodates in the C-terminal tetratricopeptide repeat domain of RapF by inducing its constriction, a conformational change propagated by a pronounced rotation to the N-terminal ComA-binding domain. This movement partially disrupts the ComA binding site by triggering the ComA disassociation, whose interaction with RapF is also sterically impaired in the PhrF-induced conformation of RapF. Sequence analyses of the Rap proteins, guided by the RapF-PhrF structure, unveil the molecular basis of Phr recognition and discrimination, allowing us to relax the Phr specificity of RapF by a single residue change. PMID:23526880

  1. Coiled-coil domain of PML is essential for the aberrant dynamics of PML-RARα, resulting in sequestration and decreased mobility of SMRT

    International Nuclear Information System (INIS)

    Huang Ying; Qiu Jihui; Chen Guoqiang; Dong Shuo

    2008-01-01

    Promyelocytic leukemia-retinoic acid receptor α (PML-RARα) is the most frequent RARα fusion protein in acute promyelocytic leukemia (APL). Our previous study has demonstrated that, compared with RARα, PML-RARα had reduced intranuclear mobility accompanied with mislocalization. To understand the molecular basis for the altered dynamics of PML-RARα fusion protein, we performed FRAP analysis at a single cell level. Results indicated that three known sumoylation site mutated PML-RARα had same intracellular localization and reduced mobility as wild-type counterpart. The coiled-coil domain of PML is responsible for the aberrant dynamics of PML-RARα. In addition, we revealed that co-repressor SMRT co-localized with PML-RARα, resulting in the immobilization of SMRT while ATRA treatment eliminated their association and reversed the immobile effect of SMRT. Furthermore, co-activator CBP, co-localized with PML-RARα in an ATRA-independent way, was demonstrated as a high dynamic intranuclear molecule. These results would shed new insights for the molecular mechanisms of PML-RARα-associated leukemogenesis

  2. BECN2 interacts with ATG14 through a metastable coiled-coil to mediate autophagy: BECN2 CCD Structure and Interaction with ATG14

    Energy Technology Data Exchange (ETDEWEB)

    Su, Minfei; Li, Yue; Wyborny, Shane; Neau, David; Chakravarthy, Srinivas; Levine, Beth; Colbert, Christopher L.; Sinha, Sangita C. (NDSU); (IIT); (Cornell); (UTSMC)

    2017-03-12

    ATG14 binding to BECN/Beclin homologs is essential for autophagy, a critical catabolic homeostasis pathway. Here, we show that the α-helical, coiled-coil domain (CCD) of BECN2, a recently identified mammalian BECN1 paralog, forms an antiparallel, curved homodimer with seven pairs of nonideal packing interactions, while the BECN2 CCD and ATG14 CCD form a parallel, curved heterodimer stabilized by multiple, conserved polar interactions. Compared to BECN1, the BECN2 CCD forms a weaker homodimer, but binds more tightly to the ATG14 CCD. Mutation of nonideal BECN2 interface residues to more ideal pairs improves homodimer self-association and thermal stability. Unlike BECN1, all BECN2 CCD mutants bind ATG14, although more weakly than wild type. Thus, polar BECN2 CCD interface residues result in a metastable homodimer, facilitating dissociation, but enable better interactions with polar ATG14 residues stabilizing the BECN2:ATG14 heterodimer. These structure-based mechanistic differences in BECN1 and BECN2 homodimerization and heterodimerization likely dictate competitive ATG14 recruitment.

  3. The Chloroplastic Protein THF1 Interacts with the Coiled-Coil Domain of the Disease Resistance Protein N′ and Regulates Light-Dependent Cell Death1[OPEN

    Science.gov (United States)

    Sekine, Ken-Taro; Wallon, Thérèse; Sugiwaka, Yuji; Kobayashi, Kappei

    2016-01-01

    One branch of plant immunity is mediated through nucleotide-binding/Leu-rich repeat (NB-LRR) family proteins that recognize specific effectors encoded by pathogens. Members of the I2-like family constitute a well-conserved subgroup of NB-LRRs from Solanaceae possessing a coiled-coil (CC) domain at their N termini. We show here that the CC domains of several I2-like proteins are able to induce a hypersensitive response (HR), a form of programmed cell death associated with disease resistance. Using yeast two-hybrid screens, we identified the chloroplastic protein Thylakoid Formation1 (THF1) as an interacting partner for several I2-like CC domains. Co-immunoprecipitations and bimolecular fluorescence complementation assays confirmed that THF1 and I2-like CC domains interact in planta and that these interactions take place in the cytosol. Several HR-inducing I2-like CC domains have a negative effect on the accumulation of THF1, suggesting that the latter is destabilized by active CC domains. To confirm this model, we investigated N′, which recognizes the coat protein of most Tobamoviruses, as a prototypical member of the I2-like family. Transient expression and gene silencing data indicated that THF1 functions as a negative regulator of cell death and that activation of full-length N′ results in the destabilization of THF1. Consistent with the known function of THF1 in maintaining chloroplast homeostasis, we show that the HR induced by N′ is light-dependent. Together, our results define, to our knowledge, novel molecular mechanisms linking light and chloroplasts to the induction of cell death by a subgroup of NB-LRR proteins. PMID:26951433

  4. The Chloroplastic Protein THF1 Interacts with the Coiled-Coil Domain of the Disease Resistance Protein N' and Regulates Light-Dependent Cell Death.

    Science.gov (United States)

    Hamel, Louis-Philippe; Sekine, Ken-Taro; Wallon, Thérèse; Sugiwaka, Yuji; Kobayashi, Kappei; Moffett, Peter

    2016-05-01

    One branch of plant immunity is mediated through nucleotide-binding/Leu-rich repeat (NB-LRR) family proteins that recognize specific effectors encoded by pathogens. Members of the I2-like family constitute a well-conserved subgroup of NB-LRRs from Solanaceae possessing a coiled-coil (CC) domain at their N termini. We show here that the CC domains of several I2-like proteins are able to induce a hypersensitive response (HR), a form of programmed cell death associated with disease resistance. Using yeast two-hybrid screens, we identified the chloroplastic protein Thylakoid Formation1 (THF1) as an interacting partner for several I2-like CC domains. Co-immunoprecipitations and bimolecular fluorescence complementation assays confirmed that THF1 and I2-like CC domains interact in planta and that these interactions take place in the cytosol. Several HR-inducing I2-like CC domains have a negative effect on the accumulation of THF1, suggesting that the latter is destabilized by active CC domains. To confirm this model, we investigated N', which recognizes the coat protein of most Tobamoviruses, as a prototypical member of the I2-like family. Transient expression and gene silencing data indicated that THF1 functions as a negative regulator of cell death and that activation of full-length N' results in the destabilization of THF1. Consistent with the known function of THF1 in maintaining chloroplast homeostasis, we show that the HR induced by N' is light-dependent. Together, our results define, to our knowledge, novel molecular mechanisms linking light and chloroplasts to the induction of cell death by a subgroup of NB-LRR proteins. © 2016 American Society of Plant Biologists. All Rights Reserved.

  5. Contributed Review: Absolute spectral radiance calibration of fiber-optic shock-temperature pyrometers using a coiled-coil irradiance standard lamp

    Energy Technology Data Exchange (ETDEWEB)

    Fat’yanov, O. V., E-mail: fatyan1@gps.caltech.edu; Asimow, P. D., E-mail: asimow@gps.caltech.edu [Division of Geological and Planetary Sciences 252-21, California Institute of Technology, Pasadena, California 91125 (United States)

    2015-10-15

    We describe an accurate and precise calibration procedure for multichannel optical pyrometers such as the 6-channel, 3-ns temporal resolution instrument used in the Caltech experimental geophysics laboratory. We begin with a review of calibration sources for shock temperatures in the 3000-30 000 K range. High-power, coiled tungsten halogen standards of spectral irradiance appear to be the only practical alternative to NIST-traceable tungsten ribbon lamps, which are no longer available with large enough calibrated area. However, non-uniform radiance complicates the use of such coiled lamps for reliable and reproducible calibration of pyrometers that employ imaging or relay optics. Careful analysis of documented methods of shock pyrometer calibration to coiled irradiance standard lamps shows that only one technique, not directly applicable in our case, is free of major radiometric errors. We provide a detailed description of the modified Caltech pyrometer instrument and a procedure for its absolute spectral radiance calibration, accurate to ±5%. We employ a designated central area of a 0.7× demagnified image of a coiled-coil tungsten halogen lamp filament, cross-calibrated against a NIST-traceable tungsten ribbon lamp. We give the results of the cross-calibration along with descriptions of the optical arrangement, data acquisition, and processing. We describe a procedure to characterize the difference between the static and dynamic response of amplified photodetectors, allowing time-dependent photodiode correction factors for spectral radiance histories from shock experiments. We validate correct operation of the modified Caltech pyrometer with actual shock temperature experiments on single-crystal NaCl and MgO and obtain very good agreement with the literature data for these substances. We conclude with a summary of the most essential requirements for error-free calibration of a fiber-optic shock-temperature pyrometer using a high-power coiled tungsten halogen

  6. Structural analysis of intermolecular interactions in the kinesin adaptor complex fasciculation and elongation protein zeta 1/ short coiled-coil protein (FEZ1/SCOCO.

    Directory of Open Access Journals (Sweden)

    Marcos Rodrigo Alborghetti

    Full Text Available Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans, SCOCO (short coiled-coil protein / UNC-69 and kinesins (e.g. kinesin heavy chain / UNC116 are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth, we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance, cross-linking coupled with mass spectrometry (MS, SAXS (Small Angle X-ray Scattering and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance studies of the region involved in this process, corresponding to FEZ1 (92-194. Through studies involving the protein in its monomeric configuration (reduced and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.

  7. Immune response against the coiled coil domain of Sjögren's syndrome associated autoantigen Ro52 induces salivary gland dysfunction.

    Science.gov (United States)

    Sroka, Magdalena; Bagavant, Harini; Biswas, Indranil; Ballard, Abigail; Deshmukh, Umesh S

    2018-01-31

    The structural domains of Ro52, termed the RING, B-box, coiled coil (CC) and B30.2/SPRY are targets of anti-Ro52 in multiple autoimmune disorders. In Sjögren's syndrome patients, the presence of anti-Ro52 is associated with higher disease severity, and in mice, they induce salivary gland hypofunction. This study was undertaken to investigate whether immune responses against different domains of Ro52, influences salivary gland disease in mice. Female NZM2758 mice were immunised with Ro52 domains expressed as recombinant fusion proteins with maltose binding protein (MBP) [MBP-RING-B-box, MBP-CC, MBP-CC(ΔC19), MBP-B30.2/SPRY]. Sera from immunised mice were studied for IgG antibodies to Ro52 by immunoprecipitation, and to salivary gland cells by immunofluorescence. Pilocarpine-induced saliva production was measured to evaluate salivary gland function. Submandibular glands were investigated by histopathology for inflammation and by immune-histochemistry for IgG deposition. Mice immunised with different Ro52-domains had comparable reactivity to Ro52 and to salivary gland cells. However, only mice immunised with the CC domain and its C-terminal truncated version CC(ΔC19) showed a significant drop in saliva production. None of the mice developed severe salivary gland inflammation. The salivary gland hypofunction significantly correlated with increased intra-lobar IgG deposits in the submandibular salivary glands. Our data demonstrate that epitope specificity of anti-Ro52 antibodies plays a critical role in the induction of glandular dysfunction. Clearly, screening Sjögren's syndrome patients for relative levels of Ro52 domain specific antibodies will be more informative for associating anti-Ro52 with clinical measures of the disorder.

  8. Ancient Origin of the CARD–Coiled Coil/Bcl10/MALT1-Like Paracaspase Signaling Complex Indicates Unknown Critical Functions

    Directory of Open Access Journals (Sweden)

    Jens Staal

    2018-05-01

    Full Text Available The CARD–coiled coil (CC/Bcl10/MALT1-like paracaspase (CBM signaling complexes composed of a CARD–CC family member (CARD-9, -10, -11, or -14, Bcl10, and the type 1 paracaspase MALT1 (PCASP1 play a pivotal role in immunity, inflammation, and cancer. Targeting MALT1 proteolytic activity is of potential therapeutic interest. However, little is known about the evolutionary origin and the original functions of the CBM complex. Type 1 paracaspases originated before the last common ancestor of planulozoa (bilaterians and cnidarians. Notably in bilaterians, Ecdysozoa (e.g., nematodes and insects lacks Bcl10, whereas other lineages have a Bcl10 homolog. A survey of invertebrate CARD–CC homologs revealed such homologs only in species with Bcl10, indicating an ancient common origin of the entire CBM complex. Furthermore, vertebrate-like Syk/Zap70 tyrosine kinase homologs with the ITAM-binding SH2 domain were only found in invertebrate organisms with CARD–CC/Bcl10, indicating that this pathway might be related to the original function of the CBM complex. Moreover, the type 1 paracaspase sequences from invertebrate organisms that have CARD–CC/Bcl10 are more similar to vertebrate paracaspases. Functional analysis of protein–protein interactions, NF-κB signaling, and CYLD cleavage for selected invertebrate type 1 paracaspase and Bcl10 homologs supports this scenario and indicates an ancient origin of the CARD–CC/Bcl10/paracaspase signaling complex. By contrast, many of the known MALT1-associated activities evolved fairly recently, indicating that unknown functions are at the basis of the protein conservation. As a proof-of-concept, we provide initial evidence for a CBM- and NF-κB-independent neuronal function of the Caenorhabditis elegans type 1 paracaspase malt-1. In conclusion, this study shows how evolutionary insights may point at alternative functions of MALT1.

  9. Endosomolytic Nano-Polyplex Platform Technology for Cytosolic Peptide Delivery To Inhibit Pathological Vasoconstriction.

    Science.gov (United States)

    Evans, Brian C; Hocking, Kyle M; Kilchrist, Kameron V; Wise, Eric S; Brophy, Colleen M; Duvall, Craig L

    2015-06-23

    A platform technology has been developed and tested for delivery of intracellular-acting peptides through electrostatically complexed nanoparticles, or nano-polyplexes, formulated from an anionic endosomolytic polymer and cationic therapeutic peptides. This delivery platform has been initially tested and optimized for delivery of two unique vasoactive peptides, a phosphomimetic of heat shock protein 20 and an inhibitor of MAPKAP kinase II, to prevent pathological vasoconstriction (i.e., vasospasm) in human vascular tissue. These peptides inhibit vasoconstriction and promote vasorelaxation by modulating actin dynamics in vascular smooth muscle cells. Formulating these peptides into nano-polyplexes significantly enhances peptide uptake and retention, facilitates cytosolic delivery through a pH-dependent endosomal escape mechanism, and enhances peptide bioactivity in vitro as measured by inhibition of F-actin stress fiber formation. In comparison to treatment with the free peptides, which were endowed with cell-penetrating sequences, the nano-polyplexes significantly increased vasorelaxation, inhibited vasoconstriction, and decreased F-actin formation in the human saphenous vein ex vivo. These results suggest that these formulations have significant potential for treatment of conditions such as cerebral vasospasm following subarachnoid hemorrhage. Furthermore, because many therapeutic peptides include cationic cell-penetrating segments, this simple and modular platform technology may have broad applicability as a cost-effective approach for enhancing the efficacy of cytosolically active peptides.

  10. Molecular genetic and functional characterization implicate muscle-restricted coiled-coil gene (MURC) as a causal gene for familial dilated cardiomyopathy.

    Science.gov (United States)

    Rodriguez, Gabriela; Ueyama, Tomomi; Ogata, Takehiro; Czernuszewicz, Grazyna; Tan, Yanli; Dorn, Gerald W; Bogaev, Roberta; Amano, Katsuya; Oh, Hidemasa; Matsubara, Hiroaki; Willerson, James T; Marian, Ali J

    2011-08-01

    Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are classic forms of systolic and diastolic heart failure, respectively. Mutations in genes encoding sarcomere and cytoskeletal proteins are major causes of HCM and DCM. MURC, encoding muscle-restricted coiled-coil, a Z-line protein, regulates cardiac function in mice. We investigated potential causal role of MURC in human cardiomyopathies. We sequenced MURC in 1199 individuals, including 383 probands with DCM, 307 with HCM, and 509 healthy control subjects. We found 6 heterozygous DCM-specific missense variants (p.N128K, p.R140W, p.L153P, p.S307T, p.P324L, and p.S364L) in 8 unrelated probands. Variants p.N128K and p.S307T segregated with inheritance of DCM in small families (χ(2)=8.5, P=0.003). Variants p.N128K, p.R140W, p.L153P, and p.S364L were considered probably or possibly damaging. Variant p.P324L recurred in 3 independent probands, including 1 proband with a TPM1 mutation (p.M245T). A deletion variant (p.L232-R238del) was present in 3 unrelated HCM probands, but it did not segregate with HCM in a family who also had a MYH7 mutation (p.L907V). The phenotype in mutation carriers was notable for progressive heart failure leading to heart transplantation in 4 patients, conduction defects, and atrial arrhythmias. Expression of mutant MURC proteins in neonatal rat cardiac myocytes transduced with recombinant adenoviruses was associated with reduced RhoA activity, lower mRNA levels of hypertrophic markers and smaller myocyte size as compared with wild-type MURC. MURC mutations impart loss-of-function effects on MURC functions and probably are causal variants in human DCM. The causal role of a deletion mutation in HCM is uncertain.

  11. Inhibition of duck hepatitis B virus replication by mimic peptides in vitro.

    Science.gov (United States)

    Jia, Hongyu; Liu, Changhong; Yang, Ying; Zhu, Haihong; Chen, Feng; Liu, Jihong; Zhou, Linfu

    2015-11-01

    The aim of the present study was to investigate the inhibitory effect of specific mimic peptides targeting duck hepatitis B virus polymerase (DHBVP) on duck hepatitis B virus (DHBV) replication in primary duck hepatocytes. Phage display technology (PDT) was used to screen for mimic peptides specifically targeting DHBVP and the associated coding sequences were determined using DNA sequencing. The selected mimic peptides were then used to treat primary duck hepatocytes infected with DHBV in vitro. Infected hepatocytes expressing the mimic peptides intracellularly were also prepared. The cells were divided into mimic peptide groups (EXP groups), an entecavir-treated group (positive control) and a negative control group. The medium was changed every 48 h. Following a 10-day incubation, the cell supernatants were collected. DHBV-DNA in the cellular nucleus, cytoplasm and culture supernatant was analyzed by quantitative polymerase chain reaction (qPCR). Eight mimic peptides were selected following three PDT screening rounds for investigation in the DHBV-infected primary duck hepatocytes. The qPCR results showed that following direct treatment with mimic peptide 2 or 7, intracellular expression of mimic peptide 2 or 7, or treatment with entecavir, the DHBV-DNA levels in the culture supernatant and cytoplasm of duck hepatocytes were significantly lower than those in the negative control (Pmimic peptide 7 was lower than that in the other groups (Pmimic peptide 7 was significantly lower than that in the other groups (PMimic peptides specifically targeting DHBVP, administered directly or expressed intracellularly, can significantly inhibit DHBV replication in vitro .

  12. Hydroxyapatite-binding peptides for bone growth and inhibition

    Science.gov (United States)

    Bertozzi, Carolyn R [Berkeley, CA; Song, Jie [Shrewsbury, MA; Lee, Seung-Wuk [Walnut Creek, CA

    2011-09-20

    Hydroxyapatite (HA)-binding peptides are selected using combinatorial phage library display. Pseudo-repetitive consensus amino acid sequences possessing periodic hydroxyl side chains in every two or three amino acid sequences are obtained. These sequences resemble the (Gly-Pro-Hyp).sub.x repeat of human type I collagen, a major component of extracellular matrices of natural bone. A consistent presence of basic amino acid residues is also observed. The peptides are synthesized by the solid-phase synthetic method and then used for template-driven HA-mineralization. Microscopy reveal that the peptides template the growth of polycrystalline HA crystals .about.40 nm in size.

  13. Inhibition of Snake Venom Metalloproteinase by β-Lactoglobulin Peptide from Buffalo (Bubalus bubalis) Colostrum.

    Science.gov (United States)

    Arpitha, Ashok; Sebastin Santhosh, M; Rohit, A C; Girish, K S; Vinod, D; Aparna, H S

    2017-08-01

    Bioactive peptide research has experienced considerable therapeutic interest owing to varied physiological functions, efficacy in excretion, and tolerability of peptides. Colostrum is a rich natural source of bioactive peptides with many properties elucidated such as anti-thrombotic, anti-hypertensive, opioid, immunomodulatory, etc. In this study, a variant peptide derived from β-lactoglobulin from buffalo colostrum was evaluated for the anti-ophidian property by targeting snake venom metalloproteinases. These are responsible for rapid local tissue damages that develop after snakebite such as edema, hemorrhage, myonecrosis, and extracellular matrix degradation. The peptide identified by LC-MS/MS effectively neutralized hemorrhagic activity of the Echis carinatus venom in a dose-dependent manner. Histological examinations revealed that the peptide mitigated basement membrane degradation and accumulation of inflammatory leucocytes at the venom-injected site. Inhibition of proteolytic activity was evidenced in both casein and gelatin zymograms. Also, inhibition of fibrinolytic and fibrinogenolytic activities was seen. The UV-visible spectral study implicated Zn 2+ chelation, which was further confirmed by molecular docking and dynamic studies by assessing molecular interactions, thus implicating the probable mechanism for inhibition of venom-induced proteolytic and hemorrhagic activities. The present investigation establishes newer vista for the BLG-col peptide with anti-ophidian efficacy as a promising candidate for therapeutic interventions.

  14. Reversible pH-controlled DNA-binding peptide nanotweezers: An in-silico study

    Directory of Open Access Journals (Sweden)

    Gaurav Sharma

    2008-11-01

    Full Text Available Gaurav Sharma1, Kaushal Rege2,3, David E Budil4, Martin L Yarmush2,5, Constantinos Mavroidis11Department of Mechanical and Industrial Engineering; 4Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA; 2The Center for Engineering in Medicine (CEM, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; 3Department of Chemical Engineering, Arizona State University, Tempe, AZ, USA; 5Department of Biomedical Engineering, Rutgers University, NJ, USAAbstract: We describe the molecular dynamics (MD-aided engineering design of mutant peptides based on the α-helical coiled-coil GCN4 leucine zipper peptide (GCN4-p1 in order to obtain environmentally-responsive nanotweezers. The actuation mechanism of the nanotweezers depends on the modification of electrostatic charges on the residues along the length of the coiled coil. Modulating the solution pH between neutral and acidic values results in the reversible movement of helices toward and away from each other and creates a complete closed-open-closed transition cycle between the helices. Our results indicate that the mutants show a reversible opening of up to 15 Å (1.5 nm; approximately 150% of the initial separation upon pH actuation. Investigation on the physicochemical phenomena that influence conformational properties, structural stability, and reversibility of the coiled-coil peptide-based nanotweezers revealed that a rationale- and design-based approach is needed to engineer stable peptide or macromolecules into stimuli-responsive devices. The efficacy of the mutant that demonstrated the most significant reversible actuation for environmentally responsive modulation of DNA-binding activity was also demonstrated. Our results have significant implications in bioseparations and in the engineering of novel transcription factors.Keywords: bionanotechnology, nanotweezers, coiled-coil, GCN4, leucine zipper, molecular dynamics, environmentally

  15. PspF-binding domain PspA1-144 and the PspA·F complex: New insights into the coiled-coil-dependent regulation of AAA+ proteins.

    Science.gov (United States)

    Osadnik, Hendrik; Schöpfel, Michael; Heidrich, Eyleen; Mehner, Denise; Lilie, Hauke; Parthier, Christoph; Risselada, H Jelger; Grubmüller, Helmut; Stubbs, Milton T; Brüser, Thomas

    2015-11-01

    Phage shock protein A (PspA) belongs to the highy conserved PspA/IM30 family and is a key component of the stress inducible Psp system in Escherichia coli. One of its central roles is the regulatory interaction with the transcriptional activator of this system, the σ(54) enhancer-binding protein PspF, a member of the AAA+ protein family. The PspA/F regulatory system has been intensively studied and serves as a paradigm for AAA+ enzyme regulation by trans-acting factors. However, the molecular mechanism of how exactly PspA controls the activity of PspF and hence σ(54) -dependent expression of the psp genes is still unclear. To approach this question, we identified the minimal PspF-interacting domain of PspA, solved its structure, determined its affinity to PspF and the dissociation kinetics, identified residues that are potentially important for PspF regulation and analyzed effects of their mutation on PspF in vivo and in vitro. Our data indicate that several characteristics of AAA+ regulation in the PspA·F complex resemble those of the AAA+ unfoldase ClpB, with both proteins being regulated by a structurally highly conserved coiled-coil domain. The convergent evolution of both regulatory domains points to a general mechanism to control AAA+ activity for divergent physiologic tasks via coiled-coil domains. © 2015 John Wiley & Sons Ltd.

  16. Potential role of an antimicrobial peptide, KLK in inhibiting lipopolysaccharide-induced macrophage inflammation.

    Directory of Open Access Journals (Sweden)

    Pornpimon Jantaruk

    Full Text Available Antimicrobial peptides (AMPs are attractive alternatives to antibiotics. Due to their immune modulatory properties, AMPs are at present emerging as promising agents for controlling inflammatory-mediated diseases. In this study, anti-inflammatory potential of an antimicrobial peptide, KLK (KLKLLLLLKLK and its analogs was evaluated in lipopolysaccharide (LPS-induced RAW 264.7 macrophages. The results herein demonstrated that KLK peptide as well as its analogs significantly inhibited the pro-inflammatory mediator nitric oxide (NO, interleukin-1β (IL-1β and tumor necrosis factor-α (TNF-α production in LPS-stimulated RAW 264.7 macrophages in dose-dependent manners, and such inhibitory effects were not due to direct cytotoxicity. When considering inhibition potency, KLK among the test peptides exhibited the most effective activity. The inhibitory activity of KLK peptide also extended to include suppression of LPS-induced production of prostaglandin E2 (PGE2. KLK significantly decreased mRNA and protein expression of inducible nitric oxide synthase (iNOS and cyclooxygenase-2 (COX-2 as well as mRNA expression of IL-1β and TNF-α. Moreover, KLK inhibited nuclear translocation of nuclear factor-κB (NF-κB p65 and blocked degradation and phosphorylation of inhibitor of κB (IκB. Taken together, these results suggested that the KLK peptide inhibited inflammatory response through the down-regulation of NF-κB mediated activation in macrophages. Since peptide analogs with different amino acid sequences and arrangement were investigated for their anti-inflammatory activities, the residues/structures required for activity were also discussed. Our findings therefore proved anti-inflammatory potential of the KLK peptide and provide direct evidence for therapeutic application of KLK as a novel anti-inflammatory agent.

  17. Insect antimicrobial peptides act synergistically to inhibit a trypanosome parasite.

    Science.gov (United States)

    Marxer, Monika; Vollenweider, Vera; Schmid-Hempel, Paul

    2016-05-26

    The innate immune system provides protection from infection by producing essential effector molecules, such as antimicrobial peptides (AMPs) that possess broad-spectrum activity. This is also the case for bumblebees, Bombus terrestris, when infected by the trypanosome, Crithidia bombi Furthermore, the expressed mixture of AMPs varies with host genetic background and infecting parasite strain (genotype). Here, we used the fact that clones of C. bombi can be cultivated and kept as strains in medium to test the effect of various combinations of AMPs on the growth rate of the parasite. In particular, we used pairwise combinations and a range of physiological concentrations of three AMPs, namely Abaecin, Defensin and Hymenoptaecin, synthetized from the respective genomic sequences. We found that these AMPs indeed suppress the growth of eight different strains of C. bombi, and that combinations of AMPs were typically more effective than the use of a single AMP alone. Furthermore, the most effective combinations were rarely those consisting of maximum concentrations. In addition, the AMP combination treatments revealed parasite strain specificity, such that strains varied in their sensitivity towards the same mixtures. Hence, variable expression of AMPs could be an alternative strategy to combat highly variable infections.This article is part of the themed issue 'Evolutionary ecology of arthropod antimicrobial peptides'. © 2016 The Author(s).

  18. Inhibition of HIV-1 infection by synthetic peptides derived CCR5 fragments

    International Nuclear Information System (INIS)

    Imai, Masaki; Baranyi, Lajos; Okada, Noriko; Okada, Hidechika

    2007-01-01

    HIV-1 infection requires interaction of viral envelope protein gp160 with CD4 and a chemokine receptor, CCR5 or CXCR4 as entry coreceptor. We designed HIV-inhibitory peptides targeted to CCR5 using a novel computer program (ANTIS), which searched all possible sense-antisense amino acid pairs between proteins. Seven AHBs were found in CCR5 receptor. All AHB peptides were synthesized and tested for their ability to prevent HIV-1 infection to human T cells. A peptide fragment (LC5) which is a part of the CCR5 receptor corresponding to the loop between the fifth and sixth transmembrane regions (amino acids 222-240) proved to inhibit HIV-1 IIIB infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. LC5 effectively indicated dose-dependent manner, and the suppression was enhanced additively by T20 peptide, which inhibits infection in vitro by disrupting the gp41 conformational changes necessary for membrane fusion. Thus, these results indicate that CCR5-derived AHB peptides could provide a useful tool to define the mechanism(s) of HIV infection, and may provide insight which will contribute to the development of an anti-HIV-1 reagent

  19. Mechanism by which DHA inhibits the aggregation of KLVFFA peptides: A molecular dynamics study

    Science.gov (United States)

    Zhou, Hong; Liu, Shengtang; Shao, Qiwen; Ma, Dongfang; Yang, Zaixing; Zhou, Ruhong

    2018-03-01

    Docosahexaenoic acid (DHA) is one of the omega-3 polyunsaturated fatty acids, which has shown promising applications in lowering Aβ peptide neurotoxicity in vitro by preventing aggregation of Aβ peptides and relieving accumulation of Aβ fibrils. Unfortunately, the underlying molecular mechanisms of how DHA interferes with the aggregation of Aβ peptides remain largely enigmatic. Herein, aggregation behaviors of amyloid-β(Aβ)16-21 peptides (KLVFFA) with or without the presence of a DHA molecule were comparatively studied using extensive all-atom molecular dynamics simulations. We found that DHA could effectively suppress the aggregation of KLVFFA peptides by redirecting peptides to unstructured oligomers. The highly hydrophobic and flexible nature of DHA made it randomly but tightly entangled with Leu-17, Phe-19, and Phe-20 residues to form unstructured but stable complexes. These lower-ordered unstructured oligomers could eventually pass through energy barriers to form ordered β-sheet structures through large conformational fluctuations. This study depicts a microscopic picture for understanding the role and mechanism of DHA in inhibition of aggregation of Aβ peptides, which is generally believed as one of the important pathogenic mechanisms of Alzheimer's disease.

  20. Glucagon-like peptide 2 inhibits ghrelin secretion in humans

    DEFF Research Database (Denmark)

    Banasch, Matthias; Bulut, Kerem; Hagemann, Dirk

    2006-01-01

    INTRODUCTION: The growth hormone secretagogue receptor ligand ghrelin is known to play a pivotal role in the central nervous control of energy homeostasis. Circulating ghrelin levels are high under fasting conditions and decline after meal ingestion, but the mechanisms underlying the postprandial...... drop in ghrelin levels are poorly understood. In the present study we addressed, whether (1) exogenous GLP-2 administration decreases ghrelin levels and (2) what other endogenous factors are related to ghrelin secretion under fasting conditions. PATIENTS AND METHODS: Fifteen healthy male volunteers...... were studied with the intravenous infusion of GLP-2 (2 pmol l(-1) min(-1)) or placebo over 120 min in the fasting state. Plasma concentrations of glucose, insulin, C-peptide, glucagon, intact GLP-2 and ghrelin were determined. RESULTS: During the infusion of GLP-2, plasma concentrations of intact GLP-2...

  1. Inhibition of Vaccinia virus entry by a broad spectrum antiviral peptide

    International Nuclear Information System (INIS)

    Altmann, S.E.; Jones, J.C.; Schultz-Cherry, S.; Brandt, C.R.

    2009-01-01

    Concerns about the possible use of Variola virus, the causative agent of smallpox, as a weapon for bioterrorism have led to renewed efforts to identify new antivirals against orthopoxviruses. We identified a peptide, EB, which inhibited infection by Vaccinia virus with an EC 50 of 15 μM. A control peptide, EBX, identical in composition to EB but differing in sequence, was inactive (EC 50 > 200 μM), indicating sequence specificity. The inhibition was reversed upon removal of the peptide, and EB treatment had no effect on the physical integrity of virus particles as determined by electron microscopy. Viral adsorption was unaffected by the presence of EB, and the addition of EB post-entry had no effect on viral titers or on early gene expression. The addition of EB post-adsorption resulted in the inhibition of β-galactosidase expression from an early viral promoter with an EC 50 of 45 μM. A significant reduction in virus entry was detected in the presence of the peptide when the number of viral cores released into the cytoplasm was quantified. Electron microscopy indicated that 88% of the virions remained on the surface of cells in the presence of EB, compared to 37% in the control (p < 0.001). EB also blocked fusion-from-within, suggesting that virus infection is inhibited at the fusion step. Analysis of EB derivatives suggested that peptide length may be important for the activity of EB. The EB peptide is, to our knowledge, the first known small molecule inhibitor of Vaccinia virus entry.

  2. Sequence-specific inhibition of duck hepatitis B virus reverse transcription by peptide nucleic acids (PNA)

    DEFF Research Database (Denmark)

    Robaczewska, Magdalena; Narayan, Ramamurthy; Seigneres, Beatrice

    2005-01-01

    BACKGROUND/AIMS: Peptide nucleic acids (PNAs) appear as promising new antisense agents, that have not yet been examined as hepatitis B virus (HBV) inhibitors. Our aim was to study the ability of PNAs targeting the duck HBV (DHBV) encapsidation signal epsilon to inhibit reverse transcription (RT...... in primary duck hepatocytes (PDH). RESULTS: Both PNAs reproducibly inhibited DHBV RT in a dose-dependent manner with IC(50) of 10nM, whereas up to 600-fold higher concentration of S-ODNs was required for similar inhibition. The PNA targeting the bulge and upper stem of epsilon appeared as more efficient RT...

  3. Potent and Selective Peptide-based Inhibition of the G Protein Gαq*

    Science.gov (United States)

    Charpentier, Thomas H.; Waldo, Gary L.; Lowery-Gionta, Emily G.; Krajewski, Krzysztof; Strahl, Brian D.; Kash, Thomas L.; Harden, T. Kendall; Sondek, John

    2016-01-01

    In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gαq binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gαq within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gαq in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gαq. A representative peptide was specific for active Gαq because it did not bind inactive Gαq or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ1γ2. In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gαq; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gαq in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gαq-dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gαq in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gαq in cells. PMID:27742837

  4. Potent and Selective Peptide-based Inhibition of the G Protein Gαq.

    Science.gov (United States)

    Charpentier, Thomas H; Waldo, Gary L; Lowery-Gionta, Emily G; Krajewski, Krzysztof; Strahl, Brian D; Kash, Thomas L; Harden, T Kendall; Sondek, John

    2016-12-02

    In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gα q binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gα q within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gα q in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gα q A representative peptide was specific for active Gα q because it did not bind inactive Gα q or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ 1 γ 2 In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gα q ; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gα q in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gα q -dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gα q in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gα q in cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. A Chimeric Peptide Composed of a Dermaseptin Derivative and an RNA III-Inhibiting Peptide Prevents Graft-Associated Infections by Antibiotic-Resistant Staphylococci

    Science.gov (United States)

    Balaban, Naomi; Gov, Yael; Giacometti, Andrea; Cirioni, Oscar; Ghiselli, Roberto; Mocchegiani, Federico; Orlando, Fiorenza; D'Amato, Giuseppina; Saba, Vittorio; Scalise, Giorgio; Bernes, Sabina; Mor, Amram

    2004-01-01

    Staphylococcal bacteria are a prevalent cause of infections associated with foreign bodies and indwelling medical devices. Bacteria are capable of escaping antibiotic treatment through encapsulation into biofilms. RNA III-inhibiting peptide (RIP) is a heptapeptide that inhibits staphylococcal biofilm formation by obstructing quorum-sensing mechanisms. K4-S4(1-13)a is a 13-residue dermaseptin derivative (DD13) believed to kill bacteria via membrane disruption. We tested each of these peptides as well as a hybrid construct, DD13-RIP, for their ability to inhibit bacterial proliferation and suppress quorum sensing in vitro and for their efficacy in preventing staphylococcal infection in a rat graft infection model with methicillin-resistant Staphylococcus aureus (MRSA) or S. epidermidis (MRSE). In vitro, proliferation assays demonstrated that RIP had no inhibitory effect, while DD13-RIP and DD13 were equally effective, and that the chimeric peptide but not DD13 was slightly more effective than RIP in inhibiting RNA III synthesis, a regulatory RNA molecule important for staphylococcal pathogenesis. In vivo, the three peptides reduced graft-associated bacterial load in a dose-dependent manner, but the hybrid peptide was most potent in totally preventing staphylococcal infections at the lowest dose. In addition, each of the peptides acted synergistically with antibiotics. The data indicate that RIP and DD13 act in synergy by attacking bacteria simultaneously by two different mechanisms. Such a chimeric peptide may be useful for coating medical devices to prevent drug-resistant staphylococcal infections. PMID:15215107

  6. Potent inhibition of late stages of hepadnavirus replication by a modified cell penetrating peptide

    DEFF Research Database (Denmark)

    Abdul, Fabien; Ndeboko, Bénédicte; Buronfosse, Thierry

    2012-01-01

    Cationic cell-penetrating peptides (CPPs) and their lipid domain-conjugates (CatLip) are agents for the delivery of (uncharged) biologically active molecules into the cell. Using infection and transfection assays we surprisingly discovered that CatLip peptides were able to inhibit replication...... by confocal laser scanning microscopy indicating severe structural changes of preS/S. Sucrose gradient analysis of supernatants from Deca-(Arg)8-treated cells showed unaffected naked viral nucleocapsids release, which was concomitant with a complete arrest of virion and surface protein-containing subviral...

  7. C-peptide prevents SMAD3 binding to alpha promoters to inhibit collagen type IV synthesis.

    Science.gov (United States)

    Li, Yanning; Zhong, Yan; Gong, Wenjian; Gao, Xuehan; Qi, Huanli; Liu, Kun; Qi, Jinsheng

    2018-07-01

    Activation of transforming growth factor β1 (TGFB1)/SMAD3 signaling may lead to additional synthesis of collagen type IV (COL4), which is a major contributor to extracellular matrix (ECM) accumulation in diabetic nephropathy (DN). C-peptide can attenuate fibrosis to have unique beneficial effects in DN. However, whether and how C-peptide affects TGFB1/SMAD3-activated COL4 synthesis is unclear. In this study, pathological changes, expression of COL4 a1-a5 chains ( Col4a1-a5 ), COL4 distribution and protein and TGFB1 and SMAD3 protein were first assessed in a rat model of diabetes. Then, rat mesangial cells were treated with high glucose (HG) and/or C-peptide to investigate the underlying mechanism. Col4a1-a5 expression, COL4 protein and secretion, TGFB1 protein, SMAD3 nuclear translocation and binding of SMAD3 to its cognate sites in the promoters of Col4a1a2 , Col4a3a4 and Col4a5 were measured. It was found that C-peptide attenuated glomerular pathological changes and suppressed renal Col4a1 -a5 mRNA expression, COL4 protein content and TGFB1 protein content. C-peptide had a dose-dependent effect to inhibit Col4a1-a5 mRNA expression, COL4 protein content and secretion, in HG-stimulated mesangial cells. In addition, the HG-induced increase in TGFB1 protein content was significantly reduced by C-peptide. Although not apparently affecting SMAD3 nuclear translocation, C-peptide prevented SMAD3 from binding to its sites in the Col4a1a2 , Col4a3a4 and Col4a5 promoters in HG-stimulated mesangial cells. In conclusion, C-peptide could prevent SMAD3 from binding to its sites in the Col4a1a2 , Col4a3a4 and Col4a5 promoters, to inhibit COL4 generation. These results may provide a mechanism for the alleviation of fibrosis in DN by C-peptide. © 2018 Society for Endocrinology.

  8. Melanocortin peptides inhibit production of proinflammatory cytokines and nitric oxide by activated microglia.

    Science.gov (United States)

    Delgado, R; Carlin, A; Airaghi, L; Demitri, M T; Meda, L; Galimberti, D; Baron, P; Lipton, J M; Catania, A

    1998-06-01

    Inflammatory processes contribute to neurodegenerative disease, stroke, encephalitis, and other central nervous system (CNS) disorders. Activated microglia are a source of cytokines and other inflammatory agents within the CNS and it is therefore important to control glial function in order to preserve neural cells. Melanocortin peptides are pro-opiomelanocortin-derived amino acid sequences that include alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH). These peptides have potent and broad anti-inflammatory effects. We tested effects of alpha-MSH (1-13), alpha-MSH (11-13), and ACTH (1-24) on production of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO) in a cultured murine microglial cell line (N9) stimulated with lipopolysaccharide (LPS) plus interferon gamma (IFN-gamma). Melanocortin peptides inhibited production of these cytokines and NO in a concentration-related fashion, probably by increasing intracellular cAMP. When stimulated with LPS + IFN-gamma, microglia increased release of alpha-MSH. Production of TNF-alpha, IL-6, and NO was greater in activated microglia after innmunoneutralization of endogenous alpha-MSH. The results suggest that alpha-MSH is an autocrine factor in microglia. Because melanocortin peptides inhibit production of pro-inflammatory mediators by activated microglia they might be useful in treatment of inflammatory/degenerative brain disorders.

  9. Macrocycle peptides delineate locked-open inhibition mechanism for microorganism phosphoglycerate mutases

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Hao; Dranchak, Patricia; Li, Zhiru; MacArthur, Ryan; Munson, Matthew S.; Mehzabeen, Nurjahan; Baird, Nathan J.; Battalie, Kevin P.; Ross, David; Lovell, Scott; Carlow, Clotilde K.S.; Suga, Hiroaki; Inglese, James (U of Tokyo); (NEB); (Kansas); (NIH); (NIST); (HHMI)

    2017-04-03

    Glycolytic interconversion of phosphoglycerate isomers is catalysed in numerous pathogenic microorganisms by a cofactor-independent mutase (iPGM) structurally distinct from the mammalian cofactor-dependent (dPGM) isozyme. The iPGM active site dynamically assembles through substrate-triggered movement of phosphatase and transferase domains creating a solvent inaccessible cavity. Here we identify alternate ligand binding regions using nematode iPGM to select and enrich lariat-like ligands from an mRNA-display macrocyclic peptide library containing >1012 members. Functional analysis of the ligands, named ipglycermides, demonstrates sub-nanomolar inhibition of iPGM with complete selectivity over dPGM. The crystal structure of an iPGM macrocyclic peptide complex illuminated an allosteric, locked-open inhibition mechanism placing the cyclic peptide at the bi-domain interface. This binding mode aligns the pendant lariat cysteine thiolate for coordination with the iPGM transition metal ion cluster. The extended charged, hydrophilic binding surface interaction rationalizes the persistent challenges these enzymes have presented to small-molecule screening efforts highlighting the important roles of macrocyclic peptides in expanding chemical diversity for ligand discovery.

  10. Novel cyclo-peptides inhibit Ebola pseudotyped virus entry by targeting primed GP protein.

    Science.gov (United States)

    Li, Quanjie; Ma, Ling; Yi, Dongrong; Wang, Han; Wang, Jing; Zhang, Yongxin; Guo, Ying; Li, Xiaoyu; Zhou, Jinming; Shi, Yi; Gao, George F; Cen, Shan

    2018-07-01

    Ebola virus (EBOV) causes fatal hemorrhagic fever with high death rates in human. Currently, there are no available clinically-approved prophylactic or therapeutic treatments. The recently solved crystal structure of cleavage-primed EBOV glycoprotein (GPcl) in complex with the C domain of endosomal protein Niemann-Pick C1 (NPC1) provides a new target for the development of EBOV entry inhibitors. In this work, a computational approach using docking and molecular dynamic simulations is carried out for the rational design of peptide inhibitors. A novel cyclo-peptide (Pep-3.3) was identified to target at the late stage of EBOV entry and exhibit specific inhibitory activity against EBOV-GP pseudotyped viruses, with 50% inhibitory concentration (IC50) of 5.1 μM. In vitro binding assay and molecular simulations revealed that Pep-3.3 binds to GPcl with a KD value of 69.7 μM, through interacting with predicted residues in the hydrophobic binding pocket of GPcl. Mutation of predicted residues T83 caused resistance to Pep-3.3 inhibition in viral infectivity, providing preliminary support for the model of the peptide binding to GPcl. This study demonstrates the feasibility of inhibiting EBOV entry by targeting GPcl with peptides. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Glucagon-like peptide 1 inhibition of gastric emptying outweighs its insulinotropic effects in healthy humans

    DEFF Research Database (Denmark)

    Nauck, M A; Niedereichholz, U; Ettler, R

    1997-01-01

    Glucagon-like peptide 1 (GLP-1) has been shown to inhibit gastric emptying of liquid meals in type 2 diabetic patients. It was the aim of the present study to compare the action of physiological and pharmacological doses of intravenous GLP-1-(7-36) amide and GLP-1-(7-37) on gastric emptying...... (0-240 min), integrated incremental glucose (P inhibits gastric emptying also in normal subjects, 2) physiological doses (0.4 pmol.kg-1.min-1) still have...

  12. A Fusion-Inhibiting Peptide against Rift Valley Fever Virus Inhibits Multiple, Diverse Viruses

    Science.gov (United States)

    2013-09-12

    performance liquid chroma- tography to greater than 95% purity (Bio-synthesis, Inc., Lewisville, TX). Lyophilized peptides were initially resuspended in...lesser extent RVFV-6sc, were found to precipitate Gc (Figure 5); however, in the presence of the non- ionic detergent Triton-X, which will solubilize...Viral membrane fusion. Nat Struct Mol Biol 15: 690–698. 12. Allison SL, Schalich J, Stiasny K, Mandl CW, Kunz C, et al. (1995) Oligomeric rearrangement

  13. 2-d and 1-d Nanomaterials Construction through Peptide Computational Design and Solution Assembly

    Science.gov (United States)

    Pochan, Darrin

    Self-assembly of molecules is an attractive materials construction strategy due to its simplicity in application. By considering peptidic molecules in the bottom-up materials self-assembly design process, one can take advantage of inherently biomolecular attributes; intramolecular folding events, secondary structure, and electrostatic/H-bonding/hydrophobic interactions to define hierarchical material structure and consequent properties. Importantly, while biomimicry has been a successful strategy for the design of new peptide molecules for intermolecular assembly, computational tools have been developed to de novo design peptide molecules required for construction of pre-determined, desired nanostructures and materials. A new system comprised of coiled coil bundle motifs theoretically designed to assemble into designed, one and two-dimensional nanostructures will be introduced. The strategy provides the opportunity for arbitrary nanostructure formation, i.e. structures not observed in nature, with peptide molecules. Importantly, the desired nanostructure was chosen first while the peptides needed for coiled coil formation and subsequent nanomaterial formation were determined computationally. Different interbundle, two-dimensional nanostructures are stabilized by differences in amino acid composition exposed on the exterior of the coiled coil bundles. Computation was able to determine molecules required for different interbundle symmetries within two-dimensional sheets stabilized by subtle differences in amino acid composition of the inherent peptides. Finally, polymers were also created through covalent interactions between bundles that allowed formation of architectures spanning flexible network forming chains to ultra-stiff polymers, all with the same building block peptides. The success of the computational design strategy is manifested in the nanomaterial results as characterized by electron microscopy, scattering methods, and biophysical techniques. Support

  14. Selection of peptides interfering with protein-protein interaction.

    Science.gov (United States)

    Gaida, Annette; Hagemann, Urs B; Mattay, Dinah; Räuber, Christina; Müller, Kristian M; Arndt, Katja M

    2009-01-01

    Cell physiology depends on a fine-tuned network of protein-protein interactions, and misguided interactions are often associated with various diseases. Consequently, peptides, which are able to specifically interfere with such adventitious interactions, are of high interest for analytical as well as medical purposes. One of the most abundant protein interaction domains is the coiled-coil motif, and thus provides a premier target. Coiled coils, which consist of two or more alpha-helices wrapped around each other, have one of the simplest interaction interfaces, yet they are able to confer highly specific homo- and heterotypic interactions involved in virtually any cellular process. While there are several ways to generate interfering peptides, the combination of library design with a powerful selection system seems to be one of the most effective and promising approaches. This chapter guides through all steps of such a process, starting with library options and cloning, detailing suitable selection techniques and ending with purification for further down-stream characterization. Such generated peptides will function as versatile tools to interfere with the natural function of their targets thereby illuminating their down-stream signaling and, in general, promoting understanding of factors leading to specificity and stability in protein-protein interactions. Furthermore, peptides interfering with medically relevant proteins might become important diagnostics and therapeutics.

  15. Proline-rich peptide from the coral pathogen Vibrio shiloi that inhibits photosynthesis of Zooxanthellae.

    Science.gov (United States)

    Banin, E; Khare, S K; Naider, F; Rosenberg, E

    2001-04-01

    The coral-bleaching bacterium Vibrio shiloi biosynthesizes and secretes an extracellular peptide, referred to as toxin P, which inhibits photosynthesis of coral symbiotic algae (zooxanthellae). Toxin P was produced during the stationary phase when the bacterium was grown on peptone or Casamino Acids media at 29 degrees C. Glycerol inhibited the production of toxin P. Toxin P was purified to homogeneity, yielding the following 12-residue peptide: PYPVYAPPPVVP (molecular weight, 1,295.54). The structure of toxin P was confirmed by chemical synthesis. In the presence of 12.5 mM NH(4)Cl, pure natural or synthetic toxin P (10 microM) caused a 64% decrease in the photosynthetic quantum yield of zooxanthellae within 5 min. The inhibition was proportional to the toxin P concentration. Toxin P bound avidly to zooxanthellae, such that subsequent addition of NH(4)Cl resulted in rapid inhibition of photosynthesis. When zooxanthellae were incubated in the presence of NH(4)Cl and toxin P, there was a rapid decrease in the pH (pH 7.8 to 7.2) of the bulk liquid, suggesting that toxin P facilitates transport of NH(3) into the cell. It is known that uptake of NH(3) into cells can destroy the pH gradient and block photosynthesis. This mode of action of toxin P can help explain the mechanism of coral bleaching by V. shiloi.

  16. Venom-derived peptides inhibiting Kir channels: Past, present, and future.

    Science.gov (United States)

    Doupnik, Craig A

    2017-12-01

    Inwardly rectifying K + (Kir) channels play a significant role in vertebrate and invertebrate biology by regulating the movement of K + ions involved in membrane transport and excitability. Yet unlike other ion channels including their ancestral K + -selective homologs, there are very few venom toxins known to target and inhibit Kir channels with the potency and selectivity found for the Ca 2+ -activated and voltage-gated K + channel families. It is unclear whether this is simply due to a lack of discovery, or instead a consequence of the evolutionary processes that drive the development of venom components towards their targets based on a collective efficacy to 1) elicit pain for defensive purposes, 2) promote paralysis for prey capture, or 3) facilitate delivery of venom components into the circulation. The past two decades of venom screening has yielded three venom peptides with inhibitory activity towards mammalian Kir channels, including the discovery of tertiapin, a high-affinity pore blocker from the venom of the European honey bee Apis mellifera. Venomics and structure-based computational approaches represent exciting new frontiers for venom peptide development, where re-engineering peptide 'scaffolds' such as tertiapin may aid in the quest to expand the palette of potent and selective Kir channel blockers for future research and potentially new therapeutics. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.' Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization

    International Nuclear Information System (INIS)

    Kim, Sun Young; Song, Kyung-A; Kieff, Elliott; Kang, Myung-Soo

    2012-01-01

    Highlights: ► Evidence that targeting EBNA1 dimer, an EBV onco-antigen, can be achievable. ► A small molecule and a peptide as EBNA1 dimerization inhibitors identified. ► Both inhibitors associated with EBNA1 and blocked EBNA1 DNA binding activity. ► Also, prevented its dimerization, and repressed viral gene transcription. -- Abstract: Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)’s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459–607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-Jκ binding to the Jκ site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560–574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated with EBNA1 in vitro, and repressed EBNA1-dependent transcription in vivo. Collectively, this study describes two

  18. Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun Young; Song, Kyung-A [Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Samsung Biomedical Research Institute (SBRI), Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Kieff, Elliott [Department of Medicine, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States); Kang, Myung-Soo, E-mail: mkang@skku.edu [Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Samsung Biomedical Research Institute (SBRI), Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Department of Medicine, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States)

    2012-07-27

    Highlights: Black-Right-Pointing-Pointer Evidence that targeting EBNA1 dimer, an EBV onco-antigen, can be achievable. Black-Right-Pointing-Pointer A small molecule and a peptide as EBNA1 dimerization inhibitors identified. Black-Right-Pointing-Pointer Both inhibitors associated with EBNA1 and blocked EBNA1 DNA binding activity. Black-Right-Pointing-Pointer Also, prevented its dimerization, and repressed viral gene transcription. -- Abstract: Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)'s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459-607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-J{kappa} binding to the J{kappa} site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560-574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated

  19. A synthetic peptide blocking TRPV1 activation inhibits UV-induced skin responses.

    Science.gov (United States)

    Kang, So Min; Han, Sangbum; Oh, Jang-Hee; Lee, Young Mee; Park, Chi-Hyun; Shin, Chang-Yup; Lee, Dong Hun; Chung, Jin Ho

    2017-10-01

    Transient receptor potential type 1 (TRPV1) can be activated by ultraviolet (UV) irradiation, and mediates UV-induced matrix metalloproteinase (MMP)-1 and proinflammatory cytokines in keratinocytes. Various chemicals and compounds targeting TRPV1 activation have been developed, but are not in clinical use mostly due to their safety issues. We aimed to develop a novel TRPV1-targeting peptide to inhibit UV-induced responses in human skin. We designed and generated a novel TRPV1 inhibitory peptide (TIP) which mimics the specific site in TRPV1 (aa 701-709: Gln-Arg-Ala-Ile-Thr-Ile-Leu-Asp-Thr, QRAITILDT), Thr 705 , and tested its efficacy of blocking UV-induced responses in HaCaT, mouse, and human skin. TIP effectively inhibited capsaicin-induced calcium influx and TRPV1 activation. Treatment of HaCaT with TIP prevented UV-induced increases of MMP-1 and pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor-α. In mouse skin in vivo, TIP inhibited UV-induced skin thickening and prevented UV-induced expression of MMP-13 and MMP-9. Moreover, TIP attenuated UV-induced erythema and the expression of MMP-1, MMP-2, IL-6, and IL-8 in human skin in vivo. The novel synthetic peptide targeting TRPV1 can ameliorate UV-induced skin responses in vitro and in vivo, providing a promising therapeutic approach against UV-induced inflammation and photoaging. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

  20. Streptococcus mutans competence-stimulating peptide inhibits Candida albicans hypha formation.

    Science.gov (United States)

    Jarosz, Lucja M; Deng, Dong Mei; van der Mei, Henny C; Crielaard, Wim; Krom, Bastiaan P

    2009-11-01

    The oral cavity is colonized by microorganisms growing in biofilms in which interspecies interactions take place. Streptococcus mutans grows in biofilms on enamel surfaces and is considered one of the main etiological agents of human dental caries. Candida albicans is also commonly found in the human oral cavity, where it interacts with S. mutans. C. albicans is a polymorphic fungus, and the yeast-to-hypha transition is involved in virulence and biofilm formation. The aim of this study was to investigate interkingdom communication between C. albicans and S. mutans based on the production of secreted molecules. S. mutans UA159 inhibited C. albicans germ tube (GT) formation in cocultures even when physically separated from C. albicans. Only S. mutans spent medium collected in the early exponential phase (4-h-old cultures) inhibited the GT formation of C. albicans. During this phase, S. mutans UA159 produces a quorum-sensing molecule, competence-stimulating peptide (CSP). The role of CSP in inhibiting GT formation was confirmed by using synthetic CSP and a comC deletion strain of S. mutans UA159, which lacks the ability to produce CSP. Other S. mutans strains and other Streptococcus spp. also inhibited GT formation but to different extents, possibly reflecting differences in CSP amino acid sequences among Streptococcus spp. or differences in CSP accumulation in the media. In conclusion, CSP, an S. mutans quorum-sensing molecule secreted during the early stages of growth, inhibits the C. albicans morphological switch.

  1. Chimeric peptide-mediated siRNA transduction to inhibit HIV-1 infection.

    Science.gov (United States)

    Bivalkar-Mehla, Shalmali; Mehla, Rajeev; Chauhan, Ashok

    2017-04-01

    Persistent human immunodeficiency virus 1 (HIV-1) infection provokes immune activation and depletes CD4 +  lymphocytes, leading to acquired immunodeficiency syndrome. Uninterrupted administration of combination antiretroviral therapy (cART) in HIV-infected patients suppresses viral replication to below the detectable level and partially restores the immune system. However, cART-unresponsive residual HIV-1 infection and elusive transcriptionally silent but reactivatable viral reservoirs maintain a permanent viral DNA blue print. The virus rebounds within a few weeks after interruption of suppressive therapy. Adjunct gene therapy to control viral replication by ribonucleic acid interference (RNAi) is a post-transcriptional gene silencing strategy that could suppress residual HIV-1 burden and overcome viral resistance. Small interfering ribonucleic acids (siRNAs) are efficient transcriptional inhibitors, but need delivery systems to reach inside target cells. We investigated the potential of chimeric peptide (FP-PTD) to deliver specific siRNAs to HIV-1-susceptible and permissive cells. Chimeric FP-PTD peptide was designed with an RNA binding domain (PTD) to bind siRNA and a cell fusion peptide domain (FP) to enter cells. FP-PTD-siRNA complex entered and inhibited HIV-1 replication in susceptible cells, and could be a candidate for in vivo testing.

  2. A peptide of heparin cofactor II inhibits endotoxin-mediated shock and invasive Pseudomonas aeruginosa infection.

    Directory of Open Access Journals (Sweden)

    Martina Kalle

    Full Text Available Sepsis and septic shock remain important medical problems with high mortality rates. Today's treatment is based mainly on using antibiotics to target the bacteria, without addressing the systemic inflammatory response, which is a major contributor to mortality in sepsis. Therefore, novel treatment options are urgently needed to counteract these complex sepsis pathologies. Heparin cofactor II (HCII has recently been shown to be protective against Gram-negative infections. The antimicrobial effects were mapped to helices A and D of the molecule. Here we show that KYE28, a 28 amino acid long peptide representing helix D of HCII, is antimicrobial against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida albicans. Moreover, KYE28 binds to LPS and thereby reduces LPS-induced pro-inflammatory responses by decreasing NF-κB/AP-1 activation in vitro. In mouse models of LPS-induced shock, KYE28 significantly enhanced survival by dampening the pro-inflammatory cytokine response. Finally, in an invasive Pseudomonas infection model, the peptide inhibited bacterial growth and reduced the pro-inflammatory response, which lead to a significant reduction of mortality. In summary, the peptide KYE28, by simultaneously targeting bacteria and LPS-induced pro-inflammatory responses represents a novel therapeutic candidate for invasive infections.

  3. A peptide of heparin cofactor II inhibits endotoxin-mediated shock and invasive Pseudomonas aeruginosa infection.

    Science.gov (United States)

    Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath; van der Plas, Mariena J A; Mörgelin, Matthias; Malmsten, Martin; Schmidtchen, Artur

    2014-01-01

    Sepsis and septic shock remain important medical problems with high mortality rates. Today's treatment is based mainly on using antibiotics to target the bacteria, without addressing the systemic inflammatory response, which is a major contributor to mortality in sepsis. Therefore, novel treatment options are urgently needed to counteract these complex sepsis pathologies. Heparin cofactor II (HCII) has recently been shown to be protective against Gram-negative infections. The antimicrobial effects were mapped to helices A and D of the molecule. Here we show that KYE28, a 28 amino acid long peptide representing helix D of HCII, is antimicrobial against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida albicans. Moreover, KYE28 binds to LPS and thereby reduces LPS-induced pro-inflammatory responses by decreasing NF-κB/AP-1 activation in vitro. In mouse models of LPS-induced shock, KYE28 significantly enhanced survival by dampening the pro-inflammatory cytokine response. Finally, in an invasive Pseudomonas infection model, the peptide inhibited bacterial growth and reduced the pro-inflammatory response, which lead to a significant reduction of mortality. In summary, the peptide KYE28, by simultaneously targeting bacteria and LPS-induced pro-inflammatory responses represents a novel therapeutic candidate for invasive infections.

  4. Simultaneous inhibition of key growth pathways in melanoma cells and tumor regression by a designed bidentate constrained helical peptide.

    Science.gov (United States)

    Dhar, Amlanjyoti; Mallick, Shampa; Ghosh, Piya; Maiti, Atanu; Ahmed, Israr; Bhattacharya, Seemana; Mandal, Tapashi; Manna, Asit; Roy, Koushik; Singh, Sandeep; Nayak, Dipak Kumar; Wilder, Paul T; Markowitz, Joseph; Weber, David; Ghosh, Mrinal K; Chattopadhyay, Samit; Guha, Rajdeep; Konar, Aditya; Bandyopadhyay, Santu; Roy, Siddhartha

    2014-07-01

    Protein-protein interactions are part of a large number of signaling networks and potential targets for drug development. However, discovering molecules that can specifically inhibit such interactions is a major challenge. S100B, a calcium-regulated protein, plays a crucial role in the proliferation of melanoma cells through protein-protein interactions. In this article, we report the design and development of a bidentate conformationally constrained peptide against dimeric S100B based on a natural tight-binding peptide, TRTK-12. The helical conformation of the peptide was constrained by the substitution of α-amino isobutyric acid--an amino acid having high helical propensity--in positions which do not interact with S100B. A branched bidentate version of the peptide was bound to S100B tightly with a dissociation constant of 8 nM. When conjugated to a cell-penetrating peptide, it caused growth inhibition and rapid apoptosis in melanoma cells. The molecule exerts antiproliferative action through simultaneous inhibition of key growth pathways, including reactivation of wild-type p53 and inhibition of Akt and STAT3 phosphorylation. The apoptosis induced by the bidentate constrained helix is caused by direct migration of p53 to mitochondria. At moderate intravenous dose, the peptide completely inhibits melanoma growth in a mouse model without any significant observable toxicity. The specificity was shown by lack of ability of a double mutant peptide to cause tumor regression at the same dose level. The methodology described here for direct protein-protein interaction inhibition may be effective for rapid development of inhibitors against relatively weak protein-protein interactions for de novo drug development. © 2014 Wiley Periodicals, Inc.

  5. Preliminary study on the inhibition of nuclear internalization of Tat peptides by conjugation with a receptor-specific peptide and fluorescent dyes

    Science.gov (United States)

    Shen, Duanwen; Liang, Kexiang; Ye, Yunpeng; Tetteh, Elizabeth; Achilefu, Samuel

    2006-02-01

    Numerous studies have shown that basic Tat peptide (48-57) internalized non-specifically in cells and localized in the nucleus. However, localization of imaging agents in cellular nucleus is not desirable because of the potential mutagenesis. When conjugated to the peptides that undergo receptor-mediated endocytosis, Tat peptide could target specific cells or pathologic tissue. We tested this hypothesis by incorporating a somatostatin receptor-avid peptide (octreotate, Oct) and two different fluorescent dyes, Cypate 2 (Cy2) and fluorescein 5'-carboxlic acid (5-FAM), into the Tat-peptide sequence. In addition to the Cy2 or 5-FAM-labeled Oct conjugated to Tat peptide (Tat) to produce Tat-Oct-Cypate2 or Tat-Oct-5-FAM, we also labeled the Tat the Tat peptide with these dyes (Tat-Cy2 and Tat-5-FAM) to serve as positive control. A somatostatin receptor-positive pancreatic tumor cell line, AR42J, was used to assess cell internalization. The results show that Tat-5-FAM and Tat-Cypate2 localized in both nucleus and cytoplasm of the cells. In contrast to Tat-Oct-Cypate2, which localized in both the cytoplasm and nucleus, Tat-Oct-5-FAM internalized in the cytoplasm but not in the nucleus of AR42J cells. The internalizations were inhibited by adding non-labeled corresponding peptides, suggesting that the endocytoses of each group of labeled and the corresponding unlabeled compounds occurred through a common pathway. Thus, fluorescent probes and endocytosis complex between octreotate and somatostatin receptors in cytoplasm could control nuclear internalization of Tat peptides.

  6. Klebsazolicin inhibits 70S ribosome by obstructing the peptide exit tunnel

    Energy Technology Data Exchange (ETDEWEB)

    Metelev, Mikhail; Osterman, Ilya A.; Ghilarov, Dmitry; Khabibullina, Nelli F.; Yakimov, Alexander; Shabalin, Konstantin; Utkina, Irina; Travin, Dmitry Y.; Komarova, Ekaterina S.; Serebryakova, Marina; Artamonova, Tatyana; Khodorkovskii, Mikhail; Konevega, Andrey L.; Sergiev, Petr V.; Severinov, Konstantin; Polikanov, Yury S.

    2017-08-28

    Whereas screening of the small-molecule metabolites produced by most cultivatable microorganisms often results in the rediscovery of known compounds, genome-mining programs allow researchers to harness much greater chemical diversity, and result in the discovery of new molecular scaffolds. Here we report the genome-guided identification of a new antibiotic, klebsazolicin (KLB), from Klebsiella pneumoniae that inhibits the growth of sensitive cells by targeting ribosomes. A ribosomally synthesized post-translationally modified peptide (RiPP), KLB is characterized by the presence of a unique N-terminal amidine ring that is essential for its activity. Biochemical in vitro studies indicate that KLB inhibits ribosomes by interfering with translation elongation. Structural analysis of the ribosome–KLB complex showed that the compound binds in the peptide exit tunnel overlapping with the binding sites of macrolides or streptogramin-B. KLB adopts a compact conformation and largely obstructs the tunnel. Engineered KLB fragments were observed to retain in vitro activity, and thus have the potential to serve as a starting point for the development of new bioactive compounds.

  7. Selective inhibition by a synthetic hirudin peptide of fibrin-dependent thrombosis in baboons

    International Nuclear Information System (INIS)

    Cadroy, Y.; Hanson, S.R.; Harker, L.A.; Maraganore, J.M.

    1991-01-01

    To determine the importance of the thrombin substrate recognition exosite for fibrinogen binding in the formation of both arterial and venous thrombi the authors evaluated the antithrombotic effects of the tyrosine-sulfated dodecapeptide from residues 53-64 of hirudin (H peptide) in a nonhuman primate model. This peptide was studied because it inhibits thrombin cleavages of fibrinogen by simple competition without blocking enzyme catalytic-site function. When an exteriorized arteriovenous access shunt model was used in baboons (Papio anubis), thrombus formation was induced by placing a thrombogenic device made of (i) a segment of tubing coated covalently with type I collagen, which generated platelet-rich thrombi under arterial flow conditions, and (ii) two subsequent annular regions of flow expansion that produced fibrin-rich thrombi typically associated with venous valves and veins. Thrombus formation was quantified by measurements of 111 In-labeled platelet and 125 I-labeled fibrinogen deposition in both arterial-flow and venous-flow portions of the device. These finding suggest that, by competitive inhibition of fibrinogen binding to thrombin, fibrin-rich venous-type thrombus formation may be selectively prevented. This strategy may be therapeutically attractive for preserving normal platelet function when conventional anticoagulant therapy is contraindicated

  8. Inhibition of the myostatin/Smad signaling pathway by short decorin-derived peptides.

    Science.gov (United States)

    El Shafey, Nelly; Guesnon, Mickaël; Simon, Françoise; Deprez, Eric; Cosette, Jérémie; Stockholm, Daniel; Scherman, Daniel; Bigey, Pascal; Kichler, Antoine

    2016-02-15

    Myostatin, also known as growth differentiation factor 8, is a member of the transforming growth factor-beta superfamily that has been shown to play a key role in the regulation of the skeletal muscle mass. Indeed, while myostatin deletion or loss of function induces muscle hypertrophy, its overexpression or systemic administration causes muscle atrophy. Since myostatin blockade is effective in increasing skeletal muscle mass, myostatin inhibitors have been actively sought after. Decorin, a member of the small leucine-rich proteoglycan family is a metalloprotein that was previously shown to bind and inactivate myostatin in a zinc-dependent manner. Furthermore, the myostatin-binding site has been shown to be located in the decorin N-terminal domain. In the present study, we investigated the anti-myostatin activity of short and soluble fragments of decorin. Our results indicate that the murine decorin peptides DCN48-71 and 42-65 are sufficient for inactivating myostatin in vitro. Moreover, we show that the interaction of mDCN48-71 to myostatin is strictly zinc-dependent. Binding of myostatin to activin type II receptor results in the phosphorylation of Smad2/3. Addition of the decorin peptide 48-71 decreased in a dose-dependent manner the myostatin-induced phosphorylation of Smad2 demonstrating thereby that the peptide inhibits the activation of the Smad signaling pathway. Finally, we found that mDCN48-71 displays a specificity towards myostatin, since it does not inhibit other members of the transforming growth factor-beta family. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Dynamic behavior of small heat shock protein inhibition on amyloid fibrillization of a small peptide (SSTSAA) from RNase A

    International Nuclear Information System (INIS)

    Xi, Dong; Dong, Xiao; Deng, Wei; Lai, Luhua

    2011-01-01

    Highlights: ► Mechanism of small heat shock protein inhibition on fibril formation was studied. ► Peptide SSTSAA with modified ends was used for amyloid fibril formation. ► FRET signal was followed during the fibril formation. ► Mj HSP16.5 inhibits fibril formation when introduced in the lag phase. ► Mj HSP16.5 slows down fibril formation when introduced after the lag phase. -- Abstract: Small heat shock proteins, a class of molecular chaperones, are reported to inhibit amyloid fibril formation in vitro, while the mechanism of inhibition remains unknown. In the present study, we investigated the mechanism by which Mj HSP16.5 inhibits amyloid fibril formation of a small peptide (SSTSAA) from RNase A. A model peptide (dansyl-SSTSAA-W) was designed by introducing a pair of fluorescence resonance energy transfer (FRET) probes into the peptide, allowing for the monitoring of fibril formation by this experimental model. Mj HSP16.5 completely inhibited fibril formation of the model peptide at a molar ratio of 1:120. The dynamic process of fibril formation, revealed by FRET, circular dichroism, and electron microscopy, showed a lag phase of about 2 h followed by a fast growth period. The effect of Mj HSP16.5 on amyloid fibril formation was investigated by adding it into the incubation solution during different growth phases. Adding Mj HSP16.5 to the incubating peptide before or during the lag phase completely inhibited fibril formation. However, introducing Mj HSP16.5 after the lag phase only slowed down the fibril formation process by adhering to the already formed fibrils. These findings provide insight into the inhibitory roles of small heat shock proteins on amyloid fibril formation at the molecular level.

  10. Dynamic behavior of small heat shock protein inhibition on amyloid fibrillization of a small peptide (SSTSAA) from RNase A

    Energy Technology Data Exchange (ETDEWEB)

    Xi, Dong [BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Center for Theoretical Biology, Peking University, Beijing 100871 (China); Dong, Xiao; Deng, Wei [BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Lai, Luhua, E-mail: lhlai@pku.edu.cn [BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Center for Theoretical Biology, Peking University, Beijing 100871 (China)

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Mechanism of small heat shock protein inhibition on fibril formation was studied. Black-Right-Pointing-Pointer Peptide SSTSAA with modified ends was used for amyloid fibril formation. Black-Right-Pointing-Pointer FRET signal was followed during the fibril formation. Black-Right-Pointing-Pointer Mj HSP16.5 inhibits fibril formation when introduced in the lag phase. Black-Right-Pointing-Pointer Mj HSP16.5 slows down fibril formation when introduced after the lag phase. -- Abstract: Small heat shock proteins, a class of molecular chaperones, are reported to inhibit amyloid fibril formation in vitro, while the mechanism of inhibition remains unknown. In the present study, we investigated the mechanism by which Mj HSP16.5 inhibits amyloid fibril formation of a small peptide (SSTSAA) from RNase A. A model peptide (dansyl-SSTSAA-W) was designed by introducing a pair of fluorescence resonance energy transfer (FRET) probes into the peptide, allowing for the monitoring of fibril formation by this experimental model. Mj HSP16.5 completely inhibited fibril formation of the model peptide at a molar ratio of 1:120. The dynamic process of fibril formation, revealed by FRET, circular dichroism, and electron microscopy, showed a lag phase of about 2 h followed by a fast growth period. The effect of Mj HSP16.5 on amyloid fibril formation was investigated by adding it into the incubation solution during different growth phases. Adding Mj HSP16.5 to the incubating peptide before or during the lag phase completely inhibited fibril formation. However, introducing Mj HSP16.5 after the lag phase only slowed down the fibril formation process by adhering to the already formed fibrils. These findings provide insight into the inhibitory roles of small heat shock proteins on amyloid fibril formation at the molecular level.

  11. Shape-specific nanostructured protein mimics from de novo designed chimeric peptides.

    Science.gov (United States)

    Jiang, Linhai; Yang, Su; Lund, Reidar; Dong, He

    2018-01-30

    Natural proteins self-assemble into highly-ordered nanoscaled architectures to perform specific functions. The intricate functions of proteins have provided great impetus for researchers to develop strategies for designing and engineering synthetic nanostructures as protein mimics. Compared to the success in engineering fibrous protein mimetics, the design of discrete globular protein-like nanostructures has been challenging mainly due to the lack of precise control over geometric packing and intermolecular interactions among synthetic building blocks. In this contribution, we report an effective strategy to construct shape-specific nanostructures based on the self-assembly of chimeric peptides consisting of a coiled coil dimer and a collagen triple helix folding motif. Under salt-free conditions, we showed spontaneous self-assembly of the chimeric peptides into monodisperse, trigonal bipyramidal-like nanoparticles with precise control over the stoichiometry of two folding motifs and the geometrical arrangements relative to one another. Three coiled coil dimers are interdigitated on the equatorial plane while the two collagen triple helices are located in the axial position, perpendicular to the coiled coil plane. A detailed molecular model was proposed and further validated by small angle X-ray scattering experiments and molecular dynamics (MD) simulation. The results from this study indicated that the molecular folding of each motif within the chimeric peptides and their geometric packing played important roles in the formation of discrete protein-like nanoparticles. The peptide design and self-assembly mechanism may open up new routes for the construction of highly organized, discrete self-assembling protein-like nanostructures with greater levels of control over assembly accuracy.

  12. Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc.

    Science.gov (United States)

    Barriga, Gonzalo P; Villalón-Letelier, Fernando; Márquez, Chantal L; Bignon, Eduardo A; Acuña, Rodrigo; Ross, Breyan H; Monasterio, Octavio; Mardones, Gonzalo A; Vidal, Simon E; Tischler, Nicole D

    2016-07-01

    Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses.

  13. Signal peptide-dependent inhibition of MHC class I heavy chain translation by rhesus cytomegalovirus.

    Directory of Open Access Journals (Sweden)

    Colin J Powers

    2008-10-01

    Full Text Available The US2-11 region of human and rhesus cytomegalovirus encodes a conserved family of glycoproteins that inhibit MHC-I assembly with viral peptides, thus preventing cytotoxic T cell recognition. Since HCMV lacking US2-11 is no longer able to block assembly and transport of MHC-I, we examined whether this is also observed for RhCMV lacking the corresponding region. Unexpectedly, recombinant RhCMV lacking US2-11 was still able to inhibit MHC-I expression in infected fibroblasts, suggesting the presence of an additional MHC-I evasion mechanism. Progressive deletion analysis of RhCMV-specific genomic regions revealed that MHC-I expression is fully restored upon additional deletion of rh178. The protein encoded by this RhCMV-specific open reading frame is anchored in the endoplasmic reticulum membrane. In the presence of rh178, RhCMV prevented MHC-I heavy chain (HC expression, but did not inhibit mRNA transcription or association of HC mRNA with translating ribosomes. Proteasome inhibitors stabilized a HC degradation intermediate in the absence of rh178, but not in its presence, suggesting that rh178 prevents completion of HC translation. This interference was signal sequence-dependent since replacing the signal peptide with that of CD4 or murine HC rendered human HCs resistant to rh178. We have identified an inhibitor of antigen presentation encoded by rhesus cytomegalovirus unique in both its lack of homology to any other known protein and in its mechanism of action. By preventing signal sequence-dependent HC translocation, rh178 acts prior to US2, US3 and US11 which attack MHC-I proteins after protein synthesis is completed. Rh178 is the first viral protein known to interfere at this step of the MHC-I pathway, thus taking advantage of the conserved nature of HC leader peptides, and represents a new mechanism of translational interference.

  14. Engineered chimeric peptides with antimicrobial and titanium-binding functions to inhibit biofilm formation on Ti implants.

    Science.gov (United States)

    Geng, Hongjuan; Yuan, Yang; Adayi, Aidina; Zhang, Xu; Song, Xin; Gong, Lei; Zhang, Xi; Gao, Ping

    2018-01-01

    Titanium (Ti) implants have been commonly used in oral medicine. However, despite their widespread clinical application, these implants are susceptible to failure induced by microbial infection due to bacterial biofilm formation. Immobilization of chimeric peptides with antibacterial properties on the Ti surface may be a promising antimicrobial approach to inhibit biofilm formation. Here, chimeric peptides were designed by connecting three sequences (hBD-3-1/2/3) derived from human β-defensin-3 (hBD-3) with Ti-binding peptide-l (TBP-l: RKLPDAGPMHTW) via a triple glycine (G) linker to modify Ti surfaces. Using X-ray photoelectron spectroscopy (XPS), the properties of individual domains of the chimeric peptides were evaluated for their binding activity toward the Ti surface. The antimicrobial and anti-biofilm efficacy of the peptides against initial settlers, Streptococcus oralis (S. oralis), Streptococcus gordonii (S. gordonii) and Streptococcus sanguinis (S. sanguinis), was evaluated with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Transmission electron microscopy (TEM) and real-time quantitative PCR (qRT-PCR) were used to study cell membrane changes and the underlying antimicrobial mechanism. Compared with the other two peptides, TBP-1-GGG-hBD3-3 presented stronger antibacterial activity and remained stable in saliva and serum. Therefore, it was chosen as the best candidate to modify Ti surfaces in this study. This peptide inhibited the growth of initial streptococci and biofilm formation on Ti surfaces with no cytotoxicity to MC3T3-E1 cells. Disruption of the integrity of bacterial membranes and decreased expression of adhesion protein genes from S. gordonii revealed aspects of the antibacterial mechanism of TBP-1-GGG-hBD3-3. We conclude that engineered chimeric peptides with antimicrobial activity provide a potential solution for inhibiting biofilm formation on Ti surfaces to reduce or prevent the occurrence of peri

  15. Transition metal ions mediated tyrosine based short peptide amphiphile nanostructures inhibit bacterial growth.

    Science.gov (United States)

    Joshi, Khashti Ballabh; Singh, Ramesh; Mishra, Narendra Kumar; Kumar, Vikas; Vinayak, Vandana

    2018-05-17

    We report the design and synthesis of biocompatible small peptide based molecule for the controlled and targeted delivery of the encapsulated bioactive metal ions via transforming their internal nanostructures. Tyrosine based short peptide amphiphile (sPA) was synthesized which self-assembled into β-sheet like secondary structures. The self assembly of the designed sPA was modulated by using different bioactive transition metal ions which is confirmed by spectroscopic and microscopic techniques. These bioactive metal ions conjugated sPA hybrid structures are further used to develop antibacterial materials. It is due to the excellent antibacterial activity of zinc ions that the growth of clinically relevant bacteria such as E. Coli was inhibited in the presence of zinc-sPA conjugate. The bacterial test demonstrated that owing to high biocompatibility with bacterial cell, the designed sPA worked as metal ions delivery agent and therefore it can show great potential in locally addressing bacterial infections. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Small stress molecules inhibit aggregation and neurotoxicity of prion peptide 106-126

    International Nuclear Information System (INIS)

    Kanapathipillai, Mathumai; Ku, Sook Hee; Girigoswami, Koyeli; Park, Chan Beum

    2008-01-01

    In prion diseases, the posttranslational modification of host-encoded prion protein PrP c yields a high β-sheet content modified protein PrP sc , which further polymerizes into amyloid fibrils. PrP106-126 initiates the conformational changes leading to the conversion of PrP c to PrP sc . Molecules that can defunctionalize such peptides can serve as a potential tool in combating prion diseases. In microorganisms during stressed conditions, small stress molecules (SSMs) are formed to prevent protein denaturation and maintain protein stability and function. The effect of such SSMs on PrP106-126 amyloid formation is explored in the present study using turbidity, atomic force microscopy (AFM), and cellular toxicity assay. Turbidity and AFM studies clearly depict that the SSMs-ectoine and mannosylglyceramide (MGA) inhibit the PrP106-126 aggregation. Our study also connotes that ectoine and MGA offer strong resistance to prion peptide-induced toxicity in human neuroblastoma cells, concluding that such molecules can be potential inhibitors of prion aggregation and toxicity

  17. Madumycin II inhibits peptide bond formation by forcing the peptidyl transferase center into an inactive state

    Energy Technology Data Exchange (ETDEWEB)

    Osterman, Ilya A.; Khabibullina, Nelli F.; Komarova, Ekaterina S.; Kasatsky, Pavel; Kartsev, Victor G.; Bogdanov, Alexey A.; Dontsova, Olga A.; Konevega, Andrey L.; Sergiev, Petr V.; Polikanov, Yury S. (InterBioScreen); (UIC); (MSU-Russia); (Kurchatov)

    2017-05-13

    The emergence of multi-drug resistant bacteria is limiting the effectiveness of commonly used antibiotics, which spurs a renewed interest in revisiting older and poorly studied drugs. Streptogramins A is a class of protein synthesis inhibitors that target the peptidyl transferase center (PTC) on the large subunit of the ribosome. In this work, we have revealed the mode of action of the PTC inhibitor madumycin II, an alanine-containing streptogramin A antibiotic, in the context of a functional 70S ribosome containing tRNA substrates. Madumycin II inhibits the ribosome prior to the first cycle of peptide bond formation. It allows binding of the tRNAs to the ribosomal A and P sites, but prevents correct positioning of their CCA-ends into the PTC thus making peptide bond formation impossible. We also revealed a previously unseen drug-induced rearrangement of nucleotides U2506 and U2585 of the 23S rRNA resulting in the formation of the U2506•G2583 wobble pair that was attributed to a catalytically inactive state of the PTC. The structural and biochemical data reported here expand our knowledge on the fundamental mechanisms by which peptidyl transferase inhibitors modulate the catalytic activity of the ribosome.

  18. Efficient inhibition of tumor angiogenesis and growth by a synthetic peptide blocking S100A4-methionine aminopeptidase 2 interaction

    Directory of Open Access Journals (Sweden)

    Takahiro Ochiya

    Full Text Available The prometastatic calcium-binding protein, S100A4, is expressed in endothelial cells, and its downregulation markedly suppresses tumor angiogenesis in a xenograft cancer model. Given that endothelial S100A4 can be a molecular target for inhibiting tumor angiogenesis, we addressed here whether synthetic peptide capable of blocking S100A4-effector protein interaction could be a novel antiangiogenic agent. To examine this hypothesis, we focused on the S100A4-binding domain of methionine aminopeptidase 2, an effector protein, which plays a role in endothelial cell growth. Overexpression of the domain in mouse endothelial MSS31 cells reduced DNA synthesis, and the corresponding synthetic peptide (named NBD indeed interacted with S100A4 and inhibited capillary formation in vitro and new blood vessel formation in vivo. Intriguingly, a single intra-tumor administration of the NBD peptide in human prostate cancer xenografts significantly reduced vascularity, resulting in tumor regression. Mechanistically, the NBD peptide enhanced assembly of nonmuscle myosin IIA filaments along with Ser1943 phosphorylation, stimulated formation of focal adhesions without phosphorylation of focal adhesion kinase, and provoked G1/S arrest of the cell cycle. Altogether, the NBD peptide is a potent inhibitor for tumor angiogenesis, and is the first example of an anticancer peptide drug developed on the basis of an endothelial S100A4-targeted strategy.

  19. A Macrocyclic Peptide that Serves as a Cocrystallization Ligand and Inhibits the Function of a MATE Family Transporter

    Directory of Open Access Journals (Sweden)

    Hiroaki Suga

    2013-08-01

    Full Text Available The random non-standard peptide integrated discovery (RaPID system has proven to be a powerful approach to discover de novo natural product-like macrocyclic peptides that inhibit protein functions. We have recently reported three macrocyclic peptides that bind to Pyrococcus furiosus multidrug and toxic compound extrusion (PfMATE transporter and inhibit the transport function. Moreover, these macrocyclic peptides were successfully employed as cocrystallization ligands of selenomethionine-labeled PfMATE. In this report, we disclose the details of the RaPID selection strategy that led to the identification of these three macrocyclic peptides as well as a fourth macrocyclic peptide, MaD8, which is exclusively discussed in this article. MaD8 was found to bind within the cleft of PfMATE’s extracellular side and blocked the path of organic small molecules being extruded. The results of an ethidium bromide efflux assay confirmed the efflux inhibitory activity of MaD8, whose behavior was similar to that of previously reported MaD5.

  20. Bovine Lactoferrin and Lactoferricin, a Peptide Derived from Bovine Lactoferrin, Inhibit Tumor Metastasis in Mice

    Science.gov (United States)

    Watanabe, Shikiko; Watanabe, Ryosuke; Hata, Katsusuke; Shimazaki, Kei–ichi; Azuma, Ichiro

    1997-01-01

    We investigated the effect of a bovine milk protein, lactoferrin (LF–B), and a pepsin–generated peptide of LF–B, lactoferricin (Lfcin–B), on inhibition of tumor metastasis produced by highly metastatic murine tumor cells, B16–BL6 melanoma and L5178Y–ML25 lymphoma cells, using experimental and spontaneous metastasis models in syngeneic mice. The subcutaneous (s.c.) administration of bovine apo–lactoferrin (apo–LF–B, 1 mg/mouse) and Lfcin–B (0.5 mg/monse) 1 day after tumor inoculation significantly inhibited liver and lung metastasis of L5178Y–ML25 cells. However, human apo–lactoferrin (apo–LF–H) and bovine holo–lactoferrin (holo–LF–B) at the dose of 1 mg/mouse failed to inhibit tumor metastasis of L5178Y–ML25 cells. Similarly, the s.c. administration of apo–LF–B as well as Lfcin–B, but not apo–LF–H and holo–LF–B, 1 day after tumor inoculation resulted in significant inhibition of lung metastasis of B16–BL6 cells in an experimental metastasis model. Furthermore, in in vivo analysis for tumor–induced angiogenesis, both apo–LF–B and Lfcin–B inhibited the number of tumor–induced blood vessels and suppressed tumor growth on day 8 after tumor inoculation. However, in a long–term analysis of tumor growth for up to 21 days after tumor inoculation, single administration of apo–LF–B significantly suppressed the growth of B16–BL6 cells throughout the examination period, whereas Lfcin–B showed inhibitory activity only during the early period (8 days). In spontaneous metastasis of B16–BL6 melanoma cells, multiple administration of both apo–LF–B and Lfcin–B into tumor–bearing mice significantly inhibited lung metastasis produced by B16–BL6 cells, though only apo–LF–B exhibited an inhibitory effect on tumor growth at the time of primary tumor amputation (on day 21) after tumor inoculation. These results suggest that apo–LF–B and Lfcin–B inhibit tumor metastasis through different

  1. Inhibition of serotonin release by bombesin-like peptides in rat hypothalamus in vitro

    International Nuclear Information System (INIS)

    Saporito, M.S.; Warwick, R.O. Jr.

    1989-01-01

    We investigated the activity of bombesin (BN), neuromedin-C (NM-C) and neuromedin-B (NM-B) on serotonin (5-HT) release and reuptake in rat hypothalamus (HYP) in vitro. BN and NM-C but not NM-B decreased K + evoked 3 H-5-HT release from superfused HYP slices by 25%. Bacitracin, a nonspecific peptidase inhibitor, reversed the inhibitory effect of BN on K + evoked 3 H-5-HT release. Phosphoramidon (PAN, 10 μM) an endopeptidase 24.11 inhibitor, abolished the inhibitory effect of BN, but not NM-C, on K + evoked 3 H-5-HT release. The peptidyl dipeptidase A inhibitor enalaprilat (ENP, 10 μM), enhanced both BN and NM-C inhibition of 3 H-5-HT release. Bestatin (BST, 10 μM) had no effect on BN or NM-C inhibitory activity on 3 H-5-HT release. Neither BN, NM-C nor NM-B affected reuptake of 3 H-5-HT into HYP synaptosomes alone or in combination with any of the peptidase inhibitors, nor did these peptides alter the ability of fluoxetine to inhibit 3 H-5-HT uptake

  2. Atrial natriuretic peptide: a possible mediator involved in dexamethasone's inhibition of cell proliferation in multiple myeloma.

    Science.gov (United States)

    Ding, Jiang-Hua; Chang, Yu-Sui

    2012-08-01

    Atrial natriuretic peptide (ANP) has been recognized for several decades for its role of regulating blood pressure. Recently, cumulating evidences show that ANP plays an anticancer role in various solid tumors via blocking the kinase cascade of Ras-MEK1/2-ERK1/2 with the result of inhibition of DNA synthesis. ANP, as well as its receptors (NPR-A and NPR-C) has been identified present in the embryonic stem cell and a wide range of cancer cells. Various lymphoid organs, such as lymph nodes, have been detected the presence of ANP. Multiple myeloma (MM), though the therapies have evolved significantly, is still an incurable disease as B lymphocyte cell neoplasm. Dexamethasone is the cornerstone in treatment of MM via inactivation of Ras-MEK1/2-ERK1/2 cascade reaction. Coincidently, dexamethasone can increase the expression of ANP markedly. Nevertheless, the role of ANP in MM is unclear. Based on these results above, we raise the hypothesis that ANP is involved in mediating dexamethasone's inhibition of proliferation in MM cells, which suggests that ANP may be a potential agent to treat MM. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  3. Identification of constrained peptides that bind to and preferentially inhibit the activity of the hepatitis C viral RNA-dependent RNA polymerase

    International Nuclear Information System (INIS)

    Amin, Anthony; Zaccardi, Joe; Mullen, Stanley; Olland, Stephane; Orlowski, Mark; Feld, Boris; Labonte, Patrick; Mak, Paul

    2003-01-01

    A class of disulfide constrained peptides containing a core motif FPWG was identified from a screen of phage displayed library using the HCV RNA-dependent RNA polymerase (NS5B) as a bait. Surface plasmon resonance studies showed that three highly purified synthetic constrained peptides bound to immobilized NS5B with estimated K d values ranging from 30 to 60 μM. In addition, these peptides inhibited the NS5B activity in vitro with IC 50 ranging from 6 to 48 μM, whereas in contrast they had no inhibitory effect on the enzymatic activities of calf thymus polymerase α, human polymerase β, RSV polymerase, and HIV reverse transcriptase in vitro. Two peptides demonstrated conformation-dependent inhibition since their synthetic linear versions were not inhibitory in the NS5B assay. A constrained peptide with the minimum core motif FPWG retained selective inhibition of NS5B activity with an IC 50 of 50 μM. Alanine scan analyses of a representative constrained peptide, FPWGNTW, indicated that residues F1 and W7 were critical for the inhibitory effect of this peptide, although residues P2 and N5 had some measurable inhibitory effect as well. Further analyses of the mechanism of inhibition indicated that these peptides inhibited the formation of preelongation complexes required for the elongation reaction. However, once the preelongation complex was formed, its activity was refractory to peptide inhibition. Furthermore, the constrained peptide FPWGNTW inhibited de novo initiated RNA synthesis by NS5B from a poly(rC) template. These data indicate that the peptides confer selective inhibition of NS5B activity by binding to the enzyme and perturbing an early step preceding the processive elongation step of RNA synthesis

  4. Virtual Screening of Peptide and Peptidomimetic Fragments Targeted to Inhibit Bacterial Dithiol Oxidase DsbA.

    Directory of Open Access Journals (Sweden)

    Wilko Duprez

    Full Text Available Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately needed to address the growing problem of antibiotic resistance. The periplasmic oxidative folding system in Gram-negative bacteria represents a possible target for anti-virulence antibacterials. By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota might be minimized. Here, we undertook the design of peptidomimetic inhibitors targeting the interaction between the two key enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence factor assembly. Structures of DsbB--or peptides--complexed with DsbA revealed key interactions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the active site. The present work aimed to discover peptidomimetics that target the hydrophobic groove to generate non-covalent DsbA inhibitors. The previously reported structure of a Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the heptapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomimetic fragment library. The highest scoring fragment compound and nine derivatives were synthesized and evaluated for DsbA binding and inhibition. These experiments discovered peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors.

  5. Inhibition of enterovirus 71 (EV-71 infections by a novel antiviral peptide derived from EV-71 capsid protein VP1.

    Directory of Open Access Journals (Sweden)

    Chee Wah Tan

    Full Text Available Enterovirus 71 (EV-71 is the main causative agent of hand, foot and mouth disease (HFMD. In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC(50 values ranging from 6-9.3 µM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71.

  6. Engineering D-Amino Acid Containing Collagen Like Peptide at the Cleavage Site of Clostridium histolyticum Collagenase for Its Inhibition.

    Directory of Open Access Journals (Sweden)

    Punitha Velmurugan

    Full Text Available Collagenase is an important enzyme which plays an important role in degradation of collagen in wound healing, cancer metastasis and even in embryonic development. However, the mechanism of this degradation has not yet been completely understood. In the field of biomedical and protein engineering, the design and development of new peptide based materials is of main concern. In the present work an attempt has been made to study the effect of DAla in collagen like peptide (imino-poor region of type I collagen on the structure and stability of peptide against enzyme hydrolysis. Effect of replacement of DAla in the collagen like peptide has been studied using circular dichroic spectroscopy (CD. Our findings suggest that, DAla substitution leads to conformational changes in the secondary structure and favours the formation of polyproline II conformation than its L-counterpart in the imino-poor region of collagen like peptides. Change in the chirality of alanine at the cleavage site of collagenase in the imino-poor region inhibits collagenolytic activity. This may find application in design of peptides and peptidomimics for enzyme-substrate interaction, specifically with reference to collagen and other extra cellular matrix proteins.

  7. Empirical and bioinformatic characterization of buffalo (Bubalus bubalis) colostrum whey peptides & their angiotensin I-converting enzyme inhibition.

    Science.gov (United States)

    Ashok, N R; Aparna, H S

    2017-08-01

    Whey based peptides are well known for their nutritional and multifunctional properties. In this context, whey proteins from buffalo colostrum & milk were digested by in vitro simulation digestion and analyzed by nano-LC-MS/MS. Functional protein association networks, gene annotations and localization of identified proteins were carried out. An ACE inhibitory peptide sorted from the library was custom synthesized and an in vitro ACE assay was performed. The study led to the identification of 74 small peptides which were clustered into 5 gene functional groups and majority of them were secretory proteins. Among the identified peptides, majority of them were found identical to angiotensin I-converting enzyme (ACE) inhibitors, antioxidant, antimicrobial, immunomodulatory and opioidal peptides. An octapeptide (m/z - 902.51, IQKVAGTW) synthesized was found to inhibit ACE with an IC 50 of 300±2µM. The present investigation thus establishes newer vista for food derived peptides having ACE inhibitory potential for nutraceutical or therapeutic applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Complete covalent structure of statherin, a tyrosine-rich acidic peptide which inhibits calcium phosphate precipitation from human parotid saliva.

    Science.gov (United States)

    Schlesinger, D H; Hay, D I

    1977-03-10

    The complete amino acid sequence of human salivary statherin, a peptide which strongly inhibits precipitation from supersaturated calcium phosphate solutions, and therefore stabilizes supersaturated saliva, has been determined. The NH2-terminal half of this Mr=5380 (43 amino acids) polypeptide was determined by automated Edman degradations (liquid phase) on native statherin. The peptide was digested separately with trypsin, chymotrypsin, and Staphylococcus aureus protease, and the resulting peptides were purified by gel filtration. Manual Edman degradations on purified peptide fragments yielded peptides that completed the amino acid sequence through the penultimate COOH-terminal residue. These analyses, together with carboxypeptidase digestion of native statherin and of peptide fragments of statherin, established the complete sequence of the molecule. The 2 serine residues (positions 2 and 3) in statherin were identified as phosphoserine. The amino acid sequence of human salivary statherin is striking in a number of ways. The NH2-terminal one-third is highly polar and includes three polar dipeptides: H2PO3-Ser-Ser-H2PO3-Arg-Arg-, and Glu-Glu-. The COOH-terminal two-thirds of the molecule is hydrophobic, containing several repeating dipeptides: four of -Gn-Pro-, three of -Tyr-Gln-, two of -Gly-Tyr-, two of-Gln-Tyr-, and two of the tetrapeptide sequence -Pro-Tyr-Gln-Pro-. Unusual cleavage sites in the statherin sequence obtained with chymotrypsin and S. aureus protease were also noted.

  9. Inhibition of Plant-Pathogenic Bacteria by Short Synthetic Cecropin A-Melittin Hybrid Peptides

    OpenAIRE

    Ferre, Rafael; Badosa, Esther; Feliu, Lidia; Planas, Marta; Montesinos, Emili; Bardají, Eduard

    2006-01-01

    Short peptides of 11 residues were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Pseudomonas syringae, and Xanthomonas vesicatoria and compared to the previously described peptide Pep3 (WKLFKKILKVL-NH2). The antimicrobial activity of Pep3 and 22 analogues was evaluated in terms of the MIC and the 50% effective dose (ED50) for growth. Peptide cytotoxicity against human red blood cells and peptide stability toward protease degradation wer...

  10. Combined oral administration of bovine collagen peptides with calcium citrate inhibits bone loss in ovariectomized rats.

    Science.gov (United States)

    Liu, JunLi; Wang, YiHu; Song, ShuJun; Wang, XiJie; Qin, YaYa; Si, ShaoYan; Guo, YanChuan

    2015-01-01

    Collagen peptides (CPs) and calcium citrate are commonly used as bone health supplements for treating osteoporosis. However, it remains unknown whether the combination of oral bovine CPs with calcium citrate is more effective than administration of either agent alone. Forty 12-week-old Sprague-Dawley rats were randomly divided into five groups (n = 8) for once-daily intragastric administration of different treatments for 3 months at 3 months after ovariectomy (OVX) as follows: sham + vehicle; OVX + vehicle; OVX + 750 mg/kg CP; OVX + CP-calcium citrate (75 mg/kg); OVX + calcium citrate (75 mg/kg). After euthanasia, the femurs were removed and analyzed by dual energy X-ray absorptiometry and micro-computed tomography, and serum samples were analyzed for bone metabolic markers. OVX rats supplemented with CPs or CP-calcium citrate showed osteoprotective effects, with reductions in the OVX-induced decreases in their femoral bone mineral density. Moreover, CP-calcium citrate prevented trabecular bone loss, improved the microarchitecture of the distal femur, and significantly inhibited bone loss with increased bone volume, connectivity density, and trabecular number compared with OVX control rats. CP or CP-calcium citrate administration significantly increased serum procollagen type I N-terminal propeptide levels and reduced serum bone-specific alkaline phosphatase, osteocalcin, and C-telopeptide of type I collagen levels. Our data indicate that combined oral administration of bovine CPs with calcium citrate inhibits bone loss in OVX rats. The present findings suggest that combined oral administration of bovine CPs with calcium citrate is a promising alternative for reducing bone loss in osteopenic postmenopausal women.

  11. Combined oral administration of bovine collagen peptides with calcium citrate inhibits bone loss in ovariectomized rats.

    Directory of Open Access Journals (Sweden)

    JunLi Liu

    Full Text Available Collagen peptides (CPs and calcium citrate are commonly used as bone health supplements for treating osteoporosis. However, it remains unknown whether the combination of oral bovine CPs with calcium citrate is more effective than administration of either agent alone.Forty 12-week-old Sprague-Dawley rats were randomly divided into five groups (n = 8 for once-daily intragastric administration of different treatments for 3 months at 3 months after ovariectomy (OVX as follows: sham + vehicle; OVX + vehicle; OVX + 750 mg/kg CP; OVX + CP-calcium citrate (75 mg/kg; OVX + calcium citrate (75 mg/kg. After euthanasia, the femurs were removed and analyzed by dual energy X-ray absorptiometry and micro-computed tomography, and serum samples were analyzed for bone metabolic markers.OVX rats supplemented with CPs or CP-calcium citrate showed osteoprotective effects, with reductions in the OVX-induced decreases in their femoral bone mineral density. Moreover, CP-calcium citrate prevented trabecular bone loss, improved the microarchitecture of the distal femur, and significantly inhibited bone loss with increased bone volume, connectivity density, and trabecular number compared with OVX control rats. CP or CP-calcium citrate administration significantly increased serum procollagen type I N-terminal propeptide levels and reduced serum bone-specific alkaline phosphatase, osteocalcin, and C-telopeptide of type I collagen levels.Our data indicate that combined oral administration of bovine CPs with calcium citrate inhibits bone loss in OVX rats. The present findings suggest that combined oral administration of bovine CPs with calcium citrate is a promising alternative for reducing bone loss in osteopenic postmenopausal women.

  12. AWRK6, A Synthetic Cationic Peptide Derived from Antimicrobial Peptide Dybowskin-2CDYa, Inhibits Lipopolysaccharide-Induced Inflammatory Response

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    Qiuyu Wang

    2018-02-01

    Full Text Available Lipopolysaccharides (LPS are major outer membrane components of Gram-negative bacteria and produce strong inflammatory responses in animals. Most antibiotics have shown little clinical anti-endotoxin activity while some antimicrobial peptides have proved to be effective in blocking LPS. Here, the anti-LPS activity of the synthetic peptide AWRK6, which is derived from antimicrobial peptide dybowskin-2CDYa, has been investigated in vitro and in vivo. The positively charged α-helical AWRK6 was found to be effective in blocking the binding of LBP (LPS binding protein with LPS in vitro using ELISA. In a murine endotoxemia model, AWRK6 offered satisfactory protection efficiency against endotoxemia death, and the serum levels of LPS, IL-1β, IL-6, and TNF-α were found to be attenuated using ELISA. Further, histopathological analysis suggested that AWRK6 could improve the healing of liver and lung injury in endotoxemia mice. The results of real-time PCR and Western blotting showed that AWRK6 significantly reversed LPS-induced TLR4 overexpression and IκB depression, as well as the enhanced IκB phosphorylation. Additionally, AWRK6 did not produce any significant toxicity in vivo and in vitro. In summary, AWRK6 showed efficacious protection from LPS challenges in vivo and in vitro, by blocking LPS binding to LBP, without obvious toxicity, providing a promising strategy against LPS-induced inflammatory responses.

  13. Delivery of AAV2/9-microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of α1-syntrophin and α-dystrobrevin in skeletal muscles of mdx mice.

    Science.gov (United States)

    Koo, Taeyoung; Malerba, Alberto; Athanasopoulos, Takis; Trollet, Capucine; Boldrin, Luisa; Ferry, Arnaud; Popplewell, Linda; Foster, Helen; Foster, Keith; Dickson, George

    2011-11-01

    Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signaling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain-extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction-induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.

  14. Inhibition of HLA-DM mediated MHC class II peptide loading by HLA-DO promotes self tolerance

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    Lisa K. Denzin

    2013-12-01

    Full Text Available Major histocompatibility class II (MHCII molecules are loaded with peptides derived from foreign and self-proteins within the endosomes and lysosomes of antigen presenting cells (APCs. This process is mediated by interaction of MHCII with the conserved, nonpolymorphic MHCII-like molecule HLA-DM (DM. DM activity is directly opposed by HLA-DO (DO, another conserved, non-polymorphic MHCII like molecule. DO is an MHCII substrate mimic. Binding of DO to DM prevents MHCII from binding to DM, thereby inhibiting peptide loading. Inhibition of DM function enables low stability MHC complexes to survive and populate the surface of APCS. As a consequence, DO promotes the display of a broader pool of low abundance self-peptides. Broadening the peptide repertoire theoretically reduces the likelihood of inadvertently acquiring a density of self-ligands that is sufficient to activate self-reactive T cells. One function of DO, therefore, is to promote T cell tolerance by shaping the visible image of self. Recent data also shows that DO influences the adaptive immune response by controlling B cell entry into the germinal center reaction. This review explores the data supporting these concepts.

  15. Bovine Lactoferrin and Lactoferrin-Derived Peptides Inhibit the Growth of Vibrio cholerae and Other Vibrio species

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    Erika Acosta-Smith

    2018-01-01

    Full Text Available Vibrio is a genus of Gram-negative bacteria, some of which can cause serious infectious diseases. Vibrio infections are associated with the consumption of contaminated food and classified in Vibrio cholera infections and non-cholera Vibrio infections. In the present study, we investigate whether bovine lactoferrin (bLF and several synthetic peptides corresponding to bLF sequences, are able to inhibit the growth or have bactericidal effect against V. cholerae and other Vibrio species. The antibacterial activity of LF and LF-peptides was assessed by kinetics of growth or determination of colony forming unit in bacteria treated with the peptides and antibiotics. To get insight in the mode of action, the interaction between bLF and bLF-peptides (coupled to FITC and V. cholera was evaluated. The damage of effector-induced bacterial membrane permeability was measured by inclusion of the fluorescent dye propidium iodide using flow cytometry, whereas the bacterial ultrastructural damage in bacteria treated was observed by transmission electron microscopy. The results showed that bLF and LFchimera inhibited the growth of the V. cholerae strains; LFchimera permeabilized the bacteria which membranes were seriously damaged. Assays with a multidrug-resistant strain of Vibrio species indicated that combination of sub-lethal doses of LFchimera with ampicillin or tetracycline strongly reduced the concentration of the antibiotics to reach 95% growth inhibition. Furthermore, LFchimera were effective to inhibit the V. cholerae counts and damage due to this bacterium in a model mice. These data suggest that LFchimera and bLF are potential candidates to combat the V. cholerae and other multidrug resistant Vibrio species.

  16. Bovine Lactoferrin and Lactoferrin-Derived Peptides Inhibit the Growth of Vibrio cholerae and Other Vibrio species

    Science.gov (United States)

    Acosta-Smith, Erika; Viveros-Jiménez, Karina; Canizalez-Román, Adrian; Reyes-Lopez, Magda; Bolscher, Jan G. M.; Nazmi, Kamran; Flores-Villaseñor, Hector; Alapizco-Castro, Gerardo; de la Garza, Mireya; Martínez-Garcia, Jesús J.; Velazquez-Roman, Jorge; Leon-Sicairos, Nidia

    2018-01-01

    Vibrio is a genus of Gram-negative bacteria, some of which can cause serious infectious diseases. Vibrio infections are associated with the consumption of contaminated food and classified in Vibrio cholera infections and non-cholera Vibrio infections. In the present study, we investigate whether bovine lactoferrin (bLF) and several synthetic peptides corresponding to bLF sequences, are able to inhibit the growth or have bactericidal effect against V. cholerae and other Vibrio species. The antibacterial activity of LF and LF-peptides was assessed by kinetics of growth or determination of colony forming unit in bacteria treated with the peptides and antibiotics. To get insight in the mode of action, the interaction between bLF and bLF-peptides (coupled to FITC) and V. cholera was evaluated. The damage of effector-induced bacterial membrane permeability was measured by inclusion of the fluorescent dye propidium iodide using flow cytometry, whereas the bacterial ultrastructural damage in bacteria treated was observed by transmission electron microscopy. The results showed that bLF and LFchimera inhibited the growth of the V. cholerae strains; LFchimera permeabilized the bacteria which membranes were seriously damaged. Assays with a multidrug-resistant strain of Vibrio species indicated that combination of sub-lethal doses of LFchimera with ampicillin or tetracycline strongly reduced the concentration of the antibiotics to reach 95% growth inhibition. Furthermore, LFchimera were effective to inhibit the V. cholerae counts and damage due to this bacterium in a model mice. These data suggest that LFchimera and bLF are potential candidates to combat the V. cholerae and other multidrug resistant Vibrio species. PMID:29375503

  17. The anticancer activity of lytic peptides is inhibited by heparan sulfate on the surface of the tumor cells

    Science.gov (United States)

    2009-01-01

    Background Cationic antimicrobial peptides (CAPs) with antitumor activity constitute a promising group of novel anticancer agents. These peptides induce lysis of cancer cells through interactions with the plasma membrane. It is not known which cancer cell membrane components influence their susceptibility to CAPs. We have previously shown that CAPs interact with the two glycosaminoglycans (GAGs), heparan sulfate (HS) and chondroitin sulfate (CS), which are present on the surface of most cells. The purpose of this study was to investigate the role of the two GAGs in the cytotoxic activity of CAPs. Methods Various cell lines, expressing different levels of cell surface GAGs, were exposed to bovine lactoferricin (LfcinB) and the designer peptide, KW5. The cytotoxic effect of the peptides was investigated by use of the colorimetric MTT viability assay. The cytotoxic effect on wild type CHO cells, expressing normal amounts of GAGs on the cell surface, and the mutant pgsA-745, that has no expression of GAGs on the cell surface, was also investigated. Results We show that cells not expressing HS were more susceptible to CAPs than cells expressing HS at the cell surface. Further, exogenously added heparin inhibited the cytotoxic effect of the peptides. Chondroitin sulfate had no effect on the cytotoxic activity of KW5 and only minor effects on LfcinB cytotoxicity. Conclusion Our results show for the first time that negatively charged molecules at the surface of cancer cells inhibit the cytotoxic activity of CAPs. Our results indicate that HS at the surface of cancer cells sequesters CAPs away from the phospholipid bilayer and thereby impede their ability to induce cytolysis. PMID:19527490

  18. The anticancer activity of lytic peptides is inhibited by heparan sulfate on the surface of the tumor cells

    Directory of Open Access Journals (Sweden)

    Rekdal Øystein

    2009-06-01

    Full Text Available Abstract Background Cationic antimicrobial peptides (CAPs with antitumor activity constitute a promising group of novel anticancer agents. These peptides induce lysis of cancer cells through interactions with the plasma membrane. It is not known which cancer cell membrane components influence their susceptibility to CAPs. We have previously shown that CAPs interact with the two glycosaminoglycans (GAGs, heparan sulfate (HS and chondroitin sulfate (CS, which are present on the surface of most cells. The purpose of this study was to investigate the role of the two GAGs in the cytotoxic activity of CAPs. Methods Various cell lines, expressing different levels of cell surface GAGs, were exposed to bovine lactoferricin (LfcinB and the designer peptide, KW5. The cytotoxic effect of the peptides was investigated by use of the colorimetric MTT viability assay. The cytotoxic effect on wild type CHO cells, expressing normal amounts of GAGs on the cell surface, and the mutant pgsA-745, that has no expression of GAGs on the cell surface, was also investigated. Results We show that cells not expressing HS were more susceptible to CAPs than cells expressing HS at the cell surface. Further, exogenously added heparin inhibited the cytotoxic effect of the peptides. Chondroitin sulfate had no effect on the cytotoxic activity of KW5 and only minor effects on LfcinB cytotoxicity. Conclusion Our results show for the first time that negatively charged molecules at the surface of cancer cells inhibit the cytotoxic activity of CAPs. Our results indicate that HS at the surface of cancer cells sequesters CAPs away from the phospholipid bilayer and thereby impede their ability to induce cytolysis.

  19. Streptococcal adhesin SspA/B analogue peptide inhibits adherence and impacts biofilm formation of Streptococcus mutans.

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    Tatsuro Ito

    Full Text Available Streptococcus mutans, the major causative agent of dental caries, adheres to tooth surfaces via the host salivary glycoprotein-340 (gp340. This adherence can be competitively inhibited by peptides derived from the SspA/B adhesins of Streptococcus gordonii, a human commensal microbe that competes for the same binding sites. Ssp(A4K-A11K, a double-lysine substituted SspA/B peptide analogue, has been shown to exhibit superior in vitro binding affinity for a gp340-derived peptide (SRCRP2, suggesting that Ssp(A4K-A11K may be of clinical interest. In the present work, we tested the inhibitory effects of Ssp(A4K-A11K on adherence and biofilm formation of S. mutans by reconstructing an artificial oral environment using saliva-coated polystyrene plates and hydroxyapatite disks. Bacterial adherence (adherence period: 1 h was assessed by an enzyme-linked immunosorbent assay using biotinylated bacterial cells. Biofilm formation (periods: 8, 11, or 14 h was assessed by staining and imaging of the sessile cells, or by recovering biofilm cells and plating for cell counts. The pH values of the culture media were measured as a biofilm acidogenicity indicator. Bactericidality was measured by loss of optical density during culturing in the presence of the peptide. We observed that 650 μM Ssp(A4K-A11K significantly inhibited adherence of S. mutans to saliva-coated polystyrene; a similar effect was seen on bacterial affinity for SRCRP2. Ssp(A4K-A11K had lesser effects on the adherence of commensal streptococci. Pretreatment of polystyrene and hydroxyapatite with 650 μM Ssp(A4K-A11K significantly attenuated biofilm formation, whether tested with glucose- or sucrose-containing media. The SspA/B peptide's activity did not reflect bactericidality. Strikingly, pH in Ssp-treated 8-h (6.8 ± 0.06 and 11-h (5.5 ± 0.06 biofilms showed higher values than the critical pH. Thus, Ssp(A4K-A11K acts by inhibiting bacterial adherence and cariogrnic biofilm formation. We further

  20. Galectin-1-asialofetuin interaction is inhibited by peptides containing the tyr-xxx-tyr motif acting on the glycoprotein.

    Science.gov (United States)

    Wéber, Edit; Hetényi, Anasztázia; Váczi, Balázs; Szolnoki, Eva; Fajka-Boja, Roberta; Tubak, Vilmos; Monostori, Eva; Martinek, Tamás A

    2010-01-25

    Galectin-1 (Gal-1), a ubiquitous beta-galactoside-binding protein expressed by various normal and pathological tissues, has been implicated in cancer and autoimmune/inflammatory diseases in consequence of its regulatory role in adhesion, cell viability, proliferation, and angiogenesis. The functions of Gal-1 depend on its affinity for beta-galactoside-containing glycoconjugates; accordingly, the inhibition of sugar binding blocks its functions, hence promising potential therapeutic tools. The Tyr-Xxx-Tyr peptide motifs have been reported to be glycomimetic sequences, mainly on the basis of their inhibitory effect on the Gal-1-asialofetuin (ASF) interaction. However, the results regarding the efficacy of the Tyr-Xxx-Tyr motif as a glycomimetic inhibitor are still controversial. The present STD and trNOE NMR experiments reveal that the Tyr-Xxx-Tyr peptides studied do not bind to Gal-1, whereas their binding to ASF is clearly detected. (15)N,(1)H HSQC titrations with (15)N-labeled Gal-1 confirm the absence of any peptide-Gal-1 interaction. These data indicate that the Tyr-Xxx-Tyr peptides tested in this work are not glycomimetics as they interact with ASF via an unrevealed molecular linkage.

  1. Peptide Inhibitor of Complement C1 (PIC1 Rapidly Inhibits Complement Activation after Intravascular Injection in Rats.

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    Julia A Sharp

    Full Text Available The complement system has been increasingly recognized to play a pivotal role in a variety of inflammatory and autoimmune diseases. Consequently, therapeutic modulators of the classical, lectin and alternative pathways of the complement system are currently in pre-clinical and clinical development. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement and is referred to as Peptide Inhibitor of Complement C1 (PIC1. In this study, we determined that the lead PIC1 variant demonstrates a salt-dependent binding to C1q, the initiator molecule of the classical pathway. Additionally, this peptide bound to the lectin pathway initiator molecule MBL as well as the ficolins H, M and L, suggesting a common mechanism of PIC1 inhibitory activity occurs via binding to the collagen-like tails of these collectin molecules. We further analyzed the effect of arginine and glutamic acid residue substitution on the complement inhibitory activity of our lead derivative in a hemolytic assay and found that the original sequence demonstrated superior inhibitory activity. To improve upon the solubility of the lead derivative, a pegylated, water soluble variant was developed, structurally characterized and demonstrated to inhibit complement activation in mouse plasma, as well as rat, non-human primate and human serum in vitro. After intravenous injection in rats, the pegylated derivative inhibited complement activation in the blood by 90% after 30 seconds, demonstrating extremely rapid function. Additionally, no adverse toxicological effects were observed in limited testing. Together these results show that PIC1 rapidly inhibits classical complement activation in vitro and in vivo and is functional for a variety of animal species, suggesting its utility in animal models of classical complement-mediated diseases.

  2. Characterization of angiotensin-I converting enzyme inhibiting peptide from Venerupis philippinarum with nano-liquid chromatography in combination with orbitrap mass spectrum detection and molecular docking

    Science.gov (United States)

    Shi, Lei; Wu, Tizhi; Sheng, Naijuan; Yang, Li; Wang, Qian; Liu, Rui; Wu, Hao

    2017-06-01

    The complexity and diversity of peptide mixture from protein hydrolysates make their characterization difficult. In this study, a method combining nano LC-MS/MS with molecular docking was applied to identifying and characterizing a peptide with angiotensin-I converting enzyme (ACE-I) inhibiting activity from Venerupis philippinarum hydrolysate. Firstly, ethanol supernatant of V. philippinarum hydrolysate was separated into active fractions with chromatographic methods such as ion-exchange chromatography and high performance liquid chromatography in combination. Then seven peptides from active fraction were identified according to the searching result of the MS/MS spectra against protein databases. Peptides were synthesized and subjected to ACE-I-inhibition assay. The peptide NTLTLIDTGIGMTK showed the highest potency with an IC50 of 5.75 μmol L-1. The molecular docking analysis showed that the ACE-I inhibiting peptide NTLTLIDTGIGMTK bond with residues Glu123, Glu403, Arg522, Glu376, Gln281 and Asn285 of ACE-I. Therefore, active peptides could be identified with the present method rather than the traditional purification and identification strategies. It may also be feasible to identify other food-derived peptides which target other enzymes and receptors with the method developed in this study.

  3. Modification of Titanium Substrates with Chimeric Peptides Comprising Antimicrobial and Titanium-Binding Motifs Connected by Linkers To Inhibit Biofilm Formation.

    Science.gov (United States)

    Liu, Zihao; Ma, Shiqing; Duan, Shun; Xuliang, Deng; Sun, Yingchun; Zhang, Xi; Xu, Xinhua; Guan, Binbin; Wang, Chao; Hu, Meilin; Qi, Xingying; Zhang, Xu; Gao, Ping

    2016-03-02

    Bacterial adhesion and biofilm formation are the primary causes of implant-associated infection, which is difficult to eliminate and may induce failure in dental implants. Chimeric peptides with both binding and antimicrobial motifs may provide a promising alternative to inhibit biofilm formation on titanium surfaces. In this study, chimeric peptides were designed by connecting an antimicrobial motif (JH8194: KRLFRRWQWRMKKY) with a binding motif (minTBP-1: RKLPDA) directly or via flexible/rigid linkers to modify Ti surfaces. We evaluated the binding behavior of peptides using quartz crystal microbalance (QCM) and atomic force microscopy (AFM) techniques and investigated the effect of the modification of titanium surfaces with these peptides on the bioactivity of Streptococcus gordonii (S. gordonii) and Streptococcus sanguis (S. sanguis). Compared with the flexible linker (GGGGS), the rigid linker (PAPAP) significantly increased the adsorption of the chimeric peptide on titanium surfaces (p chimeric peptide with the rigid linker exhibited more effective antimicrobial ability than the peptide with the flexible linker. This finding was ascribed to the ability of the rigid linker to separate functional domains and reduce their interference to the maximum extent. Consequently, the performance of chimeric peptides with specific titanium-binding motifs and antimicrobial motifs against bacteria can be optimized by the proper selection of linkers. This rational design of chimeric peptides provides a promising alternative to inhibit the formation of biofilms on titanium surfaces with the potential to prevent peri-implantitis and peri-implant mucositis.

  4. A carboxylated Zn-phthalocyanine inhibits fibril formation of Alzheimer's amyloid β peptide.

    Science.gov (United States)

    Tabassum, Shatera; Sheikh, Abdullah M; Yano, Shozo; Ikeue, Takafumi; Handa, Makoto; Nagai, Atsushi

    2015-02-01

    Amyloid β (Aβ), a 39-42 amino acid peptide derived from amyloid precursor protein, is deposited as fibrils in Alzheimer's disease brains, and is considered to play a major role in the pathogenesis of the disease. We have investigated the effects of a water-soluble Zn-phthalocyanine, ZnPc(COONa)₈, a macrocyclic compound with near-infrared optical properties, on Aβ fibril formation in vitro. A thioflavin T fluorescence assay showed that ZnPc(COONa)₈ significantly inhibited Aβ fibril formation, increasing the lag time and dose-dependently decreasing the plateau level of fibril formation. Moreover, it destabilized pre-formed Aβ fibrils, resulting in an increase in low-molecular-weight species. After fibril formation in the presence of ZnPc(COONa)₈, immunoprecipitation of Aβ₁₋₄₂ using Aβ-specific antibody followed by near-infrared scanning demonstrated binding of ZnPc(COONa)₈ to Aβ₁₋₄₂. A study using the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid showed that ZnPc(COONa)8 decreased the hydrophobicity during Aβ₁₋₄₂ fibril formation. CD spectroscopy showed an increase in the α helix structure and a decrease in the β sheet structure of Aβ₁₋₄₀ in fibril-forming buffer containing ZnPc(COONa)₈. SDS/PAGE and a dot-blot immunoassay showed that ZnPc(COONa)₈ delayed the disappearance of low-molecular-weight species and the appearance of higher-molecular-weight oligomeric species of Aβ₁₋₄₂. A cell viability assay showed that ZnPc(COONa)₈ was not toxic to a neuronal cell line (A1), but instead protected A1 cells against Aβ₁₋₄₂-induced toxicity. Overall, our results indicate that ZnPc(COONa)₈ binds to Aβ and decreases the hydrophobicity, and this change is unfavorable for Aβ oligomerization and fibril formation. © 2014 FEBS.

  5. Selective albumin-binding surfaces modified with a thrombin-inhibiting peptide.

    Science.gov (United States)

    Freitas, Sidónio C; Maia, Sílvia; Figueiredo, Ana C; Gomes, Paula; Pereira, Pedro J B; Barbosa, Mário A; Martins, M Cristina L

    2014-03-01

    Blood-contacting medical devices have been associated with severe clinical complications, such as thrombus formation, triggered by the activation of the coagulation cascade due to the adsorption of certain plasma proteins on the surface of biomaterials. Hence, the coating of such surfaces with antithrombotic agents has been used to increase biomaterial haemocompatibility. Biomaterial-induced clotting may also be decreased by albumin adsorption from blood plasma in a selective and reversible way, since this protein is not involved in the coagulation cascade. In this context, this paper reports that the immobilization of the thrombin inhibitor D-Phe-Pro-D-Arg-D-Thr-CONH2 (fPrt) onto nanostructured surfaces induces selective and reversible adsorption of albumin, delaying the clotting time when compared to peptide-free surfaces. fPrt, synthesized with two glycine residues attached to the N-terminus (GGfPrt), was covalently immobilized onto self-assembled monolayers (SAMs) having different ratios of carboxylate-hexa(ethylene glycol)- and tri(ethylene glycol)-terminated thiols (EG6-COOH/EG3) that were specifically designed to control GGfPrt orientation, exposure and density at the molecular level. In solution, GGfPrt was able to inactivate the enzymatic activity of thrombin and to delay plasma clotting time in a concentration-dependent way. After surface immobilization, and independently of its concentration, GGfPrt lost its selectivity to thrombin and its capacity to inhibit thrombin enzymatic activity against the chromogenic substrate n-p-tosyl-Gly-Pro-Arg-p-nitroanilide. Nevertheless, surfaces with low concentrations of GGfPrt could delay the capacity of adsorbed thrombin to cleave fibrinogen. In contrast, GGfPrt immobilized in high concentrations was found to induce the procoagulant activity of the adsorbed thrombin. However, all surfaces containing GGfPrt have a plasma clotting time similar to the negative control (empty polystyrene wells), showing resistance to

  6. A peptide antagonist of ErbB receptors, Inherbin3, induces neurite outgrowth from rat cerebellar granule neurons through ErbB1 inhibition

    DEFF Research Database (Denmark)

    Xu, Ruodan; Pankratova, Stanislava; Christiansen, Søren Hofman

    2013-01-01

    ErbB receptors not only function in cancer, but are also key developmental regulators in the nervous system. We previously identified an ErbB1 peptide antagonist, Inherbin3, that is capable of inhibiting tumor growth in vitro and in vivo. In this study, we found that inhibition of ErbB1 kinase ac...

  7. The antimicrobial peptide, lactoferricin B, is cytotoxic to neuroblastoma cells in vitro and inhibits xenograft growth in vivo.

    Science.gov (United States)

    Eliassen, Liv Tone; Berge, Gerd; Leknessund, Arild; Wikman, Mari; Lindin, Inger; Løkke, Cecilie; Ponthan, Frida; Johnsen, John Inge; Sveinbjørnsson, Baldur; Kogner, Per; Flaegstad, Trond; Rekdal, Øystein

    2006-08-01

    Antimicrobial peptides have been shown to exert cytotoxic activity towards cancer cells through their ability to interact with negatively charged cell membranes. In this study the cytotoxic effect of the antimicrobial peptide, LfcinB was tested in a panel of human neuroblastoma cell lines. LfcinB displayed a selective cytotoxic activity against both MYCN-amplified and non-MYCN-amplified cell lines. Non-transformed fibroblasts were not substantially affected by LfcinB. Treatment of neuroblastoma cells with LfcinB induced rapid destabilization of the cytoplasmic membrane and formation of membrane blebs. Depolarization of the mitochondria membranes and irreversible changes in the mitochondria morphology was also evident. Immuno- and fluorescence-labeled LfcinB revealed that the peptide co-localized with mitochondria. Furthermore, treatment of neuroblastoma cells with LfcinB induced cleavage of caspase-6, -7 and -9 followed by cell death. However, neither addition of the pan-caspase inhibitor, zVAD-fmk, or specific caspase inhibitors could reverse the cytotoxic effect induced by LfcinB. Treatment of established SH-SY-5Y neuroblastoma xenografts with repeated injections of LfcinB resulted in significant tumor growth inhibition. These results revealed a selective destabilizing effect of LfcinB on two important targets in the neuroblastoma cells, the cytoplasmic- and the mitochondria membrane. Copyright (c) 2006 Wiley-Liss, Inc.

  8. The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis.

    Science.gov (United States)

    Mardirossian, Mario; Grzela, Renata; Giglione, Carmela; Meinnel, Thierry; Gennaro, Renato; Mergaert, Peter; Scocchi, Marco

    2014-12-18

    Antimicrobial peptides (AMPs) are molecules from innate immunity with high potential as novel anti-infective agents. Most of them inactivate bacteria through pore formation or membrane barrier disruption, but others cross the membrane without damages and act inside the cells, affecting vital processes. However, little is known about their intracellular bacterial targets. Here we report that Bac71-35, a proline-rich AMP belonging to the cathelicidin family, can reach high concentrations (up to 340 μM) inside the E. coli cytoplasm. The peptide specifically and completely inhibits in vitro translation in the micromolar concentration range. Experiments of incorporation of radioactive precursors in macromolecules with E. coli cells confirmed that Bac71-35 affects specifically protein synthesis. Ribosome coprecipitation and crosslinking assays showed that the peptide interacts with ribosomes, binding to a limited subset of ribosomal proteins. Overall, these results indicate that the killing mechanism of Bac71-35 is based on a specific block of protein synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. A small molecule inhibitor of signal peptide peptidase inhibits Plasmodium development in the liver and decreases malaria severity.

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    Iana Parvanova

    Full Text Available The liver stage of Plasmodium's life cycle is the first, obligatory step in malaria infection. Decreasing the hepatic burden of Plasmodium infection decreases the severity of disease and constitutes a promising strategy for malaria prophylaxis. The efficacy of the gamma-secretase and signal peptide peptidase inhibitor LY411,575 in targeting Plasmodium liver stages was evaluated both in human hepatoma cell lines and in mouse primary hepatocytes. LY411,575 was found to prevent Plasmodium's normal development in the liver, with an IC(50 of approximately 80 nM, without affecting hepatocyte invasion by the parasite. In vivo results with a rodent model of malaria showed that LY411,575 decreases the parasite load in the liver and increases by 55% the resistance of mice to cerebral malaria, one of the most severe malaria-associated syndromes. Our data show that LY411,575 does not exert its effect via the Notch signaling pathway suggesting that it may interfere with Plasmodium development through an inhibition of the parasite's signal peptide peptidase. We therefore propose that selective signal peptide peptidase inhibitors could be potentially used for preventive treatment of malaria in humans.

  10. Peptide aptamers as new tools to modulate clathrin-mediated internalisation — inhibition of MT1-MMP internalisation

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    Ferrigno Paul

    2010-07-01

    Full Text Available Abstract Background Peptide aptamers are combinatorial protein reagents that bind to targets with a high specificity and a strong affinity thus providing a molecular tool kit for modulating the function of their targets in vivo. Results Here we report the isolation of a peptide aptamer named swiggle that interacts with the very short (21 amino acid long intracellular domain of membrane type 1-metalloproteinase (MT1-MMP, a key cell surface protease involved in numerous and crucial physiological and pathological cellular events. Expression of swiggle in mammalian cells was found to increase the cell surface expression of MT1-MMP by impairing its internalisation. Swiggle interacts with the LLY573 internalisation motif of MT1-MMP intracellular domain, thus disrupting the interaction with the μ2 subunit of the AP-2 internalisation complex required for endocytosis of the protease. Interestingly, swiggle-mediated inhibition of MT1-MMP clathrin-mediated internalisation was also found to promote MT1-MMP-mediated cell migration. Conclusions Taken together, our results provide further evidence that peptide aptamers can be used to dissect molecular events mediated by individual protein domains, in contrast to the pleiotropic effects of RNA interference techniques.

  11. Peptide aptamers as new tools to modulate clathrin-mediated internalisation--inhibition of MT1-MMP internalisation.

    Science.gov (United States)

    Wickramasinghe, Rochana D; Ko Ferrigno, Paul; Roghi, Christian

    2010-07-23

    Peptide aptamers are combinatorial protein reagents that bind to targets with a high specificity and a strong affinity thus providing a molecular tool kit for modulating the function of their targets in vivo. Here we report the isolation of a peptide aptamer named swiggle that interacts with the very short (21 amino acid long) intracellular domain of membrane type 1-metalloproteinase (MT1-MMP), a key cell surface protease involved in numerous and crucial physiological and pathological cellular events. Expression of swiggle in mammalian cells was found to increase the cell surface expression of MT1-MMP by impairing its internalisation. Swiggle interacts with the LLY573 internalisation motif of MT1-MMP intracellular domain, thus disrupting the interaction with the mu2 subunit of the AP-2 internalisation complex required for endocytosis of the protease. Interestingly, swiggle-mediated inhibition of MT1-MMP clathrin-mediated internalisation was also found to promote MT1-MMP-mediated cell migration. Taken together, our results provide further evidence that peptide aptamers can be used to dissect molecular events mediated by individual protein domains, in contrast to the pleiotropic effects of RNA interference techniques.

  12. A new class of synthetic anti-lipopolysaccharide peptides inhibits influenza A virus replication by blocking cellular attachment.

    Science.gov (United States)

    Hoffmann, Julia; Schneider, Carola; Heinbockel, Lena; Brandenburg, Klaus; Reimer, Rudolph; Gabriel, Gülsah

    2014-04-01

    Influenza A viruses are a continuous threat to human health as illustrated by the 2009 H1N1 pandemic. Since circulating influenza virus strains become increasingly resistant against currently available drugs, the development of novel antivirals is urgently needed. Here, we have evaluated a recently described new class of broad-spectrum antiviral peptides (synthetic anti-lipopolysaccharide peptides; SALPs) for their potential to inhibit influenza virus replication in vitro and in vivo. We found that particularly SALP PEP 19-2.5 shows high binding affinities for the influenza virus receptor molecule, N-Acetylneuraminic acid, leading to impaired viral attachment and cellular entry. As a result, replication of several influenza virus subtypes (H7N7, H3N2 and 2009 pandemic H1N1) was strongly reduced. Furthermore, mice co-treated with PEP 19-2.5 were protected against an otherwise 100% lethal H7N7 influenza virus infection. These findings show that SALPs exhibit antiviral activity against influenza viruses by blocking virus attachment and entry into host cells. Thus, SALPs present a new class of broad-spectrum antiviral peptides for further development for influenza virus therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Transgenic tobacco expressing a modified spider peptide inhibits the growth of plant pathogens and insect larvae

    Science.gov (United States)

    The gene encoding lycotoxin I, an amphipathic pore-forming peptide, was modified to increase oral toxicity to insects. One of the most active modified genes was then constitutively expressed in tobacco (Nicotiana tabacum) and transformants were evaluated for insect and disease resistance. Pathogenic...

  14. TRAM-Derived Decoy Peptides inhibits the inflammatory response in mouse mammary epithelial cells and a mastitis model in mice.

    Science.gov (United States)

    Hu, Xiaoyu; Tian, Yuan; Wang, Tiancheng; Zhang, Wenlong; Wang, Wei; Gao, Xuejiao; Qu, Shihui; Cao, Yongguo; Zhang, Naisheng

    2015-10-05

    It has been proved that TRAM-Derived Decoy peptides have anti-inflammatory properties. In this study, we synthesized a TRAM-Derived decoy peptide (TM6), belongs to TRAM TIR domain, of which sequence is "N"-RQIKIWFQNRRMKWK, KENFLRDTWCNFQFY-"C" and evaluated the effects of TM6 on lipopolysaccharide-induced mastitis in mice. In vivo, LPS-induced mice mastitis model was established by injection of LPS through the duct of mammary gland. TM6 was injected 1h before or after LPS treatment. In vitro, primary mouse mammary epithelial cells were used to investigate the effects of TM6 on LPS-induced inflammatory responses. The results showed that TM6 inhibited LPS-induced mammary gland histopathologic changes, MPO activity, and TNF-α, IL-1β and IL-6 production in mice. In vitro, TM6 significantly inhibited LPS-induced TNF-α and IL-6 production, as well as NF-κB and MAPKs activation. In conclusion, this study demonstrated that TM6 had protective effects on LPS-mastitis and may be a promising therapeutic reagent for mastitis treatment. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Inhibition of Candida albicans Biofilm Formation by the Synthetic Lactoferricin Derived Peptide hLF1-11.

    Science.gov (United States)

    Morici, Paola; Fais, Roberta; Rizzato, Cosmeri; Tavanti, Arianna; Lupetti, Antonella

    2016-01-01

    The aim of this study was to evaluate the in vitro activity of the synthetic peptide hLF1-11 against biofilm produced by clinical isolates of Candida albicans with different fluconazole susceptibility. The antibiofilm activity of the peptide hLF1-11 was assessed in terms of reduction of biofilm cellular density, metabolic activity and sessile cell viability. The extent of morphogenesis in hLF1-11 treated and untreated biofilms was also investigated microscopically. Transcription levels of genes related to cell adhesion, hyphal development and extracellular matrix production were analysed by qRT-PCR in hLF1-11 treated and untreated biofilms. Exogenous dibutyryl-cAMP (db-cAMP) was used to rescue morphogenesis in cells exposed to the peptide. The results revealed that hLF1-11 exhibited an inhibitory effect on biofilm formation by all C. albicans isolates tested in a dose-dependent manner, regardless of their fluconazole susceptibility. Visual inspection of treated or untreated biofilm cells with an inverted microscope revealed a significant reduction in hyphal formation by hLF1-11 treated cells, as early as 3 hours of incubation. Moreover, hLF1-11 showed a reduced activity on preadherent cells. hLF1-11 induced the down-regulation of biofilm and hyphal-associated genes, which were predominantly regulated via the Ras1-cAMP-Efg1 pathway. Indeed, exogenous db-cAMP restored morphogenesis in hLF1-11 treated cells. The hLF1-11 peptide significantly inhibited biofilm formation by C. albicans mainly at early stages, interfering with biofilm cellular density and metabolic activity, and affected morphogenesis through the Ras1-cAMP-Efg1 pathway. Our findings provide the first evidence that hLF1-11 could represent a potential candidate for the prevention of biofilm formation by C. albicans.

  16. CopA3 Peptide Prevents Ultraviolet-Induced Inhibition of Type-I Procollagen and Induction of Matrix Metalloproteinase-1 in Human Skin Fibroblasts

    Directory of Open Access Journals (Sweden)

    Dong-Hee Kim

    2014-05-01

    Full Text Available Ultraviolet (UV exposure is well-known to induce premature aging, which is mediated by matrix metalloproteinase-1 (MMP-1 activity. A 9-mer peptide, CopA3 (CopA3 was synthesized from a natural peptide, coprisin, which is isolated from the dung beetle Copris tripartitus. As part of our continuing search for novel bioactive natural products, CopA3 was investigated for its in vitro anti-skin photoaging activity. UV-induced inhibition of type-I procollagen and induction of MMP-1 were partially prevented in human skin fibroblasts by CopA3 peptide in a dose-dependent manner. At a concentration of 25 μM, CopA3 nearly completely inhibited MMP-1 expression. These results suggest that CopA3, an insect peptide, is a potential candidate for the prevention and treatment of skin aging.

  17. The structure of salt bridges between Arg(+) and Glu(-) in peptides investigated with 2D-IR spectroscopy: Evidence for two distinct hydrogen-bond geometries.

    Science.gov (United States)

    Huerta-Viga, Adriana; Amirjalayer, Saeed; Domingos, Sérgio R; Meuzelaar, Heleen; Rupenyan, Alisa; Woutersen, Sander

    2015-06-07

    Salt bridges play an important role in protein folding and in supramolecular chemistry, but they are difficult to detect and characterize in solution. Here, we investigate salt bridges between glutamate (Glu(-)) and arginine (Arg(+)) using two-dimensional infrared (2D-IR) spectroscopy. The 2D-IR spectrum of a salt-bridged dimer shows cross peaks between the vibrational modes of Glu(-) and Arg(+), which provide a sensitive structural probe of Glu(-)⋯Arg(+) salt bridges. We use this probe to investigate a β-turn locked by a salt bridge, an α-helical peptide whose structure is stabilized by salt bridges, and a coiled coil that is stabilized by intra- and intermolecular salt bridges. We detect a bidentate salt bridge in the β-turn, a monodentate one in the α-helical peptide, and both salt-bridge geometries in the coiled coil. To our knowledge, this is the first time 2D-IR has been used to probe tertiary side chain interactions in peptides, and our results show that 2D-IR spectroscopy is a powerful method for investigating salt bridges in solution.

  18. Lithium chloride increases the production of amyloid-beta peptide independently from its inhibition of glycogen synthase kinase 3.

    Science.gov (United States)

    Feyt, Christine; Kienlen-Campard, Pascal; Leroy, Karelle; N'Kuli, Francisca; Courtoy, Pierre J; Brion, Jean-Pierre; Octave, Jean-Noël

    2005-09-30

    Glycogen synthase kinase 3 (GSK3) is able to phosphorylate tau at many sites that are found to be phosphorylated in paired helical filaments in Alzheimer disease. Lithium chloride (LiCl) efficiently inhibits GSK3 and was recently reported to also decrease the production of amyloid-beta peptide (Abeta) from its precursor, the amyloid precursor protein. Therefore, lithium has been proposed as a combined therapeutic agent, inhibiting both the hyperphosphorylation of tau and the production of Abeta. Here, we demonstrate that the inhibition of GSK3 by LiCl induced the nuclear translocation of beta-catenin in Chinese hamster ovary cells and rat cultured neurons, in which a decrease in tau phosphorylation was observed. In both cellular models, a nontoxic concentration of LiCl increased the production of Abeta by increasing the beta-cleavage of amyloid precursor protein, generating more substrate for an unmodified gamma-secretase activity. SB415286, another GSK3 inhibitor, induced the nuclear translocation of beta-catenin and slightly decreased Abeta production. It is concluded that the LiCl-mediated increase in Abeta production is not related to GSK3 inhibition.

  19. Symbiotic Plant Peptides Eliminate Candida albicans Both In Vitro and in an Epithelial Infection Model and Inhibit the Proliferation of Immortalized Human Cells

    Directory of Open Access Journals (Sweden)

    Lilla Ördögh

    2014-01-01

    Full Text Available The increasing number of multidrug-resistant microbes now emerging necessitates the identification of novel antimicrobial agents. Plants produce a great variety of antimicrobial peptides including hundreds of small, nodule-specific cysteine-rich NCR peptides that, in the legume Medicago truncatula, govern the differentiation of endosymbiotic nitrogen fixing bacteria and, in vitro, can display potent antibacterial activities. In this study, the potential candidacidal activity of 19 NCR peptides was investigated. Cationic NCR peptides having an isoelectric point above 9 were efficient in killing Candida albicans, one of the most common fungal pathogens of humans. None of the tested NCR peptides were toxic for immortalized human epithelial cells at concentrations that effectively killed the fungus; however, at higher concentrations, some of them inhibited the division of the cells. Furthermore, the cationic peptides successfully inhibited C. albicans induced human epithelial cell death in an in vitro coculture model. These results highlight the therapeutic potential of cationic NCR peptides in the treatment of candidiasis.

  20. Stabilization of a β-hairpin in monomeric Alzheimer's amyloid-β peptide inhibits amyloid formation

    OpenAIRE

    Hoyer, Wolfgang; Grönwall, Caroline; Jonsson, Andreas; Ståhl, Stefan; Härd, Torleif

    2008-01-01

    According to the amyloid hypothesis, the pathogenesis of Alzheimer's disease is triggered by the oligomerization and aggregation of the amyloid-β (Aβ) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar Aβ assemblies is accompanied by a conformational change toward a high content of β-structure. Here, we report the solution structure of Aβ(1–40) in complex with the phage-display selected affibody protein ZAβ3, a binding protein of nanomolar affinity. Boun...

  1. Membrane-tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity.

    Science.gov (United States)

    Valente, Pierluigi; Fernández-Carvajal, Asia; Camprubí-Robles, María; Gomis, Ana; Quirce, Susana; Viana, Félix; Fernández-Ballester, Gregorio; González-Ros, José M; Belmonte, Carlos; Planells-Cases, Rosa; Ferrer-Montiel, Antonio

    2011-05-01

    The transient receptor potential vanilloid 1 (TRPV1) channel is a thermosensory receptor implicated in diverse physiological and pathological processes. The TRP domain, a highly conserved region in the C terminus adjacent to the internal channel gate, is critical for subunit tetramerization and channel gating. Here, we show that cell-penetrating, membrane-anchored peptides patterned after this protein domain are moderate and selective TRPV1 antagonists both in vitro and in vivo, blocking receptor activity in intact rat primary sensory neurons and their peripheral axons with mean decline time of 30 min. The most potent lipopeptide, TRP-p5, blocked all modes of TRPV1 gating with micromolar efficacy (IC(50)100 μM). TRP-p5 did not affect the capsaicin sensitivity of the vanilloid receptor. Our data suggest that TRP-p5 interferes with protein-protein interactions at the level of the TRP domain that are essential for the "conformational" change that leads to gate opening. Therefore, these palmitoylated peptides, which we termed TRPducins, are noncompetitive, voltage-independent, sequence-specific TRPV1 blockers. Our findings indicate that TRPducin-like peptides may embody a novel molecular strategy that can be exploited to generate a selective pharmacological arsenal for the TRP superfamily of ion channels.

  2. Generation of Dipeptidyl Peptidase-IV-Inhibiting Peptides from β-Lactoglobulin Secreted by Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Suguru Shigemori

    2014-01-01

    Full Text Available Previous studies showed that hydrolysates of β-lactoglobulin (BLG prepared using gastrointestinal proteases strongly inhibit dipeptidyl peptidase-IV (DPP-IV activity in vitro. In this study, we developed a BLG-secreting Lactococcus lactis strain as a delivery vehicle and in situ expression system. Interestingly, trypsin-digested recombinant BLG from L. lactis inhibited DPP-IV activity, suggesting that BLG-secreting L. lactis may be useful in the treatment of type 2 diabetes mellitus.

  3. RGD peptide-modified multifunctional dendrimer platform for drug encapsulation and targeted inhibition of cancer cells.

    Science.gov (United States)

    He, Xuedan; Alves, Carla S; Oliveira, Nilsa; Rodrigues, João; Zhu, Jingyi; Bányai, István; Tomás, Helena; Shi, Xiangyang

    2015-01-01

    Development of multifunctional nanoscale drug-delivery systems for targeted cancer therapy still remains a great challenge. Here, we report the synthesis of cyclic arginine-glycine-aspartic acid (RGD) peptide-conjugated generation 5 (G5) poly(amidoamine) dendrimers for anticancer drug encapsulation and targeted therapy of cancer cells overexpressing αvβ3 integrins. In this study, amine-terminated G5 dendrimers were used as a platform to be sequentially modified with fluorescein isothiocyanate (FI) via a thiourea linkage and RGD peptide via a polyethylene glycol (PEG) spacer, followed by acetylation of the remaining dendrimer terminal amines. The developed multifunctional dendrimer platform (G5.NHAc-FI-PEG-RGD) was then used to encapsulate an anticancer drug doxorubicin (DOX). We show that approximately six DOX molecules are able to be encapsulated within each dendrimer platform. The formed complexes are water-soluble, stable, and able to release DOX in a sustained manner. One- and two-dimensional NMR techniques were applied to investigate the interaction between dendrimers and DOX, and the impact of the environmental pH on the release rate of DOX from the dendrimer/DOX complexes was also explored. Furthermore, cell biological studies demonstrate that the encapsulation of DOX within the G5.NHAc-FI-PEG-RGD dendrimers does not compromise the anticancer activity of DOX and that the therapeutic efficacy of the dendrimer/DOX complexes is solely related to the encapsulated DOX drug. Importantly, thanks to the role played by RGD-mediated targeting, the developed dendrimer/drug complexes are able to specifically target αvβ3 integrin-overexpressing cancer cells and display specific therapeutic efficacy to the target cells. The developed RGD peptide-targeted multifunctional dendrimers may thus be used as a versatile platform for targeted therapy of different types of αvβ3 integrin-overexpressing cancer cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Enhanced striatial 3H-spiroperidol binding induced by chronic haloperidol treatment inhibited by peptides administered during the withdrawal phase

    International Nuclear Information System (INIS)

    Bhargava, H.N.

    1984-01-01

    Chronic intragastric administration of haloperidol (1.5 mg/kg/day) for 21 days followed by a 3-day withdrawal period resulted in the development of enhanced locomotor activity response to apomorphine, and an increase in the number of binding sites for 3 H-spiroperidol in the striatal membranes of the rat brain. Subcutaneous administration of Pro-Leu-Gly-NH 2 or cyclo-(Leu-Gly) in doses of 2 mg/kg/day given for 3-days after termination of haloperidol treatment inhibited the enhanced response to apomorphine, as well as the increases in the number of 3 H-spiroperidol binding sites in the striatum. If indeed, the supersensitivity of striatal dopamine receptors is one of the mechanisms in the development of tardive dyskinesia symptoms, the present results suggest that the above peptides may be helpful in ameliorating some of the symptoms of tardive dyskinesia induced by neuroleptic drugs. 31 references, 3 figures

  5. MCR-1 Inhibition with Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers Restores Sensitivity to Polymyxin in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Seth M. Daly

    2017-11-01

    Full Text Available In late 2015, the first example of a transferrable polymyxin resistance mechanism in Gram-negative pathogens, MCR-1, was reported. Since that report, MCR-1 has been described to occur in many Gram-negative pathogens, and the mechanism of MCR-1-mediated resistance was rapidly determined: an ethanolamine is attached to lipid A phosphate groups, rendering the membrane more electropositive and repelling positively charged polymyxins. Acquisition of MCR-1 is clinically significant because polymyxins are frequently last-line antibiotics used to treat extensively resistant organisms, so acquisition of this mechanism might lead to pan-resistant strains. Therefore, the ability to inhibit MCR-1 and restore polymyxin sensitivity would be an important scientific advancement. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs are antisense molecules that were designed to target mRNA, preventing translation. Peptide conjugation enhances cellular entry, but they are positively charged, so we tested our lead antibacterial PPMOs by targeting an essential Escherichia coli gene, acpP, and demonstrated that they were still effective in mcr-1-positive E. coli strains. We then designed and synthesized two PPMOs targeted to mcr-1 mRNA. Five clinical mcr-1-positive E. coli strains were resensitized to polymyxins by MCR-1 inhibition, reducing MICs 2- to 16-fold. Finally, therapeutic dosing of BALB/c mice with MCR-1 PPMO combined with colistin in a sepsis model reduced morbidity and bacterial burden in the spleen at 24 h and offered a survival advantage out to 5 days. This is the first example of a way to modulate colistin resistance with an antisense approach and may be a viable strategy to combat this globally emerging antibiotic resistance threat.

  6. Benzothiazole aniline tetra(ethylene glycol) and 3-amino-1,2,4-triazole inhibit neuroprotection against amyloid peptides by catalase overexpression in vitro.

    Science.gov (United States)

    Chilumuri, Amrutha; Odell, Mark; Milton, Nathaniel G N

    2013-11-20

    Alzheimer's disease, Familial British dementia, Familial Danish dementia, Type 2 diabetes mellitus, plus Creutzfeldt-Jakob disease are associated with amyloid fibril deposition and oxidative stress. The antioxidant enzyme catalase is a neuroprotective amyloid binding protein. Herein the effects of catalase overexpression in SH-SY5Y neuronal cells on the toxicity of amyloid-β (Aβ), amyloid-Bri (ABri), amyloid-Dan (ADan), amylin (IAPP), and prion protein (PrP) peptides were determined. Results showed catalase overexpression was neuroprotective against Aβ, ABri, ADan, IAPP, and PrP peptides. The catalase inhibitor 3-amino-1,2,4-triazole (3-AT) and catalase-amyloid interaction inhibitor benzothiazole aniline tetra(ethylene glycol) (BTA-EG4) significantly enhanced neurotoxicity of amyloid peptides in catalase overexpressing neuronal cells. This suggests catalase neuroprotection involves breakdown of hydrogen peroxide (H2O2) plus a direct binding interaction between catalase and the Aβ, ABri, ADan, IAPP, and PrP peptides. Kisspeptin 45-50 had additive neuroprotective actions against the Aβ peptide in catalase overexpressing cells. The effects of 3-AT had an intracellular site of action, while catalase-amyloid interactions had an extracellular component. These results suggest that the 3-AT and BTA-EG4 compounds may be able to inhibit endogenous catalase mediated neuroprotection. Use of BTA-EG4, or compounds that inhibit catalase binding to amyloid peptides, as potential therapeutics for Neurodegenerative diseases may therefore result in unwanted effects.

  7. Benzothiazole Aniline Tetra(ethylene glycol) and 3-Amino-1,2,4-triazole Inhibit Neuroprotection against Amyloid Peptides by Catalase Overexpression in Vitro

    Science.gov (United States)

    2013-01-01

    Alzheimer’s disease, Familial British dementia, Familial Danish dementia, Type 2 diabetes mellitus, plus Creutzfeldt-Jakob disease are associated with amyloid fibril deposition and oxidative stress. The antioxidant enzyme catalase is a neuroprotective amyloid binding protein. Herein the effects of catalase overexpression in SH-SY5Y neuronal cells on the toxicity of amyloid-β (Aβ), amyloid-Bri (ABri), amyloid-Dan (ADan), amylin (IAPP), and prion protein (PrP) peptides were determined. Results showed catalase overexpression was neuroprotective against Aβ, ABri, ADan, IAPP, and PrP peptides. The catalase inhibitor 3-amino-1,2,4-triazole (3-AT) and catalase-amyloid interaction inhibitor benzothiazole aniline tetra(ethylene glycol) (BTA-EG4) significantly enhanced neurotoxicity of amyloid peptides in catalase overexpressing neuronal cells. This suggests catalase neuroprotection involves breakdown of hydrogen peroxide (H2O2) plus a direct binding interaction between catalase and the Aβ, ABri, ADan, IAPP, and PrP peptides. Kisspeptin 45–50 had additive neuroprotective actions against the Aβ peptide in catalase overexpressing cells. The effects of 3-AT had an intracellular site of action, while catalase-amyloid interactions had an extracellular component. These results suggest that the 3-AT and BTA-EG4 compounds may be able to inhibit endogenous catalase mediated neuroprotection. Use of BTA-EG4, or compounds that inhibit catalase binding to amyloid peptides, as potential therapeutics for Neurodegenerative diseases may therefore result in unwanted effects. PMID:23968537

  8. Inhibition of Candida albicans Biofilm Formation by the Synthetic Lactoferricin Derived Peptide hLF1-11

    Science.gov (United States)

    Morici, Paola; Fais, Roberta; Rizzato, Cosmeri

    2016-01-01

    The aim of this study was to evaluate the in vitro activity of the synthetic peptide hLF1-11 against biofilm produced by clinical isolates of Candida albicans with different fluconazole susceptibility. The antibiofilm activity of the peptide hLF1-11 was assessed in terms of reduction of biofilm cellular density, metabolic activity and sessile cell viability. The extent of morphogenesis in hLF1-11 treated and untreated biofilms was also investigated microscopically. Transcription levels of genes related to cell adhesion, hyphal development and extracellular matrix production were analysed by qRT-PCR in hLF1-11 treated and untreated biofilms. Exogenous dibutyryl-cAMP (db-cAMP) was used to rescue morphogenesis in cells exposed to the peptide. The results revealed that hLF1-11 exhibited an inhibitory effect on biofilm formation by all C. albicans isolates tested in a dose-dependent manner, regardless of their fluconazole susceptibility. Visual inspection of treated or untreated biofilm cells with an inverted microscope revealed a significant reduction in hyphal formation by hLF1-11 treated cells, as early as 3 hours of incubation. Moreover, hLF1-11 showed a reduced activity on preadherent cells. hLF1-11 induced the down-regulation of biofilm and hyphal-associated genes, which were predominantly regulated via the Ras1-cAMP-Efg1 pathway. Indeed, exogenous db-cAMP restored morphogenesis in hLF1-11 treated cells. The hLF1-11 peptide significantly inhibited biofilm formation by C. albicans mainly at early stages, interfering with biofilm cellular density and metabolic activity, and affected morphogenesis through the Ras1-cAMP-Efg1 pathway. Our findings provide the first evidence that hLF1-11 could represent a potential candidate for the prevention of biofilm formation by C. albicans. PMID:27902776

  9. Inhibition of Candida albicans Biofilm Formation by the Synthetic Lactoferricin Derived Peptide hLF1-11.

    Directory of Open Access Journals (Sweden)

    Paola Morici

    Full Text Available The aim of this study was to evaluate the in vitro activity of the synthetic peptide hLF1-11 against biofilm produced by clinical isolates of Candida albicans with different fluconazole susceptibility. The antibiofilm activity of the peptide hLF1-11 was assessed in terms of reduction of biofilm cellular density, metabolic activity and sessile cell viability. The extent of morphogenesis in hLF1-11 treated and untreated biofilms was also investigated microscopically. Transcription levels of genes related to cell adhesion, hyphal development and extracellular matrix production were analysed by qRT-PCR in hLF1-11 treated and untreated biofilms. Exogenous dibutyryl-cAMP (db-cAMP was used to rescue morphogenesis in cells exposed to the peptide. The results revealed that hLF1-11 exhibited an inhibitory effect on biofilm formation by all C. albicans isolates tested in a dose-dependent manner, regardless of their fluconazole susceptibility. Visual inspection of treated or untreated biofilm cells with an inverted microscope revealed a significant reduction in hyphal formation by hLF1-11 treated cells, as early as 3 hours of incubation. Moreover, hLF1-11 showed a reduced activity on preadherent cells. hLF1-11 induced the down-regulation of biofilm and hyphal-associated genes, which were predominantly regulated via the Ras1-cAMP-Efg1 pathway. Indeed, exogenous db-cAMP restored morphogenesis in hLF1-11 treated cells. The hLF1-11 peptide significantly inhibited biofilm formation by C. albicans mainly at early stages, interfering with biofilm cellular density and metabolic activity, and affected morphogenesis through the Ras1-cAMP-Efg1 pathway. Our findings provide the first evidence that hLF1-11 could represent a potential candidate for the prevention of biofilm formation by C. albicans.

  10. A cyclic peptide derived from alpha-fetoprotein inhibits the proliferative effects of the epidermal growth factor and estradiol in MCF7 cells.

    Science.gov (United States)

    Torres, Cristian; Antileo, Elmer; Epuñán, Maráa José; Pino, Ana María; Valladares, Luis Emilio; Sierralta, Walter Daniel

    2008-06-01

    A cyclic peptide derived from the active domain of alpha-fetoprotein (AFP) significantly inhibited the proliferation of MCF7 cells stimulated with the epidermal growth factor (EGF) or estradiol (E2). The action of these three agents on cell growth was independent of the presence of calf serum in the culture medium. Our results demonstrated that the cyclic peptide interfered markedly with the regulation of MAPK by activated c-erbB2. The cyclic peptide showed no effect on the E2-stimulated release of matrix metalloproteinases 2 and 9 nor on the shedding of heparin-binding EGF into the culture medium. We propose that the AFP-derived cyclic peptide represents a valuable novel antiproliferative agent for treating breast cancer.

  11. Antioxidative, DPP-IV and ACE inhibiting peptides from fish protein hydrolysed with intestinal proteases

    DEFF Research Database (Denmark)

    Falkenberg, Susan Skanderup; Stagsted, Jan; Nielsen, Henrik Hauch

    amino groups, antioxidative capacity by ABTS (2,2-azinobis(3-ethylbenzothiazoline-6-sulfonicacid)), DPP-IV and ACE inhibiting activity. Degree of hydrolysis (DH) of hydrolysates was approximately 13% and 10% for belly flap and skin respectively. No clear difference was observed in DH between pancreatin...

  12. A class of DNA-binding peptides from wheat bud causes growth inhibition, G2 cell cycle arrest and apoptosis induction in HeLa cells

    Directory of Open Access Journals (Sweden)

    Elgjo Kjell

    2009-07-01

    Full Text Available Abstract Background Deproteinized DNA from eukaryotic and prokaryotic cells still contains a low-molecular weight peptidic fraction which can be dissociated by alkalinization of the medium. This fraction inhibits RNA transcription and tumor cell growth. Removal from DNA of normal cells causes amplification of DNA template activity. This effect is lower or absent in several cancer cell lines. Likewise, the amount of active peptides in cancer cell DNA extracts is lower than in DNA preparation of the corresponding normal cells. Such evidence, and their ubiquitous presence, suggests that they are a regulatory, conserved factor involved in the control of normal cell growth and gene expression. Results We report that peptides extracted from wheat bud chromatin induce growth inhibition, G2 arrest and caspase-dependent apoptosis in HeLa cells. The growth rate is decreased in cells treated during the S phase only and it is accompanied by DNA damage and DNA synthesis inhibition. In G2 cells, this treatment induces inactivation of the CDK1-cyclin B1 complex and an increase of active chk1 kinase expression. Conclusion The data indicate that the chromatin peptidic pool inhibits HeLa cell growth by causing defective DNA replication which, in turn, arrests cell cycle progression to mitosis via G2 checkpoint pathway activation.

  13. Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low density lipoprotein receptor family by a peptide isolated from a phage displayed library

    DEFF Research Database (Denmark)

    Jensen, Jan K.; Malmendal, Anders; Schiøtt, Birgit

    2006-01-01

    (DVPCFGWCQDA) was determined by NMR. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the u...

  14. Glucagon-like peptide-1 inhibits blood-brain glucose transfer in humans

    DEFF Research Database (Denmark)

    Lerche, Susanne; Brock, Birgitte; Rungby, Jørgen

    2008-01-01

    OBJECTIVE: Glucagon-like peptide-1 (GLP-1) has many effects on glucose homeostasis, and GLP-1 receptors are broadly represented in many tissues including the brain. Recent research in rodents suggests a protective effect of GLP-1 on brain tissue. The mechanism is unknown. We therefore tested......-independent effect of GLP-1 on unidirectional glucose transport into the brain during a pituitary-pancreatic normoglycemic (plasma glucose approximately 4.5 mmol/l) clamp with 18-fluoro-deoxy-glucose as tracer. RESULTS: On average, GLP-1 reduced cerebral glucose transport by 27% in total cerebral gray matter (P = 0...... that a hormone involved in postprandial glucose regulation also limits glucose delivery to brain tissue and hence provides a possible regulatory mechanism for the link between plasma glucose and brain glucose. Because GLP-1 reduces glucose uptake across the intact blood-brain barrier at normal glycemia, GLP-1...

  15. Inhibiting effects of Streptococcus salivarius on competence-stimulating peptide-dependent biofilm formation by Streptococcus mutans.

    Science.gov (United States)

    Tamura, S; Yonezawa, H; Motegi, M; Nakao, R; Yoneda, S; Watanabe, H; Yamazaki, T; Senpuku, H

    2009-04-01

    The effects of Streptococcus salivarius on the competence-stimulating peptide (CSP)-dependent biofilm formation by Streptococcus mutans were investigated. Biofilms were grown on 96-well microtiter plates coated with salivary components in tryptic soy broth without dextrose supplemented with 0.25% sucrose. Biofilm formations were stained using safranin and quantification of stained biofilms was performed by measuring absorbance at 492 nm. S. mutans formed substantial biofilms, whereas biofilms of S. salivarius were formed poorly in the medium conditions used. Furthermore, in combination cultures, S. salivarius strongly inhibited biofilm formation when cultured with S. mutans. This inhibition occurred in the early phase of biofilm formation and was dependent on inactivation of the CSP of S. mutans, which is associated with competence, biofilm formation, and antimicrobial activity of the bacterium, and is induced by expression of the comC gene. Comparisons between the S. mutans clinical strains FSC-3 and FSC-3DeltaglrA in separate dual-species cultures with S. salivarius indicated that the presence of the bacitracin transport ATP-binding protein gene glrA caused susceptibility to inhibition of S. mutans biofilm formation by S. salivarius, and was also associated with the regulation of CSP production by com gene-dependent quorum sensing systems. It is considered that regulation of CSP by glrA in S. mutans and CSP inactivation by S. salivarius are important functions for cell-to-cell communication between biofilm bacteria and oral streptococci such as S. salivarius. Our results provide useful information for understanding the ecosystem of oral streptococcal biofilms, as well as the competition between and coexistence of multiple species in the oral cavity.

  16. Electroacupuncture Inhibits Inflammation Reaction by Upregulating Vasoactive Intestinal Peptide in Rats with Adjuvant-Induced Arthritis

    Directory of Open Access Journals (Sweden)

    Tian-Feng He

    2011-01-01

    Full Text Available Acupuncture is emerging as an alternative therapy for rheumatoid arthritis (RA. However, the molecular mechanism underlying this beneficial effect of acupuncture has not been fully understood. Here, we demonstrated that electroacupuncture at acupoints Zusanli (ST36, Xuanzhong (GB39; and Shenshu (BL23 markedly decreased the paw swelling and the histologic scores of inflammation in the synovial tissue, and reduced the body weight loss in an adjuvant-induced arthritis rat model. However, the electrical stimulation at nonacupoint did not produce any beneficial effects against the experimental arthritis. Most interestingly, the electroacupuncture treatment resulted in an enhanced immunostaining for vasoactive intestinal peptide (VIP, a potent anti-inflammatory neuropeptide, in the synovial tissue. Moreover, the VIP-immunostaining intensity was significantly negatively correlated with the scores of inflammation in the synovial tissue (r=−0.483, P=.0026. In conclusion, these findings suggest that electroacupuncture may offer therapeutic benefits for the treatment of RA, at least partially through the induction of VIP expression.

  17. Stabilization of a β-hairpin in monomeric Alzheimer's amyloid-β peptide inhibits amyloid formation

    Science.gov (United States)

    Hoyer, Wolfgang; Grönwall, Caroline; Jonsson, Andreas; Ståhl, Stefan; Härd, Torleif

    2008-01-01

    According to the amyloid hypothesis, the pathogenesis of Alzheimer's disease is triggered by the oligomerization and aggregation of the amyloid-β (Aβ) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar Aβ assemblies is accompanied by a conformational change toward a high content of β-structure. Here, we report the solution structure of Aβ(1–40) in complex with the phage-display selected affibody protein ZAβ3, a binding protein of nanomolar affinity. Bound Aβ(1–40) features a β-hairpin comprising residues 17–36, providing the first high-resolution structure of Aβ in β conformation. The positions of the secondary structure elements strongly resemble those observed for fibrillar Aβ. ZAβ3 stabilizes the β-sheet by extending it intermolecularly and by burying both of the mostly nonpolar faces of the Aβ hairpin within a large hydrophobic tunnel-like cavity. Consequently, ZAβ3 acts as a stoichiometric inhibitor of Aβ fibrillation. The selected Aβ conformation allows us to suggest a structural mechanism for amyloid formation based on soluble oligomeric hairpin intermediates. PMID:18375754

  18. Theoretical study of Escherichia coli peptide deformylase inhibition by several drugs.

    Science.gov (United States)

    Chikhi, Abdelouahab; Bensegueni, Abderrahmane; Boulahrouf, Abderrahmane; Bencharif, Mustapha

    2006-01-01

    Because peptide deformylase (PDF) is essential for the initiation of translation in eubacteria but not in eukaryotes, it is a potentially interesting target for antibiotics. Computer simulation using docking software can be used to model protein-ligand interactions, and in this brief report we describe its use in optimizing the design in PDF-directed inhibitors. PDF was used as target for a set of five inhibitors with substantial structural differences. Docking results show that the compound 1BB2 (actinonin) binds with high affinity to the enzyme and produces the most stable complex, forming nine hydrogen bonds with the enzyme active site. Its binding energy is DeltaG = -31.880 kJ/mol. The modeling study shows that when the methyl group of 1BB2 is replaced with an amine group, the binding energy is increased to -35.316 kJ/mole. This enhancement is more marked (DeltaG = -41.141 kJ/mol) when the propyl group and the five-membered ring of 1BB2 are replaced by an amide group and a phenyl ring, respectively. We describe an attempt to design better antibiotics on the basis of a computer-aided simulation of the interaction between a drug and its target molecule.

  19. Inhibiting effects of fructanase on competence-stimulating peptide-dependent quorum sensing system in Streptococcus mutans.

    Science.gov (United States)

    Suzuki, Yusuke; Nagasawa, Ryo; Senpuku, Hidenobu

    2017-09-01

    Streptococcus mutans produces glucosyltransferases encoded by the gtfB and gtfC genes, which synthesize insoluble glucan, and both insoluble and soluble glucans by conversion of sucrose, and are known as principal agents to provide strong biofilm formation and demineralization on tooth surfaces. S. mutans possess a Com-dependent quorum sensing (QS) system, which is important for survival in severe conditions. The QS system is stimulated by the interaction between ComD {Receptor to competence-stimulating peptide (CSP)} encoded by the comD and CSP encoded by the comC, and importantly associated with bacteriocin production and genetic competence. Previously, we found enzyme fructanase (FruA) as a new inhibitor for the glucan-dependent biofilm formation. In the present study, inhibiting effects by FruA on glucan-independent biofilm formation of S. mutans UA159, UA159.gtfB - , UA159.gtfC - , and UA159.gtfBC - were observed in sucrose and no sucrose sugars-supplemented conditions using the plate assay. The reduction of UA159.comC - and UA159.comD - biofilm formation were also observed as compared with UA159 in same conditions. These results suggested that inhibitions of glucan-independent and Com-dependent biofilm formation were involved in the inhibiting mechanism by FruA. To more thoroughly investigate effects by FruA on the QS system, we examined on CSP-stimulated and Com-dependent bacteriocin production and genetic transformation. FruA inhibited bacteriocin production in collaboration with CSP and genetic transformation in bacterial cell conditions treated with FruA. Our findings show that FruA has multiple effects that inhibit survival functions of S. mutans, including biofilm formation and CSP-dependent QS responses, indicating its potential use as an agent for prevention of dental caries. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  20. Effective intracellular inhibition of the cAMP-dependent protein kinase by microinjection of a modified form of the specific inhibitor peptide PKi in living fibroblasts.

    Science.gov (United States)

    Fernandez, A; Mery, J; Vandromme, M; Basset, M; Cavadore, J C; Lamb, N J

    1991-08-01

    In order to obtain a peptide retaining its biological activity following microinjection into living cells, we have modified a synthetic peptide [PKi(m)(6-24)], derived from the specific inhibitor protein of the cAMP-dependent protein kinase (A-kinase) in two ways: (1) substitution of the arginine at position 18 for a D-arginine; (2) blockade of the side chain on the C-terminal aspartic acid by a cyclohexyl ester group. In an in vitro assay, PKi(m) has retained a specific inhibitory activity against A-kinase as assessed against six other kinases, with similar efficiency to that of the unmodified PKi(5-24) peptide. Microinjection of PKi(m) into living fibroblasts reveals its capacity to prevent the changes in cell morphology and cytoskeleton induced by drugs which activate endogenous A-kinase, whereas the original PKi peptide failed to do so. This inhibition of A-kinase in vivo by PKi(m) lasts between 4 and 6 h after injection. In light of its effective half-life, this modified peptide opens a route for the use of biologically active peptides in vivo, an approach which has been hampered until now by the exceedingly short half-life of peptides inside living cells. By providing a direct means of inhibiting A-kinase activity for sufficiently long periods to observe effects on cellular functions in living cells, PKi(m) represents a powerful tool in studying the potential role of cAMP-dependent phosphorylation in vivo.

  1. Short Stat5-interacting peptide derived from phospholipase C-β3 inhibits hematopoietic cell proliferation and myeloid differentiation.

    Directory of Open Access Journals (Sweden)

    Hiroki Yasudo

    Full Text Available Constitutive activation of the transcription factor Stat5 in hematopoietic stem/progenitor cells leads to various hematopoietic malignancies including myeloproliferative neoplasm (MPN. Our recent study found that phospholipase C (PLC-β3 is a novel tumor suppressor involved in MPN, lymphoma and other tumors. Stat5 activity is negatively regulated by the SH2 domain-containing protein phosphatase SHP-1 in a PLC-β3-dependent manner. PLC-β3 can form the multimolecular SPS complex together with SHP-1 and Stat5. The close physical proximity of SHP-1 and Stat5 brought about by interacting with the C-terminal segment of PLC-β3 (PLC-β3-CT accelerates SHP-1-mediated dephosphorylation of Stat5. Here we identify the minimal sequences within PLC-β3-CT required for its tumor suppressor function. Two of the three Stat5-binding noncontiguous regions, one of which also binds SHP-1, substantially inhibited in vitro proliferation of Ba/F3 cells. Surprisingly, an 11-residue Stat5-binding peptide (residues 988-998 suppressed Stat5 activity in Ba/F3 cells and in vivo proliferation and myeloid differentiation of hematopoietic stem/progenitor cells. Therefore, this study further defines PLC-β3-CT as the Stat5- and SHP-1-binding domain by identifying minimal functional sequences of PLC-β3 for its tumor suppressor function and implies their potential utility in the control of hematopoietic malignancies.

  2. A cyclized peptide derived from alpha fetoprotein inhibits the proliferation of ER-positive canine mammary cancer cells.

    Science.gov (United States)

    Torres, Cristian Gabriel; Pino, Ana María; Sierralta, Walter Daniel

    2009-06-01

    The effects of estradiol (E2) and of an AFP-derived cyclized peptide (cP) on the proliferation of primary cultures of cancer cells isolated from spontaneous canine mammary tumors were studied. The cellular response to E2 and cP was related to the expression of estradiol receptor (isoforms alpha and beta). In ER-positive cells, 2 nM estradiol increased cell proliferation and the phosphorylation of ERK1/2; 2 microg/ml cP inhibited all these effects. Estradiol also increased HER2 immunoreactivity in ER-positive cells, an effect that was reverted to its basal values by cP. Estradiol stimulated in these cells the release of MMP2 and MMP9 and the shedding of HB-EGF, effects that the cP did not affect. ER-negative cells were refractory to estradiol or cP. All canine mammary tumor cells in culture responded to treatments analogously to human mammary cancer cells. Our results support the proposal of cP as a new, potentially effective therapeutic agent for the management of mammary cancer.

  3. A peptide derived from alpha-fetoprotein inhibits the proliferation induced by estradiol in mammary tumor cells in culture.

    Science.gov (United States)

    Sierralta, Walter D; Epuñan, Maria J; Reyes, José M; Valladares, Luis E; Andersen, Thomas T; Bennett, James A; Jacobson, Herbert I; Pino, Ana M

    2008-01-01

    This study was aimed to obtain additional information on the activity of a cyclized 9-amino acid peptide (cP) containing the active site of alpha fetoprotein, which inhibits the estrogen-stimulated proliferation of tumor cells in culture and of xenografts in immunodeficient mice. Breast cancer cells cultured in the presence of 2 nM estradiol were exposed to cP for different periods and their proliferation, estradiol binding parameters, clustering tendency and expression of E-cadherin and p21Cip1 were analyzed by biochemical and cell biology methods. The proliferation of MCF7 cells was significantly decreased by the addition of 2 microg/ml cP to the medium. cP did not increase cell death rate nor alter the number of binding sites for estradiol nor the endogenous aromatase activity of MCF7 cells. cP also decreased the proliferation of estrogen-dependent ZR75-1 cells but had no effect on estrogen-independent MDA-MB-231 cells. An increased nuclear p21Cip1 expression detected after cP treatment suggests that cP slows MCF7 cell proliferation via this regulator. We propose that cP could represent a novel breast cancer therapeutic agent whose mechanism of action is different from that of tamoxifen or of inhibitors of aromatase.

  4. Bacterial Peptide Deformylase Inhibition of Tetrazole-Substituted Biaryl Acid Analogs: Synthesis, Biological Evaluations, and Molecular Docking Study.

    Science.gov (United States)

    Khan, Firoz A Kalam; Patil, Rajendra H; Patil, Manjiri; Arote, Rohidas; Shinde, Devanand B; Sangshetti, Jaiprakash N

    2016-12-01

    The synthesis and screening of tetrazole-substituted biaryl acid analogs 7a-l as bacterial peptide deformylase (PDF) enzyme inhibitors is reported. The compounds 7e (IC 50 value = 5.50 μM) and 7g (IC 50 value = 7.25 μM) showed good PDF inhibition activity. The compounds 7e (MIC range = 10.75-11.66 μg/mL) and 7g (MIC range = 8.91-12.83 μg/mL) also showed potent antibacterial activity when compared with the standard ciprofloxacin (MIC range = 25-50 μg/mL). Thus, the active derivatives were not only potent PDF enzyme inhibitors but also efficient antibacterial agents. In order to gain more insight into the binding mode of the compounds with the PDF enzyme, the most active compounds 7e and 7g, the moderately active compound 7k, and the least active compound 7h were docked against the PDF enzyme of Escherichia coli. The docking study of the most active compounds 7e and 7g against the PDF enzyme exhibited good binding properties. Hence, we believe our synthesized compounds 7a-l could serve as reservoir for bacterial PDF inhibitor development. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Platelet factor-4 and its p17-70 peptide inhibit myeloma proliferation and angiogenesis in vivo

    International Nuclear Information System (INIS)

    Yang, Longjiang; Du, Juan; Hou, Jian; Jiang, Hua; Zou, Jianfeng

    2011-01-01

    Angiogenesis plays an important role in the development of multiple myeloma (MM). The interaction between MM cells and the bone marrow microenvironment stimulates the proliferation and migration of endothelial progenitor cells (EPCs). Vascular endothelial growth factor (VEGF) contributes to the formation of new blood vessels by actively recruiting circulating EPCs. The production of proangiogenic and antiangiogenic factors is also dysregulated in MM. Platelet factor 4 (PF4) is a potent angiostatic cytokine that inhibits angiogenesis and tumor growth in several animal models. In this study, we stably transfected human myeloma cell lines with the PF4 gene or the sequence encoding its more potent p17-70 peptide and investigated the effects of PF4 and p17-70 on angiogenesis and tumor growth in vitro and in a SCID-rab myeloma model. PF4 and p17-70 significantly attenuated VEGF production, both in vitro and in vivo. In a migration study using a Transwell system, PF4 or p17-70 markedly suppressed the migration of co-cultured human endothelial progenitor cells. PF4 or p17-70 also caused a significant reduction in microvessel densities in myeloma xenografts and markedly reduced the tumor volume in the SCID mice. Kaplan-Meier analysis demonstrated that PF4 and p17-70 significantly extended the overall survival of SCID mice bearing human myeloma xenografts. Our findings indicate that PF4 or p17-70 could be valuable in combating multiple myeloma by disrupting tumor angiogenesis

  6. Characterization and Molecular Mechanism of Peptide-Conjugated Gold Nanoparticle Inhibiting p53-HDM2 Interaction in Retinoblastoma

    Directory of Open Access Journals (Sweden)

    Sushma Kalmodia

    2017-12-01

    Full Text Available Inhibition of the interaction between p53 and HDM2 is an effective therapeutic strategy in cancers that harbor a wild-type p53 protein such as retinoblastoma (RB. Nanoparticle-based delivery of therapeutic molecules has been shown to be advantageous in localized delivery, including to the eye, by overcoming ocular barriers. In this study, we utilized biocompatible gold nanoparticles (GNPs to deliver anti-HDM2 peptide to RB cells. Characterization studies suggested that GNP-HDM2 was stable in biologically relevant solvents and had optimal cellular internalization capability, the primary requirement of any therapeutic molecule. GNP-HDM2 treatment in RB cells in vitro suggested that they function by arresting RB cells at the G2M phase of the cell cycle and initiating apoptosis. Analysis of molecular changes in GNP-HDM2-treated cells by qRT-PCR and western blotting revealed that the p53 protein was upregulated; however, transactivation of its downstream targets was minimal, except for the PUMA-BCl2 and Bax axis. Global gene expression and in silico bioinformatic analysis of GNP-HDM2-treated cells suggested that upregulation of p53 might presumptively mediate apoptosis through the induction of p53-inducible miRNAs.

  7. Inhibition of 125I-labeled ristocetin binding to Micrococcus luteus cells by the peptides related to bacterial cell wall mucopeptide precursors: quantitative structure-activity relationships

    International Nuclear Information System (INIS)

    Kim, K.H.; Martin, Y.; Otis, E.; Mao, J.

    1989-01-01

    Quantitative structure-activity relationships (QSAR) of N-Ac amino acids, N-Ac dipeptides, and N-Ac tripeptides in inhibition of 125 I-labeled ristocetin binding to Micrococcus luteus cell wall have been developed to probe the details of the binding between ristocetin and N-acetylated peptides. The correlation equations indicate that (1) the binding is stronger for peptides in which the side chain of the C-terminal amino acid has a large molar refractivity (MR) value, (2) the binding is weaker for peptides with polar than for those with nonpolar C-terminal side chains, (3) the N-terminal amino acid in N-Ac dipeptides contributes 12 times that of the C-terminal amino acid to binding affinity, and (4) the interactions between ristocetin and the N-terminal amino acid of N-acetyl tripeptides appear to be much weaker than those with the first two amino acids

  8. Antimicrobial peptides secreted by equine mesenchymal stromal cells inhibit the growth of bacteria commonly found in skin wounds.

    Science.gov (United States)

    Harman, Rebecca M; Yang, Steven; He, Megan K; Van de Walle, Gerlinde R

    2017-07-04

    The prevalence of chronic skin wounds in humans is high, and treatment is often complicated by the presence of pathogenic bacteria. Therefore, safe and innovative treatments to reduce the bacterial load in cutaneous wounds are needed. Mesenchymal stromal cells (MSC) are known to provide paracrine signals that act on resident skin cells to promote wound healing, but their potential antibacterial activities are not well described. The present study was designed to examine the antibacterial properties of MSC from horses, as this animal model offers a readily translatable model for MSC therapies in humans. Specifically, we aimed to (i) evaluate the in vitro effects of equine MSC on the growth of representative gram-negative and gram-positive bacterial species commonly found in skin wounds and (ii) define the mechanisms by which MSC inhibit bacterial growth. MSC were isolated from the peripheral blood of healthy horses. Gram-negative E. coli and gram-positive S. aureus were cultured in the presence of MSC and MSC conditioned medium (CM), containing all factors secreted by MSC. Bacterial growth was measured by plating bacteria and counting viable colonies or by reading the absorbance of bacterial cultures. Bacterial membrane damage was detected by incorporation of N-phenyl-1-naphthylamine (NPN). Antimicrobial peptide (AMP) gene and protein expression by equine MSC were determined by RT-PCR and Western blot analysis, respectively. Blocking of AMP activity of MSC CM was achieved using AMP-specific antibodies. We found that equine MSC and MSC CM inhibit the growth of E. coli and S. aureus, and that MSC CM depolarizes the cell membranes of these bacteria. In addition, we found that equine MSC CM contains AMPs, and blocking these AMPs with antibodies reduces the effects of MSC CM on bacteria. Our results demonstrate that equine MSC inhibit bacterial growth and secrete factors that compromise the membrane integrity of bacteria commonly found in skin wounds. We also identified

  9. Synthetic peptides derived from salivary proteins and the control of surface charge densities of dental surfaces improve the inhibition of dental calculus formation.

    Science.gov (United States)

    Grohe, Bernd

    2017-08-01

    Peptides descended from the salivary proteins statherin and histatin were recently identified in saliva and the acquired enamel pellicle (AEP), a proteomic layer coated on enamel. In particular, the statherin phosphopeptide DpSpSEEKFLR (DSS) was found to adsorb to enamel-like hydroxyapatite and inhibit plaque-related crystal formation. To determine the mechanism of these processes, we studied peptide-crystal interactions based on the sequences DSS and RKFHEKHHSHRGYR (RKF). The latter is a basic histatin sequence showing antimicrobial effects. To initiate crystallization we used calcium oxalate monohydrate (COM), a rather secondary phase in the oral environment, however highly amenable to experimental analyses of nucleation and growth processes. Using electron microscopy we found that the peptides DSS, DSS-RKF and DSS-DSS all inhibit crystal formation; with DSS-DSS showing the strongest effects while RKF showed no effect. In addition, using either enamel-like or mica substrates, we found that the ratio of the substrate's surface charge densities was directly correlated with the ratio of COM nucleation rates on theses surfaces. The findings suggest that mineralization processes on enamel/AEP-films are controllable by the degree of peptide phosphorylation/acidity and the level of the enamel surface charge density. Both parameters can, when well adjusted, help to overcome periodontal disease and dental calculus formation. In addition, the presence of antimicrobial RKF will reduce the buildup of bacterial plaque. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Peptide Fraction pOh2 Exerts Antiadipogenic Activity through Inhibition of C/EBP-α and PPAR-γ Expression in 3T3-L1 Adipocytes

    Directory of Open Access Journals (Sweden)

    Thi Tuyet Nhung Nguyen

    2017-01-01

    Full Text Available Many studies have comprehensively examined the venom of Ophiophagus hannah snake. Its venom comprises different compounds exhibiting a wide range of pharmacological activities. In this investigation, four peptide fractions (PFs, ranging from 3 kDa to 10 kDa, isolated from the Vietnamese snake venom of O. hannah were separated by HPLC and investigated for their inhibitory activity on adipogenesis in 3T3-L1 adipocytes. The most effective PF was then further purified, generating two peptides, pOh1 and pOh2. Upon investigation of these two peptides on 3T3-L1 adipocytes, it was revealed that, at 10 μg/mL, pOh2 was able to inhibit the lipid accumulation in 3T3-L1 adipocytes by up to 56%, without affecting cell viability. Furthermore, the pOh2 downregulated the gene expression of important transcription factors C/EBP-α and PPAR-γ. In addition, aP2 and GPDH adipocyte-specific markers were also significantly reduced compared to untreated differentiated cells. Taken together, pOh2 inhibited the expression of key transcription factors C/EBP-α and PPAR-γ and their target genes, aP2 and GPDH, thereby blocking the adipocyte differentiation. In conclusion, this novel class of peptide might have potential for in vivo antiobesity effects.

  11. Short communication: Is consumption of a cheese rich in angiotensin-converting enzyme-inhibiting peptides, such as the Norwegian cheese Gamalost, associated with reduced blood pressure?

    Science.gov (United States)

    Nilsen, R; Pripp, A H; Høstmark, A T; Haug, A; Skeie, S

    2014-05-01

    Epidemiological and clinical studies have shown that angiotensin-converting enzyme (ACE)-inhibiting peptides derived from dairy products may decrease blood pressure. These peptides have been identified in many cheeses, and Gamalost, a traditional Norwegian cheese, is particularly rich in these peptides. The aim of this cross-sectional study was to examine whether frequency of Gamalost intake was associated with blood pressure in a Norwegian population sample. Blood pressure and other clinical measurements, including the factors of metabolic syndrome, were obtained from 168 participants (56% female, mean age = 51 yr) who completed a questionnaire about dietary habits and other health-related factors. Mean Gamalost intake was 2 servings per week. The prevalence of hypertension was 23.8% in the population, with mean systolic and diastolic blood pressures of 128 and 78 mmHg, respectively. Intake of Gamalost was inversely associated with systolic blood pressure. Each increase in frequency unit of Gamalost intake corresponded to a reduction in systolic blood pressure of 0.72 mmHg, after controlling for sex, age, education, waist circumference, physical activity, smoking status, and dairy food intake. Results from this study indicate that consumption of Gamalost (or other foods rich in ACE-inhibiting peptides) may reduce blood pressure. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  12. Identification of the peptide derived from S1 domain that inhibits type I and type II feline infectious peritonitis virus infection.

    Science.gov (United States)

    Doki, Tomoyoshi; Takano, Tomomi; Koyama, Yusuke; Hohdatsu, Tsutomu

    2015-06-02

    Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). A therapeutic drug that is effective against FIP has not yet been developed. Peptides based on viral protein amino acid sequences have recently been attracting attention as new antiviral drugs. In the present study, we synthesized 30 overlapping peptides based on the amino acid sequence of the S1 domain of the type I FIPV strain KU-2 S protein, and investigated their inhibitory effects on FIPV infection. To evaluate the inhibitory effects on type I FIPV infection of these peptides, we investigated a method to increase the infection efficiency of poorly replicative type I FIPV. The efficiency of type I FIPV infection was increased by diluting the virus with medium containing a polycation. Of the 30 peptides, I-S1-8 (S461-S480), I-S1-9 (S471-S490), I-S1-10 (S481-S500), I-S1-16 (S541-S560), and I-S1-22 (S601-S620) significantly decreased the infectivity of FIPV strain KU-2 while I-S1-9 and I-S1-16 exhibited marked inhibitory effects on FIPV infection. The inhibitory effects on FIPV infection of these 2 peptides on other type I and type II FIPV strains, feline herpesvirus (FHV), and feline calicivirus (FCV) were also examined. These 2 peptides specifically inhibited type I and type II FIPV, but did FHV or FCV infection. In conclusion, the possibility of peptides derived from the S protein of type I FIPV strain KU-2 as anti-FIPV agents effective not only for type I, but also type II FIPV was demonstrated in vitro. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Orlistat inhibition of intestinal lipase acutely increases appetite and attenuates postprandial glucagon-like peptide-1-(7-36)-amide-1, cholecystokinin, and peptide YY concentrations

    DEFF Research Database (Denmark)

    Ellrichmann, Mark; Kapelle, Mario; Ritter, Peter R

    2008-01-01

    of Orlistat or placebo. Gastric emptying, gallbladder volume and the plasma levels of CCK, PYY, GLP-1, and ghrelin were determined and appetite sensations were measured using visual analogue scales. RESULTS: Gastric emptying was accelerated by Orlistat administration (P emptying.......0001), whereas appetite and prospective food consumption increased (P gastric and gallbladder emptying and reduces...... whether Orlistat alters the secretion of glucagon-like peptide-1-(7-36)-amide (GLP-1), cholecystokinin (CCK), peptide YY (PYY), and ghrelin as well as postprandial appetite sensations. METHODS: Twenty-five healthy human volunteers were examined with a solid-liquid test meal after the oral administration...

  14. Inhibition of neurotensin-stimulated mast cell secretion and carboxypeptidase A activity by the peptide inhibitor of carboxypeptidase A and neurotensin-receptor antagonist SR 48692.

    Science.gov (United States)

    Miller, L A; Cochrane, D E; Feldberg, R S; Carraway, R E

    1998-06-01

    Neurotensin (NT), a peptide found in brain and several peripheral tissues, is a potent stimulus for mast cell secretion and its actions are blocked by the specific NT receptor antagonist, SR 48692. Subsequent to stimulation, NT is rapidly degraded by mast cell carboxypeptidase A (CPA). In the experiments described here, we tested for the involvement of CPA activity in the activation of mast cell secretion by the peptide, NT. Mast cells were isolated from the peritoneal and pleural cavities of rats, purified over metrizamide gradients and incubated at 37 degrees C in Locke solution or Locke containing the appropriate inhibitors. For some experiments, media derived from mast cells stimulated by compound 48/80 were used as a source of mast cell CPA activity. Treatment of mast cells with the highly specific peptide inhibitor of CPA derived from potato (PCI) inhibited histamine release in response to NT and NT8-13 (the biologically active region of NT). This inhibition required some 20 min to develop and was only partially reversed by a 20-min wash period. PCI (10 microM) did not inhibit histamine release in response to NT1-12, bradykinin, compound 48/80, the calcium ionophore, A23187, or anti-IgE serum. PCI also inhibited mast cell CPA activity. SR 48692, a highly selective antagonist of the brain NT receptor and of NT-stimulated mast cell secretion, also inhibited mast cell CPA activity as well as bovine pancreatic CPA activity in a concentration-dependent manner. It is suggested that the mast cell binding site for NT and the active site for CPA may share similar characteristics. The results are discussed in terms of NT mechanism of action on the mast cell.

  15. Alpha-helical destabilization of the Bcl-2-BH4-domain peptide abolishes its ability to inhibit the IP3 receptor.

    Directory of Open Access Journals (Sweden)

    Giovanni Monaco

    Full Text Available The anti-apoptotic Bcl-2 protein is the founding member and namesake of the Bcl-2-protein family. It has recently been demonstrated that Bcl-2, apart from its anti-apoptotic role at mitochondrial membranes, can also directly interact with the inositol 1,4,5-trisphosphate receptor (IP3R, the primary Ca(2+-release channel in the endoplasmic reticulum (ER. Bcl-2 can thereby reduce pro-apoptotic IP3R-mediated Ca(2+ release from the ER. Moreover, the Bcl-2 homology domain 4 (Bcl-2-BH4 has been identified as essential and sufficient for this IP3R-mediated anti-apoptotic activity. In the present study, we investigated whether the reported inhibitory effect of a Bcl-2-BH4 peptide on the IP 3R1 was related to the distinctive α-helical conformation of the BH4 domain peptide. We therefore designed a peptide with two glycine "hinges" replacing residues I14 and V15, of the wild-type Bcl-2-BH4 domain (Bcl-2-BH4-IV/GG. By comparing the structural and functional properties of the Bcl-2-BH4-IV/GG peptide with its native counterpart, we found that the variant contained reduced α-helicity, neither bound nor inhibited the IP 3R1 channel, and in turn lost its anti-apoptotic effect. Similar results were obtained with other substitutions in Bcl-2-BH4 that destabilized the α-helix with concomitant loss of IP3R inhibition. These results provide new insights for the further development of Bcl-2-BH4-derived peptides as specific inhibitors of the IP3R with significant pharmacological implications.

  16. The proteolytically stable peptidomimetic Pam-(Lys-βNSpe)6-NH2 selectively inhibits human neutrophil activation via formyl peptide receptor 2.

    Science.gov (United States)

    Skovbakke, Sarah Line; Heegaard, Peter M H; Larsen, Camilla J; Franzyk, Henrik; Forsman, Huamei; Dahlgren, Claes

    2015-01-15

    Immunomodulatory host defense peptides (HDPs) are considered to be lead compounds for novel anti-sepsis and anti-inflammatory agents. However, development of drugs based on HDPs has been hampered by problems with toxicity and low bioavailability due to in vivo proteolysis. Here, a subclass of proteolytically stable HDP mimics consisting of lipidated α-peptide/β-peptoid oligomers was investigated for their effect on neutrophil function. The most promising compound, Pam-(Lys-βNSpe)6-NH2, was shown to inhibit formyl peptide receptor 2 (FPR2) agonist-induced neutrophil granule mobilization and release of reactive oxygen species. The potency of Pam-(Lys-βNSpe)6-NH2 was comparable to that of PBP10, the most potent FPR2-selective inhibitor known. The immunomodulatory effects of structural analogs of Pam-(Lys-βNSpe)6-NH2 emphasized the importance of both the lipid and peptidomimetic parts. By using imaging flow cytometry in primary neutrophils and FPR-transfected cell lines, we found that a fluorescently labeled analog of Pam-(Lys-βNSpe)6-NH2 interacted selectively with FPR2. Furthermore, the interaction between Pam-(Lys-βNSpe)6-NH2 and FPR2 was found to prevent binding of the FPR2-specific activating peptide agonist Cy5-WKYMWM, while the binding of an FPR1-selective agonist was not inhibited. To our knowledge, Pam-(Lys-βNSpe)6-NH2 is the first HDP mimic found to inhibit activation of human neutrophils via direct interaction with FPR2. Hence, we consider Pam-(Lys-βNSpe)6-NH2 to be a convenient tool in the further dissection of the role of FPR2 in inflammation and homeostasis as well as for investigation of the importance of neutrophil stimulation in anti-infective therapy involving HDPs. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Alanine rich peptide from Populus trichocarpa inhibit growth of Staphylococcus aureus via targetting its extracellular domain of Sensor Histidine Kinase YycGex protein.

    Science.gov (United States)

    Al Akeel, Raid; Mateen, Ayesha; Syed, Rabbani; Al-Qahtani, Mohammed S; Alqahtani, A

    2018-05-11

    Due to growing concern towards microbial resistance, ongoing search for developing novel bioactive compounds such as peptides is on rise. The aim of this study was to evaluate antimicrobial effect of Populus trichocarpa extract, chemically identify the active peptide fraction and finds its target in Staphylococcus aureus. In this study the active fraction of P. trichocarpa crude extract was purified and characterized using MS/MS. This peptide PT13 antimicrobial activity was confirmed by in-vitro agar based disk diffusion and in-vivo infection model of G. mellonella. The proteomic expression analysis of S. aureus under influence of PT13 was studied using LTQ-Orbitrap-MS in-solution digestion and identity of target protein was acquired with their quantified expression using label-free approach of Progenesis QI software. Docking study was performed with peptide PT13 and its target YycG protein using CABS-dock. The active fraction PT13 sequence was identified as KVPVAAAAAAAAAVVASSMVVAAAK, with 25 amino acid including 13 alanine having M/Z 2194.2469. PT13 was uniformly inhibited growth S. aureus SA91 and MIC was determined 16 μg/mL for SA91 S. aureus strain. Sensor histidine kinase (YycG) was most significant target found differentially expressed under influence of PT13. G. mellonella larvae were killed rapidly due to S aureus infection, whereas death in protected group was insignificant in compare to control. The docking models showed ten docking models with RMSD value 1.89 for cluster 1 and RMSD value 3.95 for cluster 2 which is predicted to be high quality model. Alanine rich peptide could be useful in constructing as antimicrobial peptide for targeting extracellular Domain of Sensor Histidine Kinase YycG from S. aureus used in the study. Copyright © 2018. Published by Elsevier Ltd.

  18. Natriuretic peptide receptor A inhibition suppresses gastric cancer development through reactive oxygen species-mediated G2/M cell cycle arrest and cell death.

    Science.gov (United States)

    Li, Zheng; Wang, Ji-Wei; Wang, Wei-Zhi; Zhi, Xiao-Fei; Zhang, Qun; Li, Bo-Wen; Wang, Lin-Jun; Xie, Kun-Ling; Tao, Jin-Qiu; Tang, Jie; Wei, Song; Zhu, Yi; Xu, Hao; Zhang, Dian-Cai; Yang, Li; Xu, Ze-Kuan

    2016-10-01

    Natriuretic peptide receptor A (NPRA), the major receptor for atrial natriuretic peptide (ANP), has been implicated in tumorigenesis; however, the role of ANP-NPRA signaling in the development of gastric cancer remains unclear. Immunohistochemical analyses indicated that NPRA expression was positively associated with gastric tumor size and cancer stage. NPRA inhibition by shRNA induced G2/M cell cycle arrest, cell death, and autophagy in gastric cancer cells, due to accumulation of reactive oxygen species (ROS). Either genetic or pharmacologic inhibition of autophagy led to caspase-dependent cell death. Therefore, autophagy induced by NPRA silencing may represent a cytoprotective mechanism. ROS accumulation activated c-Jun N-terminal kinase (JNK) and AMP-activated protein kinase (AMPK). ROS-mediated activation of JNK inhibited cell proliferation by disturbing cell cycle and decreased cell viability. In addition, AMPK activation promoted autophagy in NPRA-downregulated cancer cells. Overall, our results indicate that the inhibition of NPRA suppresses gastric cancer development and targeting NPRA may represent a promising strategy for the treatment of gastric cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. [Chromogranin A derived peptide CGA47-66 inhibits hyper-permeability of blood brain barrier in mice with sepsis].

    Science.gov (United States)

    Zeng, Yan; Zhang, Dan; Jiang, Liping; Wei, Fu; Xu, Shan

    2016-02-01

    group. In CHR pretreatment groups, EB fluorescence was decreased in brain parenchyma, indicating that EB leakage was significantly less marked, while it was more obvious in high dose CHR group. It was shown by HE staining that cerebral blood vessel structure was intact in NS group, and the gap around blood vessel was not significant increased. On the other hand, brain structure in LPS group appeared loose, with widening of small perivascular spaces and obvious edema. Brain edema in CHR pretreatment groups was improved as compared with that of the LPS group, and it was more apparent in high dose CHR group. LPS induced change in blood brain barrier permeability in mice in a time-dependent manner. Exogenous CGA derived peptides CHR can inhibit LPS induced hyper-permeability of blood brain barrier in septic mice, thus reduces brain edema, protects the brain tissue, and the effect is more obvious with a high dose of CHR (77.5 μg/kg).

  20. A peptide antagonist of the ErbB1 receptor inhibits receptor activation, tumor cell growth and migration in vitro and xenograft tumor growth in vivo

    DEFF Research Database (Denmark)

    Xu, Ruodan; Povlsen, Gro Klitgaard; Soroka, Vladislav

    2010-01-01

    The epidermal growth factor family of receptor tyrosine kinases (ErbBs) plays essential roles in tumorigenesis and cancer disease progression, and therefore has become an attractive target for structure-based drug design. ErbB receptors are activated by ligand-induced homo- and heterodimerization...... constitutes part of the dimerization arm of ErbB3. Inherbin3 binds to the extracellular domains of all four ErbB receptors, with the lowest peptide binding affinity for ErbB4. Inherbin3 functions as an antagonist of epidermal growth factor (EGF)-ErbB1 signaling. We show that Inherbin3 inhibits EGF-induced Erb....... Structural studies have revealed that ErbB receptor dimers are stabilized by receptor-receptor interactions, primarily mediated by a region in the second extracellular domain, termed the "dimerization arm". The present study is the first biological characterization of a peptide, termed Inherbin3, which...

  1. Discovery and characterization of a novel cyclic peptide that effectively inhibits ephrin binding to the EphA4 receptor and displays anti-angiogenesis activity.

    Directory of Open Access Journals (Sweden)

    Xiaofeng Han

    Full Text Available The EphA4 receptor tyrosine kinase regulates a variety of physiological and pathological processes during neural development and the formation of tumor blood vessels; thus, it represents a new and promising therapeutic target. We used a combination of phage peptide display and computer modeling/docking approaches and discovered a novel cyclic nonapeptide, now designated TYY. This peptide selectively inhibits the binding of the ephrinA5 ligand with EphA4 and significantly blocks angiogenesis in a 3D matrigel culture system. Molecular docking reveals that TYY recognizes the same binding pocket on EphA4 that the natural ephrin ligand binds to and that the Tyr3 and Tyr4 side chains of TYY are both critical for the TYY/EphA4 interaction. The discovery of TYY introduces a valuable probe of EphA4 function and a new lead for EphA4-targeted therapeutic development.

  2. Inhibition of dehydration-induced water intake by glucocorticoids is associated with activation of hypothalamic natriuretic peptide receptor-A in rat.

    Directory of Open Access Journals (Sweden)

    Chao Liu

    Full Text Available Atrial natriuretic peptide (ANP provides a potent defense mechanism against volume overload in mammals. Its primary receptor, natriuretic peptide receptor-A (NPR-A, is localized mostly in the kidney, but also is found in hypothalamic areas involved in body fluid volume regulation. Acute glucocorticoid administration produces potent diuresis and natriuresis, possibly by acting in the renal natriuretic peptide system. However, chronic glucocorticoid administration attenuates renal water and sodium excretion. The precise mechanism underlying this paradoxical phenomenon is unclear. We assume that chronic glucocorticoid administration may activate natriuretic peptide system in hypothalamus, and cause volume depletion by inhibiting dehydration-induced water intake. Volume depletion, in turn, compromises renal water excretion. To test this postulation, we determined the effect of dexamethasone on dehydration-induced water intake and assessed the expression of NPR-A in the hypothalamus. The rats were deprived of water for 24 hours to have dehydrated status. Prior to free access to water, the water-deprived rats were pretreated with dexamethasone or vehicle. Urinary volume and water intake were monitored. We found that dexamethasone pretreatment not only produced potent diuresis, but dramatically inhibited the dehydration-induced water intake. Western blotting analysis showed the expression of NPR-A in the hypothalamus was dramatically upregulated by dexamethasone. Consequently, cyclic guanosine monophosphate (the second messenger for the ANP content in the hypothalamus was remarkably increased. The inhibitory effect of dexamethasone on water intake presented in a time- and dose-dependent manner, which emerged at least after 18-hour dexamethasone pretreatment. This effect was glucocorticoid receptor (GR mediated and was abolished by GR antagonist RU486. These results indicated a possible physiologic role for glucocorticoids in the hypothalamic control of

  3. A chimeric peptide of intestinal trefoil factor containing cholesteryl ester transfer protein B cell epitope significantly inhibits atherosclerosis in rabbits after oral administration.

    Science.gov (United States)

    Qi, Gaofu; Li, Jingjing; Wang, Shengying; Xin, Shanshan; Du, Peng; Zhang, Qingye; Zhao, Xiuyun

    2011-04-01

    Vaccination against cholesteryl ester transfer protein (CETP) is proven to be effective for inhibiting atherosclerosis in animal models. In this study, the proteases-resistant intestinal trefoil factor (TFF3) was used as a molecular vehicle to construct chimeric TFF3 (cTFF3) containing CETP B cell epitope and tetanus toxin helper T cell epitope. It was found that cTFF3 still preserved a trefoil structure, and can resist proteases digestion in vitro. After oral immunization with cTFF3, the CETP-specific IgA and IgG could be found in intestine lavage fluid and serum, and the anti-CETP antibodies could inhibit partial CETP activity to increase high-density lipoprotein cholesterol, decrease low-density lipoprotein cholesterol, and inhibit atherosclerosis in animals. Therefore, TFF3 is a potential molecular vehicle for developing oral peptide vaccines. Our research highlights a novel strategy for developing oral peptide vaccines in the future. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system.

    Science.gov (United States)

    Taniguchi, Masayuki; Ochiai, Akihito; Kondo, Hiroshi; Fukuda, Shun; Ishiyama, Yohei; Saitoh, Eiichi; Kato, Tetsuo; Tanaka, Takaaki

    2016-05-01

    Previous studies have shown that pyrrhocoricin, a proline-rich antimicrobial peptide (PrAMP), killed sensitive species in a dose-dependent manner by specifically binding to DnaK. Here, on the basis of the finding that DnaK-deficient Escherichia coli strains are susceptible to PrAMPs, we used pyrrhocoricin to investigate internal targets other than DnaK. Using conventional antibiotics (bleomycin, streptomycin, and fosfomycin) that have known modes of action, first, we validated the availability of an assay using a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on E. coli lysate, for evaluating inhibition of protein synthesis. We found that, similarly to bleomycin and streptomycin, pyrrhocoricin inhibited GFP synthesis in RTS in a concentration-dependent manner. In addition, blockage of transcription and translation steps in RTS was individually estimated using RT-PCR after gene expression to determine mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that this inhibition of GFP synthesis by pyrrhocoricin did not occur at the transcription step but rather at the translation step, in a manner similar to that of GFP synthesis by streptomycin, an inhibitor of the translation step by causing misreading of tRNA. These results suggest that RTS is a powerful assay system for determining if antimicrobial peptides inhibit protein synthesis and its transcription and/or translation steps. This is the first study to have shown that pyrrhocoricin inhibited protein synthesis by specifically repressing the translation step. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. Genetic selection of peptide aptamers that interact and inhibit both Small protein B and alternative ribosome-rescue factor A of Aeromonas veronii C4

    Directory of Open Access Journals (Sweden)

    Peng Liu

    2016-08-01

    Full Text Available Aeromonas veronii is a pathogenic gram-negative bacterium, which infects a variety of animals and results in mass mortality. The stalled-ribosome rescues are reported to ensure viability and virulence under stress conditions, of which primarily include trans-translation and alternative ribosome-rescue factor A (ArfA in A. veronii. For identification of specific peptides that interact and inhibit the stalled-ribosome rescues, peptide aptamer library (pTRG-SN-peptides was constructed using pTRG as vector and Staphylococcus aureus nuclease (SN as scaffold protein, in which 16 random amino acids were introduced to form an exposed surface loop. In the meantime both Small Protein B (SmpB which acts as one of the key components in trans-translation, and alternative ribosome-rescue factor A (ArfA were inserted to pBT to constitute pBT-SmpB and pBT-ArfA, respectively. The peptide aptamer PA-2 was selected from pTRG-SN-peptides by bacterial two-hybrid system (B2H employing pBT-SmpB or pBT-ArfA as baits. The conserved sites G133K134 and D138K139R140 of C-terminal SmpB were identified by interacting with N-terminal SN, and concurrently the residue K62 of ArfA was recognized by interacting with the surface loop of the specific peptide aptamer PA-2. The expression plasmids pN-SN or pN-PA-2, which combined the duplication origin of pRE112 with the neokanamycin promoter expressing SN or PA-2, were created and transformed into A. veronii C4, separately. The engineered A. veronii C4 which endowing SN or PA-2 expression impaired growth capabilities under stress conditions including temperatures, sucrose, glucose, potassium chloride (KCl and antibiotics, and the stress-related genes rpoS and nhaP were down-regulated significantly by Quantitative Real-time PCR (qRT-PCR when treating in 2.0% KCl. Thus,the engineered A. veronii C4 conferring PA-2 expression might be potentially attenuated vaccine, and also the peptide aptamer PA-2 could develop as anti

  6. Genetic Selection of Peptide Aptamers That Interact and Inhibit Both Small Protein B and Alternative Ribosome-Rescue Factor A of Aeromonas veronii C4.

    Science.gov (United States)

    Liu, Peng; Chen, Yong; Wang, Dan; Tang, Yanqiong; Tang, Hongqian; Song, Haichao; Sun, Qun; Zhang, Yueling; Liu, Zhu

    2016-01-01

    Aeromonas veronii is a pathogenic gram-negative bacterium, which infects a variety of animals and results in mass mortality. The stalled-ribosome rescues are reported to ensure viability and virulence under stress conditions, of which primarily include trans-translation and alternative ribosome-rescue factor A (ArfA) in A. veronii. For identification of specific peptides that interact and inhibit the stalled-ribosome rescues, peptide aptamer library (pTRG-SN-peptides) was constructed using pTRG as vector and Staphylococcus aureus nuclease (SN) as scaffold protein, in which 16 random amino acids were introduced to form an exposed surface loop. In the meantime both Small Protein B (SmpB) which acts as one of the key components in trans-translation, and ArfA were inserted to pBT to constitute pBT-SmpB and pBT-ArfA, respectively. The peptide aptamer PA-2 was selected from pTRG-SN-peptides by bacterial two-hybrid system (B2H) employing pBT-SmpB or pBT-ArfA as baits. The conserved sites G133K134 and D138K139R140 of C-terminal SmpB were identified by interacting with N-terminal SN, and concurrently the residue K62 of ArfA was recognized by interacting with the surface loop of the specific peptide aptamer PA-2. The expression plasmids pN-SN or pN-PA-2, which combined the duplication origin of pRE112 with the neokanamycin promoter expressing SN or PA-2, were created and transformed into A. veronii C4, separately. The engineered A. veronii C4 which endowing SN or PA-2 expression impaired growth capabilities under stress conditions including temperatures, sucrose, glucose, potassium chloride (KCl) and antibiotics, and the stress-related genes rpoS and nhaP were down-regulated significantly by Quantitative Real-time PCR (qRT-PCR) when treating in 2.0% KCl. Thus, the engineered A. veronii C4 conferring PA-2 expression might be potentially attenuated vaccine, and also the peptide aptamer PA-2 could develop as anti-microbial drugs targeted to the ribosome rescued factors in A

  7. Anti-Biofilm and Immunomodulatory Activities of Peptides That Inhibit Biofilms Formed by Pathogens Isolated from Cystic Fibrosis Patients

    Directory of Open Access Journals (Sweden)

    César de la Fuente-Núñez

    2014-10-01

    Full Text Available Cystic fibrosis (CF patients often acquire chronic respiratory tract infections due to Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc species. In the CF lung, these bacteria grow as multicellular aggregates termed biofilms. Biofilms demonstrate increased (adaptive resistance to conventional antibiotics, and there are currently no available biofilm-specific therapies. Using plastic adherent, hydroxyapatite and flow cell biofilm models coupled with confocal and scanning electron microscopy, it was demonstrated that an anti-biofilm peptide 1018 prevented biofilm formation, eradicated mature biofilms and killed biofilms formed by a wide range of P. aeruginosa and B. cenocepacia clinical isolates. New peptide derivatives were designed that, compared to their parent peptide 1018, showed similar or decreased anti-biofilm activity against P. aeruginosa biofilms, but increased activity against biofilms formed by the Gram-positive bacterium methicillin resistant Staphylococcus aureus. In addition, some of these new peptide derivatives retained the immunomodulatory activity of 1018 since they induced the production of the chemokine monocyte chemotactic protein-1 (MCP-1 and suppressed lipopolysaccharide-mediated tumor necrosis factor-α (TNF-α production by human peripheral blood mononuclear cells (PBMC and were non-toxic towards these cells. Peptide 1018 and its derivatives provide promising leads for the treatment of chronic biofilm infections and hyperinflammatory lung disease in CF patients.

  8. Glucagon-like peptide 2 stimulates glucagon secretion, enhances lipid absorption, and inhibits gastric acid secretion in humans

    DEFF Research Database (Denmark)

    Meier, Juris J; Nauck, Michael A; Pott, Andrea

    2006-01-01

    or placebo during the ingestion of a solid test meal. Gastric emptying was determined using a 13C-sodium-octanote breath test. Plasma concentrations of glucose, insulin, C-peptide, glucagon, GLP-2, free fatty acids, free glycerol, and triglycerides were determined. RESULTS: GLP-2 administration led...... (P = .07). GLP-2 administration caused an approximately 15% reduction in pentagastrin-stimulated gastric acid and chloride secretion (P gastric emptying was not affected (P = .99). CONCLUSIONS: GLP-2 reduces gastric acid secretion but does not seem to have an influence on gastric......BACKGROUND & AIMS: The gut-derived peptide glucagon-like peptide 2 (GLP-2) has been suggested as a potential drug candidate for the treatment of various intestinal diseases. However, the acute effects of GLP-2 on gastric functions as well as on glucose and lipid homeostasis in humans are less well...

  9. A new peptide (Ruviprase) purified from the venom of Daboia russelii russelii shows potent anticoagulant activity via non-enzymatic inhibition of thrombin and factor Xa.

    Science.gov (United States)

    Thakur, Rupamoni; Kumar, Ashok; Bose, Biplab; Panda, Dulal; Saikia, Debashree; Chattopadhyay, Pronobesh; Mukherjee, Ashis K

    2014-10-01

    Compounds showing dual inhibition of thrombin and factor Xa (FXa) are the subject of great interest owing to their broader specificity for effective anticoagulation therapy against cardiovascular disorders. This is the first report on the functional characterization and assessment of therapeutic potential of a 4423.6 Da inhibitory peptide (Ruviprase) purified from Daboia russelii russelii venom. The secondary structure of Ruviprase is composed of α-helices (61.9%) and random coils (38.1%). The partial N-terminal sequence (E(1)-V(2)-X(3)-W(4)-W(5)-W(6)-A(7)-Q(8)-L(9)-S(10)) of Ruviprase demonstrated significant similarity (80.0%) with an internal sequence of apoptosis-stimulating protein reported from the venom of Ophiophagus hannah and Python bivittatus; albeit Ruviprase did not show sequence similarity with existing thrombin/FXa inhibitors, suggesting its uniqueness. Ruviprase demonstrated a potent in vitro anticoagulant property and inhibited both thrombin and FXa following slow binding kinetics. Ruviprase inhibited thrombin by binding to its active site via an uncompetitive mechanism with a Ki value and dissociation constant (KD) of 0.42 μM and 0.46 μM, respectively. Conversely, Ruviprase demonstrated mixed inhibition (Ki = 0.16 μM) of FXa towards its physiological substrate prothrombin. Furthermore, the biological properties of Ruviprase could not be neutralized by commercial polyvalent or monovalent antivenom. Ruviprase at a dose of 2.0 mg/kg was non-toxic and showed potent in vivo anticoagulant activity after 6 h of intraperitoneal treatment in mice. Because of the potent anticoagulant property as well as non-toxic nature of Ruviprase, the possible application of the peptide as an antithrombotic agent for combating thrombosis-associated ailments appears promising. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  10. Pseudomonas aeruginosa elastase cleaves a C-terminal peptide from human thrombin that inhibits host inflammatory responses

    DEFF Research Database (Denmark)

    van der Plas, Mariena J A; Bhongir, Ravi K V; Kjellström, Sven

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen known for its immune evasive abilities amongst others by degradation of a large variety of host proteins. Here we show that digestion of thrombin by P. aeruginosa elastase leads to the release of the C-terminal thrombin-derived peptide FYT21...

  11. Exenatide, a Glucagon-like Peptide-1 Receptor Agonist, Acutely Inhibits Intestinal Lipoprotein Production in Healthy Humans

    NARCIS (Netherlands)

    Xiao, Changting; Bandsma, Robert H. J.; Dash, Satya; Szeto, Linda; Lewis, Gary F.

    Objective-Incretin-based therapies for the treatment of type 2 diabetes mellitus improve plasma lipid profiles and postprandial lipemia, but their exact mechanism of action remains unclear. Here, we examined the acute effect of the glucagon-like peptide-1 receptor agonist, exenatide, on intestinal

  12. Dipeptidyl peptidase IV inhibition enhances the intestinotrophic effect of glucagon-like peptide-2 in rats and mice

    DEFF Research Database (Denmark)

    Hartmann, B; Thulesen, J; Kissow, Hannelouise

    2000-01-01

    Glucagon-like peptide-2 (GLP-2) induces intestinal growth in mice; but in normal rats, it seems less potent, possibly because of degradation of GLP-2 by the enzyme dipeptidyl peptidase IV (DPP-IV). The purpose of this study was to investigate the survival and effect of GLP-2 in rats and mice afte...

  13. BG-4, a novel bioactive peptide from Momordica charantia, inhibits lipopolysaccharide-induced inflammation in THP-1 human macrophages

    Science.gov (United States)

    Background: Bitter melon (Momordica charantia) is a commonly used food crop for management of a variety of diseases most notably for control of diabetes, a disease associated with aberrant inflammation. Purpose: To evaluate the anti-inflammatory property of BG-4, a novel bioactive peptide isolated f...

  14. Peptide-derivatized SB105-A10 dendrimer inhibits the infectivity of R5 and X4 HIV-1 strains in primary PBMCs and cervicovaginal histocultures.

    Directory of Open Access Journals (Sweden)

    Isabella Bon

    Full Text Available Peptide dendrimers are a class of molecules that exhibit a large array of biological effects including antiviral activity. In this report, we analyzed the antiviral activity of the peptide-derivatized SB105-A10 dendrimer, which is a tetra-branched dendrimer synthetized on a lysine core, in activated peripheral blood mononuclear cells (PBMCs that were challenged with reference and wild-type human immunodeficiency virus type 1 (HIV-1 strains. SB105-A10 inhibited infections by HIV-1 X4 and R5 strains, interfering with the early phases of the viral replication cycle. SB105-A10 targets heparan sulfate proteoglycans (HSPGs and, importantly, the surface plasmon resonance (SPR assay revealed that SB105-A10 strongly binds gp41 and gp120, most likely preventing HIV-1 attachment/entry through multiple mechanisms. Interestingly, the antiviral activity of SB105-A10 was also detectable in an organ-like structure of human cervicovaginal tissue, in which SB105-A10 inhibited the HIV-1ada R5 strain infection without altering the tissue viability. These results demonstrated the strong antiviral activity of SB105-A10 and suggest a potential microbicide use of this dendrimer to prevent the heterosexual transmission of HIV-1.

  15. Feedback suppression of meal-induced glucagon-like peptide-1 (GLP-1) secretion mediated through elevations in intact GLP-1 caused by dipeptidyl peptidase-4 inhibition

    DEFF Research Database (Denmark)

    Baranov, Oleg; Kahle, Melanie; Deacon, Carolyn F

    2016-01-01

    AIM: To compare directly the clinical effects of vildagliptin and sitagliptin in patients with type 2 diabetes, with a special emphasis on incretin hormones and L-cell feedback inhibition induced by dipeptidyl peptidase (DPP-4) inhibition. METHODS: A total of 24 patients (12 on a diet/exercise re......AIM: To compare directly the clinical effects of vildagliptin and sitagliptin in patients with type 2 diabetes, with a special emphasis on incretin hormones and L-cell feedback inhibition induced by dipeptidyl peptidase (DPP-4) inhibition. METHODS: A total of 24 patients (12 on a diet....../exercise regimen, 12 on metformin) were treated, in randomized order, for 7-9 days, with either vildagliptin (50 mg twice daily = 100 mg/d), sitagliptin (100 mg once daily in those on diet, 50 mg twice daily in those on metformin treatment = 100 mg/d) or placebo (twice daily). A mixed-meal test was performed....... RESULTS: Intact glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide concentrations were doubled by both DPP-4 inhibitors. Meal-related total GLP-1 responses were reduced by vildagliptin and sitagliptin treatment alike in the majority of patients (vildagliptin: p = 0...

  16. Glucagon-like peptide-2 inhibits antral emptying in man, but is not as potent as glucagon-like peptide-1

    DEFF Research Database (Denmark)

    Nagell, C F; Wettergren, A; Pedersen, J F

    2004-01-01

    with GLP-1 inhibits gastric emptying and the sensation of hunger in man. METHODS: Eight healthy volunteers were tested in a double-blind, placebo-controlled fashion. Antral emptying of a liquid meal and hunger ratings were determined using ultrasound technology and visual analogue scales scoring during...... infusions of saline, GLP-2 (0.5, and 1.0 pmol kg body wt(-1) min(-1)), GLP-1 (0.5 pmol kg body wt(-1) min(-1)) or GLP-1 and GLP-2 (0.5 pmol kg body wt(-1) min(-1)). RESULTS: The GLP-2 infusions resulted in a dose-dependent increase in antral emptying time (35%; ns and 75%; P = 0.049) compared to saline...

  17. The analgesic agent tapentadol inhibits calcitonin gene-related peptide release from isolated rat brainstem via a serotonergic mechanism.

    Science.gov (United States)

    Greco, Maria Cristina; Navarra, Pierluigi; Tringali, Giuseppe

    2016-01-15

    In this study we tested the hypothesis that tapentadol inhibits GGRP release from the rat brainstem through a mechanism mediated by the inhibition of NA reuptake; as a second alternative hypothesis, we investigated whether tapentadol inhibits GGRP release via the inhibition of 5-HT reuptake. Rat brainstems were explanted and incubated in short-term experiments. CGRP released in the incubation medium was taken as a marker of CGRP release from the central terminals of trigeminal neurons within the brainstem. CGRP levels were measured by radioimmunoassay under basal conditions or in the presence of tapentadol; NA, 5-HT, clonidine, yohimbine and ondansetron were used as pharmacological tools to investigate the action mechanism of tapentadol. The α2-antagonist yohimbine failed to counteract the effects of tapentadol. Moreover, neither NA nor the α2-agonist clonidine per se inhibited K(+)-stimulated CGRP release, thereby indicating that the effects of tapentadol are nor mediated through the block of NA reuptake. Further experiments showed that 5-HT and tramadol, which inhibits both NA and 5-HT reuptake, significantly reduced K(+)-stimulated CGRP release. Moreover, the 5-HT3 antagonist ondansetron was able to counteract the effects of tapentadol in this system. This study provided pharmacological evidence that tapentadol inhibits stimulated CGRP release from the rat brainstem in vitro through a mechanism involving an increase in 5-HT levels in the system and the subsequent activation of 5-HT3 receptors. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Hydrogen sulfide inhibits opioid withdrawal-induced pain sensitization in rats by down-regulation of spinal calcitonin gene-related peptide expression in the spine.

    Science.gov (United States)

    Yang, Hai-Yu; Wu, Zhi-Yuan; Bian, Jin-Song

    2014-09-01

    Hyperalgesia often occurs in opioid-induced withdrawal syndrome. In the present study, we found that three hourly injections of DAMGO (a μ-opioid receptor agonist) followed by naloxone administration at the fourth hour significantly decreased rat paw nociceptive threshold, indicating the induction of withdrawal hyperalgesia. Application of NaHS (a hydrogen sulfide donor) together with each injection of DAMGO attenuated naloxone-precipitated withdrawal hyperalgesia. RT-PCR and Western blot analysis showed that NaHS significantly reversed the gene and protein expression of up-regulated spinal calcitonin gene-related peptide (CGRP) in naloxone-treated animals. NaHS also inhibited naloxone-induced cAMP rebound and cAMP response element-binding protein (CREB) phosphorylation in rat spinal cord. In SH-SY5Y neuronal cells, NaHS inhibited forskolin-stimulated cAMP production and adenylate cyclase (AC) activity. Moreover, NaHS pre-treatment suppressed naloxone-stimulated activation of protein kinase C (PKC) α, Raf-1, and extracellular signal-regulated kinase (ERK) 1/2 in rat spinal cord. Our data suggest that H2S prevents the development of opioid withdrawal-induced hyperalgesia via suppression of synthesis of CGRP in spine through inhibition of AC/cAMP and PKC/Raf-1/ERK pathways.

  19. The Proteolytically Stable Peptidomimetic Pam-(Lys-ßNSpe)6-NH2 Selectively Inhibits Human Neutrophil Activation via Formyl Peptide Receptor 2

    DEFF Research Database (Denmark)

    Skovbakke, Sarah Line; Heegaard, Peter M. H.; Larsen, Camilla J.

    2015-01-01

    of proteolytically stable HDP mimics consisting of lipidated α-peptide/β-peptoid oligomers was investigated for their effect on neutrophil function. The most promising compound, Pam-(Lys-βNSpe)6-NH2, was shown to inhibit formyl peptide receptor 2 (FPR2) agonist-induced neutrophil granule mobilization and release...... of reactive oxygen species. The potency of Pam-(Lys-βNSpe)6-NH2 was comparable to that of PBP10, the most potent FPR2-selective inhibitor known. The immunomodulatory effects of structural analogues of Pam-(Lys-βNSpe)6-NH2 emphasized the importance of both the lipid and peptidomimetic parts. By using imaging...... flow cytometry in primary neutrophils and FPR-transfected cell lines we found that a fluorescently labelled analogue of Pam-(Lys-βNSpe)6-NH2 interacted selectively with FPR2. Furthermore the interaction between Pam-(Lys-βNSpe)6-NH2 and FPR2 was found to prevent binding of the FPR2-specific activating...

  20. Evidence that morphine and opioid peptides do not share a common pathway with adenosine in inhibiting acetylcholine release from isolated intestine.

    Science.gov (United States)

    Vizi, E S; Somogyi, G T; Magyar, K

    1981-12-01

    1 The release of acetylcholine from guinea-pig ileal isolated longitudinal muscle strip with intact Auerbach's plexus was measured by bioassay and by a radioisotope technique. 2 Normorphine (5 x 10(-7)M) and D-Met2, Pro5-enkephalinamide (D-Met, Pro-EA) reduced the release of acetylcholine. Theophylline, an adenosine antagonist, failed to prevent the inhibitory effect of normorphine or D-Met, Pro-EA. 3 Theophylline (1.7 x 10(-4)M) by itself enhanced the twitch responses to field stimulation (0.1 Hz) but did not prevent the inhibitory effect of normorphine and D-Met, Pro-EA. 4 From the results it can be concluded that morphine and opioid peptides do not share a common pathway with adenosine in inhibiting acetylcholine release from axon terminals of Auerbach's plexus.

  1. Inhibition of β-bungarotoxin binding to brain membranes by mast cell degranulating peptide, toxin I, and ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid

    International Nuclear Information System (INIS)

    Schmidt, R.R.; Betz, H.; Rehm, H.

    1988-01-01

    The presynaptically active snake venom neurotoxin β-bungarotoxin (β-Butx) is known to affect neurotransmitter release by binding to a subtype of voltage-activated K + channels. Here the authors show that mast cell degranulating (MCD) peptide from bee venom inhibits the binding of 125 I-labeled β-Butx to chick and rat brain membranes with apparent K/sub i/ values of 180 nM and 1100 nM, respectively. The mechanisms of inhibition of MCD peptide is noncompetitive, as is inhibition of 125 I-β-Butx binding by the protease inhibitor homologue from mamba venom, toxin I. β-Butx and its binding antagonists thus bind to different sites of the same membrane protein. Removal of Ca 2+ by ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid inhibits the binding of 125 I-β-Butx by lowering its affinity to brain membranes

  2. The Effects of Antimicrobial Peptide Nal-P-113 on Inhibiting Periodontal Pathogens and Improving Periodontal Status

    Directory of Open Access Journals (Sweden)

    Hongyan Wang

    2018-01-01

    Full Text Available Periodontal disease consists of chronic gingival inflammation characterized by both degradation of the periodontal connective tissue and alveolar bone loss. Drug therapy is used as an auxiliary treatment method in severe chronic periodontitis, aggressive periodontitis, and periodontitis-associated systemic disease. Nal-P-113, a modified antimicrobial peptide, specifically replaces the histidine residues of P-113 with the bulky amino acid β-naphthylalanine, and our previous studies have verified that this novel peptide is not toxic to the human body within a certain concentration range. The objective of the present study was to evaluate the effect of Nal-P-113 on periodontal pathogens and periodontal status in clinical studies. In a split-mouth clinical trial, the pocket depth and bleeding index values tended to decrease in the experimental group compared with those in the control group. SEM results verified that Nal-P-113 restrained the maturation of plaque. Based on real-time polymerase chain reaction, the levels of Fusobacterium nucleatum, Streptococcus gordonii, Treponema denticola, and Porphyromonas gingivalis in subgingival plaque were decreased when the subjects were given Nal-P-113. Bacterial growth curve analysis and a biofilm susceptibility assay verified that Nal-P-113 at a concentration of 20 μg/mL restrained the growth of S. gordonii, F. nucleatum, and P. gingivalis and biofilm formation. Therefore, Nal-P-113 effectively reduces periodontal pathogens and ameliorates periodontal status.

  3. A novel peptide derived from human pancreatitis-associated protein inhibits inflammation in vivo and in vitro and blocks NF-kappa B signaling pathway.

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    Xiaolu Yang

    Full Text Available BACKGROUND: Pancreatitis-associated protein (PAP is a pancreatic secretory protein belongs to the group VII of C-type lectin family. Emerging evidence suggests that PAP plays a protective effect in inflammatory diseases. In the present study, we newly identified a 16-amino-acid peptide (named PAPep derived from C-type lectin-like domain (CTLD of human PAP with potent anti-inflammatory activity using both in vivo and in vitro assays. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the anti-inflammatory effect of PAPep on endotoxin-induced uveitis (EIU in rats and demonstrated that intravitreal pretreatment of PAPep concentration-dependently attenuated clinical manifestation of EIU rats, reduced protein leakage and cell infiltration into the aqueous humor (AqH, suppressed tumor necrosis factor (TNF-α, interleukin (IL-6, intercellular adhesion molecule-1 (ICAM-1 and monocyte chemoattractant protein (MCP-1 production in ocular tissues, and improved histopathologic manifestation of EIU. Furthermore, PAPep suppressed the LPS-induced mRNA expression of TNF-α and IL-6 in RAW 264.7 cells, inhibited protein expression of ICAM-1 in TNF-α-stimulated human umbilical vein endothelial cells (HUVECs as well as U937 cells adhesion to HUVECs. Western blot analysis in ocular tissues and different cell lines revealed that the possible mechanism for this anti-inflammatory effect of PAPep may depend on its ability to inhibit the activation of NF-kB signaling pathway. CONCLUSIONS/SIGNIFICANCE: Our studies provide the first evidence that the sequence of PAPep is within the critically active region for the anti-inflammatory function of PAP and the peptide may be a promising candidate for the management of ocular inflammatory diseases.

  4. Anti-Inflammatory Action of an Antimicrobial Model Peptide That Suppresses the TRIF-Dependent Signaling Pathway via Inhibition of Toll-Like Receptor 4 Endocytosis in Lipopolysaccharide-Stimulated Macrophages.

    Directory of Open Access Journals (Sweden)

    Do-Wan Shim

    Full Text Available Antimicrobial peptides (AMPs, also called host defense peptides, particularly those with amphipathic helical structures, are emerging as target molecules for therapeutic development due to their immunomodulatory properties. Although the antimicrobial activity of AMPs is known to be exerted primarily by permeation of the bacterial membrane, the mechanism underlying its anti-inflammatory activity remains to be elucidated. We report potent anti-inflammatory activity of WALK11.3, an antimicrobial model peptide with an amphipathic helical conformation, in lipopolysaccharide (LPS-stimulated RAW264.7 cells. This peptide inhibited the expression of inflammatory mediators, including nitric oxide, COX-2, IL-1β, IL-6, INF-β, and TNF-α. Although WALK11.3 did not exert a major effect on all downstream signaling in the MyD88-dependent pathway, toll-like receptor 4 (TLR4- mediated pro-inflammatory signals were markedly attenuated in the TRIF-dependent pathway due to inhibition of the phosphorylation of STAT1 by attenuation of IRF3 phosphorylation. WALK11.3 specifically inhibited the endocytosis of TLR4, which is essential for triggering TRIF-mediated signaling in macrophage cells. Hence, we suggest that specific interference with TLR4 endocytosis could be one of the major modes of the anti-inflammatory action of AMPs. Our designed WALK11 peptides, which possess both antimicrobial and anti-inflammatory activities, may be promising molecules for the development of therapies for infectious inflammation.

  5. Inhibition of both HIV-1 reverse transcription and gene expression by a cyclic peptide that binds the Tat-transactivating response element (TAR RNA.

    Directory of Open Access Journals (Sweden)

    Matthew S Lalonde

    2011-05-01

    Full Text Available The RNA response element TAR plays a critical role in HIV replication by providing a binding site for the recruitment of the viral transactivator protein Tat. Using a structure-guided approach, we have developed a series of conformationally-constrained cyclic peptides that act as structural mimics of the Tat RNA binding region and block Tat-TAR interactions at nanomolar concentrations in vitro. Here we show that these compounds block Tat-dependent transcription in cell-free systems and in cell-based reporter assays. The compounds are also cell permeable, have low toxicity, and inhibit replication of diverse HIV-1 strains, including both CXCR4-tropic and CCR5-tropic primary HIV-1 isolates of the divergent subtypes A, B, C, D and CRF01_AE. In human peripheral blood mononuclear cells, the cyclic peptidomimetic L50 exhibited an IC(50 ∼250 nM. Surprisingly, inhibition of LTR-driven HIV-1 transcription could not account for the full antiviral activity. Timed drug-addition experiments revealed that L-50 has a bi-phasic inhibition curve with the first phase occurring after HIV-1 entry into the host cell and during the initiation of HIV-1 reverse transcription. The second phase coincides with inhibition of HIV-1 transcription. Reconstituted reverse transcription assays confirm that HIV-1 (- strand strong stop DNA synthesis is blocked by L50-TAR RNA interactions in-vitro. These findings are consistent with genetic evidence that TAR plays critical roles both during reverse transcription and during HIV gene expression. Our results suggest that antiviral drugs targeting TAR RNA might be highly effective due to a dual inhibitory mechanism.

  6. Antimicrobial Peptides in 2014

    Directory of Open Access Journals (Sweden)

    Guangshun Wang

    2015-03-01

    Full Text Available This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms.

  7. Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides

    International Nuclear Information System (INIS)

    Sluss, P.M.; Krystek, S.R. Jr.; Andersen, T.T.; Melson, B.E.; Huston, J.S.; Ridge, R.; Reichert, L.E. Jr.

    1986-01-01

    Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of 125 I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of 125 I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of 125 I-labeled human FSH to testis receptor

  8. A new class of HIV-1 protease inhibitor: the crystallographic structure, inhibition and chemical synthesis of an aminimide peptide isostere.

    Science.gov (United States)

    Rutenber, E E; McPhee, F; Kaplan, A P; Gallion, S L; Hogan, J C; Craik, C S; Stroud, R M

    1996-09-01

    The essential role of HIV-1 protease (HIV-1 PR) in the viral life cycle makes it an attractive target for the development of substrate-based inhibitors that may find efficacy as anti-AIDS drugs. However, resistance has arisen to potent peptidomimetic drugs necessitating the further development of novel chemical backbones for diversity based chemistry focused on probing the active site for inhibitor interactions and binding modes that evade protease resistance. AQ148 is a potent inhibitor of HIV-1 PR and represents a new class of transition state analogues incorporating an aminimide peptide isostere. A 3-D crystallographic structure of AQ148, a tetrapeptide isostere, has been determined in complex with its target HIV-1 PR to a resolution of 2.5 A and used to evaluate the specific structural determinants of AQ148 potency and to correlate structure-activity relationships within the class of related compounds. AQ148 is a competitive inhibitor of HIV-1 PR with a Ki value of 137 nM. Twenty-nine derivatives have been synthesized and chemical modifications have been made at the P1, P2, P1', and P2' sites. The atomic resolution structure of AQ148 bound to HIV-1 PR reveals both an inhibitor binding mode that closely resembles that of other peptidomimetic inhibitors and specific protein/inhibitor interactions that correlate with structure-activity relationships. The structure provides the basis for the design, synthesis and evaluation of the next generation of hydroxyethyl aminimide inhibitors. The aminimide peptide isostere is a scaffold with favorable biological properties well suited to both the combinatorial methods of peptidomimesis and the rational design of potent and specific substrate-based analogues.

  9. Purification of a peptide from seahorse, that inhibits TPA-induced MMP, iNOS and COX-2 expression through MAPK and NF-kappaB activation, and induces human osteoblastic and chondrocytic differentiation.

    Science.gov (United States)

    Ryu, BoMi; Qian, Zhong-Ji; Kim, Se-Kwon

    2010-03-30

    Ongoing efforts to search for naturally occurring, bioactive substances for the amelioration of arthritis have led to the discovery of natural products with substantial bioactive properties. The seahorse (Hippocampus kuda Bleeler), a telelost fish, is one source of known beneficial products, yet has not been utilized for arthritis research. In the present work, we have purified and characterized a bioactive peptide from seahorse hydrolysis. Among the hydrolysates tested, pronase E-derived hydrolysate exhibited the highest alkaline phosphatase (ALP) activity, a phenotype marker of osteoblast and chondrocyte differentiation. After its separation from the hydrolysate by several purification steps, the peptide responsible for the ALP activity was isolated and its sequence was identified as LEDPFDKDDWDNWK (1821Da). We have shown that the isolated peptide induces differentiation of osteoblastic MG-63 and chondrocytic SW-1353 cells by measuring ALP activity, mineralization and collagen synthesis. Our results indicate that the peptide acts during early to late stages of differentiation in MG-63 and SW-1353 cells. We also assessed the concentration dependence of the peptide's inhibition of MMP (-1, -3 and -13), iNOS and COX-2 expression after treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a common form of phorbol ester. The peptide also inhibited NO production in MG-63 and SW-1353 cells. To elucidate the mechanisms by which the peptide acted, we examined its effects on TPA-induced MAPKs/NF-kappaB activation and determined that the peptide treatment significantly reduced p38 kinase/NF-kappaB in MG-63 cells and MAPKs/NF-kappaB in SW-1353 cells.

  10. Cocaine- and amphetamine-regulated transcript peptide in the nucleus accumbens shell inhibits cocaine-induced locomotor sensitization to transient over-expression of α-Ca2+ /calmodulin-dependent protein kinase II.

    Science.gov (United States)

    Xiong, Lixia; Meng, Qing; Sun, Xi; Lu, Xiangtong; Fu, Qiang; Peng, Qinghua; Yang, Jianhua; Oh, Ki-Wan; Hu, Zhenzhen

    2018-01-04

    Cocaine- and amphetamine-regulated transcript (CART) peptide is a widely distributed neurotransmitter that attenuates cocaine-induced locomotor activity when injected into the nucleus accumbens (NAc). Our previous work first confirmed that the inhibitory mechanism of the CART peptide on cocaine-induced locomotor activity is related to a reduction in cocaine-enhanced phosphorylated Ca 2+ /calmodulin-dependent protein kinaseIIα (pCaMKIIα) and the enhancement of cocaine-induced D3R function. This study investigated whether CART peptide inhibited cocaine-induced locomotor activity via inhibition of interactions between pCaMKIIα and the D3 dopamine receptor (D3R). We demonstrated that lentivirus-mediated gene transfer transiently increased pCaMKIIα expression, which peaked at 10 days after microinjection into the rat NAc shell, and induced a significant increase in Ca 2+ influx along with greater behavioral sensitivity in the open field test after intraperitoneal injections of cocaine (15 mg/kg). However, western blot analysis and coimmunoprecipitation demonstrated that CART peptide treatment in lentivirus-transfected CaMKIIα-over-expressing NAc rat tissues or cells prior to cocaine administration inhibited the cocaine-induced Ca 2+ influx and attenuated the cocaine-increased pCaMKIIα expression in lentivirus-transfected CaMKIIα-over-expressing cells. CART peptide decreased the cocaine-enhanced phosphorylated cAMP response element binding protein (pCREB) expression via inhibition of the pCaMKIIα-D3R interaction, which may account for the prolonged locomotor sensitization induced by repeated cocaine treatment in lentivirus-transfected CaMKIIα-over-expressing cells. These results provide strong evidence for the inhibitory modulation of CART peptide in cocaine-induced locomotor sensitization. © 2018 International Society for Neurochemistry.

  11. Cellular and molecular insight into the inhibition of primary root growth of Arabidopsis induced by peptaibols, a class of linear peptide antibiotics mainly produced by Trichoderma spp.

    Science.gov (United States)

    Shi, Wei-Ling; Chen, Xiu-Lan; Wang, Li-Xia; Gong, Zhi-Ting; Li, Shuyu; Li, Chun-Long; Xie, Bin-Bin; Zhang, Wei; Shi, Mei; Li, Chuanyou; Zhang, Yu-Zhong; Song, Xiao-Yan

    2016-04-01

    Trichoderma spp. are well known biocontrol agents that produce a variety of antibiotics. Peptaibols are a class of linear peptide antibiotics mainly produced by Trichoderma Alamethicin, the most studied peptaibol, is reported as toxic to plants at certain concentrations, while the mechanisms involved are unclear. We illustrated the toxic mechanisms of peptaibols by studying the growth-inhibitory effect of Trichokonin VI (TK VI), a peptaibol from Trichoderma longibrachiatum SMF2, on Arabidopsis primary roots. TK VI inhibited root growth by suppressing cell division and cell elongation, and disrupting root stem cell niche maintenance. TK VI increased auxin content and disrupted auxin response gradients in root tips. Further, we screened the Arabidopsis TK VI-resistant mutant tkr1. tkr1 harbors a point mutation in GORK, which encodes gated outwardly rectifying K(+)channel proteins. This mutation alleviated TK VI-induced suppression of K(+)efflux in roots, thereby stabilizing the auxin gradient. The tkr1 mutant also resisted the phytotoxicity of alamethicin. Our results indicate that GORK channels play a key role in peptaibol-plant interaction and that there is an inter-relationship between GORK channels and maintenance of auxin homeostasis. The cellular and molecular insight into the peptaibol-induced inhibition of plant root growth advances our understanding of Trichoderma-plant interactions. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  12. Cdk5 inhibitory peptide (CIP inhibits Cdk5/p25 activity induced by high glucose in pancreatic beta cells and recovers insulin secretion from p25 damage.

    Directory of Open Access Journals (Sweden)

    Ya-Li Zheng

    Full Text Available Cdk5/p25 hyperactivity has been demonstrated to lead to neuron apoptosis and degenerations. Chronic exposure to high glucose (HG results in hyperactivity of Cdk5 and reduced insulin secretion. Here, we set out to determine whether abnormal upregulation of Cdk5/p25 activity may be induced in a pancreatic beta cell line, Min6 cells. We first confirmed that p25 were induced in overexpressed p35 cells treated with HG and increased time course dependence. Next, we showed that no p25 was detected under short time HG stimulation (4-12 hrs, however was detectable in the long exposure in HG cells (24 hrs and 48 hrs. Cdk5 activity in the above cells was much higher than low glucose treated cells and resulted in more than 50% inhibition of insulin secretion. We confirmed these results by overexpression of p25 in Min6 cells. As in cortical neurons, CIP, a small peptide, inhibited Cdk5/p25 activity and restored insulin secretion. The same results were detected in co-infection of dominant negative Cdk5 (DNCdk5 with p25. CIP also reduced beta cells apoptosis induced by Cdk5/p25. These studies indicate that Cdk5/p25 hyperactivation deregulates insulin secretion and induces cell death in pancreatic beta cells and suggests that CIP may serve as a therapeutic agent for type 2 diabetes.

  13. A novel peptide sansalvamide analogue inhibits pancreatic cancer cell growth through G0/G1 cell-cycle arrest

    International Nuclear Information System (INIS)

    Ujiki, Michael B.; Milam, Ben; Ding Xianzhong; Roginsky, Alexandra B.; Salabat, M. Reza; Talamonti, Mark S.; Bell, Richard H.; Gu Wenxin; Silverman, Richard B.; Adrian, Thomas E.

    2006-01-01

    Patients with pancreatic cancer have little hope for cure because no effective therapies are available. Sansalvamide A is a cyclic depsipeptide produced by a marine fungus. We investigated the effect of a novel sansalvamide A analogue on growth, cell-cycle phases, and induction of apoptosis in human pancreatic cancer cells in vitro. The sansalvamide analogue caused marked time- and concentration-dependent inhibition of DNA synthesis and cell proliferation of two human pancreatic cancer cell lines (AsPC-1 and S2-013). The analogue induced G0/G1 phase cell-cycle arrest and morphological changes suggesting induction of apoptosis. Apoptosis was confirmed by annexin V binding. This novel sansalvamide analogue inhibits growth of pancreatic cancer cells through G0/G1 arrest and induces apoptosis. Sansalvamide analogues may be valuable for the treatment of pancreatic cancer

  14. Targeted recombinant fusion proteins of IFNγ and mimetic IFNγ with PDGFβR bicyclic peptide inhibits liver fibrogenesis in vivo.

    Directory of Open Access Journals (Sweden)

    Ruchi Bansal

    Full Text Available Hepatic stellate cells (HSCs, following transdifferentiation to myofibroblasts plays a key role in liver fibrosis. Therefore, attempts to attenuate this myofibroblastic phenotype would be a promising therapeutic approach. Interferon gamma (IFNγ is a potent anti-fibrotic cytokine, but its pleiotropic receptor expression leading to severe adverse effects has limited its clinical application. Since, activated HSC express high-level of platelet derived growth factor beta receptor (PDGFβR, we investigated the potential of PDGFβR-specific targeting of IFNγ and its signaling peptide that lacks IFNγR binding site (mimetic IFNγ or mimIFNγ in liver fibrosis. We prepared DNA constructs expressing IFNγ, mimIFNγ or BiPPB (PDGFβR-specific bicyclic peptide-IFNγ, BiPPB-mimIFNγ fusion proteins. Both chimeric proteins alongwith IFNγ and mimIFNγ were produced in E.coli. The expressed proteins were purified and analyzed for PDGFβR-specific binding and in vitro effects. Subsequently, these recombinant proteins were investigated for the liver uptake (pSTAT1α signaling pathway, for anti-fibrotic effects and adverse effects (platelet counts in CCl4-induced liver fibrogenesis in mice. The purified HSC-targeted IFNγ and mimIFNγ fusion proteins showed PDGFβR-specific binding and significantly reduced TGFβ-induced collagen-I expression in human HSC (LX2 cells, while mouse IFNγ and mimIFNγ did not show any effect. Conversely, mouse IFNγ and BiPPB-IFNγ induced activation and dose-dependent nitric oxide release in mouse macrophages (express IFNγR while lack PDGFβR, which was not observed with mimIFNγ and BiPPB-mimIFNγ, due to the lack of IFNγR binding sites. In vivo, targeted BiPPB-IFNγ and BiPPB-mimIFNγ significantly activated intrahepatic IFNγ-signaling pathway compared to IFNγ and mimIFNγ suggesting increased liver accumulation. Furthermore, the targeted fusion proteins ameliorated liver fibrogenesis in mice by significantly reducing

  15. Insights on the mechanism of thioredoxin reductase inhibition by gold N-heterocyclic carbene compounds using the synthetic linear selenocysteine containing C-terminal peptide hTrxR(488-499): an ESI-MS investigation.

    Science.gov (United States)

    Pratesi, Alessandro; Gabbiani, Chiara; Michelucci, Elena; Ginanneschi, Mauro; Papini, Anna Maria; Rubbiani, Riccardo; Ott, Ingo; Messori, Luigi

    2014-07-01

    Gold-based drugs typically behave as strong inhibitors of the enzyme thioredoxin reductase (hTrxR), possibly as the consequence of direct Gold(I) coordination to its active site selenocysteine. To gain a deeper insight into the molecular basis of enzyme inhibition and prove gold-selenocysteine coordination, the reactions of three parent Gold(I) NHC compounds with the synthetic C-terminal dodecapeptide of hTrxR containing Selenocysteine at position 498, were investigated by electrospray ionization mass spectrometry (ESI-MS). Formation of 1:1 Gold-peptide adducts, though in highly different amounts, was demonstrated in all cases. In these adducts the same [Au-NHC](+) moiety is always associated to the intact peptide. Afterward, tandem MS experiments, conducted on a specific Gold-peptide complex, pointed out that Gold is coordinated to the selenolate group. The relatively large strength of the Gold-selenolate coordinative bond well accounts for potent enzyme inhibition typically afforded by these Gold(I) compounds. In a selected case, the time course of enzyme inhibition was explored. Interestingly, enzyme inhibition turned out to show up very quickly and reached its maximum just few minutes after mixing. Overall, the present results offer some clear insight into the process of thioredoxin reductase inhibition by Gold-based compounds. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. A PEDF-Derived Peptide Inhibits Retinal Neovascularization and Blocks Mobilization of Bone Marrow-Derived Endothelial Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Richard Longeras

    2012-01-01

    Full Text Available Proliferative diabetic retinopathy is characterized by pathological retinal neovascularization, mediated by both angiogenesis (involving mature endothelial cells and vasculogenesis (involving bone marrow-derived circulating endothelial progenitor cells (EPCs. Pigment epithelium-derived factor (PEDF contains an N-terminal 34-amino acid peptide (PEDF-34 that has antiangiogenic properties. Herein, we present a novel finding that PEDF-34 also possesses antivasculogenic activity. In the oxygen-induced retinopathy (OIR model using transgenic mice that have Tie2 promoter-driven GFP expression, we quantified Tie2GFP+ cells in bone marrow and peripheral blood by fluorescence-activated cell sorting (FACS. OIR significantly increased the number of circulating Tie2-GFP+ at P16, correlating with the peak progression of neovascularization. Daily intraperitoneal injections of PEDF-34 into OIR mice decreased the number of Tie2-GFP+ cells in the circulation at P16 by 65% but did not affect the number of Tie2-GFP+ cells in the bone marrow. These studies suggest that PEDF-34 attenuates EPC mobilization from the bone marrow into the blood circulation during retinal neovascularization.

  17. Drosophila male sex peptide inhibits siesta sleep and promotes locomotor activity in the post-mated female.

    Science.gov (United States)

    Isaac, R Elwyn; Li, Chenxi; Leedale, Amy E; Shirras, Alan D

    2010-01-07

    Quiescence, or a sleep-like state, is a common and important feature of the daily lives of animals from both invertebrate and vertebrate taxa, suggesting that sleep appeared early in animal evolution. Recently, Drosophila melanogaster has been shown to be a relevant and powerful model for the genetic analysis of sleep behaviour. The sleep architecture of D. melanogaster is sexually dimorphic, with females sleeping much less than males during day-time, presumably because reproductive success requires greater foraging activity by the female as well as the search for egg-laying sites. However, this loss of sleep and increase in locomotor activity will heighten the risk for the female from environmental and predator hazards. In this study, we show that virgin females can minimize this risk by behaving like males, with an extended afternoon 'siesta'. Copulation results in the female losing 70 per cent of day-time sleep and becoming more active. This behaviour lasts for at least 8 days after copulation and is abolished if the mating males lack sex peptide (SP), normally present in the seminal fluid. Our results suggest that SP is the molecular switch that promotes wakefulness in the post-mated female, a change of behaviour compatible with increased foraging and egg-laying activity. The stress resulting from SP-dependent sleep deprivation might be an important contribution to the toxic side-effects of male accessory gland products that are known to reduce lifespan in post-mated females.

  18. Inhibition of cytosolic Phospholipase A2 prevents prion peptide-induced neuronal damage and co-localisation with Beta III Tubulin

    Directory of Open Access Journals (Sweden)

    Last Victoria

    2012-08-01

    Full Text Available Abstract Background Activation of phospholipase A2 (PLA2 and the subsequent metabolism of arachidonic acid (AA to prostaglandins have been shown to play an important role in neuronal death in neurodegenerative disease. Here we report the effects of the prion peptide fragment HuPrP106-126 on the PLA2 cascade in primary cortical neurons and translocation of cPLA2 to neurites. Results Exposure of primary cortical neurons to HuPrP106-126 increased the levels of phosphorylated cPLA2 and caused phosphorylated cPLA2 to relocate from the cell body to the cellular neurite in a PrP-dependent manner, a previously unreported observation. HuPrP106-126 also induced significant AA release, an indicator of cPLA2 activation; this preceded synapse damage and subsequent cellular death. The novel translocation of p-cPLA2 postulated the potential for exposure to HuPrP106-126 to result in a re-arrangement of the cellular cytoskeleton. However p-cPLA2 did not colocalise significantly with F-actin, intermediate filaments, or microtubule-associated proteins. Conversely, p-cPLA2 did significantly colocalise with the cytoskeletal protein beta III tubulin. Pre-treatment with the PLA2 inhibitor, palmitoyl trifluoromethyl ketone (PACOCF3 reduced cPLA2 activation, AA release and damage to the neuronal synapse. Furthermore, PACOCF3 reduced expression of p-cPLA2 in neurites and inhibited colocalisation with beta III tubulin, resulting in protection against PrP-induced cell death. Conclusions Collectively, these findings suggest that cPLA2 plays a vital role in the action of HuPrP106-126 and that the colocalisation of p-cPLA2 with beta III tubulin could be central to the progress of neurodegeneration caused by prion peptides. Further work is needed to define exactly how PLA2 inhibitors protect neurons from peptide-induced toxicity and how this relates to intracellular structural changes occurring in neurodegeneration.

  19. Dipeptidyl peptidase IV inhibition enhances the intestinotrophic effect of glucagon-like peptide-2 in rats and mice

    DEFF Research Database (Denmark)

    Hartmann, B; Thulesen, J; Kissow, Hannelouise

    2000-01-01

    ; 15 mg VP; 40 microg GLP-2, 40 microg GLP-2+15 mg VP; 40 microg GLP-2 (3-33). Mice were treated for 10 days with: saline; 5 microg GLP-2; 5 microg GLP-2+1.5 mg VP; 25 microg GLP-2; 25 microg GLP-2 (3-33). In both cases, body weight, intestinal weight, length, and morphometric data were measured. After...... (4.68 +/- 0.11%, relative to body weight), compared with the two control groups, [3.01 +/- 0.06% (VP) and 2.94 +/- 0.07% (NaCl)] and GLP-2 alone (3.52 +/- 0.10%). In mice, the growth effect of 5 microg GLP-2+VP was comparable with that of 25 microg GLP-2. GLP-2 (3-33) had no effect in rats......, but it had a weak effect on intestinal growth in mice. The extensive GLP-2 degradation in rats can be reduced by VP, and DPP-IV inhibition markedly enhances the intestinotrophic effect of GLP-2 in both rats and mice. We propose that DPP-IV inhibition may be considered to enhance the efficacy of GLP-2...

  20. Inhibition of xyloglucanase from an alkalothermophilic Thermomonospora sp. by a peptidic aspartic protease inhibitor from Penicillium sp. VM24.

    Science.gov (United States)

    Menon, Vishnu; Rao, Mala

    2012-11-01

    A bifunctional inhibitor from Penicillium sp VM24 causing inactivation of xyloglucanase from Thermomonospora sp and an aspartic protease from Aspergillus saitoi was identified. Steady state kinetics studies of xyloglucanase and the inhibitor revealed an irreversible, non-competitive, two-step inhibition mechanism with IC(50) and K(i) values of 780 and 500nM respectively. The interaction of o-phthalaldehyde (OPTA)-labeled xyloglucanase with the inhibitor revealed that the inhibitor binds to the active site of the enzyme. Far- and near-UV spectrophotometric analysis suggests that the conformational changes induced in xyloglucanase by the inhibitor may be due to irreversible denaturation of enzyme. The bifunctional inhibitor may have potential as a biocontrol agent for the protection of plants against phytopathogenic fungi. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Early events elicited by Bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. Activation of protein kinase C and inhibition of epidermal growth factor binding

    International Nuclear Information System (INIS)

    Zachary, I.; Sinnett-Smith, J.W.; Rozengurt, E.

    1986-01-01

    Addition of bombesin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an M/sub r/ 80,000 cellular protein (designated 80k). The effect was both concentration and time dependent. The 80k phosphoproteins generated in response to bombesin and to phorbol 12,13-dibutyrate were identical as judged by one- and two-dimensional PAGE and by peptide mapping after partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with phorbol 12,13-dibutyrate, which leads to the disappearance of protein kinase C activity, blocked the ability of bombesin to stimulate 80k. Bombesin also caused a rapid (1 min) inhibition of 125 I-labeled epidermal growth factor ( 125 I-EGF) binding to Swiss 3T3 cells. The inhibition was both concentration and temperature dependent and resulted from a marked decrease in the affinity of the EGF receptor for its ligand. These results strongly suggest that these responses are mediated by specific high-affinity receptors that recognize the peptides of the bombesin family in Swiss 3T3 cells. While an increase in cytosolic Ca 2+ concentration does not mediate the bombesin inhibition of 125 I-EGF binding, the activation of protein kinase C in intact Swiss 3T3 cells by peptides of the bombesin family may lead to rapid inhibition of the binding of 125 I-EGF to its cellular receptor

  2. Inhibition of Grape Crown Gall by Agrobacterium vitis F2/5 Requires Two Nonribosomal Peptide Synthetases and One Polyketide Synthase.

    Science.gov (United States)

    Zheng, Desen; Burr, Thomas J

    2016-02-01

    Agrobacterium vitis nontumorigenic strain F2/5 is able to inhibit crown gall disease on grapevines. The mechanism of grape tumor inhibition (GTI) by F2/5 has not been fully determined. In this study, we demonstrate that two nonribosomal peptide synthetase (NRPS) genes (F-avi3342 and F-avi5730) and one polyketide synthase gene (F-avi4330) are required for GTI. Knockout of any one of them resulted in F/25 losing GTI capacity. We previously reported that F-avi3342 and F-avi4330 but not F-avi5730 are required for induction of grape tissue necrosis and tobacco hypersensitive response. F-avi5730 is predicted to encode a single modular NRPS. It is located in a cluster that is homologous to the siderophore vicibactin biosynthesis locus in Rhizobium species. Individual disruption of F-avi5730 and two immediate downstream genes, F-avi5731 and F-avi5732, all resulted in reduced siderophore production; however, only F-avi5730 was found to be required for GTI. Complemented F-avi5730 mutant (ΔF-avi5730(+)) restored a wild-type level of GTI activity. It was determined that, over time, populations of ΔF-avi4330, ΔF-avi3342, and ΔF-avi5730 at inoculated wound sites on grapevine did not differ from those of ΔF-avi5730(+) indicating that loss of GTI was not due to reduced colonization of wound sites by mutants.

  3. cGMP inhibition of type 3 phosphodiesterase is the major mechanism by which C-type natriuretic peptide activates CFTR in the shark rectal gland

    Science.gov (United States)

    De Jonge, Hugo R.; Tilly, Ben C.; Hogema, Boris M.; Pfau, Daniel J.; Kelley, Catherine A.; Kelley, Megan H.; Melita, August M.; Morris, Montana T.; Viola, Ryan M.

    2013-01-01

    The in vitro perfused rectal gland of the dogfish shark (Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride secretion. CNP binds to natriuretic receptors in the basolateral membrane, elevates cellular cGMP, and opens cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in the apical membrane. CNP-provoked chloride secretion was completely inhibitable by the nonspecific protein kinase inhibitor staurosporine and the PKA inhibitor H89 but insensitive to H8, an inhibitor of type I and II isoforms of cGMP-dependent protein kinase (cGKI and cGKII). CNP-induced secretion could not be mimicked by nonhydrolyzable cGMP analogs added alone or in combination with the protein kinase C activator phorbolester, arguing against a role for cGK or for cGMP-induced PKC signaling. We failed to detect a dogfish ortholog of cGKII by molecular cloning and affinity chromatography. However, inhibitors of the cGMP-inhibitable isoform of phosphodiesterase (PDE3) including milrinone, amrinone, and cilostamide but not inhibitors of other PDE isoenzymes mimicked the effect of CNP on chloride secretion in perfused glands and monolayers. CNP raised cGMP and cAMP levels in the SRG epithelial cells. This rise in cAMP as well as the CNP and amrinone-provoked chloride secretion, but not the rise in cGMP, was almost completely blocked by the Gαi-coupled adenylyl cyclase inhibitor somatostatin, arguing against a role for cGMP cross-activation of PKA in CNP action. These data provide molecular, functional, and pharmacological evidence for a CNP/cGMP/PDE3/cAMP/PKA signaling cascade coupled to CFTR in the SRG. PMID:24259420

  4. Milk-derived peptide Val-Pro-Pro (VPP) inhibits obesity-induced adipose inflammation via an angiotensin-converting enzyme (ACE) dependent cascade.

    Science.gov (United States)

    Sawada, Yoko; Sakamoto, Yuri; Toh, Mariko; Ohara, Nozomi; Hatanaka, Yuiko; Naka, Ayano; Kishimoto, Yoshimi; Kondo, Kazuo; Iida, Kaoruko

    2015-12-01

    This study aimed to examine the effects of Val-Pro-Pro (VPP), a food-derived peptide with an angiotensin-converting enzyme (ACE) inhibitory property, on obesity-linked insulin resistance, and adipose inflammation in vivo and in vitro. C57BL/6J mice were fed high-fat high-sucrose diet and VPP (0.1% in water) for 4 months. For in vitro analysis, coculture of 3T3-L1 adipocytes overexpressing either ACE (3T3-ACE) or green fluorescent protein (3T3-GFP) and RAW264 macrophages was conducted with VPP. In diet-induced obese mice, VPP improved insulin sensitivity, concomitant with a significant decrease in tumor necrosis factor α (TNF-α) and IL-1β expression in adipose tissue, with a tendency (p = 0.06) toward decreased CC chemokine ligand 5 expression. Additionally, VPP administration inhibited macrophage accumulation and activation in fat tissues. In vitro, VPP attenuated TNF-α mRNA induced by ACE overexpression in 3T3-L1 adipocytes. TNF-α and IL-1β expression decreased following VPP treatment of RAW264 macrophage and 3T3-ACE adipocyte cocultures, but not in RAW264-3T3-GFP adipocyte cocultures. Our data suggest that VPP inhibits adipose inflammation in the interaction between adipocytes and macrophages, acting as an ACE inhibitor, thereby improving obesity-related insulin resistance. Thus, ingestion of VPP may be a viable protective and therapeutic strategy for insulin resistance and obesity-associated adipose inflammation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Conformational changes in fragments D and double-D from human fibrin(ogen) upon binding the peptide ligand Gly-His-Arg-Pro-amide.

    Science.gov (United States)

    Everse, S J; Spraggon, G; Veerapandian, L; Doolittle, R F

    1999-03-09

    The structure of fragment double-D from human fibrin has been solved in the presence and absence of the peptide ligands that simulate the two knobs exposed by the removal of fibrinopeptides A and B, respectively. All told, six crystal structures have been determined, three of which are reported here for the first time: namely, fragments D and double-D with the peptide GHRPam alone and double-D in the absence of any peptide ligand. Comparison of the structures has revealed a series of conformational changes that are brought about by the various knob-hole interactions. Of greatest interest is a moveable "flap" of two negatively charged amino acids (Glubeta397 and Aspbeta398) whose side chains are pinned back to the coiled coil with a calcium atom bridge until GHRPam occupies the beta-chain pocket. Additionally, in the absence of the peptide ligand GPRPam, GHRPam binds to the gamma-chain pocket, a new calcium-binding site being formed concomitantly.

  6. Inhibition of peptide bond formation by pleuromutilins: the structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin.

    Science.gov (United States)

    Schlünzen, Frank; Pyetan, Erez; Fucini, Paola; Yonath, Ada; Harms, Jörg M

    2004-12-01

    Tiamulin, a prominent member of the pleuromutilin class of antibiotics, is a potent inhibitor of protein synthesis in bacteria. Up to now the effect of pleuromutilins on the ribosome has not been determined on a molecular level. The 3.5 A structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin provides for the first time a detailed picture of its interactions with the 23S rRNA, thus explaining the molecular mechanism of the antimicrobial activity of the pleuromutilin class of antibiotics. Our results show that tiamulin is located within the peptidyl transferase center (PTC) of the 50S ribosomal subunit with its tricyclic mutilin core positioned in a tight pocket at the A-tRNA binding site. Also, the extension, which protrudes from its mutilin core, partially overlaps with the P-tRNA binding site. Thereby, tiamulin directly inhibits peptide bond formation. Comparison of the tiamulin binding site with other PTC targeting drugs, like chloramphenicol, clindamycin and streptogramins, may facilitate the design of modified or hybridized drugs that extend the applicability of this class of antibiotics.

  7. Some commonly used brominated flame retardants cause Ca2+-ATPase inhibition, beta-amyloid peptide release and apoptosis in SH-SY5Y neuronal cells.

    Directory of Open Access Journals (Sweden)

    Fawaz Al-Mousa

    Full Text Available Brominated flame retardants (BFRs are chemicals commonly used to reduce the flammability of consumer products and are considered pollutants since they have become widely dispersed throughout the environment and have also been shown to bio-accumulate within animals and man. This study investigated the cytotoxicity of some of the most commonly used groups of BFRs on SH-SY5Y human neuroblastoma cells. The results showed that of the BFRs tested, hexabromocyclododecane (HBCD, tetrabromobisphenol-A (TBBPA and decabromodiphenyl ether (DBPE, all are cytotoxic at low micromolar concentrations (LC(50 being 2.7 ± 0.7 µM, 15 ± 4 µM and 28 ± 7 µM, respectively. They induced cell death, at least in part, by apoptosis through activation of caspases. They also increased intracellular [Ca(2+] levels and reactive-oxygen-species within these neuronal cells. Furthermore, these BFRs also caused rapid depolarization of the mitochondria and cytochrome c release in these neuronal cells. Elevated intracellular [Ca(2+] levels appear to occur through a mechanism involving microsomal Ca(2+-ATPase inhibition and this maybe responsible for Ca(2+-induced mitochondrial dysfunction. In addition, µM levels of these BFRs caused β-amyloid peptide (Aβ-42 processing and release from these cells with a few hours of exposure. These results therefore shows that these pollutants are both neurotoxic and amyloidogenic in-vitro.

  8. Bacterial Peptide deformylase inhibition of cyano substituted biaryl analogs: Synthesis, in vitro biological evaluation, molecular docking study and in silico ADME prediction.

    Science.gov (United States)

    Khan, Firoz A Kalam; Patil, Rajendra H; Shinde, Devanand B; Sangshetti, Jaiprakash N

    2016-08-15

    Herein, we report the synthesis and screening of cyano substituted biaryl analogs 5(a-m) as Peptide deformylase (PDF) enzyme inhibitors. The compounds 5a (IC50 value=13.16μM), 5d (IC50 value=15.66μM) and 5j (IC50 value=19.16μM) had shown good PDF inhibition activity. The compounds 5a (MIC range=11.00-15.83μg/mL), 5b (MIC range=23.75-28.50μg/mL) and 5j (MIC range=7.66-16.91μg/mL) had also shown potent antibacterial activity when compared with ciprofloxacin (MIC range=25-50μg/mL). Thus, the active derivatives were not only potent PDF inhibitors but also efficient antibacterial agents. In order to gain more insight on the binding mode of the compounds with PDF, the synthesized compounds 5(a-m) were docked against PDF enzyme of Escherichia coli and compounds exhibited good binding properties. In silico ADME properties of synthesized compounds were also analyzed and showed potential to develop as good oral drug candidates. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Inhibition of transforming growth factor-beta1-induced signaling and epithelial-to-mesenchymal transition by the Smad-binding peptide aptamer Trx-SARA.

    Science.gov (United States)

    Zhao, Bryan M; Hoffmann, F Michael

    2006-09-01

    Overexpression of the inhibitory Smad, Smad7, is used frequently to implicate the Smad pathway in cellular responses to transforming growth factor beta (TGF-beta) signaling; however, Smad7 regulates several other proteins, including Cdc42, p38MAPK, and beta-catenin. We report an alternative approach for more specifically disrupting Smad-dependent signaling using a peptide aptamer, Trx-SARA, which comprises a rigid scaffold, the Escherichia coli thioredoxin A protein (Trx), displaying a constrained 56-amino acid Smad-binding motif from the Smad anchor for receptor activation (SARA) protein. Trx-SARA bound specifically to Smad2 and Smad3 and inhibited both TGF-beta-induced reporter gene expression and epithelial-to-mesenchymal transition in NMuMG murine mammary epithelial cells. In contrast to Smad7, Trx-SARA had no effect on the Smad2 or 3 phosphorylation levels induced by TGF-beta1. Trx-SARA was primarily localized to the nucleus and perturbed the normal cytoplasmic localization of Smad2 and 3 to a nuclear localization in the absence of TGF-beta1, consistent with reduced Smad nuclear export. The key mode of action of Trx-SARA was to reduce the level of Smad2 and Smad3 in complex with Smad4 after TGF-beta1 stimulation, a mechanism of action consistent with the preferential binding of SARA to monomeric Smad protein and Trx-SARA-mediated disruption of active Smad complexes.

  10. Folate-targeted amphiphilic cyclodextrin nanoparticles incorporating a fusogenic peptide deliver therapeutic siRNA and inhibit the invasive capacity of 3D prostate cancer tumours.

    Science.gov (United States)

    Evans, James C; Malhotra, Meenakshi; Sweeney, Katrina; Darcy, Raphael; Nelson, Colleen C; Hollier, Brett G; O'Driscoll, Caitriona M

    2017-10-30

    The main barrier to the development of an effective RNA interference (RNAi) therapy is the lack of a suitable delivery vector. Modified cyclodextrins have emerged in recent years for the delivery of siRNA. In the present study, a folate-targeted amphiphilic cyclodextrin was formulated using DSPE-PEG 5000 -folate to target prostate cancer cells. The fusogenic peptide GALA was included in the formulation to aid in the endosomal release of siRNA. Targeted nanoparticles were less than 200nm in size with a neutral surface charge. The complexes were able to bind siRNA and protect it from serum nucleases. Incubation with excess free folate resulted in a significant decrease in the uptake of targeted nanoparticles in LNCaP and PC3 cells, both of which have been reported to have differing pathways of folate uptake. There was a significant reduction in the therapeutic targets, ZEB1 and NRP1 at mRNA and protein level following treatment with targeted complexes. In preliminary functional assays using 3D spheroids, treatment of PC3 tumours with targeted complexes with ZEB1 and NRP1 siRNA resulted in more compact colonies relative to the untargeted controls and inhibited infiltration into the Matrigel™ layer. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. (99m)Tc-labeled gastrins of varying peptide chain length: Distinct impact of NEP/ACE-inhibition on stability and tumor uptake in mice.

    Science.gov (United States)

    Kaloudi, Aikaterini; Nock, Berthold A; Lymperis, Emmanouil; Krenning, Eric P; de Jong, Marion; Maina, Theodosia

    2016-06-01

    In situ inhibition of neutral endopeptidase (NEP) has been recently shown to impressively increase the bioavailability and tumor uptake of biodegradable gastrin radioligands. Furthermore, angiotensin converting enzyme (ACE) has been previously shown to cleave gastrin analogs in vitro. In the present study, we have assessed the effects induced by single or dual NEP/ACE-inhibition on the pharmacokinetic profile of three (99m)Tc-labeled gastrins of varying peptide chain length: [(99m)Tc]SG6 ([(99m)Tc-N4-Gln(1)]gastrin(1-17)), [(99m)Tc]DG2 ([(99m)Tc-N4-Gly(4),DGlu(5)]gastrin(4-17)) and [(99m)Tc]DG4 ([(99m)Tc-N4-DGlu(10)]gastrin(10-17)). Mouse blood samples were collected 5min after injection of each of [(99m)Tc]SG6/DG2/DG4 together with: a) vehicle, b) the NEP-inhibitor phosphoramidon (PA), c) the ACE-inhibitor lisinopril (Lis), or d) PA plus Lis and were analyzed by RP-HPLC for radiometabolite detection. Biodistribution was studied in SCID mice bearing A431-CCK2R(+/-) xenografts at 4h postinjection (pi). [(99m)Tc]SG6 or [(99m)Tc]DG4 was coinjected with either vehicle or the above described NEP/ACE-inhibitor regimens; for [(99m)Tc]DG2 control and PA animal groups were only included. Treatment of mice with PA induced significant stabilization of (99m)Tc-radiotracers in peripheral blood, while treatment with Lis or Lis+PA affected the stability of des(Glu)5 [(99m)Tc]DG4 only. In line with these findings, PA coinjection led to notable amplification of tumor uptake of radiopeptides compared to controls (PTc]DG4 profited by single Lis (2.06±0.39%ID/g vs 0.99±0.13%ID/g in controls) or combined Lis+PA coinjection (8.91±1.61%ID/g vs 4.89±1.33%ID/g in PA-group). Furthermore, kidney uptake remained favourably low and unaffected by PA and/or Lis coinjection only in the case of [(99m)Tc]DG4 (Tc-radioligands based on different-length gastrins. Truncated [(99m)Tc]DG4 exhibited overall the most attractive profile during combined NEP/ACE-inhibition in mouse models, providing new

  12. SHP-1, a novel peptide isolated from seahorse inhibits collagen release through the suppression of collagenases 1 and 3, nitric oxide products regulated by NF-kappaB/p38 kinase.

    Science.gov (United States)

    Ryu, BoMi; Qian, Zhong-Ji; Kim, Se-Kwon

    2010-01-01

    Considerable efforts have been taken to identify natural peptides as potential bioactive substances. In this study, novel peptide (SHP-1) derived from seahorse (Hippocampus, Syngnathidae) hydrolysate was explored for its inhibitory effects on collagen release in arthritis with the investigation of its underlying mechanism of action. The efficacy of SHP-1 was determined on cartilage protective effects such as inhibition of collagen and GAG release. SHP-1 was able to suppress not only the expression of collagenases 1 and 3, but also the production of NO via down-regulation of iNOS. However, it presented an irrelevant effect on the level of GAG release in chondrocytic and osteoblastic cells. Inhibition of collagen release by SHP-1 is associated with restraining the phosphorylation of NF-kappaB and p38 kinase cascade. Therefore, it could be suggested that SHP-1 has a potential to be used in arthritis treatment.

  13. [Distiller Yeasts Producing Antibacterial Peptides].

    Science.gov (United States)

    Klyachko, E V; Morozkina, E V; Zaitchik, B Ts; Benevolensky, S V

    2015-01-01

    A new method of controlling lactic acid bacteria contamination was developed with the use of recombinant Saccharomyces cerevisiae strains producing antibacterial peptides. Genes encoding the antibacterial peptides pediocin and plantaricin with codons preferable for S. cerevisiae were synthesized, and a system was constructed for their secretory expression. Recombinant S. cerevisiae strains producing antibacterial peptides effectively inhibit the growth of Lactobacillus sakei, Pediacoccus pentasaceus, Pediacoccus acidilactici, etc. The application of distiller yeasts producing antibacterial peptides enhances the ethanol yield in cases of bacterial contamination. Recombinant yeasts producing the antibacterial peptides pediocin and plantaricin can successfully substitute the available industrial yeast strains upon ethanol production.

  14. 99mTc-labeled gastrins of varying peptide chain length: Distinct impact of NEP/ACE-inhibition on stability and tumor uptake in mice

    International Nuclear Information System (INIS)

    Kaloudi, Aikaterini; Nock, Berthold A.; Lymperis, Emmanouil; Krenning, Eric P.; Jong, Marion de; Maina, Theodosia

    2016-01-01

    Introduction: In situ inhibition of neutral endopeptidase (NEP) has been recently shown to impressively increase the bioavailability and tumor uptake of biodegradable gastrin radioligands. Furthermore, angiotensin converting enzyme (ACE) has been previously shown to cleave gastrin analogs in vitro. In the present study, we have assessed the effects induced by single or dual NEP/ACE-inhibition on the pharmacokinetic profile of three 99m Tc-labeled gastrins of varying peptide chain length: [ 99m Tc]SG6 ([ 99m Tc-N 4 -Gln 1 ]gastrin(1–17)), [ 99m Tc]DG2 ([ 99m Tc-N 4 -Gly 4 ,DGlu 5 ]gastrin(4–17)) and [ 99m Tc]DG4 ([ 99m Tc-N 4 -DGlu 10 ]gastrin(10–17)). Methods: Mouse blood samples were collected 5 min after injection of each of [ 99m Tc]SG6/DG2/DG4 together with: a) vehicle, b) the NEP-inhibitor phosphoramidon (PA), c) the ACE-inhibitor lisinopril (Lis), or d) PA plus Lis and were analyzed by RP-HPLC for radiometabolite detection. Biodistribution was studied in SCID mice bearing A431-CCK2R(+/−) xenografts at 4 h postinjection (pi). [ 99m Tc]SG6 or [ 99m Tc]DG4 was coinjected with either vehicle or the above described NEP/ACE-inhibitor regimens; for [ 99m Tc]DG2 control and PA animal groups were only included. Results: Treatment of mice with PA induced significant stabilization of 99m Tc-radiotracers in peripheral blood, while treatment with Lis or Lis + PA affected the stability of des(Glu) 5 [ 99m Tc]DG4 only. In line with these findings, PA coinjection led to notable amplification of tumor uptake of radiopeptides compared to controls (P < 0.01). Only [ 99m Tc]DG4 profited by single Lis (2.06 ± 0.39%ID/g vs 0.99 ± 0.13%ID/g in controls) or combined Lis + PA coinjection (8.91 ± 1.61%ID/g vs 4.89 ± 1.33%ID/g in PA-group). Furthermore, kidney uptake remained favourably low and unaffected by PA and/or Lis coinjection only in the case of [ 99m Tc]DG4 (< 1.9%ID/g) resulting in the most optimal tumor-to-kidney ratios. Conclusions: In situ NEP/ACE-inhibition

  15. The antibacterial activity of peptides derived from human beta-2 glycoprotein I is inhibited by protein H and M1 protein from Streptococcus pyogenes

    NARCIS (Netherlands)

    Nilsson, Maria; Wasylik, Sylwia; Mörgelin, Matthias; Olin, Anders I.; Meijers, Joost C. M.; Derksen, Ronald H. W. M.; de Groot, Philip G.; Herwald, Heiko

    2008-01-01

    During the last years, the importance of antibacterial peptides has attracted considerable attention. We report here that peptides derived from the fifth domain of beta-2 glycoprotein I (beta(2)GPI), a human heparin binding plasma protein, have antibacterial activities against Gram-positive and

  16. Glucagon-like peptide-1 (GLP-1) receptor agonism or DPP-4 inhibition does not accelerate neoplasia in carcinogen treated mice

    DEFF Research Database (Denmark)

    Kissow, Hannelouise; Hartmann, Bolette; Holst, Jens Juul

    2012-01-01

    Glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) are secreted in parallel from the intestinal endocrine cells after nutrient intake. GLP-1 is an incretin hormone and analogues are available for the treatment of type 2 diabetes mellitus (T2DM). GLP-2 is an intestinal growth horm...

  17. Rabbit IgG directed to a synthetic C-terminal peptide of the major grass pollen allergen Lol p I inhibits human basophil histamine release induced by natural Lol p I.

    Science.gov (United States)

    van Ree, R; Aalberse, R C

    1995-03-01

    The potential role of allergen-specific IgG antibodies as 'blocking' antibodies in allergen-induced human basophil histamine release was investigated. This was studied in a model with the major grass pollen allergen Lol p I and polyclonal rabbit antisera directed against this allergen and against a synthetic peptide of its C terminus. When allergen and antibodies were allowed to preincubate, Lol p I induced histamine release was inhibited up to 85% by the antiserum against Lol p I. By omitting preincubation, and thereby more closely mimicking an in vivo situation, up to 55% inhibition was realized. This indicates that allergen-specific IgG can act as 'blocking' antibody without preincubation. Immunization of rabbits with a synthetic C-terminal peptide of Lol p I resulted in antibodies reactive with natural Lol p I. Despite their 100-fold lower avidity for Lol p I (as compared with antinatural Lol p I), these antibodies had the capacity to inhibit Lol p I induced histamine release for > 90% (up to 50% without preincubation). This indicates that it is possible to block histamine release induced by a major allergen with low-avidity IgG antibodies directed against a minor proportion of the allergen (25 amino acids). IgE antibodies from the donors studied were unreactive with this synthetic peptide, indicating that for blocking activity identical epitope specificity of IgE and IgG is not essential. This opens interesting perspectives for application of synthetic peptides in immunotherapy, distinct from their effects on T cell reactivity.

  18. Dissect Kif5b in nuclear positioning during myogenesis: The light chain binding domain and the autoinhibitory peptide are both indispensable

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zai, E-mail: wangzai81@hotmail.com [Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam (Hong Kong); Institute of Clinical Medical Sciences, China–Japan Friendship Hospital, Beijing (China); Xue, Wenqian; Li, Xiuling; Lin, Raozhou [Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam (Hong Kong); Cui, Ju [Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam (Hong Kong); Beijing Institute of Geriatrics, Beijing Hospital, Ministry of Health (China); Huang, Jian-Dong, E-mail: jdhuang@hku.hk [Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam (Hong Kong)

    2013-03-08

    Highlights: ► Kif5b localizes at myonuclear membrane and is responsible for nuclear dispersion. ► Kif5b stalk/tail domain contains signal for nuclear membrane targeting. ► Kif5b stalk/tail domain directly binds to a nesprin 4 in vitro. ► KLC binding domain and autoinhibitory peptide are both functionally indispensable. -- Abstract: The microtubule motor kinesin-1 is responsible for the nuclear positioning during myogenesis. Here we show that the coiled-coil stalk/tail domain containing the kinesin light chain (KLC) binding sites targets to the perinuclear region like endogenous Kif5b, while the globular tail domain cannot. To investigate which fragments of kinesin heavy chain (Kif5b) is responsible for the myonuclear positioning, we transfect Kif5b expression constructs into Kif5b deficient myoblasts and test their ability to rescue the myonuclear phenotype. We find that the KLC binding domain and the autoinhibitory peptide in the globular tail region are both indispensable for the nuclear membrane localization of Kif5b and the kinesin-1-mediated myonuclear positioning. These results suggest that while the KLC binding domain may directly targets Kif5b to the myonuclear membrane, the autoinhibitory peptide may play an indirect role in regulating the kinesin-1-mediated myonuclear positioning.

  19. Inhibition of. beta. -bungarotoxin binding to brain membranes by mast cell degranulating peptide, toxin I, and ethylene glycol bis(. beta. -aminoethyl ether)-N,N,N',N'-tetraacetic acid

    Energy Technology Data Exchange (ETDEWEB)

    Schmidt, R.R.; Betz, H.; Rehm, H.

    1988-02-09

    The presynaptically active snake venom neurotoxin ..beta..-bungarotoxin (..beta..-Butx) is known to affect neurotransmitter release by binding to a subtype of voltage-activated K/sup +/ channels. Here the authors show that mast cell degranulating (MCD) peptide from bee venom inhibits the binding of /sup 125/I-labeled ..beta..-Butx to chick and rat brain membranes with apparent K/sub i/ values of 180 nM and 1100 nM, respectively. The mechanisms of inhibition of MCD peptide is noncompetitive, as is inhibition of /sup 125/I-..beta..-Butx binding by the protease inhibitor homologue from mamba venom, toxin I. ..beta..-Butx and its binding antagonists thus bind to different sites of the same membrane protein. Removal of Ca/sup 2 +/ by ethylene glycol bis(..beta..-aminoethyl ether)-N,N,N',N'-tetraacetic acid inhibits the binding of /sup 125/I-..beta..-Butx by lowering its affinity to brain membranes.

  20. Rational design and synthesis of altered peptide ligands based on human myelin oligodendrocyte glycoprotein 35-55 epitope: inhibition of chronic experimental autoimmune encephalomyelitis in mice.

    Science.gov (United States)

    Tselios, Theodore; Aggelidakis, Mihalis; Tapeinou, Anthi; Tseveleki, Vivian; Kanistras, Ioannis; Gatos, Dimitrios; Matsoukas, John

    2014-11-04

    Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). Although the etiology of MS remains unclear, there is evidence T-cell recognition of immunodominant epitopes of myelin proteins, such as the 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG), plays a pathogenic role in the induction of chronic EAE. Cyclization of peptides is of great interest since the limited stability of linear peptides restricts their potential use as therapeutic agents. Herein, we have designed and synthesized a number of linear and cyclic peptides by mutating crucial T cell receptor (TCR) contact residues of the human MOG35-55 epitope. In particular, we have designed and synthesized cyclic altered peptide ligands (APLs) by mutating Arg41 with Ala or Arg41 and Arg46 with Ala. The peptides were synthesized in solid phase on 2-chlorotrityl chloride resin (CLTR-Cl) using the Fmoc/t-Bu methodology. The purity of final products was verified by RP-HPLC and their identification was achieved by ESI-MS. It was found that the substitutions of Arg at positions 41 and 46 with Ala results in peptide analogues that reduce the severity of MOG-induced EAE clinical symptoms in C57BL/6 mice when co-administered with mouse MOG35-55 peptide at the time of immunization.

  1. Milk fat globule-epidermal growth factor-factor VIII-derived peptide MSP68 is a cytoskeletal immunomodulator of neutrophils that inhibits Rac1.

    Science.gov (United States)

    Hendricks, Louie; Aziz, Monowar; Yang, Weng-Lang; Nicastro, Jeffrey; Coppa, Gene F; Symons, Marc; Wang, Ping

    2017-02-01

    Prolonged neutrophil infiltration leads to exaggerated inflammation and tissue damage during sepsis. Neutrophil migration requires rearrangement of their cytoskeleton. Milk fat globule-epidermal growth factor-factor VIII-derived short peptide 68 (MSP68) has recently been shown to be beneficial in sepsis-induced tissue injury and mortality. We hypothesize that MSP68 inhibits neutrophil migration by modulating small GTPase Rac1-dependent cytoskeletal rearrangements. Bone marrow-derived neutrophils (BMDNs) or whole lung digest isolated neutrophils were isolated from 8 to 10 wk old C57BL/6 mice by Percoll density gradient centrifugation. The purity of BMDN was verified by flow cytometry with CD11b/Gr-1 staining. Neutrophils were stimulated with N-formylmethionine-leucine-phenylalanine (f-MLP) (10 nM) in the presence or absence of MSP68 at 10 nM or cecal ligation and puncture (CLP) was used to induce sepsis, and MSP68 was administered at 1 mg/kg intravenously. Cytoskeletal organization was assessed by phalloidin staining, followed by analysis using fluorescence microscopy. Activity of the Rac1 GTPase in f-MLP or CLP-activated BMDN in the presence or absence of MSP68 was assessed by GTPase enzyme-linked immunosorbent assay. Mitogen-activated protein (MAP) kinase activity was determined by western blot densitometry. BMDN treatment with f-MLP increased cytoskeletal remodeling as revealed by the localization of filamentous actin to the periphery of the neutrophil. By contrast, cells pretreated with MSP68 had considerably reduced filamentous actin polymerization. Cytoskeletal spreading is associated with the activation of the small GTPase Rac1. We found BMDN-treated with f-MLP or that were exposed to sepsis by CLP had increased Rac1 signaling, whereas the cells pretreated with MSP68 had significantly reduced Rac1 activation (P Rac1-MAP kinase-mediated neutrophil motility. Thus, MSP68 is a novel therapeutic candidate for regulating inflammation and tissue damage caused

  2. Enteral peptide formulas inhibit radiation induced enteritis and apoptosis in intestinal epithelial cells and suppress the expression and function of Alzheimer's and cell division control gene products

    International Nuclear Information System (INIS)

    Cope, F.O.; Issinger, O.G.; McArdle, A.H.; Shapiro, J.; Tomei, L.D.

    1991-01-01

    Studies have shown that patients receiving enteral peptide formulas prior to irradiation have a significantly reduced incidence of enteritis and express a profound increase in intestinal cellularity. Two conceptual approaches were taken to describe this response. First was the evaluation in changes in programmed intestinal cell death and secondly the evaluation of a gene product controlling cell division cycling. This study provided a relationship between the ratio of cell death to cell formulations. The results indicate that in the canine and murine models, irradiation induces expression of the Alzheimer's gene in intestinal crypt cells, while the incidence of apoptosis in apical cells is significantly increased. The use of peptide enteral formulations suppresses the expression of the Alzheimer's gene in crypt cells, while apoptosis is eliminated in the apical cells of the intestine. Concomitantly, enteral peptide formulations suppress the function of the CK-II gene product in the basal and baso-lateral cells of the intestine. These data indicate that although the mitotic index is significantly reduced in enterocytes, this phenomenon alone is not sufficient to account for the peptide-induced radio-resistance of the intestine. The data also indicate a significant reduction of normal apoptosis in the upper lateral and apical cells of the intestinal villi. Thus, the ratio of cell death to cell replacement is significantly decreased resulting in an increase in villus height and hypertrophy of the apical villus cells. Thus, peptide solutions should be considered as an adjunct treatment both in radio- and chemotherapy

  3. Peptide deformylase as an antibacterial drug target: assays for detection of its inhibition in Escherichia coli cell homogenates and intact cells.

    Science.gov (United States)

    Apfel, C M; Evers, S; Hubschwerlen, C; Pirson, W; Page, M G; Keck, W

    2001-04-01

    An assay was developed to determine the activity of peptide deformylase (PDF) inhibitors under conditions as close as possible to the physiological situation. The assay principle is the detection of N-terminal [35S]methionine labeling of a protein that contains no internal methionine. If PDF is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the N-terminal methionine label. In the presence of a PDF inhibitor, the deformylation is blocked so that the N-formylated peptide is not processed and the label is detected. Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation system as well as in intact bacterial cells.

  4. Peptide aldehyde inhibitors of bacterial peptide deformylases.

    Science.gov (United States)

    Durand, D J; Gordon Green, B; O'Connell, J F; Grant, S K

    1999-07-15

    Bacterial peptide deformylases (PDF, EC 3.5.1.27) are metalloenzymes that cleave the N-formyl groups from N-blocked methionine polypeptides. Peptide aldehydes containing a methional or norleucinal inhibited recombinant peptide deformylase from gram-negative Escherichia coli and gram-positive Bacillus subtilis. The most potent inhibitor was calpeptin, N-CBZ-Leu-norleucinal, which was a competitive inhibitor of the zinc-containing metalloenzymes, E. coli and B. subtilis PDF with Ki values of 26.0 and 55.6 microM, respectively. Cobalt-substituted E. coli and B. subtilis deformylases were also inhibited by these aldehydes with Ki values for calpeptin of 9.5 and 12.4 microM, respectively. Distinct spectral changes were observed upon binding of calpeptin to the Co(II)-deformylases, consistent with the noncovalent binding of the inhibitor rather than the formation of a covalent complex. In contrast, the chelator 1,10-phenanthroline caused the time-dependent inhibition of B. subtilis Co(II)-PDF activity with the loss of the active site metal. The fact that calpeptin was nearly equipotent against deformylases from both gram-negative and gram-positive bacterial sources lends further support to the idea that a single deformylase inhibitor might have broad-spectrum antibacterial activity. Copyright 1999 Academic Press.

  5. Peptide dendrimers

    Czech Academy of Sciences Publication Activity Database

    Niederhafner, Petr; Šebestík, Jaroslav; Ježek, Jan

    2005-01-01

    Roč. 11, - (2005), 757-788 ISSN 1075-2617 R&D Projects: GA ČR(CZ) GA203/03/1362 Institutional research plan: CEZ:AV0Z40550506 Keywords : multiple antigen peptides * peptide dendrimers * synthetic vaccine * multipleantigenic peptides Subject RIV: CC - Organic Chemistry Impact factor: 1.803, year: 2005

  6. cGMP inhibition of type 3 phosphodiesterase is the major mechanism by which C-type natriuretic peptide activates CFTR in the shark rectal gland

    NARCIS (Netherlands)

    H.R. de Jonge (Hugo); B.C. Tilly (Bernard); B.M. Hogema (Boris); D.J. Pfau (Daniel); C.A. Kelley (Catherine); M.H. Kelley (Megan); A.M. Melita (August); M.T. Morris (Montana); M.S. Viola (Maria); J.N. Forrest Jr. (John)

    2014-01-01

    textabstractThe in vitro perfused rectal gland of the dogfish shark (Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride

  7. Saccharomyces cerevisiae CCMI 885 secretes peptides that inhibit the growth of some non-Saccharomyces wine-related strains

    DEFF Research Database (Denmark)

    Albergaria, Helena; Francisco, Diana; Gori, Klaus

    2010-01-01

    of mixed supernatants towards H. guilliermondii was inactivated by protease treatments, thus revealing the proteinaceous nature of the toxic compounds. Analysis of the protein pattern of mixed supernatants on Tricine SDS-PAGE showed that this S. cerevisiae strain secretes peptides (

  8. GLP-1 (glucagon-like peptide 1) and truncated GLP-1, fragments of human proglucagon, inhibit gastric acid secretion in humans

    DEFF Research Database (Denmark)

    Schjoldager, B T; Mortensen, P E; Christiansen, J

    1989-01-01

    Glucagon-like peptide 1 amide (GLP-1 amide), a predicted product of the glucagon gene (proglucagon 72-107-amide), and truncated GLP-1 (proglucagon 78-107-amide), recently isolated from porcine small intestine, were infused in doses of 100 and 400 ng/kg/hr and 12.5 and 50 ng/kg/hr, respectively...

  9. Rosuvastatin ameliorates cognitive impairment in rats fed with high-salt and cholesterol diet via inhibiting acetylcholinesterase activity and amyloid beta peptide aggregation.

    Science.gov (United States)

    Husain, I; Akhtar, M; Abdin, M Zainul; Islamuddin, M; Shaharyar, M; Najmi, A K

    2018-04-01

    Amyloid beta (Aβ) peptide aggregation and cholinergic neurodegeneration are involved in the development of cognitive impairment. Therefore, in this article, we examined rosuvastatin (RSV), an oral hypolipidemic drug, to determine its potential as a dual inhibitor of acetylcholinesterase (AChE) and Aβ peptide aggregation for the treatment of cognitive impairment. Molecular docking study was done to examine the affinity of RSV with Aβ 1-42 and AChE in silico. We also employed neurobehavioral activity tests, biochemical estimation, and histopathology to study the anti-Aβ 1-42 aggregation capability of RSV in vivo. Molecular docking study provided evidence that RSV has the best binding conformer at its receptor site or active site of an enzyme. The cognitive impairment in female Wistar rats was induced by high-salt and cholesterol diet (HSCD) ad libitum for 8 weeks. RSV ameliorated serum cholesterol level, AChE activity, and Aβ 1-42 peptide aggregations in HSCD induced cognitive impairment. In addition, RSV-treated rats showed greater scores in the open field (locomotor activity) test. Moreover, the histopathological studies in the hippocampus and cortex of rat brain also supported that RSV markedly reduced the cognitive impairment and preserved the normal histoarchitectural pattern of the hippocampus and cortex. Taken together, these data indicate that RSV may act as a dual inhibitor of AChE and Aβ 1-42 peptide aggregation, therefore suggesting a therapeutic strategy for cognitive impairment treatment.

  10. Soluble γ-secretase modulators selectively inhibit the production of the 42-amino acid amyloid β peptide variant and augment the production of multiple carboxy-truncated amyloid β species.

    Science.gov (United States)

    Wagner, Steven L; Zhang, Can; Cheng, Soan; Nguyen, Phuong; Zhang, Xulun; Rynearson, Kevin D; Wang, Rong; Li, Yueming; Sisodia, Sangram S; Mobley, William C; Tanzi, Rudolph E

    2014-02-04

    Alzheimer's disease (AD) is characterized pathologically by an abundance of extracellular neuritic plaques composed primarily of the 42-amino acid amyloid β peptide variant (Aβ42). In the majority of familial AD (FAD) cases, e.g., those harboring mutations in presenilin 1 (PS1), there is a relative increase in the levels of Aβ42 compared to the levels of Aβ40. We previously reported the characterization of a series of aminothiazole-bridged aromates termed aryl aminothiazole γ-secretase modulators or AGSMs [Kounnas, M. Z., et al. (2010) Neuron 67, 769-780] and showed their potential for use in the treatment of FAD [Wagner, S. L., et al. (2012) Arch. Neurol. 69, 1255-1258]. Here we describe a series of GSMs with physicochemical properties improved compared to those of AGSMs. Specific heterocycle replacements of the phenyl rings in AGSMs provided potent molecules with improved aqueous solubilities. A number of these soluble γ-secretase modulators (SGSMs) potently lowered Aβ42 levels without inhibiting proteolysis of Notch or causing accumulation of amyloid precursor protein carboxy-terminal fragments, even at concentrations approximately 1000-fold greater than their IC50 values for reducing Aβ42 levels. The effects of one potent SGSM on Aβ peptide production were verified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, showing enhanced production of a number of carboxy-truncated Aβ species. This SGSM also inhibited Aβ42 peptide production in a highly purified reconstituted γ-secretase in vitro assay system and retained the ability to modulate γ-secretase-mediated proteolysis in a stably transfected cell culture model overexpressing a human PS1 mutation validating the potential for use in FAD.

  11. Therapeutic peptides for cancer therapy. Part II - cell cycle inhibitory peptides and apoptosis-inducing peptides.

    Science.gov (United States)

    Raucher, Drazen; Moktan, Shama; Massodi, Iqbal; Bidwell, Gene L

    2009-10-01

    Therapeutic peptides have great potential as anticancer agents owing to their ease of rational design and target specificity. However, their utility in vivo is limited by low stability and poor tumor penetration. The authors review the development of peptide inhibitors with potential for cancer therapy. Peptides that arrest the cell cycle by mimicking CDK inhibitors or induce apoptosis directly are discussed. The authors searched Medline for articles concerning the development of therapeutic peptides and their delivery. Inhibition of cancer cell proliferation directly using peptides that arrest the cell cycle or induce apoptosis is a promising strategy. Peptides can be designed that interact very specifically with cyclins and/or cyclin-dependent kinases and with members of apoptotic cascades. Use of these peptides is not limited by their design, as a rational approach to peptide design is much less challenging than the design of small molecule inhibitors of specific protein-protein interactions. However, the limitations of peptide therapy lie in the poor pharmacokinetic properties of these large, often charged molecules. Therefore, overcoming the drug delivery hurdles could open the door for effective peptide therapy, thus making an entirely new class of molecules useful as anticancer drugs.

  12. A sensitive and selective electrochemical biosensor for the determination of beta-amyloid oligomer by inhibiting the peptide-triggered in situ assembly of silver nanoparticles

    Directory of Open Access Journals (Sweden)

    Xing Y

    2017-04-01

    Full Text Available Yun Xing,1,2 Xiao-Zhen Feng,2 Lipeng Zhang,1 Jiating Hou,2 Guo-Cheng Han,2 Zhencheng Chen2 1Henan Province of Key Laboratory of New Optoelectronic Functional Materials, College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, 2School of Life and Environmental Sciences, Guilin University of Electronic Technology, Guilin, Guangxi, People’s Republic of China Abstract: Soluble beta-amyloid (Aβ oligomer is believed to be the most important toxic species in the brain of Alzheimer’s disease (AD patients. Thus, it is critical to develop a simple method for the selective detection of Aβ oligomer with low cost and high sensitivity. In this paper, we report an electrochemical method for the detection of Aβ oligomer with a peptide as the bioreceptor and silver nanoparticle (AgNP aggregates as the redox reporters. This strategy is based on the conversion of AgNP-based colorimetric assay into electrochemical analysis. Specifically, the peptide immobilized on the electrode surface and presented in solution triggered together the in situ formation of AgNP aggregates, which produced a well-defined electrochemical signal. However, the specific binding of Aβ oligomer to the immobilized peptide prevented the in situ assembly of AgNPs. As a result, a poor electrochemical signal was observed. The detection limit of the method was found to be 6 pM. Furthermore, the amenability of this method for the analysis of Aβ oligomer in serum and artificial cerebrospinal fluid (aCSF samples was demonstrated. Keywords: electrochemical biosensors, Alzheimer’s disease, beta-amyloid oligomer, peptide, silver nanoparticles

  13. Therapeutic peptides for cancer therapy. Part I - peptide inhibitors of signal transduction cascades.

    Science.gov (United States)

    Bidwell, Gene L; Raucher, Drazen

    2009-10-01

    Therapeutic peptides have great potential as anticancer agents owing to their ease of rational design and target specificity. However, their utility in vivo is limited by low stability and poor tumor penetration. The authors review the development of peptide inhibitors with potential for cancer therapy. Peptides that inhibit signal transduction cascades are discussed. The authors searched Medline for articles concerning the development of therapeutic peptides and their delivery. Given our current knowledge of protein sequences, structures and interaction interfaces, therapeutic peptides that inhibit interactions of interest are easily designed. These peptides are advantageous because they are highly specific for the interaction of interest, and they are much more easily developed than small molecule inhibitors of the same interactions. The main hurdle to application of peptides for cancer therapy is their poor pharmacokinetic and biodistribution parameters. Therefore, successful development of peptide delivery vectors could potentially make possible the use of this new and very promising class of anticancer agents.

  14. Nuclear protein IκB-ζ inhibits the activity of STAT3

    International Nuclear Information System (INIS)

    Wu, Zhihao; Zhang, Xiaoai; Yang, Juntao; Wu, Guangzhou; Zhang, Ying; Yuan, Yanzhi; Jin, Chaozhi; Chang, Zhijie; Wang, Jian; Yang, Xiaoming; He, Fuchu

    2009-01-01

    STAT3 (Signal transducer and activator of transcription 3) is a key transcription factor of the JAK-STAT (Janus kinase/signal transducer and activator of transcription) pathway that regulates cell proliferation and apoptosis. Activation of STAT3 is under tight regulation, and yet the different signaling pathways and the mechanisms that regulate its activity remain to be elucidated. Using a yeast two-hybrid screening, we have identified a nuclear protein IκB-ζ that interacts in a novel way with STAT3. This physical interaction was further confirmed by co-immunoprecipitation assays. The interaction regions were mapped to the coiled-coil domain of STAT3 and the C-terminal of IκB-ζ. Overexpression of IκB-ζ inhibited the transcriptional activity of STAT3. It also suppressed cell growth and induced cell apoptosis in SRC-simulated cells, which is partially mediated by down-regulation of expression of a known STAT3 target gene, MCL1. Our results suggest that IκB-ζ is a negative regulator of STAT3, and demonstrate a novel mechanism in which a component of the NF-κB signaling pathway inhibits the activation of STAT3.

  15. Reconstitution of a physical complex between the N-formyl chemotactic peptide receptor and G protein. Inhibition by pertussis toxin-catalyzed ADP ribosylation.

    Science.gov (United States)

    Bommakanti, R K; Bokoch, G M; Tolley, J O; Schreiber, R E; Siemsen, D W; Klotz, K N; Jesaitis, A J

    1992-04-15

    Photoaffinity-labeled N-formyl chemotactic peptide receptors from human neutrophils solubilized in octyl glucoside exhibit two forms upon sucrose density gradient sedimentation, with apparent sedimentation coefficients of approximately 4 and 7 S. The 7 S form can be converted to the 4 S form by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with an EC50 of approximately 20 nM, suggesting that the 7 S form may represent a physical complex of the receptor with endogenous G protein (Jesaitis, A. J., Tolley, J. O., Bokoch, G. M., and Allen, R. A. (1989) J. Cell Biol. 109, 2783-2790). To probe the nature of the 7 S form, we reconstituted the 7 S form from the 4 S form by adding purified G protein. The 4 S form, obtained by solubilizing GTP gamma S-treated neutrophil plasma membranes, was incubated with purified (greater than 95%) Gi protein from bovine brain (containing both Gi alpha 1 and Gi alpha 2) or with neutrophil G protein (Gn), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC50 of 7 S complex formation induced by the two G proteins was 70 +/- 25 and 170 +/- 40 nM for Gn and Gi, respectively. No complexation was measurable when bovine transducin (Gt) was used up to 30 times the EC50 for Gn. The EC50 for Gi was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 microM GTP gamma S to the reconstituted 7 S complex caused a complete revision of the receptor to the 4 S form, and anti-Gi peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gi prevented formation of the 7 S form even at 20 times the concentration of unribosylated Gi normally used to attain 50% conversion to the 7 S form. These observations suggest that the 7 S species is a physical complex containing N-formyl chemotactic peptide receptor and G protein.

  16. Vascular targeting with peptide libraries

    Energy Technology Data Exchange (ETDEWEB)

    Pasqualini, R. [La Jolla Cancer Research Center The Burnham Inst., La Jolla CA (United States)

    1999-06-01

    The authors have developed an 'in vivo' selection system in which phage capable of selective homing to different tissues are recovered from a phage display peptide library following intravenous administration. Using this strategy, they have isolate several organ and tumor-homing peptides. They have shown that each of those peptides binds of different receptors that are selectively expressed on the vasculature of the target tissue. The tumor-homing peptides bind to receptors that are up regulated in tumor angiogenic vasculature. Targeted delivery of doxorubicin to angiogenic vasculature using these peptides in animals models decrease toxicity and increased the therapeutic efficacy of the drug. Vascular targeting may facilitate the development of other treatment strategies that rely on inhibition of angio genesis and lead to advances to extend the potential for targeting of drugs, genes and radionuclides in the context of many diseases.

  17. A Tat-conjugated Peptide Nucleic Acid Tat-PNA-DR Inhibits Hepatitis B Virus Replication In Vitro and In Vivo by Targeting LTR Direct Repeats of HBV RNA

    Science.gov (United States)

    Zeng, Zhengyang; Han, Shisong; Hong, Wei; Lang, Yange; Li, Fangfang; Liu, Yongxiang; Li, Zeyong; Wu, Yingliang; Li, Wenxin; Zhang, Xianzheng; Cao, Zhijian

    2016-01-01

    Hepatitis B virus (HBV) infection is a major cause of chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma, all of which are severe threats to human health. However, current clinical therapies for HBV are limited by potential side effects, toxicity, and drug-resistance. In this study, a cell-penetrating peptide-conjugated peptide nucleic acid (PNA), Tat-PNA-DR, was designed to target the direct repeat (DR) sequences of HBV. Tat-PNA-DR effectively inhibited HBV replication in HepG2.2.15 cells. Its anti-HBV effect relied on the binding of Tat-PNA-DR to the DR, whereby it suppressed the translation of hepatitis B e antigen (HBeAg), HBsAg, HBV core, hepatitis B virus x protein, and HBV reverse transcriptase (RT) and the reverse transcription of the HBV genome. Furthermore, Tat-PNA-DR administered by intravenous injection efficiently cleared HBeAg and HBsAg in an acute hepatitis B mouse model. Importantly, it induced an 80% decline in HBV DNA in mouse serum, which was similar to the effect of the widely used clinical drug Lamivudine (3TC). Additionally, a long-term hydrodynamics HBV mouse model also demonstrated Tat-PNA-DR's antiviral effect. Interestingly, Tat-PNA-DR displayed low cytotoxicity, low mouse acute toxicity, low immunogenicity, and high serum stability. These data indicate that Tat-PNA-DR is a unique PNA and a promising drug candidate against HBV. PMID:26978579

  18. A Tat-conjugated Peptide Nucleic Acid Tat-PNA-DR Inhibits Hepatitis B Virus Replication In Vitro and In Vivo by Targeting LTR Direct Repeats of HBV RNA

    Directory of Open Access Journals (Sweden)

    Zhengyang Zeng

    2016-01-01

    Full Text Available Hepatitis B virus (HBV infection is a major cause of chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma, all of which are severe threats to human health. However, current clinical therapies for HBV are limited by potential side effects, toxicity, and drug-resistance. In this study, a cell-penetrating peptide-conjugated peptide nucleic acid (PNA, Tat-PNA-DR, was designed to target the direct repeat (DR sequences of HBV. Tat-PNA-DR effectively inhibited HBV replication in HepG2.2.15 cells. Its anti-HBV effect relied on the binding of Tat-PNA-DR to the DR, whereby it suppressed the translation of hepatitis B e antigen (HBeAg, HBsAg, HBV core, hepatitis B virus x protein, and HBV reverse transcriptase (RT and the reverse transcription of the HBV genome. Furthermore, Tat-PNA-DR administered by intravenous injection efficiently cleared HBeAg and HBsAg in an acute hepatitis B mouse model. Importantly, it induced an 80% decline in HBV DNA in mouse serum, which was similar to the effect of the widely used clinical drug Lamivudine (3TC. Additionally, a long-term hydrodynamics HBV mouse model also demonstrated Tat-PNA-DR's antiviral effect. Interestingly, Tat-PNA-DR displayed low cytotoxicity, low mouse acute toxicity, low immunogenicity, and high serum stability. These data indicate that Tat-PNA-DR is a unique PNA and a promising drug candidate against HBV.

  19. In vitro angiotensin I converting enzyme inhibition by a peptide isolated from Chiropsalmus quadrigatus Haeckel (box jellyfish) venom hydrolysate.

    Science.gov (United States)

    So, Pamela Berilyn T; Rubio, Peter; Lirio, Stephen; Macabeo, Allan Patrick; Huang, Hsi-Ya; Corpuz, Mary Jho-Anne T; Villaflores, Oliver B

    2016-09-01

    The anti-angiotensin I converting enzyme activity of box jellyfish, Chiropsalmus quadrigatus Haeckel venom hydrolysate was studied. The venom extract was obtained by centrifugation and ultrasonication. Protein concentration of 12.99 μg/mL was determined using Bradford assay. The pepsin and papain hydrolysate was tested for its toxicity by Limit test following the OECD Guideline 425 using 5 female Sprague-Dawley rats. Results showed that the hydrolysate is nontoxic with an LD50 above 2000 mg/kg. In vitro angiotensin I converting enzyme (ACE) inhibitory activity was determined using ACE kit-WST. Isolation of ACE inhibitory peptides using column chromatography with SP-Sephadex G-25 yielded 8 pooled fractions with fraction 3 (86.5%) exhibiting the highest activity. This was followed by reverse phase - high performance liquid chromatography (RP-HPLC) with an octadecyl silica column (Inertsil ODS-3) using methanol:water 15:85 at a flow rate of 1.0 mL/min. Among the 13 fractions separated with the RP-HPLC, fraction 3.5 exhibited the highest ACE inhibitory activity (84.1%). The peptide sequence ACPGPNPGRP (IC50 2.03 μM) from fraction 3.5 was identified using Matrix-assisted laser desorption/ionization with time-of-flight tandem mass spectroscopy analysis (MALDI-TOF/MS). Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Recombinant human brain natriuretic peptide attenuates trauma-/haemorrhagic shock-induced acute lung injury through inhibiting oxidative stress and the NF-κB-dependent inflammatory/MMP-9 pathway.

    Science.gov (United States)

    Song, Zhi; Zhao, Xiu; Liu, Martin; Jin, Hongxu; Wang, Ling; Hou, Mingxiao; Gao, Yan

    2015-12-01

    Acute lung injury (ALI) is one of the most serious complications in traumatic patients and is an important part of multiple organ dysfunction syndrome (MODS). Recombinant human brain natriuretic peptide (rhBNP) is a peptide with a wide range of biological activity. In this study, we investigated local changes in oxidative stress and the NF-κB-dependent matrix metalloproteinase-9 (MMP-9) pathway in rats with trauma/haemorrhagic shock (TH/S)-induced ALI and evaluated the effects of pretreatment with rhBNP. Forty-eight rats were randomly divided into four groups: sham operation group, model group, low-dosage rhBNP group and high-dosage rhBNP group (n = 12 for each group). Oxidative stress and MPO activity were measured by ELISA kits. MMP-9 activity was detected by zymography analysis. NF-κB activity was determined using Western blot assay. With rhBNP pretreatment, TH/S-induced protein leakage, increased MPO activity, lipid peroxidation and metalloproteinase (MMP)-9 activity were inhibited. Activation of antioxidative enzymes was reversed. The phosphorylation of NF-κB and the degradation of its inhibitor IκB were suppressed. The results suggested that the protection mechanism of rhBNP is possibly mediated through upregulation of anti-oxidative enzymes and inhibition of NF-κB activation. More studies are needed to further evaluate whether rhBNP is a suitable candidate as an effective inhaling drug to reduce the incidence of TH/S-induced ALI. © 2016 The Authors. International Journal of Experimental Pathology © 2016 International Journal of Experimental Pathology.

  1. An antisense peptide nucleic acid against Pseudomonas aeruginosa inhibiting bacterial-induced inflammatory responses in the cystic fibrosis IB3-1 cellular model system

    DEFF Research Database (Denmark)

    Montagner, Giulia; Bezzerri, Valentino; Cabrini, Giulio

    2017-01-01

    of the essential acpP gene of P. aeruginosa, and previously shown to inhibit bacterial growth, concomitantly also strongly inhibits induced up-regulation of the pro-inflammatory markers IL-8, IL-6, G-CSF, IFN-γ, IP-10, MCP-1 and TNF-α in IB3-1 cystic fibrosis cells infected by P. aeruginosa PAO1. Remarkably...... are significant considering the key role of this protein in the cystic fibrosis inflammatory process exacerbated by P. aeruginosa infection....

  2. Improvement of cardiac contractile function by peptide-based inhibition of NF-κB in the utrophin/dystrophin-deficient murine model of muscular dystrophy

    Directory of Open Access Journals (Sweden)

    Guttridge Denis C

    2011-05-01

    Full Text Available Abstract Background Duchenne muscular dystrophy (DMD is an inherited and progressive disease causing striated muscle deterioration. Patients in their twenties generally die from either respiratory or cardiac failure. In order to improve the lifespan and quality of life of DMD patients, it is important to prevent or reverse the progressive loss of contractile function of the heart. Recent studies by our labs have shown that the peptide NBD (Nemo Binding Domain, targeted at blunting Nuclear Factor κB (NF-κB signaling, reduces inflammation, enhances myofiber regeneration, and improves contractile deficits in the diaphragm in dystrophin-deficient mdx mice. Methods To assess whether cardiac function in addition to diaphragm function can be improved, we investigated physiological and histological parameters of cardiac muscle in mice deficient for both dystrophin and its homolog utrophin (double knockout = dko mice treated with NBD peptide. These dko mice show classic pathophysiological hallmarks of heart failure, including myocyte degeneration, an impaired force-frequency response and a severely blunted β-adrenergic response. Cardiac contractile function at baseline and frequencies and pre-loads throughout the in vivo range as well as β-adrenergic reserve was measured in isolated cardiac muscle preparations. In addition, we studied histopathological and inflammatory markers in these mice. Results At baseline conditions, active force development in cardiac muscles from NBD treated dko mice was more than double that of vehicle-treated dko mice. NBD treatment also significantly improved frequency-dependent behavior of the muscles. The increase in force in NBD-treated dko muscles to β-adrenergic stimulation was robustly restored compared to vehicle-treated mice. However, histological features, including collagen content and inflammatory markers were not significantly different between NBD-treated and vehicle-treated dko mice. Conclusions We conclude

  3. Protective Effects of Chlorella-Derived Peptide Against UVC-Induced Cytotoxicity through Inhibition of Caspase-3 Activity and Reduction of the Expression of Phosphorylated FADD and Cleaved PARP-1 in Skin Fibroblasts

    Directory of Open Access Journals (Sweden)

    Jong Yuh Cherng

    2012-08-01

    Full Text Available UVC irradiation induces oxidative stress and leads to cell death through an apoptotic pathway. This apoptosis is caused by activation of caspase-3 and formation of poly(ADP-ribose polymerase-1 (PARP-1. In this study, the underlying mechanisms of Chlorella derived peptide (CDP activity against UVC-induced cytotoxicity were investigated. Human skin fibroblasts were treated with CDP, vitamin C, or vitamin E after UVC irradiation for a total energy of 15 J/cm2. After the UVC exposure, cell proliferation and caspase-3 activity were measured at 12, 24, 48, and 72 h later. Expression of phosphorylated FADD and cleaved PARP-1 were measured 16 h later. DNA damage (expressed as pyrimidine (6-4 pyrimidone photoproducts DNA concentration and fragmentation assay were performed 24 h after the UVC exposure. Results showed that UVC irradiation induced cytotoxicity in all groups except those treated with CDP. The caspase-3 activity in CDP-treated cells was inhibited from 12 h onward. Expression of phosphorylated FADD and cleaved PARP-1 were also reduced in CDP-treated cells. Moreover, UVC-induced DNA damage and fragmentation were also prevented by the CDP treatment. This study shows that treatment of CDP provides protective effects against UVC-induced cytotoxicity through the inhibition of caspase-3 activity and the reduction of phosphorylated FADD and cleaved PARP-1 expression.

  4. A novel chimeric peptide with antimicrobial activity.

    Science.gov (United States)

    Alaybeyoglu, Begum; Akbulut, Berna Sariyar; Ozkirimli, Elif

    2015-04-01

    Beta-lactamase-mediated bacterial drug resistance exacerbates the prognosis of infectious diseases, which are sometimes treated with co-administration of beta-lactam type antibiotics and beta-lactamase inhibitors. Antimicrobial peptides are promising broad-spectrum alternatives to conventional antibiotics in this era of evolving bacterial resistance. Peptides based on the Ala46-Tyr51 beta-hairpin loop of beta-lactamase inhibitory protein (BLIP) have been previously shown to inhibit beta-lactamase. Here, our goal was to modify this peptide for improved beta-lactamase inhibition and cellular uptake. Motivated by the cell-penetrating pVEC sequence, which includes a hydrophobic stretch at its N-terminus, our approach involved the addition of LLIIL residues to the inhibitory peptide N-terminus to facilitate uptake. Activity measurements of the peptide based on the 45-53 loop of BLIP for enhanced inhibition verified that the peptide was a competitive beta-lactamase inhibitor with a K(i) value of 58 μM. Incubation of beta-lactam-resistant cells with peptide decreased the number of viable cells, while it had no effect on beta-lactamase-free cells, indicating that this peptide had antimicrobial activity via beta-lactamase inhibition. To elucidate the molecular mechanism by which this peptide moves across the membrane, steered molecular dynamics simulations were carried out. We propose that addition of hydrophobic residues to the N-terminus of the peptide affords a promising strategy in the design of novel antimicrobial peptides not only against beta-lactamase but also for other intracellular targets. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

  5. Lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFα) blunt the response of Neuropeptide Y/Agouti-related peptide (NPY/AgRP) glucose inhibited (GI) neurons to decreased glucose.

    Science.gov (United States)

    Hao, Lihong; Sheng, Zhenyu; Potian, Joseph; Deak, Adam; Rohowsky-Kochan, Christine; Routh, Vanessa H

    2016-10-01

    A population of Neuropeptide Y (NPY) neurons which co-express Agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus (ARC) are inhibited at physiological levels of brain glucose and activated when glucose levels decline (e.g. glucose-inhibited or GI neurons). Fasting enhances the activation of NPY/AgRP-GI neurons by low glucose. In the present study we tested the hypothesis that lipopolysaccharide (LPS) inhibits the enhanced activation of NPY/AgRP-GI neurons by low glucose following a fast. Mice which express green fluorescent protein (GFP) on their NPY promoter were used to identify NPY/AgRP neurons. Fasting for 24h and LPS injection decreased blood glucose levels. As we have found previously, fasting increased c-fos expression in NPY/AgRP neurons and increased the activation of NPY/AgRP-GI neurons by decreased glucose. As we predicted, LPS blunted these effects of fasting at the 24h time point. Moreover, the inflammatory cytokine tumor necrosis factor alpha (TNFα) blocked the activation of NPY/AgRP-GI neurons by decreased glucose. These data suggest that LPS and TNFα may alter glucose and energy homeostasis, in part, due to changes in the glucose sensitivity of NPY/AgRP neurons. Interestingly, our findings also suggest that NPY/AgRP-GI neurons use a distinct mechanism to sense changes in extracellular glucose as compared to our previous studies of GI neurons in the adjacent ventromedial hypothalamic nucleus. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. A synthetic peptide derived from alpha-fetoprotein inhibits the estradiol-induced proliferation of mammary tumor cells in culture through the modulation of p21.

    Science.gov (United States)

    Sierralta, Walter D; Epuñan, María J; Reyes, José M; Valladares, Luis E; Pino, Ana M

    2008-01-01

    A stable cyclized 9-mer peptide (cP) containing the active site of alpha-alpha fetoprotein (alphaFP) has been shown to be effective for prevention of estrogen-stimulated tumor cell proliferation in culture or of xenographt growth in immunodeficient mice. cP does not block 17beta-estradiol (E2) binding to its receptors, but rather appears to interfere with intracellular processing of the signal that supports growth. To obtain insight on that mechanism we studied the effect of cP on the proliferation of MCF-7 cells in culture. Proliferation in the presence of 2 microM E2 is decreased up to 40% upon addition of 2 microg ml(-1) cP to the medium; the presence of cP did not increase cell death, cP reduced also the proliferation of estrogen-dependent ZR75-1 cells but had no effect on autonomous MDA-MB-231 cells, cP did not modify the number of binding sites for labeled E2 or affected cell death. We detected increased nuclear p21Cip1 immunoreactivity after cP treatment. Our results suggest that cP acts via p21Cip1 to slow the process of MCF-7 cells through the cycle.

  7. p53 inhibits autophagy by interacting with the human ortholog of yeast Atg17, RB1CC1/FIP200.

    Science.gov (United States)

    Morselli, Eugenia; Shen, Shensi; Ruckenstuhl, Christoph; Bauer, Maria Anna; Mariño, Guillermo; Galluzzi, Lorenzo; Criollo, Alfredo; Michaud, Mickael; Maiuri, Maria Chiara; Chano, Tokuhiro; Madeo, Frank; Kroemer, Guido

    2011-08-15

    The tumor suppressor protein p53 tonically suppresses autophagy when it is present in the cytoplasm. This effect is phylogenetically conserved from mammals to nematodes, and human p53 can inhibit autophagy in yeast, as we show here. Bioinformatic investigations of the p53 interactome in relationship to the autophagy-relevant protein network underscored the possible relevance of a direct molecular interaction between p53 and the mammalian ortholog of the essential yeast autophagy protein Atg17, namely RB1-inducible coiled-coil protein 1 (RB1CC1), also called FAK family kinase-interacting protein of 200 KDa (FIP200). Mutational analyses revealed that a single point mutation in p53 (K382R) abolished its capacity to inhibit autophagy upon transfection into p53-deficient human colon cancer or yeast cells. In conditions in which wild-type p53 co-immunoprecipitated with RB1CC1/FIP200, p53 (K382R) failed to do so, underscoring the importance of the physical interaction between these proteins for the control of autophagy. In conclusion, p53 regulates autophagy through a direct molecular interaction with RB1CC1/FIP200, a protein that is essential for the very apical step of autophagy initiation.

  8. Atrial natriuretic peptide down-regulates LPS/ATP-mediated IL-1β release by inhibiting NF-kB, NLRP3 inflammasome and caspase-1 activation in THP-1 cells.

    Science.gov (United States)

    Mezzasoma, Letizia; Antognelli, Cinzia; Talesa, Vincenzo Nicola

    2016-02-01

    Atrial natriuretic peptide (ANP) is an hormone/paracrine/autocrine factor regulating cardiovascular homeostasis by guanylyl cyclase natriuretic peptide receptor (NPR-1). ANP plays an important role also in regulating inflammatory and immune systems by altering macrophages functions and cytokines secretion. Interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine involved in a wide range of biological responses, including the immunological one. Unlike other cytokines, IL-1β production is rigorously controlled. Primarily, NF-kB activation is required to produce pro-IL-1β; subsequently, NALP3 inflammasome/caspase-1 activation is required to cleave pro-IL-1β into the active secreted protein. NALP3 is a molecular platform capable of sensing a large variety of signals and a major player in innate immune defense. Due to their pleiotropism, IL-1β and NALP3 dysregulation is a common feature of a wide range of diseases. Therefore, identifying molecules regulating IL-1β/NALP3/caspase-1 expression is an important step in the development of new potential therapeutic agents. The aim of our study was to evaluate the effect of ANP on IL-1β/NALP3/caspase-1 expression in LPS/ATP-stimulated human THP1 monocytes. We provided new evidence of the direct involvement of ANP/NPR-1/cGMP axis on NF-kB/NALP3/caspase-1-mediated IL-1β release and NF-kB-mediated pro-IL-1β production. In particular, ANP inhibited both NF-kB and NALP3/caspase-1 activation leading to pro- and mature IL-1β down-regulation. Our data, pointing out a modulatory role of this endogenous peptide on IL-1β release and on NF-kB/NALP3/caspase-1 activation, indicate an important anti-inflammatory and immunomodulatory effect of ANP via these mechanisms. We suggest a possible employment of ANP for the treatment of inflammatory/immune-related diseases and IL-1β/NALP3-associated disorders, affecting millions of people worldwide.

  9. A potent and selective calcitonin gene-related peptide (CGRP) receptor antagonist, MK-8825, inhibits responses to nociceptive trigeminal activation: Role of CGRP in orofacial pain.

    Science.gov (United States)

    Romero-Reyes, Marcela; Pardi, Vanessa; Akerman, Simon

    2015-09-01

    Temporomandibular disorders (TMDs) are orofacial pains within the trigeminal distribution, which involve the masticatory musculature, the temporomandibular joint or both. Their pathophysiology remains unclear, as inflammatory mediators are thought to be involved, and clinically TMD presents pain and sometimes limitation of function, but often appears without gross indications of local inflammation, such as visible edema, redness and increase in temperature. Calcitonin gene-related peptide (CGRP) has been implicated in other pain disorders with trigeminal distribution, such as migraine, of which TMD shares a significant co-morbidity. CGRP causes activation and sensitization of trigeminal primary afferent neurons, independent of any inflammatory mechanisms, and thus may also be involved in TMD. Here we used a small molecule, selective CGRP receptor antagonist, MK-8825, to dissect the role of CGRP in inducing spontaneous nociceptive facial grooming behaviors, neuronal activation in the trigeminal nucleus, and systemic release of pro-inflammatory cytokines, in a mouse model of acute orofacial masseteric muscle pain that we have developed, as a surrogate of acute TMD. We show that CFA masseteric injection causes significant spontaneous orofacial pain behaviors, neuronal activation in the trigeminal nucleus, and release of interleukin-6 (IL-6). In mice pre-treated with MK-8825 there is a significant reduction in these spontaneous orofacial pain behaviors. Also, at 2 and 24h after CFA injection the level of Fos immunoreactivity in the trigeminal nucleus, used as a marker of neuronal activation, was much lower on both ipsilateral and contralateral sides after pre-treatment with MK-8825. There was no effect of MK-8825 on the release of IL-6. These data suggest that CGRP may be involved in TMD pathophysiology, but not via inflammatory mechanisms, at least in the acute stage. Furthermore, CGRP receptor antagonists may have therapeutic efficacy in the treatment of TMD, as they

  10. Identification of binding peptides of the ADAM15 disintegrin domain ...

    Indian Academy of Sciences (India)

    Madhsudhan

    ADAM15 disintegrin domain (RADD) that could inhibit melanoma cell adhesion by using Escherichia coli. Second, four specific binding peptides (peptides A, B, C, and D) were selected using a phage display 12-mer peptide library. The screening protocol involved 4 rounds of positive panning on RADD and 2 rounds of ...

  11. Peptide-membrane interactions of arginine-tryptophan peptides probed using quartz crystal microbalance with dissipation monitoring.

    KAUST Repository

    Rydberg, Hanna A

    2014-04-18

    Membrane-active peptides include peptides that can cross cellular membranes and deliver macromolecular cargo as well as peptides that inhibit bacterial growth. Some of these peptides can act as both transporters and antibacterial agents. It is desirable to combine the knowledge from these two different fields of membrane-active peptides into design of new peptides with tailored actions, as transporters of cargo or as antibacterial substances, targeting specific membranes. We have previously shown that the position of the amino acid tryptophan in the peptide sequence of three arginine-tryptophan peptides affects their uptake and intracellular localization in live mammalian cells, as well as their ability to inhibit bacterial growth. Here, we use quartz crystal microbalance with dissipation monitoring to assess the induced changes caused by binding of the three peptides to supported model membranes composed of POPC, POPC/POPG, POPC/POPG/cholesterol or POPC/lactosyl PE. Our results indicate that the tryptophan position in the peptide sequence affects the way these peptides interact with the different model membranes and that the presence of cholesterol in particular seems to affect the membrane interaction of the peptide with an even distribution of tryptophans in the peptide sequence. These results give mechanistic insight into the function of these peptides and may aid in the design of membrane-active peptides with specified cellular targets and actions.

  12. Peptide-membrane interactions of arginine-tryptophan peptides probed using quartz crystal microbalance with dissipation monitoring.

    KAUST Repository

    Rydberg, Hanna A; Kunze, Angelika; Carlsson, Nils; Altgä rde, Noomi; Svedhem, Sofia; Nordé n, Bengt

    2014-01-01

    Membrane-active peptides include peptides that can cross cellular membranes and deliver macromolecular cargo as well as peptides that inhibit bacterial growth. Some of these peptides can act as both transporters and antibacterial agents. It is desirable to combine the knowledge from these two different fields of membrane-active peptides into design of new peptides with tailored actions, as transporters of cargo or as antibacterial substances, targeting specific membranes. We have previously shown that the position of the amino acid tryptophan in the peptide sequence of three arginine-tryptophan peptides affects their uptake and intracellular localization in live mammalian cells, as well as their ability to inhibit bacterial growth. Here, we use quartz crystal microbalance with dissipation monitoring to assess the induced changes caused by binding of the three peptides to supported model membranes composed of POPC, POPC/POPG, POPC/POPG/cholesterol or POPC/lactosyl PE. Our results indicate that the tryptophan position in the peptide sequence affects the way these peptides interact with the different model membranes and that the presence of cholesterol in particular seems to affect the membrane interaction of the peptide with an even distribution of tryptophans in the peptide sequence. These results give mechanistic insight into the function of these peptides and may aid in the design of membrane-active peptides with specified cellular targets and actions.

  13. Oligomeric coiled-coil adhesin YadA is a double-edged sword.

    Directory of Open Access Journals (Sweden)

    Salome Casutt-Meyer

    Full Text Available Yersinia adhesin A (YadA is an essential virulence factor for the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis. Surprisingly, it is a pseudogene in Yersinia pestis. Even more intriguing, the introduction of a functional yadA gene in Y. pestis EV76 was shown to correlate with a decrease in virulence in a mouse model. Here, we report that wild type (wt Y. enterocolitica E40, as well as YadA-deprived E40 induced the synthesis of neutrophil extracellular traps (NETs upon contact with neutrophils, but only YadA-expressing Y. enterocolitica adhered to NETs and were killed. As binding seemed to be a prerequisite for killing, we searched for YadA-binding substrates and detected the presence of collagen within NETs. E40 bacteria expressing V98D,N99A mutant YadA with a severely reduced ability to bind collagen were found to be more resistant to killing, suggesting that collagen binding contributes significantly to sensitivity to NETs. Wt Y. pestis EV76 were resistant to killing by NETs, while recombinant EV76 expressing YadA from either Y. pseudotuberculosis or Y. enterocolitica were sensitive to killing by NETs, outlining the importance of YadA for susceptibility to NET-dependent killing. Recombinant EV76 endowed with YadA from Y. enterocolitica were also less virulent for the mouse than wt EV76, as shown before. In addition, EV76 carrying wt YadA were less virulent for the mouse than EV76 expressing YadA(V₉₈D,N₉₉A. The observation that YadA makes Yersinia sensitive to NETs provides an explanation as for why evolution selected for the inactivation of yadA in the flea-borne Y. pestis and clarifies an old enigma. Since YadA imposes the same cost to the food-borne Yersinia but was nevertheless conserved by evolution, this observation also illustrates the duality of some virulence functions.

  14. Synthetic peptide inhibitors of DNA replication in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Løbner-Olesen, Anders; Kjelstrup, Susanne

    F counterselection was developed to directly select for compounds able to disrupt selected interactions. We have subsequently constructed a cyclic peptide library for intracellular synthesis of cyclic peptides using known technology. Several cyclic peptides were able to interfere with oligomerization of Dna......N (), DnaB and DnaX (). Three peptides identified as inhibitors of DnaN have been purified. Two of these peptides inhibited growth as well as DNA replication in S. aureus. The minimal inhibitory concentration (MIC) of the peptides was approximately 50 g/ml. Overexpression of DnaN reduced the inhibitory...

  15. Albumin-derived peptides efficiently reduce renal uptake of radiolabelled peptides

    International Nuclear Information System (INIS)

    Vegt, Erik; Eek, Annemarie; Oyen, Wim J.G.; Gotthardt, Martin; Boerman, Otto C.; Jong, Marion de

    2010-01-01

    In peptide-receptor radionuclide therapy (PRRT), the maximum activity dose that can safely be administered is limited by high renal uptake and retention of radiolabelled peptides. The kidney radiation dose can be reduced by coinfusion of agents that competitively inhibit the reabsorption of radiolabelled peptides, such as positively charged amino acids, Gelofusine, or trypsinised albumin. The aim of this study was to identify more specific and potent inhibitors of the kidney reabsorption of radiolabelled peptides, based on albumin. Albumin was fragmented using cyanogen bromide and six albumin-derived peptides with different numbers of electric charges were selected and synthesised. The effect of albumin fragments (FRALB-C) and selected albumin-derived peptides on the internalisation of 111 In-albumin, 111 In-minigastrin, 111 In-exendin and 111 In-octreotide by megalin-expressing cells was assessed. In rats, the effect of Gelofusine and albumin-derived peptides on the renal uptake and biodistribution of 111 In-minigastrin, 111 In-exendin and 111 In-octreotide was determined. FRALB-C significantly reduced the uptake of all radiolabelled peptides in vitro. The albumin-derived peptides showed different potencies in reducing the uptake of 111 In-albumin, 111 In-exendin and 111 In-minigastrin in vitro. The most efficient albumin-derived peptide (peptide 6), was selected for in vivo testing. In rats, 5 mg of peptide 6 very efficiently inhibited the renal uptake of 111 In-minigastrin, by 88%. Uptake of 111 In-exendin and 111 In-octreotide was reduced by 26 and 33%, respectively. The albumin-derived peptide 6 efficiently inhibited the renal reabsorption of 111 In-minigastrin, 111 In-exendin and 111 In-octreotide and is a promising candidate for kidney protection in PRRT. (orig.)

  16. Peptide chemistry toolbox - Transforming natural peptides into peptide therapeutics.

    Science.gov (United States)

    Erak, Miloš; Bellmann-Sickert, Kathrin; Els-Heindl, Sylvia; Beck-Sickinger, Annette G

    2018-06-01

    The development of solid phase peptide synthesis has released tremendous opportunities for using synthetic peptides in medicinal applications. In the last decades, peptide therapeutics became an emerging market in pharmaceutical industry. The need for synthetic strategies in order to improve peptidic properties, such as longer half-life, higher bioavailability, increased potency and efficiency is accordingly rising. In this mini-review, we present a toolbox of modifications in peptide chemistry for overcoming the main drawbacks during the transition from natural peptides to peptide therapeutics. Modifications at the level of the peptide backbone, amino acid side chains and higher orders of structures are described. Furthermore, we are discussing the future of peptide therapeutics development and their impact on the pharmaceutical market. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. TRIM45, a novel human RBCC/TRIM protein, inhibits transcriptional activities of ElK-1 and AP-1

    International Nuclear Information System (INIS)

    Wang Yuequn; Li Yongqing; Qi Xinzhu; Yuan Wuzhou; Ai Jianping; Zhu Chuanbing; Cao Lei; Yang Hong; Liu Fang; Wu Xiushan; Liu Mingyao

    2004-01-01

    The tripartite motif (TRIM) proteins play important roles in a variety of cellular functions including cell proliferation, differentiation, development, oncogenesis, and apoptosis. In this study, we report the identification and characterization of the human tripartite motif-containing protein 45 (TRIM45), a novel member of the TRIM family, from a human embryonic heart cDNA library. TRIM45 has a predicted 580 amino acid open reading frame, encoding a putative 64-kDa protein. The N-terminal region harbors a RING finger, two B-boxes, and a predicted α-helical coiled-coil domain, which together form the RBCC/TRIM motif found in a large family of proteins, whereas the C-terminal region contains a filamin-type immunoglobulin (IG-FLMN) domain. Northern blot analysis indicates that TRIM45 is expressed in a variety of human adult and embryonic tissues. In the cell, TRIM45 protein is expressed both in cytoplasm and in cell nucleus. Overexpression of TRIM45 in COS-7 cells inhibits the transcriptional activities of ElK-1 and AP-1. These results suggest that TRIM45 may act as a new transcriptional repressor in mitogen-activated protein kinase signaling pathway

  18. Double quick, double click reversible peptide "stapling".

    Science.gov (United States)

    Grison, Claire M; Burslem, George M; Miles, Jennifer A; Pilsl, Ludwig K A; Yeo, David J; Imani, Zeynab; Warriner, Stuart L; Webb, Michael E; Wilson, Andrew J

    2017-07-01

    The development of constrained peptides for inhibition of protein-protein interactions is an emerging strategy in chemical biology and drug discovery. This manuscript introduces a versatile, rapid and reversible approach to constrain peptides in a bioactive helical conformation using BID and RNase S peptides as models. Dibromomaleimide is used to constrain BID and RNase S peptide sequence variants bearing cysteine (Cys) or homocysteine ( h Cys) amino acids spaced at i and i + 4 positions by double substitution. The constraint can be readily removed by displacement of the maleimide using excess thiol. This new constraining methodology results in enhanced α-helical conformation (BID and RNase S peptide) as demonstrated by circular dichroism and molecular dynamics simulations, resistance to proteolysis (BID) as demonstrated by trypsin proteolysis experiments and retained or enhanced potency of inhibition for Bcl-2 family protein-protein interactions (BID), or greater capability to restore the hydrolytic activity of the RNAse S protein (RNase S peptide). Finally, use of a dibromomaleimide functionalized with an alkyne permits further divergent functionalization through alkyne-azide cycloaddition chemistry on the constrained peptide with fluorescein, oligoethylene glycol or biotin groups to facilitate biophysical and cellular analyses. Hence this methodology may extend the scope and accessibility of peptide stapling.

  19. Incarvine C suppresses proliferation and vasculogenic mimicry of hepatocellular carcinoma cells via targeting ROCK inhibition

    International Nuclear Information System (INIS)

    Zhang, Ji-Gang; Zhang, Dan-Dan; Wu, Xin; Wang, Yu-Zhu; Gu, Sheng-Ying; Zhu, Guan-Hua; Li, Xiao-Yu; Li, Qin; Liu, Gao-Lin

    2015-01-01

    Studies have described vasculogenic mimicry (VM) as an alternative circulatory system to blood vessels in multiple malignant tumor types, including hepatocellular carcinoma (HCC). In the current study, we aimed to seek novel and more efficient treatment strategies by targeting VM and explore the underlying mechanisms in HCC cells. Cell counting kit-8 (CCK-8) assay and colony survival assay were performed to explore the inhibitory effect of incarvine C (IVC) on human cancer cell proliferation. Flow cytometry was performed to analyze the cell cycle distribution after DNA staining and cell apoptosis by the Annexin V-PE and 7-AAD assay. The effect of IVC on Rho-associated, coiled-coil-containing protein kinase (ROCK) was determined by western blotting and stress fiber formation assay. The inhibitory role of IVC on MHCC97H cell VM formation was determined by formation of tubular network structures on Matrigel in vitro, real time-qPCR, confocal microscopy and western blotting techniques. We explored an anti-metastatic HCC agent, IVC, derived from traditional Chinese medicinal herbs, and found that IVC dose-dependently inhibited the growth of MHCC97H cells. IVC induced MHCC97H cell cycle arrest at G1 transition, which was associated with cyclin-dependent kinase 2 (CDK-2)/cyclin-E1 degradation and p21/p53 up-regulation. In addition, IVC induced apoptotic death of MHCC97H cells. Furthermore, IVC strongly suppressed the phosphorylation of the ROCK substrate myosin phosphatase target subunit-1 (MYPT-1) and ROCK-mediated actin fiber formation. Finally, IVC inhibited cell-dominant tube formation in vitro, which was accompanied with the down-regulation of VM-key factors as detected by real time-qPCR and immunofluorescence. Taken together, the effective inhibitory effect of IVC on MHCC97H cell proliferation and neovascularization was associated with ROCK inhibition, suggesting that IVC may be a new potential drug candidate for the treatment of HCC

  20. ROCK inhibition abolishes the establishment of the aquiferous system in Ephydatia muelleri (Porifera, Demospongiae).

    Science.gov (United States)

    Schenkelaars, Quentin; Quintero, Omar; Hall, Chelsea; Fierro-Constain, Laura; Renard, Emmanuelle; Borchiellini, Carole; Hill, April L

    2016-04-15

    The Rho associated coiled-coil protein kinase (ROCK) plays crucial roles in development across bilaterian animals. The fact that the Rho/Rock pathway is required to initiate epithelial morphogenesis and thus to establish body plans in bilaterians makes this conserved signaling pathway key for studying the molecular mechanisms that may control early development of basally branching metazoans. The purpose of this study was to evaluate whether or not the main components of this signaling pathway exist in sponges, and if present, to investigate the possible role of the regulatory network in an early branching non-bilaterian species by evaluating ROCK function during Ephydatia muelleri development. Molecular phylogenetic analyses and protein domain predictions revealed the existence of Rho/Rock components in all studied poriferan lineages. Binding assays revealed that both Y-27632 and GSK429286A are capable of inhibiting Em-ROCK activity in vitro. Treatment with both drugs leads to impairment of growth and formation of the basal pinacoderm layer in the developing sponge. Furthermore, inhibition of Em-Rock prevents the establishment of a functional aquiferous system, including the absence of an osculum. In contrast, no effect of ROCK inhibition was observed in juvenile sponges that already possess a fully developed and functional aquiferous system. Thus, the Rho/Rock pathway appears to be essential for the proper development of the freshwater sponge, and may play a role in various cell behaviors (e.g. cell proliferation, cell adhesion and cell motility). Taken together, these data are consistent with an ancestral function of Rho/Rock signaling in playing roles in early developmental processes and may provide a new framework to study the interaction between Wnt signaling and the Rho/Rock pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. De-novo design of antimicrobial peptides for plant protection.

    Directory of Open Access Journals (Sweden)

    Benjamin Zeitler

    Full Text Available This work describes the de-novo design of peptides that inhibit a broad range of plant pathogens. Four structurally different groups of peptides were developed that differ in size and position of their charged and hydrophobic clusters and were assayed for their ability to inhibit bacterial growth and fungal spore germination. Several peptides are highly active at concentrations between 0,1 and 1 µg/ml against plant pathogenic bacteria, such as Pseudomonas syringae, Pectobacterium carotovorum, and Xanthomonas vesicatoria. Importantly, no hemolytic activity could be detected for these peptides at concentrations up to 200 µg/ml. Moreover, the peptides are also active after spraying on the plant surface demonstrating a possible way of application. In sum, our designed peptides represent new antimicrobial agents and with the increasing demand for antimicrobial compounds for production of "healthy" food, these peptides might serve as templates for novel antibacterial and antifungal agents.

  2. Peptides Interfering 3A Protein Dimerization Decrease FMDV Multiplication.

    Directory of Open Access Journals (Sweden)

    Mónica González-Magaldi

    Full Text Available Nonstructural protein 3A is involved in relevant functions in foot-and-mouth disease virus (FMDV replication. FMDV 3A can form homodimers and preservation of the two hydrophobic α-helices (α1 and α2 that stabilize the dimer interface is essential for virus replication. In this work, small peptides mimicking residues involved in the dimer interface were used to interfere with dimerization and thus gain insight on its biological function. The dimer interface peptides α1, α2 and that spanning the two hydrophobic α-helices, α12, impaired in vitro dimer formation of a peptide containing the two α-helices, this effect being higher with peptide α12. To assess the effect of dimer inhibition in cultured cells, the interfering peptides were N-terminally fused to a heptaarginine (R7 sequence to favor their intracellular translocation. Thus, when fused to R7, interference peptides (100 μM were able to inhibit dimerization of transiently expressed 3A, the higher inhibitions being found with peptides α1 and α12. The 3A dimerization impairment exerted by the peptides correlated with significant, specific reductions in the viral yield recovered from peptide-treated FMDV infected cells. In this case, α2 was the only peptide producing significant reductions at concentrations lower than 100 μM. Thus, dimer interface peptides constitute a tool to understand the structure-function relationship of this viral protein and point to 3A dimerization as a potential antiviral target.

  3. Prevention of the β-amyloid peptide-induced inflammatory process by inhibition of double-stranded RNA-dependent protein kinase in primary murine mixed co-cultures

    Directory of Open Access Journals (Sweden)

    Terro F

    2011-06-01

    Full Text Available Abstract Background Inflammation may be involved in the pathogenesis of Alzheimer's disease (AD. There has been little success with anti-inflammatory drugs in AD, while the promise of anti-inflammatory treatment is more evident in experimental models. A new anti-inflammatory strategy requires a better understanding of molecular mechanisms. Among the plethora of signaling pathways activated by β-amyloid (Aβ peptides, the nuclear factor-kappa B (NF-κB pathway could be an interesting target. In virus-infected cells, double-stranded RNA-dependent protein kinase (PKR controls the NF-κB signaling pathway. It is well-known that PKR is activated in AD. This led us to study the effect of a specific inhibitor of PKR on the Aβ42-induced inflammatory response in primary mixed murine co-cultures, allowing interactions between neurons, astrocytes and microglia. Methods Primary mixed murine co-cultures were prepared in three steps: a primary culture of astrocytes and microglia for 14 days, then a primary culture of neurons and astrocytes which were cultured with microglia purified from the first culture. Before exposure to Aβ neurotoxicity (72 h, co-cultures were treated with compound C16, a specific inhibitor of PKR. Levels of tumor necrosis factor-α (TNFα, interleukin (IL-1β, and IL-6 were assessed by ELISA. Levels of PT451-PKR and activation of IκB, NF-κB and caspase-3 were assessed by western blotting. Apoptosis was also followed using annexin V-FITC immunostaining kit. Subcellular distribution of PT451-PKR was assessed by confocal immunofluorescence and morphological structure of cells by scanning electron microscopy. Data were analysed using one-way ANOVA followed by a Newman-Keuls' post hoc test Results In these co-cultures, PKR inhibition prevented Aβ42-induced activation of IκB and NF-κB, strongly decreased production and release of tumor necrosis factor (TNFα and interleukin (IL-1β, and limited apoptosis. Conclusion In spite of the

  4. Cysteine-containing peptides having antioxidant properties

    Science.gov (United States)

    Bielicki, John K [Castro Valley, CA

    2008-10-21

    Cysteine containing amphipathic alpha helices of the exchangeable apolipoproteins, as exemplified by apolipoprotein (apo) A-I.sub.Milano (R173C) and apoA-I.sub.Paris, (R151C) were found to exhibit potent antioxidant activity on phospholipid surfaces. The addition of a free thiol, at the hydrophobic/hydrophilic interface of an amphipathic alpha helix of synthetic peptides that mimic HDL-related proteins, imparts a unique antioxidant activity to these peptides which inhibits lipid peroxidation and protects phospholipids from water-soluble free radical initiators. These peptides can be used as therapeutic agents to combat cardiovascular disease, ischemia, bone disease and other inflammatory related diseases.

  5. Human peptide transporters

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Brodin, Birger; Jørgensen, Flemming Steen

    2002-01-01

    Peptide transporters are epithelial solute carriers. Their functional role has been characterised in the small intestine and proximal tubules, where they are involved in absorption of dietary peptides and peptide reabsorption, respectively. Currently, two peptide transporters, PepT1 and PepT2, wh...

  6. Design, synthesis, and actions of a novel chimeric natriuretic peptide: CD-NP.

    Science.gov (United States)

    Lisy, Ondrej; Huntley, Brenda K; McCormick, Daniel J; Kurlansky, Paul A; Burnett, John C

    2008-07-01

    Our aim was to design, synthesize and test in vivo and in vitro a new chimeric peptide that would combine the beneficial properties of 2 distinct natriuretic peptides with a biological profile that goes beyond native peptides. Studies have established the beneficial vascular and antiproliferative properties of C-type natriuretic peptide (CNP). While lacking renal actions, CNP is less hypotensive than the cardiac peptides atrial natriuretic peptide and B-type natriuretic peptide but unloads the heart due to venodilation. Dendroaspis natriuretic peptide is a potent natriuretic and diuretic peptide that is markedly hypotensive and functions via a separate guanylyl cyclase receptor compared with CNP. Here we engineered a novel chimeric peptide CD-NP that represents the fusion of the 22-amino acid peptide CNP together with the 15-amino acid linear C-terminus of Dendroaspis natriuretic peptide. We also determined in vitro in cardiac fibroblasts cyclic guanosine monophosphate-activating and antiproliferative properties of CD-NP. Our studies demonstrate in vivo that CD-NP is natriuretic and diuretic, glomerular filtration rate enhancing, cardiac unloading, and renin inhibiting. CD-NP also demonstrates less hypotensive properties when compared with B-type natriuretic peptide. In addition, CD-NP in vitro activates cyclic guanosine monophosphate and inhibits cardiac fibroblast proliferation. The current findings advance an innovative design strategy in natriuretic peptide drug discovery and development to create therapeutic peptides with favorable properties that may be preferable to those associated with native natriuretic peptides.

  7. Methods of NMR structure refinement: molecular dynamics simulations improve the agreement with measured NMR data of a C-terminal peptide of GCN4-p1

    International Nuclear Information System (INIS)

    Dolenc, Jozica; Missimer, John H.; Steinmetz, Michel O.; Gunsteren, Wilfred F. van

    2010-01-01

    The C-terminal trigger sequence is essential in the coiled-coil formation of GCN4-p1; its conformational properties are thus of importance for understanding this process at the atomic level. A solution NMR model structure of a peptide, GCN4p16-31, encompassing the GCN4-p1 trigger sequence was proposed a few years ago. Derived using a standard single-structure refinement protocol based on 172 nuclear Overhauser effect (NOE) distance restraints, 14 hydrogen-bond and 11 φ torsional-angle restraints, the resulting set of 20 NMR model structures exhibits regular α-helical structure. However, the set slightly violates some measured NOE bounds and does not reproduce all 15 measured 3 J(H N -H Cα )-coupling constants, indicating that different conformers of GCN4p16-31 might be present in solution. With the aim to resolve structures compatible with all NOE upper distance bounds and 3 J-coupling constants, we executed several structure refinement protocols employing unrestrained and restrained molecular dynamics (MD) simulations with two force fields. We find that only configurational ensembles obtained by applying simultaneously time-averaged NOE distance and 3 J-coupling constant restraining with either force field reproduce all the experimental data. Additionally, analyses of the simulated ensembles show that the conformational variability of GCN4p16-31 in solution admitted by the available set of 187 measured NMR data is larger than represented by the set of the NMR model structures. The conformations of GCN4p16-31 in solution differ in the orientation not only of the side-chains but also of the backbone. The inconsistencies between the NMR model structures and the measured NMR data are due to the neglect of averaging effects and the inclusion of hydrogen-bond and torsional-angle restraints that have little basis in the primary, i.e. measured NMR data.

  8. Methods of NMR structure refinement: molecular dynamics simulations improve the agreement with measured NMR data of a C-terminal peptide of GCN4-p1.

    Science.gov (United States)

    Dolenc, Jozica; Missimer, John H; Steinmetz, Michel O; van Gunsteren, Wilfred F

    2010-07-01

    The C-terminal trigger sequence is essential in the coiled-coil formation of GCN4-p1; its conformational properties are thus of importance for understanding this process at the atomic level. A solution NMR model structure of a peptide, GCN4p16-31, encompassing the GCN4-p1 trigger sequence was proposed a few years ago. Derived using a standard single-structure refinement protocol based on 172 nuclear Overhauser effect (NOE) distance restraints, 14 hydrogen-bond and 11 phi torsional-angle restraints, the resulting set of 20 NMR model structures exhibits regular alpha-helical structure. However, the set slightly violates some measured NOE bounds and does not reproduce all 15 measured (3)J(H(N)-H(Calpha))-coupling constants, indicating that different conformers of GCN4p16-31 might be present in solution. With the aim to resolve structures compatible with all NOE upper distance bounds and (3)J-coupling constants, we executed several structure refinement protocols employing unrestrained and restrained molecular dynamics (MD) simulations with two force fields. We find that only configurational ensembles obtained by applying simultaneously time-averaged NOE distance and (3)J-coupling constant restraining with either force field reproduce all the experimental data. Additionally, analyses of the simulated ensembles show that the conformational variability of GCN4p16-31 in solution admitted by the available set of 187 measured NMR data is larger than represented by the set of the NMR model structures. The conformations of GCN4p16-31 in solution differ in the orientation not only of the side-chains but also of the backbone. The inconsistencies between the NMR model structures and the measured NMR data are due to the neglect of averaging effects and the inclusion of hydrogen-bond and torsional-angle restraints that have little basis in the primary, i.e. measured NMR data.

  9. Seahorse-derived peptide suppresses invasive migration of HT1080 fibrosarcoma cells by competing with intracellular α-enolase for plasminogen binding and inhibiting uPA-mediated activation of plasminogen.

    Science.gov (United States)

    Kim, Yong-Tae; Kim, Se-kwon; Jeon, You-Jin; Park, Sun Joo

    2014-12-01

    α-Enolase is a glycolytic enzyme and a surface receptor for plasminogen. α-Enolase-bound plasminogen promotes tumor cell invasion and cancer metastasis by activating plasmin and consequently degrading the extracellular matrix degradation. Therefore, α-enolase and plasminogen are novel targets for cancer therapy. We found that the amino acid sequence of a peptide purified from enzymatic hydrolysates of seahorse has striking similarities to that of α-enolase. In this study, we report that this peptide competes with cellular α-enolase for plasminogen binding and suppresses urokinase plasminogen activator (uPA)-mediated activation of plasminogen, which results in decreased invasive migration of HT1080 fibrosarcoma cells. In addition, the peptide treatment decreased the expression levels of uPA compared to that of untreated controls. These results provide new insight into the mechanism by which the seahorse-derived peptide suppresses invasive properties of human cancer cells. Our findings suggest that this peptide could emerge as a potential therapeutic agent for cancer.

  10. Peptides Derived from Type IV Collagen, CXC Chemokines, and Thrombospondin-1 Domain-Containing Proteins Inhibit Neovascularization and Suppress Tumor Growth in MDA-MB-231 Breast Cancer Xenografts

    Directory of Open Access Journals (Sweden)

    Jacob E. Koskimaki

    2009-12-01

    Full Text Available Angiogenesis or neovascularization, the process of new blood vessel formation from preexisting microvasculature, involves interactions among several cell types including parenchymal, endothelial cells, and immune cells. The formation of new vessels is tightly regulated by a balance between endogenous proangiogenic and antiangiogenic factors to maintain homeostasis in tissue; tumor progression and metastasis in breast cancer have been shown to be angiogenesis-dependent. We previously introduced a systematic methodology to identify putative endogenous antiangiogenic peptides and validated these predictions in vitro in human umbilical vein endothelial cell proliferation and migration assays. These peptides are derived from several protein families including type IV collagen, CXC chemokines, and thrombospondin-1 domain-containing proteins. On the basis of the results from the in vitro screening, we have evaluated the ability of one peptide selected from each family named pentastatin-1, chemokinostatin-1, and properdistatin, respectively, to suppress angiogenesis in an MDA-MB-231 human breast cancer orthotopic xenograft model in severe combined immunodeficient mice. Peptides were administered intraperitoneally once per day. We have demonstrated significant suppression of tumor growth in vivo and subsequent reductions in microvascular density, indicating the potential of these peptides as therapeutic agents for breast cancer.

  11. Treating autoimmune disorders with venom-derived peptides.

    Science.gov (United States)

    Shen, Bingzheng; Cao, Zhijian; Li, Wenxin; Sabatier, Jean-Marc; Wu, Yingliang

    2017-09-01

    The effective treatment of autoimmune diseases remains a challenge. Voltage-gated potassium Kv1.3 channels, which are expressed in lymphocytes, are a new therapeutic target for treating autoimmune disease. Consequently, Kv1.3 channel-inhibiting venom-derived peptides are a prospective resource for new drug discovery and clinical application. Area covered: Preclinical and clinical studies have produced a wealth of information on Kv1.3 channel-inhibiting venom-derived peptides, especially from venomous scorpions and sea anemones. This review highlights the advances in screening and design of these peptides with diverse structures and potencies. It focuses on representative strategies for improving peptide selectivity and discusses the preclinical research on those venom-derived peptides as well as their clinical developmental status. Expert opinion: Encouraging results indicate that peptides isolated from the venom of venomous animals are a large resource for discovering immunomodulators that act on Kv1.3 channels. Since the structural diversity of venom-derived peptides determines the variety of their pharmacological activities, the design and optimization of venom-peptides for improved Kv1.3 channel-specificity has been advanced through some representative strategies, such as peptide chemical modification, amino acid residue truncation and binding interface modulation. These advances should further accelerate research, development and the future clinical application of venom-derived peptides selectively targeting Kv1.3 channels.

  12. Mycobacteria attenuate nociceptive responses by formyl peptide receptor triggered opioid peptide release from neutrophils.

    Directory of Open Access Journals (Sweden)

    Heike L Rittner

    2009-04-01

    Full Text Available In inflammation, pain is regulated by a balance of pro- and analgesic mediators. Analgesic mediators include opioid peptides which are secreted by neutrophils at the site of inflammation, leading to activation of opioid receptors on peripheral sensory neurons. In humans, local opioids and opioid peptides significantly downregulate postoperative as well as arthritic pain. In rats, inflammatory pain is induced by intraplantar injection of heat inactivated Mycobacterium butyricum, a component of complete Freund's adjuvant. We hypothesized that mycobacterially derived formyl peptide receptor (FPR and/or toll like receptor (TLR agonists could activate neutrophils, leading to opioid peptide release and inhibition of inflammatory pain. In complete Freund's adjuvant-induced inflammation, thermal and mechanical nociceptive thresholds of the paw were quantified (Hargreaves and Randall-Selitto methods, respectively. Withdrawal time to heat was decreased following systemic neutrophil depletion as well as local injection of opioid receptor antagonists or anti-opioid peptide (i.e. Met-enkephalin, beta-endorphin antibodies indicating an increase in pain. In vitro, opioid peptide release from human and rat neutrophils was measured by radioimmunoassay. Met-enkephalin release was triggered by Mycobacterium butyricum and formyl peptides but not by TLR-2 or TLR-4 agonists. Mycobacterium butyricum induced a rise in intracellular calcium as determined by FURA loading and calcium imaging. Opioid peptide release was blocked by intracellular calcium chelation as well as phosphoinositol-3-kinase inhibition. The FPR antagonists Boc-FLFLF and cyclosporine H reduced opioid peptide release in vitro and increased inflammatory pain in vivo while TLR 2/4 did not appear to be involved. In summary, mycobacteria activate FPR on neutrophils, resulting in tonic secretion of opioid peptides from neutrophils and in a decrease in inflammatory pain. Future therapeutic strategies may aim

  13. Inhibition of the Rho/ROCK pathway prevents neuronal degeneration in vitro and in vivo following methylmercury exposure

    International Nuclear Information System (INIS)

    Fujimura, Masatake; Usuki, Fusako; Kawamura, Miwako; Izumo, Shuji

    2011-01-01

    Methylmercury (MeHg) is an environmental neurotoxicant which induces neuropathological changes in both the central nervous and peripheral sensory nervous systems. Our recent study demonstrated that down-regulation of Ras-related C3 botulinum toxin substrate 1 (Rac1), which is known to promote neuritic extension, preceded MeHg-induced damage in cultured cortical neurons, suggesting that MeHg-mediated axonal degeneration is due to the disturbance of neuritic extension. Therefore we hypothesized that MeHg-induced axonal degeneration might be caused by neuritic extension/retraction incoordination. This idea brought our attention to the Ras homolog gene (Rho)/Rho-associated coiled coil-forming protein kinase (ROCK) pathway because it has been known to be associated with the development of axon and apoptotic neuronal cell death. Here we show that inhibition of the Rho/ROCK pathway prevents MeHg-intoxication both in vitro and in vivo. A Rho inhibitor, C3 toxin, and 2 ROCK inhibitors, Fasudil and Y-27632, significantly protected against MeHg-induced axonal degeneration and apoptotic neuronal cell death in cultured cortical neuronal cells exposed to 100 nM MeHg for 3 days. Furthermore, Fasudil partially prevented the loss of large pale neurons in dorsal root ganglia, axonal degeneration in dorsal spinal root nerves, and vacuolar degeneration in the dorsal columns of the spinal cord in MeHg-intoxicated model rats (20 ppm MeHg in drinking water for 28 days). Hind limb crossing sign, a characteristic MeHg-intoxicated sign, was significantly suppressed in this model. The results suggest that inhibition of the Rho/ROCK pathway rescues MeHg-mediated neuritic extension/retraction incoordination and is effective for the prevention of MeHg-induced axonal degeneration and apoptotic neuronal cell death.

  14. PeptideAtlas

    Data.gov (United States)

    U.S. Department of Health & Human Services — PeptideAtlas is a multi-organism, publicly accessible compendium of peptides identified in a large set of tandem mass spectrometry proteomics experiments. Mass...

  15. A cocoa peptide protects Caenorhabditis elegans from oxidative stress and β-amyloid peptide toxicity.

    Directory of Open Access Journals (Sweden)

    Patricia Martorell

    Full Text Available BACKGROUND: Cocoa and cocoa-based products contain different compounds with beneficial properties for human health. Polyphenols are the most frequently studied, and display antioxidant properties. Moreover, protein content is a very interesting source of antioxidant bioactive peptides, which can be used therapeutically for the prevention of age-related diseases. METHODOLOGY/PRINCIPAL FINDINGS: A bioactive peptide, 13L (DNYDNSAGKWWVT, was obtained from a hydrolyzed cocoa by-product by chromatography. The in vitro inhibition of prolyl endopeptidase (PEP was used as screening method to select the suitable fraction for peptide identification. Functional analysis of 13L peptide was achieved using the transgenic Caenorhabditis elegans strain CL4176 expressing the human Aβ₁₋₄₂ peptide as a pre-clinical in vivo model for Alzheimer's disease. Among the peptides isolated, peptide 13L (1 µg/mL showed the highest antioxidant activity (P≤0.001 in the wild-type strain (N2. Furthermore, 13L produced a significant delay in body paralysis in strain CL4176, especially in the 24-47 h period after Aβ₁₋₄₂ peptide induction (P≤0.0001. This observation is in accordance with the reduction of Aβ deposits in CL4176 by western blot. Finally, transcriptomic analysis in wild-type nematodes treated with 13L revealed modulation of the proteosomal and synaptic functions as the main metabolic targets of the peptide. CONCLUSIONS/SIGNIFICANCE: These findings suggest that the cocoa 13L peptide has antioxidant activity and may reduce Aβ deposition in a C. elegans model of Alzheimer's disease; and therefore has a putative therapeutic potential for prevention of age-related diseases. Further studies in murine models and humans will be essential to analyze the effectiveness of the 13L peptide in higher animals.

  16. Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

    Science.gov (United States)

    Duffy, Fergal J; O'Donovan, Darragh; Devocelle, Marc; Moran, Niamh; O'Connell, David J; Shields, Denis C

    2015-03-23

    Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.

  17. Anti-dengue virus serotype 2 activity and mode of action of a novel peptide.

    Science.gov (United States)

    Chew, M-F; Tham, H-W; Rajik, M; Sharifah, S H

    2015-10-01

    To identify a novel antiviral peptide against dengue virus serotype 2 (DENV-2) by screening a phage display peptide library and to evaluate its in vitro antiviral activity and mode of action. A phage display peptide library was biopanned against purified DENV-2 and resulted in the identification and selection of a peptide (peptide gg-ww) for further investigation. ELISA was performed, and peptide gg-ww was shown to possess the highest binding affinity against DENV-2. Thus, peptide gg-ww was synthesized for cytotoxicity and antiviral assays. Virus plaque reduction assay, real-time PCR and immunofluorescence assay were used to investigate the inhibitory effect of peptide gg-ww on DENV-2 infection in Vero cells. Three different assays (pre-, simultaneous and post-treatments assays) were performed to investigate the peptide's mode of action. Results indicated that peptide gg-ww possessed strong antiviral activity with a ~96% inhibition rate, which was achieved at 250 μmol l(-1) . Viral replication was inhibited during a simultaneous treatment assay, indicating that the entry of the virus was impeded by this peptide. Peptide gg-ww displayed antiviral action against DENV-2 by targeting an early stage of viral replication (i.e. during viral entry). Peptide gg-ww may represent a new therapeutic candidate for the treatment of DENV infections and is a potential candidate to be developed as a peptide drug. © 2015 The Society for Applied Microbiology.

  18. Peptide-Carrier Conjugation

    DEFF Research Database (Denmark)

    Hansen, Paul Robert

    2015-01-01

    To produce antibodies against synthetic peptides it is necessary to couple them to a protein carrier. This chapter provides a nonspecialist overview of peptide-carrier conjugation. Furthermore, a protocol for coupling cysteine-containing peptides to bovine serum albumin is outlined....

  19. PH dependent adhesive peptides

    Science.gov (United States)

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  20. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2003-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  1. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  2. Peptide Nucleic Acids (PNA)

    DEFF Research Database (Denmark)

    2002-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  3. Peptide Nucleic Acid Synthons

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  4. pH dependence of MHC class I-restricted peptide presentation

    DEFF Research Database (Denmark)

    Stryhn, A; Pedersen, L O; Romme, T

    1996-01-01

    The function of MHC class I molecules is to bind and present antigenic peptides to cytotoxic T cells. Here, we report that class I-restricted peptide presentation is strongly pH dependent. The presentation of some peptides was enhanced at acidic pH, whereas the presentation of others was inhibited....... Biochemical peptide-MHC class I binding assays demonstrated that peptide-MHC class I complexes are more stable at neutral pH than at acidic pH. We suggest that acid-dependent peptide dissociation can generate empty class I molecules and that the resulting binding potential can be exploited by a subset...

  5. Structural basis for precursor protein-directed ribosomal peptide macrocyclization

    Science.gov (United States)

    Li, Kunhua; Condurso, Heather L.; Li, Gengnan; Ding, Yousong; Bruner, Steven D.

    2016-01-01

    Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides whose members target proteases with potent reversible inhibition. The product structure is constructed by three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here, we describe the detailed structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases, MdnC and MdnB, interact with a conserved α-helix of the precursor peptide using a novel precursor peptide recognition mechanism. The results provide insight into the unique protein/protein interactions key to the chemistry, suggest an origin of the natural combinatorial synthesis of microviridin peptides and provide a framework for future engineering efforts to generate designed compounds. PMID:27669417

  6. Structural basis for precursor protein-directed ribosomal peptide macrocyclization.

    Science.gov (United States)

    Li, Kunhua; Condurso, Heather L; Li, Gengnan; Ding, Yousong; Bruner, Steven D

    2016-11-01

    Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides that target proteases with potent reversible inhibition. The product structure is constructed via three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here we describe in detail the structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases MdnC and MdnB interact with a conserved α-helix of the precursor peptide using a novel precursor-peptide recognition mechanism. The results provide insight into the unique protein-protein interactions that are key to the chemistry, suggest an origin for the natural combinatorial synthesis of microviridin peptides, and provide a framework for future engineering efforts to generate designed compounds.

  7. Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3.

    Directory of Open Access Journals (Sweden)

    Wen Hwa Lee

    Full Text Available Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, p1YMESRADR8, corresponding to amino acids 313-320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384 and the β1 domain (E297 as well as an intrapeptide bond (pE315-pR317 were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.

  8. Sacubitril/valsartan: beyond natriuretic peptides.

    Science.gov (United States)

    Singh, Jagdeep S S; Burrell, Louise M; Cherif, Myriam; Squire, Iain B; Clark, Andrew L; Lang, Chim C

    2017-10-01

    Natriuretic peptides, especially B-type natriuretic peptide (BNP), have primarily been regarded as biomarkers in heart failure (HF). However, they are also possible therapeutic agents due to their potentially beneficial physiological effects. The angiotensin receptor-neprilysin inhibitor, sacubitril/valsartan, simultaneously augments the natriuretic peptide system (NPS) by inhibiting the enzyme neprilysin (NEP) and inhibits the renin-angiotensin-aldosterone system (RAAS) by blocking the angiotensin II receptor. It has been shown to improve mortality and hospitalisation outcomes in patients with HF due to left ventricular systolic dysfunction. The key advantage of sacubitril/valsartan has been perceived to be its ability to augment BNP, while its other effects have largely been overlooked. This review highlights the important effects of sacubitril/valsartan, beyond just the augmentation of BNP. First we discuss how NPS physiology differs between healthy individuals and those with HF by looking at mechanisms like the overwhelming effects of RAAS on the NPS, natriuretic peptide receptor desensitisation and absolute natriuretic deficiency. Second, this review explores other hormones that are augmented by sacubitril/valsartan such as bradykinin, substance P and adrenomedullin that may contribute to the efficacy of sacubitril/valsartan in HF. We also discuss concerns that sacubitril/valsartan may interfere with amyloid-β homeostasis with potential implications on Alzheimer's disease and macular degeneration. Finally, we explore the concept of 'autoinhibition' which is a recently described observation that humans have innate NEP inhibitory capability when natriuretic peptide levels rise above a threshold. There is speculation that autoinhibition may provide a surge of natriuretic and other vasoactive peptides to rapidly reverse decompensation. We contend that by pre-emptively inhibiting NEP, sacubitril/valsartan is inducing this surge earlier during decompensation

  9. The relation between major histocompatibility complex (MHC) restriction and the capacity of Ia to bind immunogenic peptides

    DEFF Research Database (Denmark)

    Buus, S; Sette, A; Colon, S M

    1987-01-01

    The capacity of purified I-Ad, I-Ed, I-Ak, and I-Ek to bind to protein derived peptides that have been previously reported to be T cell immunogens has been examined. For each of the 12 peptides studied strong binding to the relevant Ia restriction element was observed. All the peptides bound more...... than one Ia molecule; however, for 11 of 12 peptides, the dominant binding was to the restriction element, whereas in one instance the dominant binding was to a nonrestriction element. When the peptides were used to inhibit the presentation of antigen by prefixed accessory cells to T cells......, an excellent correlation was found between the capacity of a peptide to inhibit the binding of an antigen to purified Ia and the capacity of the peptide to inhibit accessory cell presentation of the antigen. Thus, the binding of peptide to purified Ia is immunologically relevant, and Ia seems to be the only...

  10. Soluble elastin peptides in cardiovascular homeostasis: Foe or ally.

    Science.gov (United States)

    Qin, Zhenyu

    2015-05-01

    Elastin peptides, also known as elastin-derived peptides or elastokines, are soluble polypeptides in blood and tissue. The blood levels of elastin peptides are usually low but can increase during cardiovascular diseases, such as atherosclerosis, aortic aneurysm and diabetes with vascular complications. Generally, elastin peptides are derived from the degradation of insoluble elastic polymers. The biological activities of elastin peptides are bidirectional, e.g., a pro-inflammatory effect on monocyte migration induction vs. a protective effect on vasodilation promotion. However, recent in vivo studies have demonstrated that elastin peptides promote the formation of atherosclerotic plaques in hypercholesterolemic mice and induce hyperglycemia and elevations in plasma lipid levels in fasted mice. More important, the detrimental effects induced by elastin peptides can be largely inhibited by genetic or pharmacological blockade of the elastin receptor complex or by neutralization of an antibody against elastin peptides. These studies indicate new therapeutic strategies for the treatment of cardiovascular diseases by targeting elastin peptide metabolism. Therefore, the goal of this review is to summarize current knowledge about elastin peptides relevant to cardiovascular pathologies to further delineate their potential application in cardiovascular disease. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Antimicrobial and immunomodulatory activities of PR-39 derived peptides.

    Directory of Open Access Journals (Sweden)

    Edwin J A Veldhuizen

    Full Text Available The porcine cathelicidin PR-39 is a host defence peptide that plays a pivotal role in the innate immune defence of the pig against infections. Besides direct antimicrobial activity, it is involved in immunomodulation, wound healing and several other biological processes. In this study, the antimicrobial- and immunomodulatory activity of PR-39, and N- and C-terminal derivatives of PR-39 were tested. PR-39 exhibited an unexpected broad antimicrobial spectrum including several Gram positive strains such as Bacillus globigii and Enterococcus faecalis. Of organisms tested, only Staphylococcus aureus was insensitive to PR-39. Truncation of PR-39 down to 15 (N-terminal amino acids did not lead to major loss of activity, while peptides corresponding to the C-terminal part of PR-39 were hampered in their antimicrobial activity. However, shorter peptides were all much more sensitive to inhibition by salt. Active peptides induced ATP leakage and loss of membrane potential in Bacillus globigii and Escherichia coli, indicating a lytic mechanism of action for these peptides. Finally, only the mature peptide was able to induce IL-8 production in porcine macrophages, but some shorter peptides also had an effect on TNF-α production showing differential regulation of cytokine induction by PR-39 derived peptides. None of the active peptides showed high cytotoxicity highlighting the potential of these peptides for use as an alternative to antibiotics.

  12. Antimicrobial and Immunomodulatory Activities of PR-39 Derived Peptides

    Science.gov (United States)

    Veldhuizen, Edwin J. A.; Schneider, Viktoria A. F.; Agustiandari, Herfita; van Dijk, Albert; Tjeerdsma-van Bokhoven, Johanna L. M.; Bikker, Floris J.; Haagsman, Henk P.

    2014-01-01

    The porcine cathelicidin PR-39 is a host defence peptide that plays a pivotal role in the innate immune defence of the pig against infections. Besides direct antimicrobial activity, it is involved in immunomodulation, wound healing and several other biological processes. In this study, the antimicrobial- and immunomodulatory activity of PR-39, and N- and C-terminal derivatives of PR-39 were tested. PR-39 exhibited an unexpected broad antimicrobial spectrum including several Gram positive strains such as Bacillus globigii and Enterococcus faecalis. Of organisms tested, only Staphylococcus aureus was insensitive to PR-39. Truncation of PR-39 down to 15 (N-terminal) amino acids did not lead to major loss of activity, while peptides corresponding to the C-terminal part of PR-39 were hampered in their antimicrobial activity. However, shorter peptides were all much more sensitive to inhibition by salt. Active peptides induced ATP leakage and loss of membrane potential in Bacillus globigii and Escherichia coli, indicating a lytic mechanism of action for these peptides. Finally, only the mature peptide was able to induce IL-8 production in porcine macrophages, but some shorter peptides also had an effect on TNF-α production showing differential regulation of cytokine induction by PR-39 derived peptides. None of the active peptides showed high cytotoxicity highlighting the potential of these peptides for use as an alternative to antibiotics. PMID:24755622

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

    Science.gov (United States)

    Ostrowski, Maciej; Kowalczyk, Stanisław

    2015-01-01

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

  14. Peptide stabilized amphotericin B nanodisks

    Science.gov (United States)

    Tufteland, Megan; Pesavento, Joseph B.; Bermingham, Rachelle L.; Hoeprich, Paul D.; Ryan, Robert O.

    2007-01-01

    Nanometer scale apolipoprotein A-I stabilized phospholipid disk complexes (nanodisks; ND) have been formulated with the polyene antibiotic amphotericin B (AMB). The present studies were designed to evaluate if a peptide can substitute for the function of the apolipoprotein component of ND with respect to particle formation and stability. An 18-residue synthetic amphipathic α-helical peptide, termed 4F (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2), solubilized vesicles comprised of egg phosphatidylcholine (egg PC), dipentadecanoyl PC or dimyristoylphosphatidylcholine (DMPC) at rates greater than or equal to solubilization rates observed with human apolipoprotein A-I (apoA-I; 243 amino acids). Characterization studies revealed that interaction with DMPC induced a near doubling of 4F tryptophan fluorescence emission quantum yield (excitation 280 nm) and a ~7 nm blue shift in emission wavelength maximum. Inclusion of AMB in the vesicle substrate resulted in formation of 4F AMB-ND. Spectra of AMB containing particles revealed the antibiotic is a highly effective quencher of 4F tryptophan fluorescence emission, giving rise to a Ksv = 7.7 × 104. Negative stain electron microscopy revealed that AMB-ND prepared with 4F possessed a disk shaped morphology similar to ND prepared without AMB or prepared with apoA-I. In yeast and pathogenic fungi growth inhibition assays, 4F AMB-ND was as effective as apoA-I AMB-ND. The data indicate that AMB-ND generated using an amphipathic peptide in lieu of apoA-I form a discrete population of particles that possess potent biological activity. Given their intrinsic versatility, peptides may be preferred for scale up and clinical application of AMB-ND. PMID:17293004

  15. C2360, a nuclear protein expressed in human proliferative cytotrophoblasts, is a representative member of a novel protein family with a conserved coiled coil-helix-coiled coil-helix domain.

    NARCIS (Netherlands)

    Westerman, B.A.; Poutsma, A; Steegers, EA; Oudejans, C.B.M.

    2004-01-01

    In this study, we describe the identification of nine novel genes isolated from a unique human first-trimester cDNA library generated from the placental bed. One of these clones, called C2360 and located on chromosome 10q22, was selected as it showed restricted expression in placental bed tissue as

  16. C2360, a nuclear protein expressed in human proliferative cytotrophoblasts, is a representative member of a novel protein family with a conserved coiled coil-helix-coiled coil-helix domain

    NARCIS (Netherlands)

    Westerman, Bart A.; Poutsma, Ankie; Steegers, Eric A. P.; Oudejans, Cees B. M.

    2004-01-01

    In this study, we describe the identification of nine novel genes isolated from a unique human first-trimester cDNA library generated from the placental bed. One of these clones, called C2360 and located on chromosome 10q22, was selected as it showed restricted expression in placental bed tissue as

  17. Peptides in melanoma therapy.

    Science.gov (United States)

    Mocellin, Simone

    2012-01-01

    Peptides derived from tumor associated antigens can be utilized to elicit a therapeutically effective immune response against melanoma in experimental models. However, patient vaccination with peptides - although it is often followed by the induction of melanoma- specific T lymphocytes - is rarely associated with tumor response of clinical relevance. In this review I summarize the principles of peptide design as well as the results so far obtained in the clinical setting while treating cutaneous melanoma by means of this active immunotherapy strategy. I also discuss some immunological and methodological issues that might be helpful for the successful development of peptide-based vaccines.

  18. Antimicrobial Peptides in Reptiles

    Science.gov (United States)

    van Hoek, Monique L.

    2014-01-01

    Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development. PMID:24918867

  19. Novel Coiled-Coil Cell Division Factor ZapB Stimulates Z Ring Assembly and Cell Division

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Galli, Elizabeth; Møller-Jensen, Jakob

    2008-01-01

    Formation of the Z ring is the first known event in bacterial cell division. However, it is not yet known how the assembly and contraction of the Z ring is regulated. Here, we identify a novel cell division factor ZapB in Escherichia coli that simultaneously stimulates Z ring assembly and cell...... division. Deletion of zapB resulted in delayed cell division and the formation of ectopic Z rings and spirals whereas overexpression of ZapB resulted in nucleoid condensation and aberrant cell divisions. Localization of ZapB to the divisome depended on FtsZ but not FtsA, ZipA or FtsI and ZapB interacted...... with FtsZ in a bacterial two-hybrid analysis. The simultaneous inactivation of FtsA and ZipA prevented Z ring assembly and ZapB localization. Time lapse microscopy showed that ZapB-GFP is present at mid-cell in a pattern very similar to that of FtsZ. Cells carrying a zapB deletion and the ftsZ84ts allele...

  20. A coiled coil trigger site is essential for rapid binding of synaptobrevin to the SNARE acceptor complex

    DEFF Research Database (Denmark)

    Wiederhold, Katrin; Kloepper, Tobias H; Walter, Alexander M

    2010-01-01

    Exocytosis from synaptic vesicles is driven by stepwise formation of a tight alpha-helical complex between the fusing membranes. The complex is composed of the three SNAREs: synaptobrevin 2, SNAP-25, and syntaxin 1a. An important step in complex formation is fast binding of vesicular synaptobrevi...

  1. Frog secretions and hunting magic in the upper Amazon: identification of a peptide that interacts with an adenosine receptor.

    Science.gov (United States)

    Daly, J W; Caceres, J; Moni, R W; Gusovsky, F; Moos, M; Seamon, K B; Milton, K; Myers, C W

    1992-11-15

    A frog used for "hunting magic" by several groups of Panoan-speaking Indians in the borderline between Brazil and Peru is identified as Phyllomedusa bicolor. This frog's skin secretion, which the Indians introduce into the body through fresh burns, is rich in peptides. These include vasoactive peptides, opioid peptides, and a peptide that we have named adenoregulin, with the sequence GLWSKIKEVGKEAAKAAAKAAGKAALGAVSEAV as determined from mass spectrometry and Edman degradation. The natural peptide may contain a D amino acid residue, since it is not identical in chromatographic properties to the synthetic peptide. Adenoregulin enhances binding of agonists to A1 adenosine receptors; it is accompanied in the skin secretion by peptides that inhibit binding. The vasoactive peptide sauvagine, the opioid peptides, and adenoregulin and related peptides affect behavior in mice and presumably contribute to the behavioral sequelae observed in humans.

  2. alpha-Glucosidase inhibition (acarbose) fails to enhance secretion of glucagon-like peptide 1 (7-36 amide) and to delay gastric emptying in Type 2 diabetic patients

    DEFF Research Database (Denmark)

    Hücking, K; Kostic, Z; Pox, C

    2005-01-01

    AIM: Acarbose is able to enhance GLP-1 release and delay gastric emptying in normal subjects. The effect of alpha-glucosidase inhibition on GLP-1 has been less evident in Type 2 diabetic patients. The aim of this study was to investigate the possible influence of acarbose on GLP-1 release and gas...

  3. Insulin C-peptide test

    Science.gov (United States)

    C-peptide ... the test depends on the reason for the C-peptide measurement. Ask your health care provider if ... C-peptide is measured to tell the difference between insulin the body produces and insulin someone injects ...

  4. Peptide array-based screening of human mesenchymal stem cell-adhesive peptides derived from fibronectin type III domain

    International Nuclear Information System (INIS)

    Okochi, Mina; Nomura, Shigeyuki; Kaga, Chiaki; Honda, Hiroyuki

    2008-01-01

    Human mesenchymal stem cell-adhesive peptides were screened based on the amino acid sequence of fibronectin type III domain 8-11 (FN-III 8-11 ) using a peptide array synthesized by the Fmoc-chemistry. Using hexameric peptide library of FN-III 8-11 scan, we identified the ALNGR (Ala-Leu-Asn-Gly-Arg) peptide that induced cell adhesion as well as RGDS (Arg-Gly-Asp-Ser) peptide. After incubation for 2 h, approximately 68% of inoculated cells adhere to the ALNGR peptide disk. Adhesion inhibition assay with integrin antibodies showed that the ALNGR peptide interacts with integrin β1 but not with αvβ3, indicating that the receptors for ALNGR are different from RGDS. Additionally, the ALNGR peptide expressed cell specificities for adhesion: cell adhesion was promoted for fibroblasts but not for keratinocytes or endotherial cells. The ALNGR peptide induced cell adhesion and promoted cell proliferation without changing its property. It is therefore useful for the construction of functional biomaterials

  5. Peptide Vaccines for Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Rory C. F. De Brito

    2018-05-01

    Full Text Available Due to an increase in the incidence of leishmaniases worldwide, the development of new strategies such as prophylactic vaccines to prevent infection and decrease the disease have become a high priority. Classic vaccines against leishmaniases were based on live or attenuated parasites or their subunits. Nevertheless, the use of whole parasite or their subunits for vaccine production has numerous disadvantages. Therefore, the use of Leishmania peptides to design more specific vaccines against leishmaniases seems promising. Moreover, peptides have several benefits in comparison with other kinds of antigens, for instance, good stability, absence of potentially damaging materials, antigen low complexity, and low-cost to scale up. By contrast, peptides are poor immunogenic alone, and they need to be delivered correctly. In this context, several approaches described in this review are useful to solve these drawbacks. Approaches, such as, peptides in combination with potent adjuvants, cellular vaccinations, adenovirus, polyepitopes, or DNA vaccines have been used to develop peptide-based vaccines. Recent advancements in peptide vaccine design, chimeric, or polypeptide vaccines and nanovaccines based on particles attached or formulated with antigenic components or peptides have been increasingly employed to drive a specific immune response. In this review, we briefly summarize the old, current, and future stands on peptide-based vaccines, describing the disadvantages and benefits associated with them. We also propose possible approaches to overcome the related weaknesses of synthetic vaccines and suggest future guidelines for their development.

  6. Peptide Vaccines for Leishmaniasis.

    Science.gov (United States)

    De Brito, Rory C F; Cardoso, Jamille M De O; Reis, Levi E S; Vieira, Joao F; Mathias, Fernando A S; Roatt, Bruno M; Aguiar-Soares, Rodrigo Dian D O; Ruiz, Jeronimo C; Resende, Daniela de M; Reis, Alexandre B

    2018-01-01

    Due to an increase in the incidence of leishmaniases worldwide, the development of new strategies such as prophylactic vaccines to prevent infection and decrease the disease have become a high priority. Classic vaccines against leishmaniases were based on live or attenuated parasites or their subunits. Nevertheless, the use of whole parasite or their subunits for vaccine production has numerous disadvantages. Therefore, the use of Leishmania peptides to design more specific vaccines against leishmaniases seems promising. Moreover, peptides have several benefits in comparison with other kinds of antigens, for instance, good stability, absence of potentially damaging materials, antigen low complexity, and low-cost to scale up. By contrast, peptides are poor immunogenic alone, and they need to be delivered correctly. In this context, several approaches described in this review are useful to solve these drawbacks. Approaches, such as, peptides in combination with potent adjuvants, cellular vaccinations, adenovirus, polyepitopes, or DNA vaccines have been used to develop peptide-based vaccines. Recent advancements in peptide vaccine design, chimeric, or polypeptide vaccines and nanovaccines based on particles attached or formulated with antigenic components or peptides have been increasingly employed to drive a specific immune response. In this review, we briefly summarize the old, current, and future stands on peptide-based vaccines, describing the disadvantages and benefits associated with them. We also propose possible approaches to overcome the related weaknesses of synthetic vaccines and suggest future guidelines for their development.

  7. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds known as peptide nucleic acids, bind complementary DNA and RNA strands, and generally do so more strongly than the corresponding DNA or RNA strands while exhibiting increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from...

  8. Peptide inhibitors of appressorium development in Glomerella cingulata.

    Science.gov (United States)

    Al-Samarrai, Taha H; Sullivan, Patrick A; Templeton, Matthew D; Farley, Peter C

    2002-04-09

    The phytopathogen Glomerella cingulata (anamorph: Colletotrichum gloeosporioides) infects host tissue by means of a specialised infection structure, the appressorium. The Saccharomyces cerevisiae alpha-mating factor pheromone, the Saccharomyces kluyveri alpha-mating factor pheromone and a hendecapeptide produced by G. cingulata inhibit appressorium development. The amino acid sequence of the G. cingulata peptide, GYFSYPHGNLF, is different from that of the mature pheromone peptides of other filamentous fungi. The peptide has sequence similarity with the Saccharomyces alpha-mating factor pheromones, but is unable to elicit growth arrest in S. cerevisiae.

  9. Role of SbmA in the Uptake of Peptide Nucleic Acid (PNA)-Peptide Conjugates in E. coli

    DEFF Research Database (Denmark)

    Ghosal, Anubrata; Vitali, Ally; Stach, James E M

    2013-01-01

    Antisense PNA oligomers targeting essential genes (acpP or ftsZ) and conjugated to the delivery peptide L((KFF)(3)K) show complete growth inhibition of wild type E. coli strain (MG1655) with submicromolar MIC. In this study we show that resistant mutants generated against such PNA......-peptide conjugates had disruptions in the region of sbmA, a gene encoding an inner membrane peptide transporter. The wild type sensitivity to the PNA conjugates was re-established in the resistance mutants by complementation with sbmA. Furthermore, deletion of sbmA in E. coli AS19, a strain that is sensitive...

  10. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

    Directory of Open Access Journals (Sweden)

    James Smadbeck

    Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA–protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 mM, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2

  11. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

    Directory of Open Access Journals (Sweden)

    James Smadbeck

    Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA-protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 [Formula: see text]M, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly

  12. Peptide hormones and lung cancer.

    Science.gov (United States)

    Moody, T W

    2006-03-01

    Several peptide hormones have been identified which alter the proliferation of lung cancer. Small cell lung cancer (SCLC), which is a neuroendocrine cancer, produces and secretes gastrin releasing peptide (GRP), neurotensin (NT) and adrenomedullin (AM) as autocrine growth factors. GRP, NT and AM bind to G-protein coupled receptors causing phosphatidylinositol turnover or elevated cAMP in SCLC cells. Addition of GRP, NT or AM to SCLC cells causes altered expression of nuclear oncogenes, such as c-fos, and stimulation of growth. Antagonists have been developed for GRP, NT and AM receptors which function as cytostatic agents and inhibit SCLC growth. Growth factor antagonists, such as the NT1 receptor antagonist SR48692, facilitate the ability of chemotherapeutic drugs to kill lung cancer cells. It remains to be determined if GRP, NT and AM receptors will served as molecular targets, for development of new therapies for the treatment of SCLC patients. Non-small cell lung cancer (NSCLC) cells also have a high density of GRP, NT, AM and epidermal growth factor (EGF) receptors. Several NSCLC patients with EGF receptor mutations respond to gefitinib, a tyrosine kinase inhibitor. Gefitinib relieves NSCLC symptoms, maintaining stable disease in patients who are not eligible for systemic chemotherapy. It is important to develop new therapeutic approaches using translational research techniques for the treatment of lung cancer patients.

  13. Cloning of precursors for two MIH/VIH-related peptides in the prawn, Macrobrachium rosenbergii.

    Science.gov (United States)

    Yang, W J; Rao, K R

    2001-11-30

    Two cDNA clones (634 and 1366 bp) encoding MIH/VIH (molt-inhibiting hormone/vitellogenesis-inhibiting hormone)-related peptides were isolated and sequenced from a Macrobrachium rosenbergii eyestalk ganglia cDNA library. The clones contain a 360 and 339 bp open-reading frame, and their conceptually translated peptides consist of a 41 and 34 amino acid signal peptide, respectively, and a 78 amino acid residue mature peptide hormone. The amino acid sequences of the peptides exhibit higher identities with other known MIHs and VIH (44-69%) than with CHHs (28-33%). This is the first report describing the cloning and sequencing of two MIH/VIH-related peptides in a single crustacean species. Transcription of these mRNAs was detected in the eyestalk ganglia, but not in the thoracic ganglia, hepatopancreas, gut, gill, heart, or muscle.

  14. Tandem MS Analysis of Selenamide-Derivatized Peptide Ions

    Science.gov (United States)

    Zhang, Yun; Zhang, Hao; Cui, Weidong; Chen, Hao

    2011-09-01

    Our previous study showed that selenamide reagents such as ebselen and N-(phenylseleno)phthalimide (NPSP) can be used for selective and rapid derivatization of protein/peptide thiols in high conversion yield. This paper reports the systematic investigation of MS/MS dissociation behaviors of selenamide-derivatized peptide ions upon collision induced dissociation (CID) and electron transfer dissociation (ETD). In the positive ion mode, derivatized peptide ions exhibit tag-dependent CID dissociation pathways. For instance, ebselen-derivatized peptide ions preferentially undergo Se-S bond cleavage upon CID to produce a characteristic fragment ion, the protonated ebselen ( m/z 276), which allows selective identification of thiol peptides from protein digest as well as selective detection of thiol proteins from protein mixture using precursor ion scan (PIS). In contrast, NPSP-derivatized peptide ions retain their phenylselenenyl tags during CID, which is useful in sequencing peptides and locating cysteine residues. In the negative ion CID mode, both types of tags are preferentially lost via the Se-S cleavage, analogous to the S-S bond cleavage during CID of disulfide-containing peptide anions. In consideration of the convenience in preparing selenamide-derivatized peptides and the similarity of Se-S of the tag to the S-S bond, we also examined ETD of the derivatized peptide ions to probe the mechanism for electron-based ion dissociation. Interestingly, facile cleavage of Se-S bond occurs to the peptide ions carrying either protons or alkali metal ions, while backbone cleavage to form c/z ions is severely inhibited. These results are in agreement with the Utah-Washington mechanism proposed for depicting electron-based ion dissociation processes.

  15. Diversity-oriented peptide stapling

    DEFF Research Database (Denmark)

    Tran, Thu Phuong; Larsen, Christian Ørnbøl; Røndbjerg, Tobias

    2017-01-01

    as a powerful method for peptide stapling. However, to date CuAAC stapling has not provided a simple method for obtaining peptides that are easily diversified further. In the present study, we report a new diversity-oriented peptide stapling (DOPS) methodology based on CuAAC chemistry. Stapling of peptides...

  16. PNA Peptide chimerae

    DEFF Research Database (Denmark)

    Koch, T.; Næsby, M.; Wittung, P.

    1995-01-01

    Radioactive labelling of PNA has been performed try linking a peptide segment to the PNA which is substrate for protein kinase A. The enzymatic phosphorylation proceeds in almost quantitative yields....

  17. Tumor penetrating peptides

    Directory of Open Access Journals (Sweden)

    Tambet eTeesalu

    2013-08-01

    Full Text Available Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC, contains the integrin-binding RGD motif. RGD mediates tumor homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular zip code of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is

  18. Small lytic peptides escape the inhibitory effect of heparan sulfate on the surface of cancer cells

    Science.gov (United States)

    2011-01-01

    Background Several naturally occurring cationic antimicrobial peptides (CAPs), including bovine lactoferricin (LfcinB), display promising anticancer activities. These peptides are unaffected by multidrug resistance mechanisms and have been shown to induce a protective immune response against solid tumors, thus making them interesting candidates for developing novel lead structures for anticancer treatment. Recently, we showed that the anticancer activity by LfcinB was inhibited by the presence of heparan sulfate (HS) on the surface of tumor cells. Based on extensive structure-activity relationship studies performed on LfcinB, shorter and more potent peptides have been constructed. In the present study, we have investigated the anticancer activity of three chemically modified 9-mer peptides and the influence of HS and chondroitin sulfate (CS) on their cytotoxic activity. Methods Various cell lines and red blood cells were used to investigate the anticancer activity and selectivity of the peptides. The cytotoxic effect of the peptides against the different cell lines was measured by use of a colorimetric MTT viability assay. The influence of HS and CS on their cytotoxic activity was evaluated by using HS/CS expressing and HS/CS deficient cell lines. The ability of soluble HS and CS to inhibit the cytotoxic activity of the peptides and the peptides' affinity for HS and CS were also investigated. Results The 9-mer peptides displayed selective anticancer activity. Cells expressing HS/CS were equally or more susceptible to the peptides than cells not expressing HS/CS. The peptides displayed a higher affinity for HS compared to CS, and exogenously added HS inhibited the cytotoxic effect of the peptides. Conclusions In contrast to the previously reported inhibitory effect of HS on LfcinB, the present study shows that the cytotoxic activity of small lytic peptides was increased or not affected by cell surface HS. PMID:21453492

  19. Novel MtCEP1 peptides produced in vivo differentially regulate root development in Medicago truncatula.

    Science.gov (United States)

    Mohd-Radzman, Nadiatul A; Binos, Steve; Truong, Thy T; Imin, Nijat; Mariani, Michael; Djordjevic, Michael A

    2015-08-01

    Small, post-translationally modified and secreted peptides regulate diverse plant developmental processes. Due to low natural abundance, it is difficult to isolate and identify these peptides. Using an improved peptide isolation protocol and Orbitrap mass spectrometry, nine 15-amino-acid CEP peptides were identified that corresponded to the two domains encoded by Medicago truncatula CEP1 (MtCEP1). Novel arabinosylated and hydroxylated peptides were identified in root cultures overexpressing MtCEP1. The five most abundant CEP peptides were hydroxylated and these species were detected also in low amounts in vector control samples. Synthetic peptides with different hydroxylation patterns differentially affected root development. Notably, the domain 1 peptide hydroxylated at Pro4 and Pro11 (D1:HyP4,11) imparted the strongest inhibition of lateral root emergence when grown with 5mM KNO3 and stimulated the highest increase in nodule number when grown with 0mM KNO3. Inhibition of lateral root emergence by D1:HyP4,11 was not alleviated by removing peptide exposure. In contrast, the domain 2 peptide hydroxylated at Pro11 (D2:HyP11) increased stage III-IV lateral root primordium numbers by 6-fold (P emerge. Auxin addition at levels which stimulated lateral root formation in wild-type plants had little or no ameliorating effect on CEP peptide-mediated inhibition of lateral root formation or emergence. Both peptides increased and altered the root staining pattern of the auxin-responsive reporter GH3:GUS suggesting CEPs alter auxin sensitivity or distribution. The results showed that CEP primary sequence and post-translational modifications influence peptide activities and the improved isolation procedure effectively and reproducibly identifies and characterises CEPs. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  20. Small lytic peptides escape the inhibitory effect of heparan sulfate on the surface of cancer cells

    Directory of Open Access Journals (Sweden)

    Lindin Inger

    2011-03-01

    Full Text Available Abstract Background Several naturally occurring cationic antimicrobial peptides (CAPs, including bovine lactoferricin (LfcinB, display promising anticancer activities. These peptides are unaffected by multidrug resistance mechanisms and have been shown to induce a protective immune response against solid tumors, thus making them interesting candidates for developing novel lead structures for anticancer treatment. Recently, we showed that the anticancer activity by LfcinB was inhibited by the presence of heparan sulfate (HS on the surface of tumor cells. Based on extensive structure-activity relationship studies performed on LfcinB, shorter and more potent peptides have been constructed. In the present study, we have investigated the anticancer activity of three chemically modified 9-mer peptides and the influence of HS and chondroitin sulfate (CS on their cytotoxic activity. Methods Various cell lines and red blood cells were used to investigate the anticancer activity and selectivity of the peptides. The cytotoxic effect of the peptides against the different cell lines was measured by use of a colorimetric MTT viability assay. The influence of HS and CS on their cytotoxic activity was evaluated by using HS/CS expressing and HS/CS deficient cell lines. The ability of soluble HS and CS to inhibit the cytotoxic activity of the peptides and the peptides' affinity for HS and CS were also investigated. Results The 9-mer peptides displayed selective anticancer activity. Cells expressing HS/CS were equally or more susceptible to the peptides than cells not expressing HS/CS. The peptides displayed a higher affinity for HS compared to CS, and exogenously added HS inhibited the cytotoxic effect of the peptides. Conclusions In contrast to the previously reported inhibitory effect of HS on LfcinB, the present study shows that the cytotoxic activity of small lytic peptides was increased or not affected by cell surface HS.

  1. In silico design of cyclic peptides as influenza virus, a subtype H1N1 ...

    African Journals Online (AJOL)

    Cyclic peptides can be used to make new antiviral drug design especially to inhibit neuraminidase activity by using 'structure-based design' method. Based on molecular docking, new antiviral drug designs have been found. They are DNY, NNY, DDY, DYY, RRR, RPR, RRP and LRL. These cyclic peptides showed better ...

  2. Glycotriazole-peptides derived from the peptide HSP1: synergistic effect of triazole and saccharide rings on the antifungal activity.

    Science.gov (United States)

    Junior, Eduardo F C; Guimarães, Carlos F R C; Franco, Lucas L; Alves, Ricardo J; Kato, Kelly C; Martins, Helen R; de Souza Filho, José D; Bemquerer, Marcelo P; Munhoz, Victor H O; Resende, Jarbas M; Verly, Rodrigo M

    2017-08-01

    This work proposes a strategy that uses solid-phase peptide synthesis associated with copper(I)-catalyzed azide alkyne cycloaddition reaction to promote the glycosylation of an antimicrobial peptide (HSP1) containing a carboxyamidated C-terminus (HSP1-NH 2 ). Two glycotriazole-peptides, namely [p-Glc-trz-G 1 ]HSP1-NH 2 and [p-GlcNAc-trz-G 1 ]HSP1-NH 2 , were prepared using per-O-acetylated azide derivatives of glucose and N-acetylglucosamine in the presence of copper(II) sulfate pentahydrate (CuSO 4 ·5H 2 O) and sodium ascorbate as a reducing agent. In order to investigate the synergistic action of the carbohydrate motif linked to the triazole-peptide structure, a triazole derivative [trz-G 1 ]HSP1-NH 2 was also prepared. A set of biophysical approaches such as DLS, Zeta Potential, SPR and carboxyfluorescein leakage from phospholipid vesicles confirmed higher membrane disruption and lytic activities as well as stronger peptide-LUVs interactions for the glycotriazole-peptides when compared to HSP1-NH 2 and to its triazole derivative, which is in accordance with the performed biological assays: whereas HSP1-NH 2 presents relatively low and [trz-G 1 ]HSP1-NH 2 just moderate fungicidal activity, the glycotriazole-peptides are significantly more effective antifungal agents. In addition, the glycotriazole-peptides and the triazole derivative present strong inhibition effects on ergosterol biosynthesis in Candida albicans, when compared to HSP1-NH 2 alone. In conclusion, the increased fungicidal activity of the glycotriazole-peptides seems to be the result of (A) more pronounced membrane-disruptive properties, which is related to the presence of a saccharide ring, together with (B) the inhibition of ergosterol biosynthesis, which seems to be related to the presence of both the monosaccharide and the triazole rings.

  3. Biological Applications of Designed Hairpin Peptides: As Antimicrobials and as Inhibitors of Amyloidogenesis

    Science.gov (United States)

    Sivanesam, Kalkena

    More than 40 diseases have been associated with the misfolding of peptides (or proteins) that form fibrils with a very specific morphology. These peptides classified as amyloidogenic peptides have been implicated in the development of Alzheimer's Disease, Parkinson's Disease, Type II Diabetes, Hungtinton's Disease etc. To date, these diseases have no cure, only therapies that can ameliorate the symptoms to a degree. Inhibition of the amyloidogenesis of these peptides has been proposed as a possible treatment option. While small molecules have been heavily tested as inhibitors of amyloidogenesis, peptides have emerged as potential inhibitors. In this work, the ability of a set of designed hairpin peptides to inhibit the amyloidogenesis of two different systems, alpha-synuclein (implicated in Parkinson's Disease) and human amylin (implicated in Type II Diabetes) is tested. Using circular dichroism and thioflavin T fluorescence, the ability of these peptides to inhibit amyloidogenesis is tested. The binding loci of these inhibitors to alpha-synuclein are also explored. The use of peptides as antimicrobials on the other hand is not a novel concept. However, most antimicrobial peptides, both natural and designed, rely heavily on covalent stabilizations in order to maintain secondary structure. In this study, non-covalent stabilizations are applied to a couple of natural as well as designed antimicrobials in order to study the effects of secondary structure stabilization on biological activity.

  4. Methods of NMR structure refinement: molecular dynamics simulations improve the agreement with measured NMR data of a C-terminal peptide of GCN4-p1

    Energy Technology Data Exchange (ETDEWEB)

    Dolenc, Jozica [Swiss Federal Institute of Technology, Laboratory of Physical Chemistry, ETH (Switzerland); Missimer, John H.; Steinmetz, Michel O. [Paul Scherrer Institut, Biomolecular Research (Switzerland); Gunsteren, Wilfred F. van, E-mail: wfvgn@igc.phys.chem.ethz.c [Swiss Federal Institute of Technology, Laboratory of Physical Chemistry, ETH (Switzerland)

    2010-07-15

    The C-terminal trigger sequence is essential in the coiled-coil formation of GCN4-p1; its conformational properties are thus of importance for understanding this process at the atomic level. A solution NMR model structure of a peptide, GCN4p16-31, encompassing the GCN4-p1 trigger sequence was proposed a few years ago. Derived using a standard single-structure refinement protocol based on 172 nuclear Overhauser effect (NOE) distance restraints, 14 hydrogen-bond and 11 {phi} torsional-angle restraints, the resulting set of 20 NMR model structures exhibits regular {alpha}-helical structure. However, the set slightly violates some measured NOE bounds and does not reproduce all 15 measured {sup 3}J(H{sub N}-H{sub C{alpha}})-coupling constants, indicating that different conformers of GCN4p16-31 might be present in solution. With the aim to resolve structures compatible with all NOE upper distance bounds and {sup 3}J-coupling constants, we executed several structure refinement protocols employing unrestrained and restrained molecular dynamics (MD) simulations with two force fields. We find that only configurational ensembles obtained by applying simultaneously time-averaged NOE distance and {sup 3}J-coupling constant restraining with either force field reproduce all the experimental data. Additionally, analyses of the simulated ensembles show that the conformational variability of GCN4p16-31 in solution admitted by the available set of 187 measured NMR data is larger than represented by the set of the NMR model structures. The conformations of GCN4p16-31 in solution differ in the orientation not only of the side-chains but also of the backbone. The inconsistencies between the NMR model structures and the measured NMR data are due to the neglect of averaging effects and the inclusion of hydrogen-bond and torsional-angle restraints that have little basis in the primary, i.e. measured NMR data.

  5. Novel heparan sulfate-binding peptides for blocking herpesvirus entry.

    Directory of Open Access Journals (Sweden)

    Pranay Dogra

    Full Text Available Human cytomegalovirus (HCMV infection can lead to congenital hearing loss and mental retardation. Upon immune suppression, reactivation of latent HCMV or primary infection increases morbidity in cancer, transplantation, and late stage AIDS patients. Current treatments include nucleoside analogues, which have significant toxicities limiting their usefulness. In this study we screened a panel of synthetic heparin-binding peptides for their ability to prevent CMV infection in vitro. A peptide designated, p5+14 exhibited ~ 90% reduction in murine CMV (MCMV infection. Because negatively charged, cell-surface heparan sulfate proteoglycans (HSPGs, serve as the attachment receptor during the adsorption phase of the CMV infection cycle, we hypothesized that p5+14 effectively competes for CMV adsorption to the cell surface resulting in the reduction in infection. Positively charged Lys residues were required for peptide binding to cell-surface HSPGs and reducing viral infection. We show that this inhibition was not due to a direct neutralizing effect on the virus itself and that the peptide blocked adsorption of the virus. The peptide also inhibited infection of other herpesviruses: HCMV and herpes simplex virus 1 and 2 in vitro, demonstrating it has broad-spectrum antiviral activity. Therefore, this peptide may offer an adjunct therapy for the treatment of herpes viral infections and other viruses that use HSPGs for entry.

  6. Antimicrobial peptide coatings for hydroxyapatite:Electrostatic and covalent attachment of antimicrobial peptides to surfaces

    OpenAIRE

    Townsend, Leigh; Williams, Richard L.; Anuforom, Olachi; Berwick, Matthew R.; Halstead, Fenella; Hughes, Erik; Stamboulis, Artemis; Oppenheim, Beryl; Gough, Julie; Grover, Liam; Scott, Robert A H; Webber, Mark; Peacock, Anna F A; Belli, Antonio; Logan, Ann

    2017-01-01

    The interface between implanted devices and their host tissue is complex and is often optimized for maximal integration and cell adhesion. However, this also gives a surface suitable for bacterial colonization. We have developed a novel method of modifying the surface at the material-tissue interface with an antimicrobial peptide (AMP) coating to allowcell attachment while inhibiting bacterial colonization. The technology reported here is a dual AMP coating. The dual coating consists ofAMPs c...

  7. [Ala12]MCD peptide: a lead peptide to inhibitors of immunoglobulin E binding to mast cell receptors.

    Science.gov (United States)

    Buku, A; Condie, B A; Price, J A; Mezei, M

    2005-09-01

    An effort was made to discover mast cell degranulating (MCD) peptide analogs that bind with high affinity to mast cell receptors without triggering secretion of histamine or other mediators of the allergic reaction initiated by immunoglobulin E (IgE) after mast cell activation. Such compounds could serve as inhibitors of IgE binding to mast cell receptors. An alanine scan of MCD peptide reported previously showed that the analog [Ala12]MCD was 120-fold less potent in histamine-releasing activity and fivefold more potent in binding affinity to mast cell receptors than the parent MCD peptide. Because this analog showed marginal intrinsic activity and good binding affinity it was subsequently tested in the present study as an IgE inhibitor. In contrast to MCD peptide, [Ala12]MCD showed a 50% inhibition of IgE binding to the Fc epsilon RI alpha mast cell receptor by using rat basophilic leukemia (RBL-2H3) mast cells and fluorescence polarization. Furthermore, in a beta-hexosaminidase secretory assay, the peptide also showed a 50% inhibition of the secretion of this enzyme caused by IgE. An attempt was made to relate structural changes and biologic differences between the [Ala12]MCD analog and the parent MCD peptide. The present results show that [Ala12]MCD may provide a base for designing agents to prevent IgE/Fc epsilon RI alpha interactions and, consequently, allergic conditions.

  8. Peptide Integrated Optics.

    Science.gov (United States)

    Handelman, Amir; Lapshina, Nadezda; Apter, Boris; Rosenman, Gil

    2018-02-01

    Bio-nanophotonics is a wide field in which advanced optical materials, biomedicine, fundamental optics, and nanotechnology are combined and result in the development of biomedical optical chips. Silk fibers or synthetic bioabsorbable polymers are the main light-guiding components. In this work, an advanced concept of integrated bio-optics is proposed, which is based on bioinspired peptide optical materials exhibiting wide optical transparency, nonlinear and electrooptical properties, and effective passive and active waveguiding. Developed new technology combining bottom-up controlled deposition of peptide planar wafers of a large area and top-down focus ion beam lithography provides direct fabrication of peptide optical integrated circuits. Finding a deep modification of peptide optical properties by reconformation of biological secondary structure from native phase to β-sheet architecture is followed by the appearance of visible fluorescence and unexpected transition from a native passive optical waveguiding to an active one. Original biocompatibility, switchable regimes of waveguiding, and multifunctional nonlinear optical properties make these new peptide planar optical materials attractive for application in emerging technology of lab-on-biochips, combining biomedical photonic and electronic circuits toward medical diagnosis, light-activated therapy, and health monitoring. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Antimicrobial Peptides from Plants

    Directory of Open Access Journals (Sweden)

    James P. Tam

    2015-11-01

    Full Text Available Plant antimicrobial peptides (AMPs have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic, lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms.

  10. Purification and Characterization of Antioxidant Peptide from Sunflower Protein Hydrolysate

    Directory of Open Access Journals (Sweden)

    Xi-Qun Zheng

    2010-01-01

    Full Text Available Sunflower proteins were hydrolyzed with Flavourzyme for the production of antioxidant peptide. DEAE-Sepharose Fast Flow, Sephadex G-25 gel filtration chromatography and reversed-phase HPLC were consecutively employed to purify a novel sunflower antioxidant peptide, and the ability to inhibit the autoxidation of pyrogallol was expressed as the antioxidative activity of the peptide. The amino acid sequence was identified as Ala-Cys-Ala-His-Asp-Lys-Val by a Q-Tof2 mass spectrometer. This novel peptide exhibited a high antioxidative activity of 79.42 U/mL, which is expected to protect against oxidative damage in living systems in relation to aging and carcinogenesis. Higher antioxidative activities were presumed mainly due to the presence of hydrophobic amino acids in its sequence.

  11. Peptide Based Radiopharmaceuticals: Specific Construct Approach

    Energy Technology Data Exchange (ETDEWEB)

    Som, P; Rhodes, B A; Sharma, S S

    1997-10-21

    The objective of this project was to develop receptor based peptides for diagnostic imaging and therapy. A series of peptides related to cell adhesion molecules (CAM) and immune regulation were designed for radiolabeling with 99mTc and evaluated in animal models as potential diagnostic imaging agents for various disease conditions such as thrombus (clot), acute kidney failure, and inflection/inflammation imaging. The peptides for this project were designed by the industrial partner, Palatin Technologies, (formerly Rhomed, Inc.) using various peptide design approaches including a newly developed rational computer assisted drug design (CADD) approach termed MIDAS (Metal ion Induced Distinctive Array of Structures). In this approach, the biological function domain and the 99mTc complexing domain are fused together so that structurally these domains are indistinguishable. This approach allows construction of conformationally rigid metallo-peptide molecules (similar to cyclic peptides) that are metabolically stable in-vivo. All the newly designed peptides were screened in various in vitro receptor binding and functional assays to identify a lead compound. The lead compounds were formulated in a one-step 99mTc labeling kit form which were studied by BNL for detailed in-vivo imaging using various animals models of human disease. Two main peptides usingMIDAS approach evolved and were investigated: RGD peptide for acute renal failure and an immunomodulatory peptide derived from tuftsin (RMT-1) for infection/inflammation imaging. Various RGD based metallopeptides were designed, synthesized and assayed for their efficacy in inhibiting ADP-induced human platelet aggregation. Most of these peptides displayed biological activity in the 1-100 µM range. Based on previous work by others, RGD-I and RGD-II were evaluated in animal models of acute renal failure. These earlier studies showed that after acute ischemic injury the renal cortex displays

  12. Potential of acylated peptides to target the influenza A virus

    Directory of Open Access Journals (Sweden)

    Daniel Lauster

    2015-04-01

    Full Text Available For antiviral drug design, especially in the field of influenza virus research, potent multivalent inhibitors raise high expectations for combating epidemics and pandemics. Among a large variety of covalent and non-covalent scaffold systems for a multivalent display of inhibitors, we created a simple supramolecular platform to enhance the antiviral effect of our recently developed antiviral Peptide B (PeBGF, preventing binding of influenza virus to the host cell. By conjugating the peptide with stearic acid to create a higher-order structure with a multivalent display, we could significantly enhance the inhibitory effect against the serotypes of both human pathogenic influenza virus A/Aichi/2/1968 H3N2, and avian pathogenic A/FPV/Rostock/34 H7N1 in the hemagglutination inhibition assay. Further, the inhibitory potential of stearylated PeBGF (C18-PeBGF was investigated by infection inhibition assays, in which we achieved low micromolar inhibition constants against both viral strains. In addition, we compared C18-PeBGF to other published amphiphilic peptide inhibitors, such as the stearylated sugar receptor mimicking peptide (Matsubara et al. 2010, and the “Entry Blocker” (EB (Jones et al. 2006, with respect to their antiviral activity against infection by Influenza A Virus (IAV H3N2. However, while this strategy seems at a first glance promising, the native situation is quite different from our experimental model settings. First, we found a strong potential of those peptides to form large amyloid-like supramolecular assemblies. Second, in vivo, the large excess of cell surface membranes provides an unspecific target for the stearylated peptides. We show that acylated peptides insert into the lipid phase of such membranes. Eventually, our study reveals serious limitations of this type of self-assembling IAV inhibitors.

  13. Peptides extracted from Artemisia herba alba have antimicrobial ...

    African Journals Online (AJOL)

    Background: Artemisia herba alba, classified into the family of Asteraceae, is an aromatic herb that is traditionally used as a purgative and antipyretic folk medicine by rural people of south Tunisia. This study reports the first identification of antimicrobial peptides from this medicinal plant that inhibited the growth of several ...

  14. Membrane interaction and secondary structure of de novo designed arginine-and tryptophan peptides with dual function

    KAUST Repository

    Rydberg, Hanna A.

    2012-10-01

    Cell-penetrating peptides and antimicrobial peptides are two classes of positively charged membrane active peptides with several properties in common. The challenge is to combine knowledge about the membrane interaction mechanisms and structural properties of the two classes to design peptides with membrane-specific actions, useful either as transporters of cargo or as antibacterial substances. Membrane active peptides are commonly rich in arginine and tryptophan. We have previously designed a series of arg/trp peptides and investigated how the position and number of tryptophans affect cellular uptake. Here we explore the antimicrobial properties and the interaction with lipid model membranes of these peptides, using minimal inhibitory concentrations assay (MIC), circular dichroism (CD) and linear dichroism (LD). The results show that the arg/trp peptides inhibit the growth of the two gram positive strains Staphylococcus aureus and Staphylococcus pyogenes, with some individual variations depending on the position of the tryptophans. No inhibition of the gram negative strains Proteus mirabilis or Pseudomonas aeruginosa was noticed. CD indicated that when bound to lipid vesicles one of the peptides forms an α-helical like structure, whereas the other five exhibited rather random coiled structures. LD indicated that all six peptides were somehow aligned parallel with the membrane surface. Our results do not reveal any obvious connection between membrane interaction and antimicrobial effect for the studied peptides. By contrast cell-penetrating properties can be coupled to both the secondary structure and the degree of order of the peptides. © 2012 Elsevier Inc.

  15. Acylation of Therapeutic Peptides

    DEFF Research Database (Denmark)

    Trier, Sofie; Henriksen, Jonas Rosager; Jensen, Simon Bjerregaard

    ) , which promotes intestinal growth and is used to treat bowel disorders such as inflammatory bowel diseases and short bowel syndrome, and the 32 amino acid salmon calcitonin (sCT), which lowers blood calcium and is employed in the treatment of post-menopausal osteoporosis and hypercalcemia. The two...... peptides are similar in size and structure, but oppositely charged at physiological pH. Both peptides were acylated with linear acyl chains of systematically increasing length, where sCT was furthermore acylated at two different positions on the peptide backbone. For GLP-2, we found that increasing acyl...... remained optimal overall. The results indicate that rational acylation of GLP-2 can increase its in vitro intestinal absorption, alone or in combination with permeation enhancers, and are consistent with the initial project hypothesis. For sCT, an unpredicted effect of acylation largely superseded...

  16. Synergistic gene and drug tumor therapy using a chimeric peptide.

    Science.gov (United States)

    Han, Kai; Chen, Si; Chen, Wei-Hai; Lei, Qi; Liu, Yun; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2013-06-01

    Co-delivery of gene and drug for synergistic therapy has provided a promising strategy to cure devastating diseases. Here, an amphiphilic chimeric peptide (Fmoc)2KH7-TAT with pH-responsibility for gene and drug delivery was designed and fabricated. As a drug carrier, the micelles self-assembled from the peptide exhibited a much faster doxorubicin (DOX) release rate at pH 5.0 than that at pH 7.4. As a non-viral gene vector, (Fmoc)(2)KH(7)-TAT peptide could satisfactorily mediate transfection of pGL-3 reporter plasmid with or without the existence of serum in both 293T and HeLa cell-lines. Besides, the endosome escape capability of peptide/DNA complexes was investigated by confocal laser scanning microscopy (CLSM). To evaluate the co-delivery efficiency and the synergistic anti-tumor effect of gene and drug, p53 plasmid and DOX were simultaneously loaded in the peptide micelles to form micelleplexes during the self-assembly of the peptide. Cellular uptake and intracellular delivery of gene and drug were studied by CLSM and flow cytometry respectively. And p53 protein expression was determined via Western blot analysis. The in vitro cytotoxicity and in vivo tumor inhibition effect were also studied. Results suggest that the co-delivery of gene and drug from peptide micelles resulted in effective cell growth inhibition in vitro and significant tumor growth restraining in vivo. The chimeric peptide-based gene and drug co-delivery system will find great potential for tumor therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Towards generation of bioactive peptides from meat industry waste proteins: Generation of peptides using commercial microbial proteases.

    Science.gov (United States)

    Ryder, Kate; Bekhit, Alaa El-Din; McConnell, Michelle; Carne, Alan

    2016-10-01

    Five commercially available food-grade microbial protease preparations were evaluated for their ability to hydrolyse meat myofibrillar and connective tissue protein extracts to produce bioactive peptides. A bacterial-derived protease (HT) extensively hydrolysed both meat protein extracts, producing peptide hydrolysates with significant in vitro antioxidant and ACE inhibitor activities. The hydrolysates retained bioactivity after simulated gastrointestinal hydrolysis challenge. Gel permeation chromatography sub-fractionation of the crude protein hydrolysates showed that the smaller peptide fractions exhibited the highest antioxidant and ACE inhibitor activities. OFFGEL electrophoresis of the small peptides of both hydrolysates showed that low isoelectric point peptides had antioxidant activity; however, no consistent relationship was observed between isoelectric point and ACE inhibition. Cell-based assays indicated that the hydrolysates present no significant cytotoxicity towards Vero cells. The results indicate that HT protease hydrolysis of meat myofibrillar and connective tissue protein extracts produces bioactive peptides that are non-cytotoxic, should be stable in the gastrointestinal tract and may contain novel bioactive peptide sequences. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Therapeutic HIV Peptide Vaccine

    DEFF Research Database (Denmark)

    Fomsgaard, Anders

    2015-01-01

    Therapeutic vaccines aim to control chronic HIV infection and eliminate the need for lifelong antiretroviral therapy (ART). Therapeutic HIV vaccine is being pursued as part of a functional cure for HIV/AIDS. We have outlined a basic protocol for inducing new T cell immunity during chronic HIV-1...... infection directed to subdominant conserved HIV-1 epitopes restricted to frequent HLA supertypes. The rationale for selecting HIV peptides and adjuvants are provided. Peptide subunit vaccines are regarded as safe due to the simplicity, quality, purity, and low toxicity. The caveat is reduced immunogenicity...

  19. Descriptors for antimicrobial peptides

    DEFF Research Database (Denmark)

    Jenssen, Håvard

    2011-01-01

    of these are currently being used in quantitative structure--activity relationship (QSAR) studies for AMP optimization. Additionally, some key commercial computational tools are discussed, and both successful and less successful studies are referenced, illustrating some of the challenges facing AMP scientists. Through...... examples of different peptide QSAR studies, this review highlights some of the missing links and illuminates some of the questions that would be interesting to challenge in a more systematic fashion. Expert opinion: Computer-aided peptide QSAR using molecular descriptors may provide the necessary edge...

  20. Peptide profiling and the bioactivity character of yogurt in the simulated gastrointestinal digestion.

    Science.gov (United States)

    Jin, Yan; Yu, Yang; Qi, Yanxia; Wang, Fangjun; Yan, Jiaze; Zou, Hanfa

    2016-06-01

    This study investigated the relationship between peptide profiles and the bioactivity character of yogurt in simulated gastrointestinal trials. A total of 250, 434 and 466 peptides were identified by LC-MS/MS analyses of yogurt, gastric digest and pancreatic digest. Forty peptides of yogurt survived in gastrointestinal digestion. κ-CN and β-CN contributed the diversity of peptides during the fermentation process and gastrointestinal digestion, respectively. The favorite of κ-CN by lactic acid bacteria complemented gut digestion by hydrolyzing κ-CN, the low abundance milk proteins. The potential bioactivities were evaluated by in vitro ACE and DPP-IV inhibition assays. The ACE inhibition rate of the pancreatic digests was ~4 - and ~2 - fold greater than that of yogurt and the gastric digests. The ACE inhibitory peptides generated during gastrointestinal digestion improved the ACE inhibitory activity of the gastric and pancreatic digests. The DPP-IV inhibition rate of the pancreatic digest was ~6 - and ~3 - fold greater than that of yogurt and the gastric digest. The numbers of potential DPP-IV inhibitory peptides were positively correlated to the DPP-IV inhibitory activity of the gastric and pancreatic digests. The present study describes the characters and bioactivities of peptides from yogurt in a simulated gastrointestinal digestion. The number of peptides identified from yogurt and gastrointestinal digests by LC-MS/MS increased in the simulated gastrointestinal trials. The in vitro ACE and DPP-IV inhibition bioactivities revealed that the bioactivity of yogurt was enhanced during gastrointestinal digestion. The correlation between peptides and bioactivity in vitro indicated that not only the peptides amount but also the proportion of peptides with high bioactivities contributed to increased bioactivity during gastrointestinal digestion. The study of peptides identified from yogurt and digests revealed that the number of released peptides was not determined

  1. Characterization and identification of novel antidiabetic and anti-obesity peptides from camel milk protein hydrolysates.

    Science.gov (United States)

    Mudgil, Priti; Kamal, Hina; Yuen, Gan Chee; Maqsood, Sajid

    2018-09-01

    In-vitro inhibitory properties of peptides released from camel milk proteins against dipeptidyl peptidase-IV (DPP-IV), porcine pancreatic α-amylase (PPA), and porcine pancreatic lipase (PPL) were studied. Results revealed that upon hydrolysis by different enzymes, camel milk proteins displayed dramatic increase in inhibition of DPP-IV and PPL, but slight improvement in PPA inhibition was noticed. Peptide sequencing revealed a total of 20 and 3 peptides for A9 and B9 hydrolysates respectively, obtained the score of 0.8 or more on peptide ranker and were categorized as potential DPP-IV inhibitory peptides. KDLWDDFKGL in A9 and MPSKPPLL in B9 were identified as most potent PPA inhibitory peptide. For PPL inhibition only 7 and 2 peptides qualified as PPL inhibitory peptides from hydrolysates A9 and B9, respectively. The present study report for the first time PPA and PPL inhibitory and only second for DPP-IV inhibitory potential of protein hydrolysates from camel milk. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. Structural prediction and analysis of VIH-related peptides from selected crustacean species.

    Science.gov (United States)

    Nagaraju, Ganji Purna Chandra; Kumari, Nunna Siva; Prasad, Ganji Lakshmi Vara; Rajitha, Balney; Meenu, Madan; Rao, Manam Sreenivasa; Naik, Bannoth Reddya

    2009-08-17

    The tentative elucidation of the 3D-structure of vitellogenesis inhibiting hormone (VIH) peptides is conversely underprivileged by difficulties in gaining enough peptide or protein, diffracting crystals, and numerous extra technical aspects. As a result, no structural information is available for VIH peptide sequences registered in the Genbank. In this situation, it is not surprising that predictive methods have achieved great interest. Here, in this study the molt-inhibiting hormone (MIH) of the kuruma prawn (Marsupenaeus japonicus) is used, to predict the structure of four VIHrelated peptides in the crustacean species. The high similarity of the 3D-structures and the calculated physiochemical characteristics of these peptides suggest a common fold for the entire family.

  3. Peptide Logic Circuits Based on Chemoenzymatic Ligation for Programmable Cell Apoptosis.

    Science.gov (United States)

    Li, Yong; Sun, Sujuan; Fan, Lin; Hu, Shanfang; Huang, Yan; Zhang, Ke; Nie, Zhou; Yao, Shouzhou

    2017-11-20

    A novel and versatile peptide-based bio-logic system capable of regulating cell function is developed using sortase A (SrtA), a peptide ligation enzyme, as a generic processor. By modular peptide design, we demonstrate that mammalian cells apoptosis can be programmed by peptide-based logic operations, including binary and combination gates (AND, INHIBIT, OR, and AND-INHIBIT), and a complex sequential logic circuit (multi-input keypad lock). Moreover, a proof-of-concept peptide regulatory circuit was developed to analyze the expression profile of cell-secreted protein biomarkers and trigger cancer-cell-specific apoptosis. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Stereocontrolled Synthesis of Methyl Silanediol Peptide Mimics

    DEFF Research Database (Denmark)

    Nielsen, Lone; Lindsay, Karl; Faber, Jesper

    2007-01-01

     The treatment of chiral sulfinimines with (methyldiphenylsilyl)lithium gives R-(methyldiphenylsilyl)-sulfinamides with excellent diastereoselectivity, and in good yield. The presence of α-protons on the imines is also well tolerated. The sulfinamide auxiliary is easily removed via treatment with...... corresponding bis-TMS siloxane via protection with TMSCl, and converted back to the desired silanediol via hydrolysis with aqueous KOH. Efforts to apply this approach to biologically relevant silanediol peptide mimics, with a view to protease inhibition, are described....

  5. Antimicrobial Peptides: An Introduction.

    Science.gov (United States)

    Haney, Evan F; Mansour, Sarah C; Hancock, Robert E W

    2017-01-01

    The "golden era" of antibiotic discovery has long passed, but the need for new antibiotics has never been greater due to the emerging threat of antibiotic resistance. This urgency to develop new antibiotics has motivated researchers to find new methods to combat pathogenic microorganisms resulting in a surge of research focused around antimicrobial peptides (AMPs; also termed host defense peptides) and their potential as therapeutics. During the past few decades, more than 2000 AMPs have been identified from a diverse range of organisms (animals, fungi, plants, and bacteria). While these AMPs share a number of common features and a limited number of structural motifs; their sequences, activities, and targets differ considerably. In addition to their antimicrobial effects, AMPs can also exhibit immunomodulatory, anti-biofilm, and anticancer activities. These diverse functions have spurred tremendous interest in research aimed at understanding the activity of AMPs, and various protocols have been described to assess different aspects of AMP function including screening and evaluating the activities of natural and synthetic AMPs, measuring interactions with membranes, optimizing peptide function, and scaling up peptide production. Here, we provide a general overview of AMPs and introduce some of the methodologies that have been used to advance AMP research.

  6. An HMM posterior decoder for sequence feature prediction that includes homology information

    DEFF Research Database (Denmark)

    Käll, Lukas; Krogh, Anders Stærmose; Sonnhammer, Erik L. L.

    2005-01-01

    Motivation: When predicting sequence features like transmembrane topology, signal peptides, coil-coil structures, protein secondary structure or genes, extra support can be gained from homologs. Results: We present here a general hidden Markov model (HMM) decoding algorithm that combines probabil......Motivation: When predicting sequence features like transmembrane topology, signal peptides, coil-coil structures, protein secondary structure or genes, extra support can be gained from homologs. Results: We present here a general hidden Markov model (HMM) decoding algorithm that combines......://phobius.cgb.ki.se/poly.html . An implementation of the algorithm is available on request from the authors....

  7. Cloning an artificial gene encoding angiostatic anginex: From designed peptide to functional recombinant protein

    International Nuclear Information System (INIS)

    Brandwijk, Ricardo J.M.G.E.; Nesmelova, Irina; Dings, Ruud P.M.; Mayo, Kevin H.; Thijssen, Victor L.J.L.; Griffioen, Arjan W.

    2005-01-01

    Anginex, a designed peptide 33-mer, is a potent angiogenesis inhibitor and anti-tumor agent in vivo. Anginex functions by inhibiting endothelial cell (EC) proliferation and migration leading to detachment and apoptosis of activated EC's. To better understand tumor endothelium targeting properties of anginex and enable its use in gene therapy, we constructed an artificial gene encoding the biologically exogenous peptide and produced the protein recombinantly in Pichia pastoris. Mass spectrometry shows recombinant anginex to be a dimer and circular dichroism shows the recombinant protein folds with β-strand structure like the synthetic peptide. Moreover, like parent anginex, the recombinant protein is active at inhibiting EC growth and migration, as well as inhibiting angiogenesis in vivo in the chorioallantoic membrane of the chick embryo. This study demonstrated that it is possible to produce a functionally active protein version of a rationally designed peptide, using an artificial gene and the recombinant protein approach

  8. A liver metalloendopeptidase which degrades the circulating hypotensive peptide hormones bradykinin and atrial natriuretic peptide

    Directory of Open Access Journals (Sweden)

    Carvalho K.M.

    1999-01-01

    Full Text Available A new metalloendopeptidase was purified to apparent homogeneity from a homogenate of normal human liver using successive steps of chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-200. The purified enzyme hydrolyzed the Pro7-Phe8 bond of bradykinin and the Ser25-Tyr26 bond of atrial natriuretic peptide. No cleavage was produced in other peptide hormones such as vasopressin, oxytocin or Met- and Leu-enkephalin. This enzyme activity was inhibited by 1 mM divalent cation chelators such as EDTA, EGTA and o-phenanthroline and was insensitive to 1 µM phosphoramidon and captopril, specific inhibitors of neutral endopeptidase (EC 3.4.24.11 and angiotensin-converting enzyme (EC 3.4.15.1, respectively. With Mr 85 kDa, the enzyme exhibits optimal activity at pH 7.5. The high affinity of this endopeptidase for bradykinin (Km = 10 µM and for atrial natriuretic peptide (Km = 5 µM suggests that it may play a physiological role in the inactivation of these circulating hypotensive peptide hormones.

  9. Signal peptide of eosinophil cationic protein is toxic to cells lacking signal peptide peptidase

    International Nuclear Information System (INIS)

    Wu, C.-M.; Chang, Margaret Dah-Tsyr

    2004-01-01

    Eosinophil cationic protein (ECP) is a toxin secreted by activated human eosinophils. The properties of mature ECP have been well studied but those of the signal peptide of ECP (ECPsp) are not clear. In this study, several chimeric proteins containing N-terminal fusion of ECPsp were generated, and introduced into Escherichia coli, Pichia pastoris, and human epidermoid carcinoma cell line A431 to study the function of ECPsp. We found that expression of ECPsp chimeric proteins inhibited the growth of E. coli and P. pastoris but not A431 cells. Primary sequence analysis and in vitro transcription/translation of ECPsp have revealed that it is a potential substrate for human signal peptide peptidase (hSPP), an intramembrane protease located in endoplasmic reticulum. In addition, knockdown of the hSPP mRNA expression in ECPsp-eGFP/A431 cells caused the growth inhibitory effect, whereas complementally expression of hSPP in P. pastoris system rescued the cell growth. Taken together, we have demonstrated that ECPsp is a toxic signal peptide, and expression of hSPP protects the cells from growth inhibition

  10. Novel nonpeptidic inhibitors of peptide deformylase.

    Science.gov (United States)

    Jayasekera, M M; Kendall, A; Shammas, R; Dermyer, M; Tomala, M; Shapiro, M A; Holler, T P

    2000-09-15

    A novel series of nonpeptidic compounds structurally related to the known anticholesteremic thyropropic acid were found to inhibit Escherichia coli peptide deformylase (PDF), with IC50 values in the low-micromolar range. Kinetic analysis of [4-(4-hydroxyphenoxy)-3,5-diiodophenyl]acetic acid reveals competitive inhibition, with a Ki value of 0.66 +/- 0.007 microM. A structure-activity relationship study demonstrates that the carboxylate is required for activity, while the distal phenolic function can be methylated without significant effect. Either decreasing the number of iodine atoms on the molecule to one or increasing the number of iodine atoms to four results in the loss of an order of magnitude in potency. These compounds are the first nonpeptidic inhibitors disclosed and represent a template from which better inhibitors might be designed.

  11. Ligand-regulated peptide aptamers.

    Science.gov (United States)

    Miller, Russell A

    2009-01-01

    The peptide aptamer approach employs high-throughput selection to identify members of a randomized peptide library displayed from a scaffold protein by virtue of their interaction with a target molecule. Extending this approach, we have developed a peptide aptamer scaffold protein that can impart small-molecule control over the aptamer-target interaction. This ligand-regulated peptide (LiRP) scaffold, consisting of the protein domains FKBP12, FRB, and GST, binds to the cell-permeable small-molecule rapamycin and the binding of this molecule can prevent the interaction of the randomizable linker region connecting FKBP12 with FRB. Here we present a detailed protocol for the creation of a peptide aptamer plasmid library, selection of peptide aptamers using the LiRP scaffold in a yeast two-hybrid system, and the screening of those peptide aptamers for a ligand-regulated interaction.

  12. Biosynthesis of cardiac natriuretic peptides

    DEFF Research Database (Denmark)

    Goetze, Jens Peter

    2010-01-01

    Cardiac-derived peptide hormones were identified more than 25 years ago. An astonishing amount of clinical studies have established cardiac natriuretic peptides and their molecular precursors as useful markers of heart disease. In contrast to the clinical applications, the biogenesis of cardiac...... peptides has only been elucidated during the last decade. The cellular synthesis including amino acid modifications and proteolytic cleavages has proven considerably more complex than initially perceived. Consequently, the elimination phase of the peptide products in circulation is not yet well....... An inefficient post-translational prohormone maturation will also affect the biology of the cardiac natriuretic peptide system. This review aims at summarizing the myocardial synthesis of natriuretic peptides focusing on B-type natriuretic peptide, where new data has disclosed cardiac myocytes as highly...

  13. Radiolabelled peptides for oncological diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Laverman, Peter; Boerman, Otto C.; Oyen, Wim J.G. [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, Nijmegen (Netherlands); Sosabowski, Jane K. [Queen Mary University of London, Centre for Molecular Oncology, Barts Cancer Institute, London (United Kingdom)

    2012-02-15

    Radiolabelled receptor-binding peptides targeting receptors (over)expressed on tumour cells are widely under investigation for tumour diagnosis and therapy. The concept of using radiolabelled receptor-binding peptides to target receptor-expressing tissues in vivo has stimulated a large body of research in nuclear medicine. The {sup 111}In-labelled somatostatin analogue octreotide (OctreoScan trademark) is the most successful radiopeptide for tumour imaging, and was the first to be approved for diagnostic use. Based on the success of these studies, other receptor-targeting peptides such as cholecystokinin/gastrin analogues, glucagon-like peptide-1, bombesin (BN), chemokine receptor CXCR4 targeting peptides, and RGD peptides are currently under development or undergoing clinical trials. In this review, we discuss some of these peptides and their analogues, with regard to their potential for radionuclide imaging of tumours. (orig.)

  14. Maize EMBRYO SAC family peptides interact differentially with pollen tubes and fungal cells.

    Science.gov (United States)

    Woriedh, Mayada; Merkl, Rainer; Dresselhaus, Thomas

    2015-08-01

    EMBRYO SAC1-4 (ES1-4) peptides belong to the defensin subgroup of cysteine-rich peptides known to mediate pollen tube burst in Zea mays (maize). ES1-4 are reported here to also be capable of inhibiting germination and growth of the maize fungal pathogens Fusarium graminearum and Ustilago maydis at higher concentrations. Dividing the peptides into smaller pieces showed that a 15-amino-acid peptide located in a highly variable loop region lacking similarity to other defensins or defensin-like peptides binds to maize pollen tube surfaces, causing swelling prior to burst. This peptide fragment and a second conserved neighbouring fragment showed suppression of fungal germination and growth. The two peptides caused swelling of fungal cells, production of reactive oxygen species, and finally the formation of big vacuoles prior to burst at high peptide concentration. Furthermore, peptide fragments were found to bind differently to fungal cells. In necrotrophic F. graminearum, a peptide fragment named ES-d bound only at cell surfaces whereas the peptide ES-c bound at cell surfaces and also accumulated inside cells. Conversely, in biotrophic U. maydis, both peptide fragments accumulated inside cells, but, if applied at higher concentration, ES-c but not ES-d accumulated mainly in vacuoles. Mapping of peptide interaction sites identified amino acids differing in pollen tube burst and fungal response reactions. In summary, these findings indicate that residues targeting pollen tube burst in maize are specific to the ES family, while residues targeting fungal growth are conserved within defensins and defensin-like peptides. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  15. MHC class II-derived peptides can bind to class II molecules, including self molecules, and prevent antigen presentation

    DEFF Research Database (Denmark)

    Rosloniec, E F; Vitez, L J; Buus, S

    1990-01-01

    the alpha k-3 peptide binds slightly less well. These combined data, suggesting that class II-derived peptides can bind to MHC class II molecules, including the autologous molecule from which they are derived, have important implications for the molecular basis of alloreactivity and autoreactivity. Further...... found in the first and third polymorphic regions (PMR) of the A alpha k chain (alpha k-1 and alpha k-3) were capable of inhibiting the presentation of three different HEL-derived peptide antigens to their appropriate T cells. In addition, the alpha k-1 peptide inhibited the presentation of the OVA(323......-339) immunodominant peptide to the I-Ad-restricted T cell hybridomas specific for it. Prepulsing experiments demonstrated that the PMR peptides were interacting with the APC and not with the T cell hybridomas. These observations were confirmed and extended by the demonstration that the alpha k-1 and alpha k-3...

  16. Structural Principles in the Development of Cyclic Peptidic Enzyme Inhibitors

    Science.gov (United States)

    Xu, Peng; Andreasen, Peter A.; Huang, Mingdong

    2017-01-01

    This review summarizes our studies in the development of small cyclic peptides for specifically modulating enzyme activity. Serine proteases share highly similar active sites but perform diverse physiological and pathological functions. From a phage-display peptide library, we isolated two mono-cyclic peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), which inhibit the activity of human and murine urokinase-type plasminogen activators (huPA and muPA) with Ki values in the micromolar or sub-micromolar range, respectively. The following affinity maturations significantly enhanced the potencies of the two peptides, 10-fold and >250-fold for upain-1 and mupain-1, respectively. The most potent muPA inhibitor has a potency (Ki = 2 nM) and specificity comparable to mono-clonal antibodies. Furthermore, we also found an unusual feature of mupain-1 that its inhibitory potency can be enhanced by increasing the flexibility, which challenges the traditional viewpoint that higher rigidity leading to higher affinity. Moreover, by changing a few key residues, we converted mupain-1 from a uPA inhibitor to inhibitors of other serine proteases, including plasma kallikrein (PK) and coagulation factor XIa (fXIa). PK and fXIa inhibitors showed Ki values in the low nanomolar range and high specificity. Our studies demonstrate the versatility of small cyclic peptides to engineer inhibitory potency against serine proteases and to provide a new strategy for generating peptide inhibitors of serine proteases. PMID:29104489

  17. In silico panning for a non-competitive peptide inhibitor

    Directory of Open Access Journals (Sweden)

    Ikebukuro Kazunori

    2007-01-01

    Full Text Available Abstract Background Peptide ligands have tremendous therapeutic potential as efficacious drugs. Currently, more than 40 peptides are available in the market for a drug. However, since costly and time-consuming synthesis procedures represent a problem for high-throughput screening, novel procedures to reduce the time and labor involved in screening peptide ligands are required. We propose the novel approach of 'in silico panning' which consists of a two-stage screening, involving affinity selection by docking simulation and evolution of the peptide ligand using genetic algorithms (GAs. In silico panning was successfully applied to the selection of peptide inhibitor for water-soluble quinoprotein glucose dehydrogenase (PQQGDH. Results The evolution of peptide ligands for a target enzyme was achieved by combining a docking simulation with evolution of the peptide ligand using genetic algorithms (GAs, which mimic Darwinian evolution. Designation of the target area as next to the substrate-binding site of the enzyme in the docking simulation enabled the selection of a non-competitive inhibitor. In all, four rounds of selection were carried out on the computer; the distribution of the docking energy decreased gradually for each generation and improvements in the docking energy were observed over the four rounds of selection. One of the top three selected peptides with the lowest docking energy, 'SERG' showed an inhibitory effect with Ki value of 20 μM. PQQGDH activity, in terms of the Vmax value, was 3-fold lower than that of the wild-type enzyme in the presence of this peptide. The mechanism of the SERG blockage of the enzyme was identified as non-competitive inhibition. We confirmed the specific binding of the peptide, and its equilibrium dissociation constant (KD value was calculated as 60 μM by surface plasmon resonance (SPR analysis. Conclusion We demonstrate an effective methodology of in silico panning for the selection of a non

  18. Evaluation of peptide designing strategy against subunit reassociation in mucin 1: A steered molecular dynamics approach.

    Directory of Open Access Journals (Sweden)

    J Lesitha Jeeva Kumari

    Full Text Available Subunit reassociation in mucin 1, a breast cancer tumor marker, is reported as one of the critical factors for its cytoplasmic activation. Inhibition of its heterodimeric association would therefore result in loss of its function and alter disease progression. The present study aimed at evaluating peptide inhibitor designing strategies that may serve as antagonist against this receptor-ligand alliance. Several peptides and their derivatives were designed based on native residues, subunit interface, hydrogen bonding and secondary structure. Docking studies with the peptides were carried on the receptor subunit and their binding affinities were evaluated using steered molecular dynamics simulation and umbrella sampling. Our results showed that among all the different classes of peptides evaluated, the receptor based peptide showed the highest binding affinity. This result was concurrent with the experimental observation that the receptor-ligand alliance in mucin 1 is highly specific. Our results also show that peptide ligand against this subunit association is only stabilized through native residue inter-protein interaction irrespective of the peptide structure, peptide length and number of hydrogen bonds. Consistency in binding affinity, pull force and free energy barrier was observed with only the receptor derived peptides which resulted in favorable interprotein interactions at the interface. Several observations were made and discussed which will eventually lead to designing efficient peptide inhibitors against mucin 1 heterodimeric subunit reassociation.

  19. Antimicrobial Peptides (AMPs

    Directory of Open Access Journals (Sweden)

    Mehrzad Sadredinamin

    2016-04-01

    Full Text Available Antimicrobial peptides (AMPs are extensive group of molecules that produced by variety tissues of invertebrate, plants, and animal species which play an important role in their immunity response. AMPs have different classifications such as; biosynthetic machines, biological sources, biological functions, molecular properties, covalent bonding patterns, three dimensional structures, and molecular targets.These molecules have multidimensional properties including antimicrobial activity, antiviral activity, antifungal activity, anti-parasite activity, biofilm control, antitumor activity, mitogens activity and linking innate to adaptive immunity that making them promising agents for therapeutic drugs. In spite of this advantage of AMPs, their clinical developments have some limitation for commercial development. But some of AMPs are under clinical trials for the therapeutic purpose such as diabetic foot ulcers, different bacterial infections and tissue damage. In this review, we emphasized on the source, structure, multidimensional properties, limitation and therapeutic applications of various antimicrobial peptides.

  20. Glucagon-like peptide-1 7-36 amide and peptide YY from the L-cell of the ileal mucosa are potent inhibitors of vagally induced gastric acid secretion in man

    DEFF Research Database (Denmark)

    Wettergren, A; Petersen, H; Orskov, C

    1994-01-01

    BACKGROUND: Glucagon-like peptide (GLP-1) 7-36 amide and peptide YY (PYY) from the L-cell of the ileal mucosa are potent inhibitors of gastric acid secretion in man. It is not clear, however, by which mechanism(s) they inhibit acid secretion. In dogs the inhibitory effect of PYY on acid secretion...

  1. Characterization of the anti-HIV effects of native lactoferrin and other milk proteins and protein-derived peptides

    NARCIS (Netherlands)

    Berkhout, Ben; van Wamel, Jeroen L. B.; Beljaars, Leonie; Meijer, Dirk K. F.; Visser, Servaas; Floris, René

    2002-01-01

    In a search for natural proteins with anti-HIV activity, we screened a large set of purified proteins from bovine milk and peptide fragments thereof. Because several charged proteins and peptides are known to inhibit the process of virus entry, we selected proteins with an unusual charge composition

  2. Characterization of the anti-HIV effects of native lactoferrin and other milk proteins and protein-derived peptides

    NARCIS (Netherlands)

    Berkhout, B; van Wamel, JLB; Beljaars, L; Meijer, DKF; Visser, Servaas; Floris, R

    In a search for natural proteins with anti-HIV activity, we screened a large set of purified proteins from bovine milk and peptide fragments thereof. Because several charged proteins and peptides are known to inhibit the process of virus entry, we selected proteins with an unusual charge composition

  3. Lactoferricin B-derived peptides with inhibitory effects on ECE-dependent vasoconstriction.

    Science.gov (United States)

    Fernández-Musoles, Ricardo; López-Díez, José Javier; Torregrosa, Germán; Vallés, Salvador; Alborch, Enrique; Manzanares, Paloma; Salom, Juan B

    2010-10-01

    Endothelin-converting enzyme (ECE), a key peptidase in the endothelin (ET) system, cleaves inactive big ET-1 to produce active ET-1, which binds to ET(A) receptors to exert its vasoconstrictor and pressor effects. ECE inhibition could be beneficial in the treatment of hypertension. In this study, a set of eight lactoferricin B (LfcinB)-derived peptides, previously characterized in our laboratory as angiotensin-converting enzyme (ACE) inhibitory peptides, was examined for their inhibitory effects on ECE. In vitro inhibitory effects on ECE activity were assessed using both the synthetic fluorogenic peptide substrate V (FPS V) and the natural substrate big ET-1. To study vasoactive effects, an ex vivo functional assay was developed using isolated rabbit carotid artery segments. With FPS V, only four LfcinB-derived peptides induced inhibition of ECE activity, whereas the eight peptides showed ECE inhibitory effects with big ET-1 as substrate. Regarding the ex vivo assays, six LfcinB-derived peptides showed inhibition of big ET-1-induced, ECE-dependent vasoconstriction. A positive correlation between the inhibitory effects of LfcinB-derived peptides on ECE activity when using big ET-1 and the inhibitory effects on ECE-dependent vasoconstriction was shown. ECE-independent vasoconstriction induced by ET-1 was not affected, thus discarding effects of LfcinB-derived peptides on ET(A) receptors or intracellular signal transduction mechanisms. In conclusion, a combined in vitro and ex vivo method to assess the effects of potentially antihypertensive peptides on the ET system has been developed and applied to show the inhibitory effects on ECE-dependent vasoconstriction of six LfcinB-derived peptides, five of which were dual vasopeptidase (ACE/ECE) inhibitors. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. Corrosion inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, A O

    1965-12-29

    An acid corrosion-inhibiting composition consists essentially of a sugar, and an alkali metal salt selected from the group consisting of iodides and bromides. The weight ratio of the sugar to the alkali metal salt is between 2:1 and about 20,000:1. Also, a corrosion- inhibited phosphoric acid composition comprising at least about 20 wt% of phosphoric acid and between about 0.1 wt% and about 10 wt% of molasses, and between about 0.0005 wt% and about 1 wt% of potassium iodide. The weight ratio of molasses to iodide is greater than about 2:1. (11 claims)

  5. A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

    Science.gov (United States)

    Skottrup, Peter Durand; Sørensen, Grete; Ksiazek, Miroslaw; Potempa, Jan; Riise, Erik

    2012-01-01

    Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18) by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15), shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP) was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG) could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48). Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

  6. A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

    Directory of Open Access Journals (Sweden)

    Peter Durand Skottrup

    Full Text Available Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18 by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15, shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48. Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

  7. Structure–activity relationships of the human prothrombin kringle-2 peptide derivative NSA9: anti-proliferative activity and cellular internalization

    OpenAIRE

    Hwang, Hyun Sook; Kim, Dong Won; Kim, Soung Soo

    2006-01-01

    The human prothrombin kringle-2 protein inhibits angiogenesis and LLC (Lewis lung carcinoma) growth and metastasis in mice. Additionally, the NSA9 peptide (NSAVQLVEN) derived from human prothrombin kringle-2 has been reported to inhibit the proliferation of BCE (bovine capillary endothelial) cells and CAM (chorioallantoic membrane) angiogenesis. In the present study, we examined the structure–activity relationships of the NSA9 peptide in inhibiting the proliferation of endothelial cells lines...

  8. Solid-phase peptide synthesis

    DEFF Research Database (Denmark)

    Jensen, Knud Jørgen

    2013-01-01

    This chapter provides an introduction to and overview of peptide chemistry with a focus on solid-phase peptide synthesis. The background, the most common reagents, and some mechanisms are presented. This chapter also points to the different chapters and puts them into perspective.......This chapter provides an introduction to and overview of peptide chemistry with a focus on solid-phase peptide synthesis. The background, the most common reagents, and some mechanisms are presented. This chapter also points to the different chapters and puts them into perspective....

  9. Improving Peptide Applications Using Nanotechnology.

    Science.gov (United States)

    Narayanaswamy, Radhika; Wang, Tao; Torchilin, Vladimir P

    2016-01-01

    Peptides are being successfully used in various fields including therapy and drug delivery. With advancement in nanotechnology and targeted delivery carrier systems, suitable modification of peptides has enabled achievement of many desirable goals over-riding some of the major disadvantages associated with the delivery of peptides in vivo. Conjugation or physical encapsulation of peptides to various nanocarriers, such as liposomes, micelles and solid-lipid nanoparticles, has improved their in vivo performance multi-fold. The amenability of peptides to modification in chemistry and functionalization with suitable nanocarriers are very relevant aspects in their use and have led to the use of 'smart' nanoparticles with suitable linker chemistries that favor peptide targeting or release at the desired sites, minimizing off-target effects. This review focuses on how nanotechnology has been used to improve the number of peptide applications. The paper also focuses on the chemistry behind peptide conjugation to nanocarriers, the commonly employed linker chemistries and the several improvements that have already been achieved in the areas of peptide use with the help of nanotechnology.

  10. Anticancer peptides from bacteria

    Directory of Open Access Journals (Sweden)

    Tomasz M. Karpiński

    2013-08-01

    Full Text Available Cancer is a leading cause of death in the world. The rapid development of medicine and pharmacology allows to create new and effective anticancer drugs. Among modern anticancer drugs are bacterial proteins. Until now has been shown anticancer activity among others azurin and exotoxin A from Pseudomonas aeruginosa, Pep27anal2 from Streptococcus pneumoniae, diphtheria toxin from Corynebacterium diphtheriae, and recently discovered Entap from Enterococcus sp. The study presents the current data regarding the properties, action and anticancer activity of listed peptides.

  11. Development of second generation peptides modulating cellular adiponectin receptor responses

    Directory of Open Access Journals (Sweden)

    Laszlo eOtvos

    2014-10-01

    Full Text Available The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC. In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399. The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM - low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400 was designed that counteracted 10-1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400 at similar concentrations will be an important target validation tool to study adiponectin functions.

  12. Development of second generation peptides modulating cellular adiponectin receptor responses

    Science.gov (United States)

    Otvos, Laszlo; Knappe, Daniel; Hoffmann, Ralf; Kovalszky, Ilona; Olah, Julia; Hewitson, Tim; Stawikowska, Roma; Stawikowski, Maciej; Cudic, Predrag; Lin, Feng; Wade, John; Surmacz, Eva; Lovas, Sandor

    2014-10-01

    The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC). In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML) cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399). The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM - low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400) was designed that counteracted 10-1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400) at similar concentrations will be an important target validation tool to study adiponectin functions.

  13. Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides.

    Science.gov (United States)

    Kaconis, Yani; Kowalski, Ina; Howe, Jörg; Brauser, Annemarie; Richter, Walter; Razquin-Olazarán, Iosu; Iñigo-Pestaña, Melania; Garidel, Patrick; Rössle, Manfred; Martinez de Tejada, Guillermo; Gutsmann, Thomas; Brandenburg, Klaus

    2011-06-08

    Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  14. Targeting Interleukin-4 Receptor Alpha by Hybrid Peptide for Novel Biliary Tract Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Kahori Seto

    2014-01-01

    Full Text Available It is known that the interleukin-4 receptor α (IL-4Rα is highly expressed on the surface of various human solid tumors. We previously designed novel IL-4Rα-lytic hybrid peptide composed of binding peptide to IL-4Rα and cell-lytic peptide and reported that the designed IL-4Rα-lytic hybrid peptide exhibited cytotoxic and antitumor activity both in vitro and in vivo against the human pancreatic cancer cells expressing IL-4Rα. Here, we evaluated the antitumor activity of the IL-4Rα-lytic hybrid peptide as a novel molecular targeted therapy for human biliary tract cancer (BTC. The IL-4Rα-lytic hybrid peptide showed cytotoxic activity in six BTC cell lines with a concentration that killed 50% of all cells (IC50 as low as 5 μM. We also showed that IL-4Rα-lytic hybrid peptide in combination with gemcitabine exhibited synergistic cytotoxic activity in vitro. In addition, intravenous administration of IL-4Rα-lytic hybrid peptide significantly inhibited tumor growth in a xenograft model of human BTC in vivo. Taken together, these results indicated that the IL-4Rα-lytic hybrid peptide is a potent agent that might provide a novel therapy for patients with BTC.

  15. Lactoferrin-derived Peptides Active towards Influenza: Identification of Three Potent Tetrapeptide Inhibitors.

    Science.gov (United States)

    Scala, Maria Carmina; Sala, Marina; Pietrantoni, Agostina; Spensiero, Antonia; Di Micco, Simone; Agamennone, Mariangela; Bertamino, Alessia; Novellino, Ettore; Bifulco, Giuseppe; Gomez-Monterrey, Isabel M; Superti, Fabiana; Campiglia, Pietro

    2017-09-06

    Bovine lactoferrin is a biglobular multifunctional iron binding glycoprotein that plays an important role in innate immunity against infections. We have previously demonstrated that selected peptides from bovine lactoferrin C-lobe are able to prevent both Influenza virus hemagglutination and cell infection. To deeper investigate the ability of lactoferrin derived peptides to inhibit Influenza virus infection, in this study we identified new bovine lactoferrin C-lobe derived sequences and corresponding synthetic peptides were synthesized and assayed to check their ability to prevent viral hemagglutination and infection. We identified three tetrapeptides endowed with broad anti-Influenza activity and able to inhibit viral infection in a concentration range femto- to picomolar. Our data indicate that these peptides may constitute a non-toxic tool for potential applications as anti-Influenza therapeutics.

  16. The TFPI-2 derived peptide EDC34 improves outcome of gram-negative sepsis.

    Science.gov (United States)

    Papareddy, Praveen; Kalle, Martina; Sørensen, Ole E; Malmsten, Martin; Mörgelin, Matthias; Schmidtchen, Artur

    2013-01-01

    Sepsis is characterized by a dysregulated host-pathogen response, leading to high cytokine levels, excessive coagulation and failure to eradicate invasive bacteria. Novel therapeutic strategies that address crucial pathogenetic steps during infection are urgently needed. Here, we describe novel bioactive roles and therapeutic anti-infective potential of the peptide EDC34, derived from the C-terminus of tissue factor pathway inhibitor-2 (TFPI-2). This peptide exerted direct bactericidal effects and boosted activation of the classical complement pathway including formation of antimicrobial C3a, but inhibited bacteria-induced activation of the contact system. Correspondingly, in mouse models of severe Escherichia coli and Pseudomonas aeruginosa infection, treatment with EDC34 reduced bacterial levels and lung damage. In combination with the antibiotic ceftazidime, the peptide significantly prolonged survival and reduced mortality in mice. The peptide's boosting effect on bacterial clearance paired with its inhibiting effect on excessive coagulation makes it a promising therapeutic candidate for invasive Gram-negative infections.

  17. Characterization of antidiabetic and antihypertensive properties of canary seed (Phalaris canariensis L.) peptides.

    Science.gov (United States)

    Estrada-Salas, Patricia A; Montero-Morán, Gabriela M; Martínez-Cuevas, Pedro P; González, Carmen; Barba de la Rosa, Ana P

    2014-01-15

    Canary grass is used as traditional food for diabetes and hypertension treatment. The aim of this work is to characterize the biological activity of encrypted peptides released after gastrointestinal digestion of canary seed proteins. Canary peptides showed 43.5% inhibition of dipeptidyl peptidase IV (DPPIV) and 73.5% inhibition of angiotensin-converting enzyme (ACE) activity. An isolated perfused rat heart system was used to evaluate the canary seed vasoactive effect. Nitric oxide (NO), a major vasodilator agent, was evaluated in the venous effluent from isolated perfused rat heart. Canary seed peptides (1 μg/mL) were able to induce the production of NO (12.24 μM) in amounts similar to those induced by captopril (CPT) and bradykinin (BK). These results show that encrypted peptides in canary seed have inhibitory activity against DPPIV and ACE, enzymes that are targets for diabetes and hypertension treatments.

  18. Antiviral cationic peptides as a strategy for innovation in global health therapeutics for dengue virus: high yield production of the biologically active recombinant plectasin peptide.

    Science.gov (United States)

    Rothan, Hussin A; Mohamed, Zulqarnain; Suhaeb, Abdulrazzaq M; Rahman, Noorsaadah Abd; Yusof, Rohana

    2013-11-01

    Dengue virus infects millions of people worldwide, and there is no vaccine or anti-dengue therapeutic available. Antimicrobial peptides have been shown to possess effective antiviral activity against various viruses. One of the main limitations of developing these peptides as potent antiviral drugs is the high cost of production. In this study, high yield production of biologically active plectasin peptide was inexpensively achieved by producing tandem plectasin peptides as inclusion bodies in E. coli. Antiviral activity of the recombinant peptide towards dengue serotype-2 NS2B-NS3 protease (DENV2 NS2B-NS3pro) was assessed as a target to inhibit dengue virus replication in Vero cells. Single units of recombinant plectasin were collected after applying consecutive steps of refolding, cleaving by Factor Xa, and nickel column purification to obtain recombinant proteins of high purity. The maximal nontoxic dose (MNTD) of the recombinant peptide against Vero cells was 20 μM (100 μg/mL). The reaction velocity of DENV2 NS2B-NS3pro decreased significantly after increasing concentrations of recombinant plectasin were applied to the reaction mixture. Plectasin peptide noncompetitively inhibited DENV2 NS2B-NS3pro at Ki value of 5.03 ± 0.98 μM. The percentage of viral inhibition was more than 80% at the MNTD value of plectasin. In this study, biologically active recombinant plectasin which was able to inhibit dengue protease and viral replication in Vero cells was successfully produced in E. coli in a time- and cost- effective method. These findings are potentially important in the development of potent therapeutics against dengue infection.

  19. Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin

    International Nuclear Information System (INIS)

    Kim, Dae Hong; Lee, Ik Hwan; Nam, Seung Taek; Hong, Ji; Zhang, Peng; Hwang, Jae Sam; Seok, Heon; Choi, Hyemin; Lee, Dong Gun; Kim, Jae Il; Kim, Ho

    2014-01-01

    Highlights: • 11-mer peptide Lumbricusin, a defensin like peptide, is isolated from earthworm. • We here demonstrated that Lumbricusin has neurotropic and neuroprotective effects. • p27 degradation by Lumbricusin mediates effects of Lumbricusin on neuronal cells. - Abstract: We recently isolated a polypeptide from the earthworm Lumbricus terrestris that is structurally similar to defensin, a well-known antibacterial peptide. An 11-mer antibacterial peptide (NH 2 -RNRRWCIDQQA), designated Lumbricusin, was synthesized based on the amino acid sequence of the isolated polypeptide. Since we previously reported that CopA3, a dung beetle peptide, enhanced neuronal cell proliferation, we here examined whether Lumbricusin exerted neurotropic and/or neuroprotective effects. Lumbricusin treatment induced a time-dependent increase (∼51%) in the proliferation of human neuroblastoma SH-SY5Y cells. Lumbricusin also significantly inhibited the apoptosis and decreased viability induced by treatment with 6-hydroxy dopamine, a Parkinson’s disease-mimicking agent. Immunoblot analyses revealed that Lumbricusin treatment increased ubiquitination of p27 Kip1 protein, a negative regulator of cell-cycle progression, in SH-SY5Y cells, and markedly promoted its degradation. Notably, adenoviral-mediated over-expression of p27 Kip1 significantly blocked the antiapoptotic effect of Lumbricusin in 6-hydroxy dopamine-treated SH-SY5Y cells. These results suggest that promotion of p27 Kip1 degradation may be the main mechanism underlying the neuroprotective and neurotropic effects of Lumbricusin

  20. Multimerized CHR-derived peptides as HIV-1 fusion inhibitors.

    Science.gov (United States)

    Nomura, Wataru; Hashimoto, Chie; Suzuki, Takaharu; Ohashi, Nami; Fujino, Masayuki; Murakami, Tsutomu; Yamamoto, Naoki; Tamamura, Hirokazu

    2013-08-01

    To date, several HIV-1 fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of an HIV-1 envelope protein gp41 have been discovered. We have shown that a synthetic peptide mimetic of a trimer form of the CHR-derived peptide C34 has potent inhibitory activity against the HIV-1 fusion mechanism, compared to a monomer C34 peptide. The present study revealed that a dimeric form of C34 is evidently structurally critical for fusion inhibitors, and that the activity of multimerized CHR-derived peptides in fusion inhibition is affected by the properties of the unit peptides C34, SC34EK, and T20. The fluorescence-based study suggested that the N36-interactive sites of the C34 trimer, including hydrophobic residues, are exposed outside the trimer and that trimerization of C34 caused a remarkable increase in fusion inhibitory activity. The present results could be useful in the design of fusion inhibitors against viral infections which proceed via membrane fusion with host cells. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. Buwchitin: a ruminal peptide with antimicrobial potential against Enterococcus faecalis

    Science.gov (United States)

    Oyama, Linda B.; Crochet, Jean-Adrien; Edwards, Joan E.; Girdwood, Susan E.; Cookson, Alan R.; Fernandez-Fuentes, Narcis; Hilpert, Kai; Golyshin, Peter N.; Golyshina, Olga V.; Privé, Florence; Hess, Matthias; Mantovani, Hilario C.; Creevey, Christopher J.; Huws, Sharon A.

    2017-07-01

    Antimicrobial peptides (AMPs) are gaining popularity as alternatives for treatment of bacterial infections and recent advances in omics technologies provide new platforms for AMP discovery. We sought to determine the antibacterial activity of a novel antimicrobial peptide, buwchitin, against Enterococcus faecalis. Buwchitin was identified from a rumen bacterial metagenome library, cloned, expressed and purified. The antimicrobial activity of the recombinant peptide was assessed using a broth microdilution susceptibility assay to determine the peptide's killing kinetics against selected bacterial strains. The killing mechanism of buwchitin was investigated further by monitoring its ability to cause membrane depolarization (diSC3(5) method) and morphological changes in E. faecalis cells. Transmission electron micrographs of buwchitin treated E. faecalis cells showed intact outer membranes with blebbing, but no major damaging effects and cell morphology changes. Buwchitin had negligible cytotoxicity against defibrinated sheep erythrocytes. Although no significant membrane leakage and depolarization was observed, buwchitin at minimum inhibitory concentration (MIC) was bacteriostatic against E. faecalis cells and inhibited growth in vitro by 70% when compared to untreated cells. These findings suggest that buwchitin, a rumen derived peptide, has potential for antimicrobial activity against E. faecalis.

  2. Buwchitin: A Ruminal Peptide with Antimicrobial Potential against Enterococcus faecalis

    Directory of Open Access Journals (Sweden)

    Linda B. Oyama

    2017-07-01

    Full Text Available Antimicrobial peptides (AMPs are gaining popularity as alternatives for treatment of bacterial infections and recent advances in omics technologies provide new platforms for AMP discovery. We sought to determine the antibacterial activity of a novel antimicrobial peptide, buwchitin, against Enterococcus faecalis. Buwchitin was identified from a rumen bacterial metagenome library, cloned, expressed and purified. The antimicrobial activity of the recombinant peptide was assessed using a broth microdilution susceptibility assay to determine the peptide's killing kinetics against selected bacterial strains. The killing mechanism of buwchitin was investigated further by monitoring its ability to cause membrane depolarization (diSC3(5 method and morphological changes in E. faecalis cells. Transmission electron micrographs of buwchitin treated E. faecalis cells showed intact outer membranes with blebbing, but no major damaging effects and cell morphology changes. Buwchitin had negligible cytotoxicity against defibrinated sheep erythrocytes. Although no significant membrane leakage and depolarization was observed, buwchitin at minimum inhibitory concentration (MIC was bacteriostatic against E. faecalis cells and inhibited growth in vitro by 70% when compared to untreated cells. These findings suggest that buwchitin, a rumen derived peptide, has potential for antimicrobial activity against E. faecalis.

  3. Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dae Hong; Lee, Ik Hwan; Nam, Seung Taek; Hong, Ji; Zhang, Peng [Department of Life Science, College of Natural Science, Daejin University, Pocheon, Gyeonggido 487-711 (Korea, Republic of); Hwang, Jae Sam [Department of Agricultural Biology, National Academy of Agricultural Science, RDA, Suwon 441-707 (Korea, Republic of); Seok, Heon [Department of Biomedical Engineering, Jungwon University, Goesan, Chungcheongbukdo 367-700 (Korea, Republic of); Choi, Hyemin; Lee, Dong Gun [School of Life Sciences, KNU Creative Bioresearch Group (BK21 Plus Program), College of Natural Sciences, Kyungpook National University, Daehak-ro 80, Buk-gu, Daegu 702-701 (Korea, Republic of); Kim, Jae Il [School of Life Sciences, Gwangju Institute of Science and Technology, Oryong-dong, Buk-gu, Gwangju 500-712 (Korea, Republic of); Kim, Ho, E-mail: hokim@daejin.ac.kr [Department of Life Science, College of Natural Science, Daejin University, Pocheon, Gyeonggido 487-711 (Korea, Republic of)

    2014-06-06

    Highlights: • 11-mer peptide Lumbricusin, a defensin like peptide, is isolated from earthworm. • We here demonstrated that Lumbricusin has neurotropic and neuroprotective effects. • p27 degradation by Lumbricusin mediates effects of Lumbricusin on neuronal cells. - Abstract: We recently isolated a polypeptide from the earthworm Lumbricus terrestris that is structurally similar to defensin, a well-known antibacterial peptide. An 11-mer antibacterial peptide (NH{sub 2}-RNRRWCIDQQA), designated Lumbricusin, was synthesized based on the amino acid sequence of the isolated polypeptide. Since we previously reported that CopA3, a dung beetle peptide, enhanced neuronal cell proliferation, we here examined whether Lumbricusin exerted neurotropic and/or neuroprotective effects. Lumbricusin treatment induced a time-dependent increase (∼51%) in the proliferation of human neuroblastoma SH-SY5Y cells. Lumbricusin also significantly inhibited the apoptosis and decreased viability induced by treatment with 6-hydroxy dopamine, a Parkinson’s disease-mimicking agent. Immunoblot analyses revealed that Lumbricusin treatment increased ubiquitination of p27{sup Kip1} protein, a negative regulator of cell-cycle progression, in SH-SY5Y cells, and markedly promoted its degradation. Notably, adenoviral-mediated over-expression of p27{sup Kip1} significantly blocked the antiapoptotic effect of Lumbricusin in 6-hydroxy dopamine-treated SH-SY5Y cells. These results suggest that promotion of p27{sup Kip1} degradation may be the main mechanism underlying the neuroprotective and neurotropic effects of Lumbricusin.

  4. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    Zegers, N.D.

    1995-01-01

    Synthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps that lead to the

  5. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    N.D. Zegers (Netty)

    1995-01-01

    textabstractSynthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps

  6. Peptide radiopharmaceuticals in nuclear medicine

    International Nuclear Information System (INIS)

    Blok, D.; Vermeij, P.; Feitsma, R.I.J.; Pauwels, E.J.K.

    1999-01-01

    This article reviews the labelling of peptides that are recognised to be of interest for nuclear medicine or are the subject of ongoing nuclear medicine research. Applications and approaches to the labelling of peptide radiopharmaceuticals are discussed, and drawbacks in their development considered. (orig.)