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Sample records for albumin-derived peptides efficiently

  1. An albumin-derived peptide scaffold capable of binding and catalysis.

    PubMed

    Luisi, Immacolata; Pavan, Silvia; Fontanive, Giampaolo; Tossi, Alessandro; Benedetti, Fabio; Savoini, Adriano; Maurizio, Elisa; Sgarra, Riccardo; Sblattero, Daniele; Berti, Federico

    2013-01-01

    We have identified a 101-amino-acid polypeptide derived from the sequence of the IIA binding site of human albumin. The polypeptide contains residues that make contact with IIA ligands in the parent protein, and eight cysteine residues to form disulfide bridges, that stabilize the polypeptide structure. Seventy-four amino acids are located in six α-helical regions, while the remaining thirty-seven amino acids form six connecting coil/loop regions. A soluble GST fusion protein was expressed in E. coli in yields as high as 4 mg/l. This protein retains the IIA fragment's capacity to bind typical ligands such as warfarin and efavirenz and other albumin's functional properties such as aldolase activity and the ability to direct the stereochemical outcome of a diketone reduction. This newly cloned polypeptide thus represents a valuable starting point for the construction of libraries of binders and catalysts with improved proficiency.

  2. Hyperosmotic treatment synergistically boost efficiency of cell-permeable peptides

    PubMed Central

    Wang, Hu; Zhang, Ming; Zeng, Fanhui; Liu, Changbai

    2016-01-01

    Therapeutics delivery into cells has been hurdled due to the barrier of cytoplasmic membrane. Although cell penetrating peptide (CPP) can potentially serve as an intracellular drug delivery vehicle, the application of CPP-based delivery is limited because the unsatisfactory delivery efficiency of CPP conjugated potent cargos is challenging their applications in present. Thus, the development of strategies for enhancing the penetrating efficiency of CPP would therefore urgent need to be explored to increase the scope of potential applications. We report here the effects of glucose, sucrose and manntiol (abbreviated as GSM) combination facilitating the penetration efficiency of CPP peptide alone or CPP-GFP (green fluorescence protein) conjugation in cultured cell lines or primary cells. Moreover, osmoprotectants glycerol and glycine supplementation help cells cope with the stress from GSM combination. Thus, our present study suggests that GSM combination in the presence of osmoprotectant can work as a new strategy for CPP penetration enhancement. PMID:27213591

  3. Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules.

    PubMed

    Berkers, Celia R; de Jong, Annemieke; Schuurman, Karianne G; Linnemann, Carsten; Meiring, Hugo D; Janssen, Lennert; Neefjes, Jacques J; Schumacher, Ton N M; Rodenko, Boris; Ovaa, Huib

    2015-11-01

    Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I-restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags.

  4. Efficient conformational sampling of peptides adsorbed onto inorganic surfaces: insights from a quartz binding peptide.

    PubMed

    Wright, Louise B; Walsh, Tiffany R

    2013-04-07

    Harnessing the properties of biomolecules, such as peptides, adsorbed on inorganic surfaces is of interest to many cross-disciplinary areas of science, ranging from biomineralisation to nanomedicine. Key to advancing research in this area is determination of the peptide conformation(s) in its adsorbed state, at the aqueous interface. Molecular simulation is one such approach for accomplishing this goal. In this respect, use of temperature-based replica-exchange molecular dynamics (T-REMD) can yield enhanced sampling of the interfacial conformations, but does so at great computational expense, chiefly because of the need to include an explicit representation of water at the interface. Here, we investigate a number of more economical variations on REMD, chiefly those based on Replica Exchange with Solvent Tempering (REST), using the aqueous quartz-binding peptide S1-(100) α-quartz interfacial system as a benchmark. We also incorporate additional implementation details specifically targeted at improving sampling of biomolecules at interfaces. We find the REST-based variants yield configurational sampling of the peptide-surface system comparable with T-REMD, at a fraction of the computational time and resource. Our findings also deliver novel insights into the binding behaviour of the S1 peptide at the quartz (100) surface that are consistent with available experimental data.

  5. Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation

    NASA Astrophysics Data System (ADS)

    Frey, Brian L.; Ladror, Daniel T.; Sondalle, Samuel B.; Krusemark, Casey J.; Jue, April L.; Coon, Joshua J.; Smith, Lloyd M.

    2013-11-01

    The carboxyl groups of tryptic peptides were derivatized with a tertiary or quaternary amine labeling reagent to generate more highly charged peptide ions that fragment efficiently by electron transfer dissociation (ETD). All peptide carboxyl groups—aspartic and glutamic acid side-chains as well as C-termini—were derivatized with an average reaction efficiency of 99 %. This nearly complete labeling avoids making complex peptide mixtures even more complex because of partially-labeled products, and it allows the use of static modifications during database searching. Alkyl tertiary amines were found to be the optimal labeling reagent among the four types tested. Charge states are substantially higher for derivatized peptides: a modified tryptic digest of bovine serum albumin (BSA) generates ~90% of its precursor ions with z > 2, compared with less than 40 % for the unmodified sample. The increased charge density of modified peptide ions yields highly efficient ETD fragmentation, leading to many additional peptide identifications and higher sequence coverage (e.g., 70 % for modified versus only 43 % for unmodified BSA). The utility of this labeling strategy was demonstrated on a tryptic digest of ribosomal proteins isolated from yeast cells. Peptide derivatization of this sample produced an increase in the number of identified proteins, a >50 % increase in the sequence coverage of these proteins, and a doubling of the number of peptide spectral matches. This carboxyl derivatization strategy greatly improves proteome coverage obtained from ETD-MS/MS of tryptic digests, and we anticipate that it will also enhance identification and localization of post-translational modifications.

  6. CL22 - a novel cationic peptide for efficient transfection of mammalian cells.

    PubMed

    Haines, A M; Irvine, A S; Mountain, A; Charlesworth, J; Farrow, N A; Husain, R D; Hyde, H; Ketteringham, H; McDermott, R H; Mulcahy, A F; Mustoe, T L; Reid, S C; Rouquette, M; Shaw, J C; Thatcher, D R; Welsh, J H; Williams, D E; Zauner, W; Phillips, R O

    2001-01-01

    Condensing peptide-DNA complexes have great potential as nonviral agents for gene delivery. To date, however, such complexes have given transfection activities greatly inferior to adenovirus and somewhat inferior to cationic lipid-DNA complexes, even for cell lines and primary cells in vitro. We report here the identification of a novel condensing peptide, CL22, which forms DNA complexes that efficiently transfect many cell lines, as well as primary dendritic and endothelial cells. We report studies with sequence and structure variants that define some properties of the peptide that contribute to efficient transfection. We demonstrate that the superior transfection activity of CL22 compared with other DNA condensing peptides is conferred at a step after uptake of the complexes into cells. We show that CL22-DNA complexes have transfection activity that is at least equivalent to the best available nonviral agents.

  7. FragBuilder: an efficient Python library to setup quantum chemistry calculations on peptides models.

    PubMed

    Christensen, Anders S; Hamelryck, Thomas; Jensen, Jan H

    2014-01-01

    We present a powerful Python library to quickly and efficiently generate realistic peptide model structures. The library makes it possible to quickly set up quantum mechanical calculations on model peptide structures. It is possible to manually specify a specific conformation of the peptide. Additionally the library also offers sampling of backbone conformations and side chain rotamer conformations from continuous distributions. The generated peptides can then be geometry optimized by the MMFF94 molecular mechanics force field via convenient functions inside the library. Finally, it is possible to output the resulting structures directly to files in a variety of useful formats, such as XYZ or PDB formats, or directly as input files for a quantum chemistry program. FragBuilder is freely available at https://github.com/jensengroup/fragbuilder/ under the terms of the BSD open source license.

  8. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells.

    PubMed

    Chng, Jake; Wang, Tianhua; Nian, Rui; Lau, Ally; Hoi, Kong Meng; Ho, Steven C L; Gagnon, Peter; Bi, Xuezhi; Yang, Yuansheng

    2015-01-01

    Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.

  9. Efficient and directed peptide bond formation in the gas phase via ion/ion reactions.

    PubMed

    McGee, William M; McLuckey, Scott A

    2014-01-28

    Amide linkages are among the most important chemical bonds in living systems, constituting the connections between amino acids in peptides and proteins. We demonstrate the controlled formation of amide bonds between amino acids or peptides in the gas phase using ion/ion reactions in a mass spectrometer. Individual amino acids or peptides can be prepared as reagents by (i) incorporating gas phase-labile protecting groups to silence otherwise reactive functional groups, such as the N terminus; (ii) converting the carboxyl groups to the active ester of N-hydroxysuccinimide; and (iii) incorporating a charge site. Protonation renders basic sites (nucleophiles) unreactive toward the N-hydroxysuccinimide ester reagents, resulting in sites with the greatest gas phase basicities being, in large part, unreactive. The N-terminal amines of most naturally occurring amino acids have lower gas phase basicities than the side chains of the basic amino acids (i.e., those of histidine, lysine, or arginine). Therefore, reagents may be directed to the N terminus of an existing "anchor" peptide to form an amide bond by protonating the anchor peptide's basic residues, while leaving the N-terminal amine unprotonated and therefore reactive. Reaction efficiencies of greater than 30% have been observed. We propose this method as a step toward the controlled synthesis of peptides in the gas phase.

  10. Occurrence, distribution, dereplication and efficient discovery of thiazolyl peptides by sensitive-resistant pair screening.

    PubMed

    Singh, Sheo B; Zhang, Chaowei; Zink, Deborah L; Herath, Kithsiri; Ondeyka, John; Masurekar, Prakash; Jayasuriya, Hiranthi; Goetz, Michael A; Tormo, Jose Rubén; Vicente, Francisca; Martín, Jesús; González, Ignacio; Genilloud, Olga

    2013-10-01

    Natural products have been major sources of antibacterial agents and remain very promising. Frequent rediscoveries of known compounds hampers progress of new discoveries and demands development and utilization of new methods for rapid biological and chemical dereplication. This paper describes an efficient approach for discovery of new thiazolyl peptides by sensitive-resistant pair screening and dereplication in a time and cost-effective manner at industrial scale. A highly effective library-based dereplication of thiazolyl peptides by high resolution fourier transform liquid chromatography mass spectrometry (HRFTLCMS) has been developed, which can detect and dereplicate very low levels of thiazolyl peptides particularly when combined with miniaturized high-throughput 96-well solid-phase extraction separation, and as well can be automated. Combination of sensitive (susceptible)-resistant pair screening, diversified screening collection and miniaturized high-throughput SPE and HRFTLCMS techniques were applied for discovery of new thiazolyl peptides. The combined approach allowed for identification of over 24 thiazolyl peptides represented by three of the five structural subgroups, including three novel compounds. In addition, it is possible for the first time to mechanistically group three structural subgroups of over 24 thiazolyl peptides. Furthermore, these studies helped to understand natural frequency of distribution of these compounds and helped in discovery of new producing strains of many thiazolyl compounds.

  11. Fluorescence correlation spectroscopy reveals highly efficient cytosolic delivery of certain penta-arg proteins and stapled peptides.

    PubMed

    LaRochelle, Jonathan R; Cobb, Garrett B; Steinauer, Angela; Rhoades, Elizabeth; Schepartz, Alanna

    2015-02-25

    We used fluorescence correlation spectroscopy (FCS) to accurately and precisely determine the relative efficiencies with which three families of "cell-penetrating peptides" traffic to the cytosol of mammalian cells. We find that certain molecules containing a "penta-arg" motif reach the cytosol, intact, with efficiencies greater than 50%. This value is at least 10-fold higher than that observed for the widely studied cationic sequence derived from HIV Tat or polyarginine Arg8, and equals that of hydrocarbon-stapled peptides that are active in cells and animals. Moreover, we show that the efficiency with which stapled peptides reach the cytosol, as determined by FCS, correlates directly with their efficacy in cell-based assays. We expect that these findings and the associated technology will aid the design of peptides, proteins, and peptide mimetics that predictably and efficiently reach the interior of mammalian cells.

  12. Efficient Synthesis of Peptide and Protein Functionalized Pyrrole-Imidazole Polyamides Using Native Chemical Ligation

    PubMed Central

    Janssen, Brian M. G.; van Ommeren, Sven P. F. I.; Merkx, Maarten

    2015-01-01

    The advancement of DNA-based bionanotechnology requires efficient strategies to functionalize DNA nanostructures in a specific manner with other biomolecules, most importantly peptides and proteins. Common DNA-functionalization methods rely on laborious and covalent conjugation between DNA and proteins or peptides. Pyrrole-imidazole (Py–Im) polyamides, based on natural minor groove DNA-binding small molecules, can bind to DNA in a sequence specific fashion. In this study, we explore the use of Py–Im polyamides for addressing proteins and peptides to DNA in a sequence specific and non-covalent manner. A generic synthetic approach based on native chemical ligation was established that allows efficient conjugation of both peptides and recombinant proteins to Py–Im polyamides. The effect of Py–Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR). Although the synthesis of different protein-Py–Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py–Im-polyamide conjugates. The practical use of protein-Py–Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established. PMID:26053396

  13. Efficient Synthesis of Peptide and Protein Functionalized Pyrrole-Imidazole Polyamides Using Native Chemical Ligation.

    PubMed

    Janssen, Brian M G; van Ommeren, Sven P F I; Merkx, Maarten

    2015-06-04

    The advancement of DNA-based bionanotechnology requires efficient strategies to functionalize DNA nanostructures in a specific manner with other biomolecules, most importantly peptides and proteins. Common DNA-functionalization methods rely on laborious and covalent conjugation between DNA and proteins or peptides. Pyrrole-imidazole (Py-Im) polyamides, based on natural minor groove DNA-binding small molecules, can bind to DNA in a sequence specific fashion. In this study, we explore the use of Py-Im polyamides for addressing proteins and peptides to DNA in a sequence specific and non-covalent manner. A generic synthetic approach based on native chemical ligation was established that allows efficient conjugation of both peptides and recombinant proteins to Py-Im polyamides. The effect of Py-Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR). Although the synthesis of different protein-Py-Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py-Im-polyamide conjugates. The practical use of protein-Py-Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.

  14. Construction of a high efficiency copper adsorption bacterial system via peptide display and its application on copper dye polluted wastewater.

    PubMed

    Maruthamuthu, Murali Kannan; Nadarajan, Saravanan Prabhu; Ganesh, Irisappan; Ravikumar, Sambandam; Yun, Hyungdon; Yoo, Ik-Keun; Hong, Soon Ho

    2015-11-01

    For the construction of an efficient copper waste treatment system, a cell surface display strategy was employed. The copper adsorption ability of recombinant bacterial strains displaying three different copper binding peptides were evaluated in LB Luria-Bertani medium (LB), artificial wastewater, and copper phthalocyanine containing textile dye industry wastewater samples. Structural characteristics of the three peptides were also analyzed by similarity-based structure modeling. The best binding peptide was chosen for the construction of a dimeric peptide display and the adsorption ability of the monomeric and dimeric peptide displayed strains were compared. The dimeric peptide displayed strain showed superior copper adsorption in all three tested conditions (LB, artificial wastewater, and textile dye industry wastewater). When the strains were exposed to copper phthalocyanine dye polluted wastewater, the dimeric peptide display [543.27 µmol/g DCW dry cell weight (DCW)] showed higher adsorption of copper when compared with the monomeric strains (243.53 µmol/g DCW).

  15. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection

    PubMed Central

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  16. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection.

    PubMed

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes.

  17. Downsizing the BAD BH3 peptide to small constrained α-helices with improved ligand efficiency.

    PubMed

    Shepherd, Nicholas E; Harrison, Rosemary S; Ruiz-Gomez, Gloria; Abbenante, Giovanni; Mason, Jody M; Fairlie, David P

    2016-11-22

    Bcl2 Homology (BH) proteins can either trigger or prevent programmed cell death or apoptosis. Deregulation of the BH protein family network leads to evasion of apoptosis, uncontrolled proliferation and is a hallmark of cancer. Inhibition of pro-survival BH proteins is a promising chemotherapeutic strategy for certain cancers. We have examined whether helix-constrained peptides based on the BAD BH3 domain (residues 103-127) can be downsized to much smaller more drug-like peptides. We report the preparation, structural characterisation, in vitro Bcl-xL inhibition and leukemic T-cell killing ability of 45 linear, mono-, bi- and tricyclic helical peptidomimetics between 8- and 19-residues in length. We show that the BAD BH3 can be downsized to 8-14 residues and still maintain appreciable affinity for Bcl-xL. In addition, the binding efficiency indices (BEI) of the downsized mimetics are significantly higher than the BAD BH3 and similar stapled BH3 mimetics, approaching drug-like molecules. This suggests that bicyclic and monocyclic mimetics based on BH3 domains are much more efficient binding ligands than the longer peptides which they mimic.

  18. High efficiency enrichment of low-abundance peptides by novel dual-platform graphene@SiO2@PMMA.

    PubMed

    Yin, Peng; Zhao, Man; Deng, Chunhui

    2012-11-21

    For the first time, dual-platform graphene@SiO(2)@poly(methyl methacrylate) (PMMA) material was synthesized, and successfully applied to efficiently enrich low-abundance peptides for mass spectrometry analysis.

  19. Versatile signal peptide of Flavobacterium-originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins in Escherichia coli.

    PubMed

    Kang, Dong Gyun; Seo, Jeong Hyun; Jo, Byung Hoon; Kim, Chang Sup; Choi, Suk Soon; Cha, Hyung Joon

    2016-07-08

    Organophosphorus hydrolase (OPH) from Flavobacterium species is a membrane-associated homodimeric metalloenzyme and has its own signal peptide in its N-terminus. We found that OPH was translocated into the periplasmic space when the original signal peptide-containing OPH was expressed in recombinant Escherichia coli even though its translocation efficiency was relatively low. To investigate the usability of this OPH signal peptide for periplasmic expression of heterologous proteins in an E. coli system, we employed green fluorescent protein (GFP) as a cytoplasmic folding reporter and alkaline phosphatase (ALP) as a periplasmic folding reporter. We found that the OPH signal peptide was able to use both twin-arginine translocation (Tat) and general secretory (Sec) machineries by switching translocation pathways according to the nature of target proteins in E. coli. These results might be due to the lack of Sec-avoidance sequence in the c-region and a moderate hydrophobicity of the OPH signal peptide. Interestingly, the OPH signal peptide considerably enhanced the translocation efficiencies for both GFP and ALP compared with commonly used TorA and PelB signal peptides that have Tat and Sec pathway dependences, respectively. Therefore, this OPH signal peptide could be successfully used in recombinant E. coli system for efficient periplasmic production of target protein regardless of the subcellular localization where functional folding of the protein occurs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:848-854, 2016.

  20. Efficient Cargo Delivery into Adult Brain Tissue Using Short Cell-Penetrating Peptides.

    PubMed

    Kizil, Caghan; Iltzsche, Anne; Thomas, Alvin Kuriakose; Bhattarai, Prabesh; Zhang, Yixin; Brand, Michael

    2015-01-01

    Zebrafish brains can regenerate lost neurons upon neurogenic activity of the radial glial progenitor cells (RGCs) that reside at the ventricular region. Understanding the molecular events underlying this ability is of great interest for translational studies of regenerative medicine. Therefore, functional analyses of gene function in RGCs and neurons are essential. Using cerebroventricular microinjection (CVMI), RGCs can be targeted efficiently but the penetration capacity of the injected molecules reduces dramatically in deeper parts of the brain tissue, such as the parenchymal regions that contain the neurons. In this report, we tested the penetration efficiency of five known cell-penetrating peptides (CPPs) and identified two- polyR and Trans - that efficiently penetrate the brain tissue without overt toxicity in a dose-dependent manner as determined by TUNEL staining and L-Plastin immunohistochemistry. We also found that polyR peptide can help carry plasmid DNA several cell diameters into the brain tissue after a series of coupling reactions using DBCO-PEG4-maleimide-based Michael's addition and azide-mediated copper-free click reaction. Combined with the advantages of CVMI, such as rapidness, reproducibility, and ability to be used in adult animals, CPPs improve the applicability of the CVMI technique to deeper parts of the central nervous system tissues.

  1. Efficient Cargo Delivery into Adult Brain Tissue Using Short Cell-Penetrating Peptides

    PubMed Central

    Thomas, Alvin Kuriakose; Bhattarai, Prabesh; Zhang, Yixin; Brand, Michael

    2015-01-01

    Zebrafish brains can regenerate lost neurons upon neurogenic activity of the radial glial progenitor cells (RGCs) that reside at the ventricular region. Understanding the molecular events underlying this ability is of great interest for translational studies of regenerative medicine. Therefore, functional analyses of gene function in RGCs and neurons are essential. Using cerebroventricular microinjection (CVMI), RGCs can be targeted efficiently but the penetration capacity of the injected molecules reduces dramatically in deeper parts of the brain tissue, such as the parenchymal regions that contain the neurons. In this report, we tested the penetration efficiency of five known cell-penetrating peptides (CPPs) and identified two– polyR and Trans – that efficiently penetrate the brain tissue without overt toxicity in a dose-dependent manner as determined by TUNEL staining and L-Plastin immunohistochemistry. We also found that polyR peptide can help carry plasmid DNA several cell diameters into the brain tissue after a series of coupling reactions using DBCO-PEG4-maleimide-based Michael’s addition and azide-mediated copper-free click reaction. Combined with the advantages of CVMI, such as rapidness, reproducibility, and ability to be used in adult animals, CPPs improve the applicability of the CVMI technique to deeper parts of the central nervous system tissues. PMID:25894337

  2. Convergence and sampling efficiency in replica exchange simulations of peptide folding in explicit solvent.

    PubMed

    Periole, Xavier; Mark, Alan E

    2007-01-07

    Replica exchange methods (REMs) are increasingly used to improve sampling in molecular dynamics (MD) simulations of biomolecular systems. However, despite having been shown to be very effective on model systems, the application of REM in complex systems such as for the simulation of protein and peptide folding in explicit solvent has not been objectively tested in detail. Here we present a comparison of conventional MD and temperature replica exchange MD (T-REMD) simulations of a beta-heptapeptide in explicit solvent. This system has previously been shown to undergo reversible folding on the time scales accessible to MD simulation and thus allows a direct one-to-one comparison of efficiency. The primary properties compared are the free energy of folding and the relative populations of different conformers as a function of temperature. It is found that to achieve a similar degree of precision T-REMD simulations starting from a random set of initial configurations were approximately an order of magnitude more computationally efficient than a single 800 ns conventional MD simulation for this system at the lowest temperature investigated (275 K). However, whereas it was found that T-REMD simulations are more than four times more efficient than multiple independent MD simulations at one temperature (300 K) the actual increase in conformation sampling was only twofold. The overall gain in efficiency using REMD resulted primarily from the ordering of different conformational states over temperature, as opposed to a large increase of conformational sampling. It is also shown that in this system exchanges are accepted primarily based on (random) fluctuations within the solvent and are not strongly correlated with the instantaneous peptide conformation raising questions in regard to the efficiency of T-REMD in larger systems.

  3. Efficient Covalent Bond Formation in Gas-Phase Peptide-Peptide Ion Complexes with the Photoleucine Stapler.

    PubMed

    Shaffer, Christopher J; Andrikopoulos, Prokopis C; Řezáč, Jan; Rulíšek, Lubomír; Tureček, František

    2016-04-01

    Noncovalent complexes of hydrophobic peptides GLLLG and GLLLK with photoleucine (L*) tagged peptides G(L* n L m )K (n = 1,3, m = 2,0) were generated as singly charged ions in the gas phase and probed by photodissociation at 355 nm. Carbene intermediates produced by photodissociative loss of N2 from the L* diazirine rings underwent insertion into X-H bonds of the target peptide moiety, forming covalent adducts with yields reaching 30%. Gas-phase sequencing of the covalent adducts revealed preferred bond formation at the C-terminal residue of the target peptide. Site-selective carbene insertion was achieved by placing the L* residue in different positions along the photopeptide chain, and the residues in the target peptide undergoing carbene insertion were identified by gas-phase ion sequencing that was aided by specific (13)C labeling. Density functional theory calculations indicated that noncovalent binding to GL*L*L*K resulted in substantial changes of the (GLLLK + H)(+) ground state conformation. The peptide moieties in [GL*L*LK + GLLLK + H](+) ion complexes were held together by hydrogen bonds, whereas dispersion interactions of the nonpolar groups were only secondary in ground-state 0 K structures. Born-Oppenheimer molecular dynamics for 100 ps trajectories of several different conformers at the 310 K laboratory temperature showed that noncovalent complexes developed multiple, residue-specific contacts between the diazirine carbons and GLLLK residues. The calculations pointed to the substantial fluidity of the nonpolar side chains in the complexes. Diazirine photochemistry in combination with Born-Oppenheimer molecular dynamics is a promising tool for investigations of peptide-peptide ion interactions in the gas phase. Graphical Abstract ᅟ.

  4. Efficient Covalent Bond Formation in Gas-Phase Peptide-Peptide Ion Complexes with the Photoleucine Stapler

    NASA Astrophysics Data System (ADS)

    Shaffer, Christopher J.; Andrikopoulos, Prokopis C.; Řezáč, Jan; Rulíšek, Lubomír; Tureček, František

    2016-04-01

    Noncovalent complexes of hydrophobic peptides GLLLG and GLLLK with photoleucine (L*) tagged peptides G(L* n L m )K (n = 1,3, m = 2,0) were generated as singly charged ions in the gas phase and probed by photodissociation at 355 nm. Carbene intermediates produced by photodissociative loss of N2 from the L* diazirine rings underwent insertion into X-H bonds of the target peptide moiety, forming covalent adducts with yields reaching 30%. Gas-phase sequencing of the covalent adducts revealed preferred bond formation at the C-terminal residue of the target peptide. Site-selective carbene insertion was achieved by placing the L* residue in different positions along the photopeptide chain, and the residues in the target peptide undergoing carbene insertion were identified by gas-phase ion sequencing that was aided by specific 13C labeling. Density functional theory calculations indicated that noncovalent binding to GL*L*L*K resulted in substantial changes of the (GLLLK + H)+ ground state conformation. The peptide moieties in [GL*L*LK + GLLLK + H]+ ion complexes were held together by hydrogen bonds, whereas dispersion interactions of the nonpolar groups were only secondary in ground-state 0 K structures. Born-Oppenheimer molecular dynamics for 100 ps trajectories of several different conformers at the 310 K laboratory temperature showed that noncovalent complexes developed multiple, residue-specific contacts between the diazirine carbons and GLLLK residues. The calculations pointed to the substantial fluidity of the nonpolar side chains in the complexes. Diazirine photochemistry in combination with Born-Oppenheimer molecular dynamics is a promising tool for investigations of peptide-peptide ion interactions in the gas phase.

  5. Efficient inhibition of miR-155 function in vivo by peptide nucleic acids

    PubMed Central

    Fabani, Martin M.; Abreu-Goodger, Cei; Williams, Donna; Lyons, Paul A.; Torres, Adrian G.; Smith, Kenneth G. C.; Enright, Anton J.; Gait, Michael J.; Vigorito, Elena

    2010-01-01

    MicroRNAs (miRNAs) play an important role in diverse physiological processes and are potential therapeutic agents. Synthetic oligonucleotides (ONs) of different chemistries have proven successful for blocking miRNA expression. However, their specificity and efficiency have not been fully evaluated. Here, we show that peptide nucleic acids (PNAs) efficiently block a key inducible miRNA expressed in the haematopoietic system, miR-155, in cultured B cells as well as in mice. Remarkably, miR-155 inhibition by PNA in primary B cells was achieved in the absence of any transfection agent. In mice, the high efficiency of the treatment was demonstrated by a strong overlap in global gene expression between B cells isolated from anti-miR-155 PNA-treated and miR-155-deficient mice. Interestingly, PNA also induced additional changes in gene expression. Our analysis provides a useful platform to aid the design of efficient and specific anti-miRNA ONs for in vivo use. PMID:20223773

  6. Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

    SciTech Connect

    Amand, Helene L.

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. Black-Right-Pointing-Pointer Dimer formation enhances peptiplex stability, resulting in increased transfection. Black-Right-Pointing-Pointer By dimerisation, the CPP EB1 even gain endosomal escape properties while lowering cytotoxicity. -- Abstract: Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes ('peptiplexes') enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the 'chelate effect' and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide dimer resulting from

  7. Rice seed ER-derived protein body as an efficient delivery vehicle for oral tolerogenic peptides.

    PubMed

    Takagi, Hidenori; Hiroi, Takachika; Hirose, Sakiko; Yang, Lijun; Takaiwa, Fumio

    2010-08-01

    Mucosal delivery of peptide/protein therapeutics via the oral route is a desirable strategy in human immunotherapy. A key step for enhancing the bioavailability of orally administered therapeutics is to protect them from enzymatic digestion in the gastrointestinal tract. Here, we generated transgenic rice seeds accumulating allergen-derived T cell epitopes, a model tolerogen for the control of pollen allergy, in either ER-derived protein body-I (PB-I) or protein storage vacuole protein body-II (PB-II). Compared with PB-II-localized or chemically synthesized forms, PB-I-localized T cell epitopes showed higher resistance to enzymatic digestion in simulated gastric fluid. Moreover, the dose of T cell epitope required for suppression of allergen-specific IgE in mice was about 20-fold lower when fed in PB-I localized form than when unprotected. These findings demonstrate the potential of bioencapsulation in PB-I for broad applications as a viable strategy to achieve efficient mucosal delivery of oral peptide/protein therapeutics.

  8. Efficient Methods to Generate Reproducible Mass Spectra in Matrix-Assisted Laser Desorption Ionization of Peptides

    NASA Astrophysics Data System (ADS)

    Ahn, Sung Hee; Park, Kyung Man; Bae, Yong Jin; Kim, Myung Soo

    2013-06-01

    In our previous matrix-assisted laser desorption ionization (MALDI) studies of peptides, we found that their mass spectra were virtually determined by the effective temperature in the early matrix plume, Tearly, when samples were rather homogeneous. This empirical rule allowed acquisition of quantitatively reproducible spectra. A difficulty in utilizing this rule was the complicated spectral treatment needed to get Tearly. In this work, we found another empirical rule that the total number of particles hitting the detector, or TIC, was a good measure of the spectral temperature and, hence, selection of spectra with the same TIC resulted in reproducible spectra. We also succeeded in obtaining reproducible spectra throughout a measurement by controlling TIC near a preset value through feedback adjustment of laser pulse energy. Both TIC selection and TIC control substantially reduced the shot-to-shot spectral variation in a spot, spot-to-spot variation in a sample, and even sample-to-sample variation in MALDI using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid as matrix. Based on the utilization of acquired data, TIC control was more efficient than TIC selection by an order of magnitude. Both techniques produced calibration curves with excellent linearity, suggesting their utility in quantification of peptides.

  9. A strategy for efficient cross-presentation of CTL-epitope peptides leading to enhanced induction of in vivo tumor immunity.

    PubMed

    Hayashi, Akira; Wakita, Hisashi; Yoshikawa, Tomoaki; Nakanishi, Tsuyoshi; Tsutsumi, Yasuo; Mayumi, Tadanori; Mukai, Yohei; Yoshioka, Yasuo; Okada, Naoki; Nakagawa, Shinsaku

    2007-01-22

    The activation of antitumor cytotoxic T-lymphocytes (CTLs) depends on how efficiently the relevant tumor antigen peptides are delivered into the major histocompatibility complex (MHC) class I presentation pathway in antigen presenting cells (APCs). An elegant approach to promote the peptide-MHC class I association has been described for enhanced peptide transportation into the endoplasmic reticulum (ER) by adding an ER insertion signal sequence (Eriss). Nevertheless, this approach does not appear potent enough to induce in vivo tumor protective immunity. Herein, we present a novel peptide-vaccine strategy based on the combined utilization of Eriss and fusogenic liposomes (FLs) capable of directly introducing encapsulated CTL-epitope peptides into the MHC class I pathway of APCs. APCs pulsed with free peptides, FL-encapsulated peptides, or FL-encapsulated Eriss-conjugated peptides exhibited comparable levels of antigen-presenting activity at early phases after pulsing. Interestingly, whereas in the first two methods the APC ability began to decline 40 to 60 h after pulsing, FL-encapsulated Eriss(+) peptides allowed APCs to retain peptide-presentation activity for at least 140 h. This advantage of FL-encapsulated Eriss(+) peptides correlated with the induction of more potent antitumor immunity compared with soluble Eriss(+) or Eriss(-) peptides or FL-encapsulated Eriss(-) peptides when they were administered in vivo. Thus, Eriss-conjugated CTL-epitope peptides encapsulated in FLs provide a highly efficient tumor-vaccine to enhance the induction of in vivo tumor immunity.

  10. Placental leucine aminopeptidase efficiently generates mature antigenic peptides in vitro but in patterns distinct from endoplasmic reticulum aminopeptidase 1.

    PubMed

    Georgiadou, Dimitra; Hearn, Arron; Evnouchidou, Irini; Chroni, Angeliki; Leondiadis, Leondios; York, Ian A; Rock, Kenneth L; Stratikos, Efstratios

    2010-08-01

    All three members of the oxytocinase subfamily of M1 aminopeptidases, endoplasmic reticulum aminopeptidase 1 (ERAP1), ERAP2, and placental leucine aminopeptidase (PLAP), also known as insulin-regulated aminopeptidase, have been implicated in the generation of MHC class I-presented peptides. ERAP1 and 2 trim peptides in the endoplasmic reticulum for direct presentation, whereas PLAP has been recently implicated in cross-presentation. The best characterized member of the family, ERAP1, has unique enzymatic properties that fit well with its role in Ag processing. ERAP1 can trim a large variety of long peptide sequences and efficiently accumulate mature antigenic epitopes of 8-9 aa long. In this study, we evaluate the ability of PLAP to process antigenic peptide precursors in vitro and compare it with ERAP1. We find that, similar to ERAP1, PLAP can trim a variety of long peptide sequences efficiently and, in most cases, accumulates appreciable amounts of correct length mature antigenic epitope. Again, similar to ERAP1, PLAP continued trimming some of the epitopes tested and accumulated smaller products effectively destroying the epitope. However, the intermediate accumulation properties of ERAP1 and PLAP are distinct and epitope dependent, suggesting that these two enzymes may impose different selective pressures on epitope generation. Overall, although PLAP has the necessary enzymatic properties to participate in generating or destroying MHC class I-presented peptides, its trimming behavior is distinct from that of ERAP1, something that supports a separate role for these two enzymes in Ag processing.

  11. Membrane penetrating peptides greatly enhance baculovirus transduction efficiency into mammalian cells

    SciTech Connect

    Chen, Hong-Zhang; Wu, Carol P.; Chao, Yu-Chan; Liu, Catherine Yen-Yen

    2011-02-11

    Research highlights: {yields} Ligation of CTP with GP64 enhances baculovirus transduction into mammalian cells. {yields} Fusion of PTD with VP39 enhances baculovirus transduction into mammalian cells. {yields} CTP and PTD-carrying viruses improve the transduction of co-transduced baculoviruses. {yields} Virus entry and gene expression can be separate events in different cell types. -- Abstract: The baculovirus group of insect viruses is widely used for foreign gene introduction into mammalian cells for gene expression and protein production; however, the efficiency of baculovirus entry into mammalian cells is in general still low. In this study, two recombinant baculoviruses were engineered and their ability to improve viral entry was examined: (1) cytoplasmic transduction peptide (CTP) was fused with baculovirus envelope protein, GP64, to produce a cytoplasmic membrane penetrating baculovirus (vE-CTP); and (2) the protein transduction domain (PTD) of HIV TAT protein was fused with the baculovirus capsid protein VP39 to form a nuclear membrane penetrating baculovirus (vE-PTD). Transduction experiments showed that both viruses had better transduction efficiency than vE, a control virus that only expresses EGFP in mammalian cells. Interestingly, vE-CTP and vE-PTD were also able to improve the transduction efficiency of a co-transduced baculovirus, resulting in higher levels of gene expression. Our results have described new routes to further enhance the development of baculovirus as a tool for gene delivery into mammalian cells.

  12. Efficient siRNA-peptide conjugation for specific targeted delivery into tumor cells.

    PubMed

    Gandioso, Albert; Massaguer, Anna; Villegas, Núria; Salvans, Cándida; Sánchez, Dani; Brun-Heath, Isabelle; Marchán, Vicente; Orozco, Modesto; Terrazas, Montserrat

    2017-03-02

    Despite the broad applicability of the Huisgen cycloaddition reaction, the click functionalization of RNAs with peptides still remains a challenge. Here we describe a straightforward method for the click functionalization of siRNAs with peptides of different sizes and complexities. Among them, a promising peptide carrier for the selective siRNA delivery into HER2+ breast cancer cell lines has been reported.

  13. Force field-based conformational searches: efficiency and performance for peptide receptor complexes

    NASA Astrophysics Data System (ADS)

    Grebner, Christoph; Niebling, Stephan; Schmuck, Carsten; Schlücker, Sebastian; Engels, Bernd

    2013-09-01

    Conformational search using force field methods on complex biomolecular systems is a key factor in understanding molecular and structural properties. The reliability of such investigations strongly depends on the efficiency of the conformational search algorithm as well as the accuracy of the employed force field. In the present work we compared the performance of two different approaches: the Monte-Carlo multiple minimum/low mode sampling (MCMM/LM), in combination with the OPLS2005 (MCMM/LM//OPLS2005), and Tabu-Search combined with Basin Hopping (TS/BH), employing the original OPLS-AA implementation proposed by Jorgensen (TS/BH//OPLS-AA). We investigated their performance in locating energetically low-lying structures and the efficiency in scanning the conformational phase space of non-covalently bonded complexes. As test systems we employed complexes of the artificial peptide receptor CBS-KKF with four different tetrapeptide ligands. The reliability and the accuracy of both approaches were examined by re-optimising all low-energy structures employing density functional theory with empirical dispersion correction in combination with triple zeta basis sets. Solvent effects were mimicked by a continuum solvent model. In all the four-test systems, the TS/BH//OPLS-AA approach yielded structures that are much lower in energy after the DFT optimisation. Additionally, it provided many low-lying structures that were not identified by the MCMM/LM//OPLS2005 approach.

  14. Conjugation of photosensitisers to antimicrobial peptides increases the efficiency of photodynamic therapy in cancer cells.

    PubMed

    Moret, Francesca; Gobbo, Marina; Reddi, Elena

    2015-07-01

    Some antimicrobial peptides (AMPs) have the ability to penetrate and kill not only pathogenic microorganisms but also cancer cells, while they are less active toward normal eukaryotic cells. Here we have investigated the potential of three AMPs, namely apidaecin 1b (Api), magainin 2 (Mag) and buforin II (Buf), as carriers of drugs for cancer cells by using the hydrophobic photosensitiser 5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin (cTPP) as the drug model, conjugated to the N-terminus of the peptides. Flow cytometry measurements demonstrated that conjugation of cTPP increased its rate and efficiency of uptake in A549 human lung adenocarcinoma cells in the order Mag > Buf > Api. In vitro photodynamic therapy (PDT) experiments showed that the increased uptake of the conjugated cTPP determined 100% cell killing at concentrations in the nanomolar range while micromolar concentrations were required for the same killing effect with unconjugated cTPP. Serum proteins interacted with cTPP conjugated to Buf and Api and slightly interfered with the cellular uptake of these conjugates but not with that of Mag. The data suggest electrostatic interactions of the conjugates with sialic acid and ganglioside rich domains, as lipid rafts of the plasma membrane, followed by cell internalization via non-caveolar dynamin-dependent endocytosis as indicated by the effects of inhibitors of specific endocytic pathways. Our study demonstrated that the three AMPs investigated, Mag in particular, have the ability to carry a hydrophobic cargo inside cancer cells and may therefore represent useful carriers of anticancer drugs, especially those with a poor capacity to penetrate inside the target cells.

  15. Receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient delivery system for MRTF silencing in conjunctival fibrosis

    NASA Astrophysics Data System (ADS)

    Yu-Wai-Man, Cynthia; Tagalakis, Aristides D.; Manunta, Maria D.; Hart, Stephen L.; Khaw, Peng T.

    2016-02-01

    There is increasing evidence that the Myocardin-related transcription factor/Serum response factor (MRTF/SRF) pathway plays a key role in fibroblast activation and that knocking down MRTF can lead to reduced scarring and fibrosis. Here, we have developed a receptor-targeted liposome-peptide-siRNA nanoparticle as a non-viral delivery system for MRTF-B siRNA in conjunctival fibrosis. Using 50 nM siRNA, the MRTF-B gene was efficiently silenced by 76% and 72% with LYR and LER nanoparticles, respectively. The silencing efficiency was low when non-targeting peptides or siRNA alone or liposome-siRNA alone were used. LYR and LER nanoparticles also showed higher silencing efficiency than PEGylated LYR-P and LER-P nanoparticles. The nanoparticles were not cytotoxic using different liposomes, targeting peptides, and 50 nM siRNA. Three-dimensional fibroblast-populated collagen matrices were also used as a functional assay to measure contraction in vitro, and showed that MRTF-B LYR nanoparticles completely blocked matrix contraction after a single transfection treatment. In conclusion, this is the first study to develop and show that receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient and safe non-viral siRNA delivery system that could be used to prevent fibrosis after glaucoma filtration surgery and other contractile scarring conditions in the eye.

  16. Receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient delivery system for MRTF silencing in conjunctival fibrosis

    PubMed Central

    Yu-Wai-Man, Cynthia; Tagalakis, Aristides D.; Manunta, Maria D.; Hart, Stephen L.; Khaw, Peng T.

    2016-01-01

    There is increasing evidence that the Myocardin-related transcription factor/Serum response factor (MRTF/SRF) pathway plays a key role in fibroblast activation and that knocking down MRTF can lead to reduced scarring and fibrosis. Here, we have developed a receptor-targeted liposome-peptide-siRNA nanoparticle as a non-viral delivery system for MRTF-B siRNA in conjunctival fibrosis. Using 50 nM siRNA, the MRTF-B gene was efficiently silenced by 76% and 72% with LYR and LER nanoparticles, respectively. The silencing efficiency was low when non-targeting peptides or siRNA alone or liposome-siRNA alone were used. LYR and LER nanoparticles also showed higher silencing efficiency than PEGylated LYR-P and LER-P nanoparticles. The nanoparticles were not cytotoxic using different liposomes, targeting peptides, and 50 nM siRNA. Three-dimensional fibroblast-populated collagen matrices were also used as a functional assay to measure contraction in vitro, and showed that MRTF-B LYR nanoparticles completely blocked matrix contraction after a single transfection treatment. In conclusion, this is the first study to develop and show that receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient and safe non-viral siRNA delivery system that could be used to prevent fibrosis after glaucoma filtration surgery and other contractile scarring conditions in the eye. PMID:26905457

  17. Mitochondrial transit peptide exhibits cell penetration ability and efficiently delivers macromolecules to mitochondria.

    PubMed

    Jain, Aastha; Chugh, Archana

    2016-09-01

    Mitochondrial malfunction under various circumstances can lead to a variety of disorders. Effective targeting of macromolecules (drugs) is important for restoration of mitochondrial function and treatment of related disorders. We have designed a novel cell-penetrating mitochondrial transit peptide (CpMTP) for delivery of macromolecules to mitochondria. Comparison between properties of cell-penetrating peptides (CPPs) and mitochondrial signal sequences enabled prediction of peptides with dual ability for cellular translocation and mitochondrial localization. Among the predicted peptides, CpMTP translocates across HeLa cells and shows successful delivery of noncovalently conjugated cargo molecules to mitochondria. CpMTP may have applications in transduction and transfection of mitochondria for therapeutics.

  18. Presumed LRP1-targeting transport peptide delivers β-secretase inhibitor to neurons in vitro with limited efficiency

    PubMed Central

    Kim, Jong Ah; Casalini, Tommaso; Brambilla, Davide; Leroux, Jean-Christophe

    2016-01-01

    Interfering with the activity of β-secretase to reduce the production of Aβ peptides is a conceivable therapeutic strategy for Alzheimer’s disease. However, the development of efficient yet safe inhibitors is hampered by secondary effects, usually linked to the indiscriminate inhibition of other substrates’ processing by the targeted enzyme. Based on the spatial compartmentalization of the cleavage of the amyloid precursor protein by β-secretase, we hypothesized that by exploiting the endocytosis receptor low-density lipoprotein receptor-related protein it would be possible to direct an otherwise cell-impermeable inhibitor to the endosomes of neurons, boosting the drug’s efficacy and importantly, sparing the off-target effects. We used the transport peptide Angiopep to build an endocytosis-competent conjugate and found that although the peptide facilitated the inhibitor’s internalization into neurons and delivered it to the endosomes, the delivery was not efficient enough to potently reduce β-secretase activity at the cellular level. This is likely connected to the finding that in the cell lines we used, Angiopep’s internalization was not mediated by its presumed receptor to a significant extent. Additionally, Angiopep exploited different internalization mechanisms when applied alone or when conjugated to the inhibitor, highlighting the impact that drug conjugation can have on transport peptides. PMID:27682851

  19. DeMix Workflow for Efficient Identification of Cofragmented Peptides in High Resolution Data-dependent Tandem Mass Spectrometry*

    PubMed Central

    Zhang, Bo; Pirmoradian, Mohammad; Chernobrovkin, Alexey; Zubarev, Roman A.

    2014-01-01

    Based on conventional data-dependent acquisition strategy of shotgun proteomics, we present a new workflow DeMix, which significantly increases the efficiency of peptide identification for in-depth shotgun analysis of complex proteomes. Capitalizing on the high resolution and mass accuracy of Orbitrap-based tandem mass spectrometry, we developed a simple deconvolution method of “cloning” chimeric tandem spectra for cofragmented peptides. Additional to a database search, a simple rescoring scheme utilizes mass accuracy and converts the unwanted cofragmenting events into a surprising advantage of multiplexing. With the combination of cloning and rescoring, we obtained on average nine peptide-spectrum matches per second on a Q-Exactive workbench, whereas the actual MS/MS acquisition rate was close to seven spectra per second. This efficiency boost to 1.24 identified peptides per MS/MS spectrum enabled analysis of over 5000 human proteins in single-dimensional LC-MS/MS shotgun experiments with an only two-hour gradient. These findings suggest a change in the dominant “one MS/MS spectrum - one peptide” paradigm for data acquisition and analysis in shotgun data-dependent proteomics. DeMix also demonstrated higher robustness than conventional approaches in terms of lower variation among the results of consecutive LC-MS/MS runs. PMID:25100859

  20. In Silico Template Selection of Short Antimicrobial Peptide Viscotoxin for Improving Its Antimicrobial Efficiency in Development of Potential Therapeutic Drugs.

    PubMed

    Senthilkumar, B; Rajasekaran, R

    2017-03-01

    Rapid increase in antibiotic resistance has posed a worldwide threat, due to increased mortality, morbidity, and expenditure caused by antibiotic-resistant microbes. Recent development of the antimicrobial peptides like viscotoxin (Vt) has been successfully comprehended as a substitute for classical antibiotics. A structurally stable peptide, Vt can enhance antimicrobial property and can be used for various developmental purposes. Thus, structural stability among the antimicrobial peptides, Vt A1 (3C8P), A2 (1JMN), A3 (1ED0), B (1JMP), and C (1ORL) of Viscus album was computationally analyzed. In specific, the static confirmation of VtA3 showed high number of intramolecular interactions, along with an increase in hydrophobicity than others comparatively. Further, conformational sampling was used to analyze various geometrical parameters such as root mean square deviation, root mean square fluctuation, radius of gyration, and ovality which also revealed the structural stability of VtA3. Moreover, the statistically validated contours of surface area, lipophilicity, and distance constraints of disulfide bonds also supported the priority of VtA3 with respect to stability. Finally, the functional activity of peptides was accessed by computing their free energy of membrane association and membrane interactions, which defined VtA3 as functionally stable. Currently, peptide-based antibiotics and nanoparticles have attracted the pharmaceutical industries for their potential therapeutic applications. Thereby, it is proposed that viscotoxin A3 (1ED0) could be used as a preeminent template for scaffolding potentially efficient antimicrobial peptide-based drugs and nanomaterials in future.

  1. Self-assembled cationic peptide nanoparticles as an efficient antimicrobial agent

    NASA Astrophysics Data System (ADS)

    Liu, Lihong; Xu, Kaijin; Wang, Huaying; Jeremy Tan, P. K.; Fan, Weimin; Venkatraman, Subbu S.; Li, Lanjuan; Yang, Yi-Yan

    2009-07-01

    Antimicrobial cationic peptides are of interest because they can combat multi-drug-resistant microbes. Most peptides form α-helices or β-sheet-like structures that can insert into and subsequently disintegrate negatively charged bacterial cell surfaces. Here, we show that a novel class of core-shell nanoparticles formed by self-assembly of an amphiphilic peptide have strong antimicrobial properties against a range of bacteria, yeasts and fungi. The nanoparticles show a high therapeutic index against Staphylococcus aureus infection in mice and are more potent than their unassembled peptide counterparts. Using Staphylococcus aureus-infected meningitis rabbits, we show that the nanoparticles can cross the blood-brain barrier and suppress bacterial growth in infected brains. Taken together, these nanoparticles are promising antimicrobial agents that can be used to treat brain infections and other infectious diseases.

  2. Efficient Esterification of Oxidized l-Glutathione and Other Small Peptides.

    PubMed

    Vogel, Emily R; Jackson, William; Masterson, Douglas S

    2015-06-08

    Oxidized l-glutathione was esterified to the tetra methyl ester using thionyl chloride in methanol solvent. Other alcohols were tested and the reaction progress was monitored via ESI-MS. This procedure proved to be compatible with other small peptides not containing serine and cysteine residues. In contrast to previously reported methods this procedure provided convenient access to esterified peptides requiring no purification, extended reaction times, or complicated reaction setups.

  3. Efficient Inhibition of Hepatitis B Virus Infection by Acylated Peptides Derived from the Large Viral Surface Protein†

    PubMed Central

    Gripon, Philippe; Cannie, Isabelle; Urban, Stephan

    2005-01-01

    The lack of an appropriate in vitro infection system for the major human pathogen hepatitis B virus (HBV) has prevented a molecular understanding of the early infection events of HBV. We used the novel HBV-infectible cell line HepaRG and primary human hepatocytes to investigate the interference of infection by HBV envelope protein-derived peptides. We found that a peptide consisting of the authentically myristoylated N-terminal 47 amino acids of the pre-S1 domain of the large viral envelope protein (L protein) specifically prevented HBV infection, with a 50% inhibitory concentration (IC50) of 8 nM. The replacement of myristic acid with other hydrophobic moieties resulted in changes in the inhibitory activity, most notably by a decrease in the IC50 to picomolar concentrations for longer unbranched fatty acids. The obstruction of HepaRG cell susceptibility to HBV infection after short preincubation times with the peptides suggested that the peptides efficiently target and inactivate a receptor at the hepatocyte surface. Our data both shed light on the molecular mechanism of HBV entry into hepatocytes and provide a basis for the development of potent hepadnaviral entry inhibitors as a novel therapeutic concept for the treatment of hepatitis Β. PMID:15650187

  4. Rapid and efficient assembly of functional silicone surfaces protected by PEG: cell adhesion to peptide-modified PDMS.

    PubMed

    Mikhail, Andrew S; Ranger, Jill J; Liu, Lihua; Longenecker, Ryan; Thompson, David B; Sheardown, Heather D; Brook, Michael A

    2010-01-01

    While silicone elastomers generally have excellent biomaterials properties, their hydrophobicity can elicit undesired local biological responses through adsorption and denaturation of proteins. Surface-bound poly(ethylene glycol) (PEG) can ameliorate the situation by preventing contact between the external biology and the silicone elastomer. It is further possible to manipulate the biocompatibility of the surface by linking peptides, proteins or other biological entities to the PEG. Previous synthetic approaches to PEG-protected surfaces are compromised by issues of reproducibility. We describe two rapid and efficient approaches to silicone surface modification by PEG-linked adhesion peptides that overcome this problem: SiH groups are introduced throughout a silicone elastomer during elastomer synthesis or only at the surface after cure; then, in either case, protein-repellent PEG brushes at the surface are introduced by hydrosilylation to give surfaces that can be stored for extensive periods of time without degradation. Activation of the free alcohol with an NSC group followed by immediate conjugation to relevant biological molecules occurs in high yields, as shown for RGDS and GYRGDS. High surface grafting density of the peptides was demonstrated using radiolabeling techniques. Biological activity was demonstrated by a 5-fold increase in cell adhesion on the peptide-modified surfaces when compared to unmodified PDMS control surfaces.

  5. Spatial Heterogeneity and Peptide Availability Determine CTL Killing Efficiency In Vivo

    PubMed Central

    Hogan, Thea; Kadolsky, Ulrich; Tung, Sim; Seddon, Benedict; Yates, Andrew

    2014-01-01

    The rate at which a cytotoxic T lymphocyte (CTL) can survey for infected cells is a key ingredient of models of vertebrate immune responses to intracellular pathogens. Estimates have been obtained using in vivo cytotoxicity assays in which peptide-pulsed splenocytes are killed by CTL in the spleens of immunised mice. However the spleen is a heterogeneous environment and splenocytes comprise multiple cell types. Are some cell types intrinsically more susceptible to lysis than others? Quantitatively, what impacts are made by the spatial distribution of targets and effectors, and the level of peptide-MHC on the target cell surface? To address these questions we revisited the splenocyte killing assay, using CTL specific for an epitope of influenza virus. We found that at the cell population level T cell targets were killed more rapidly than B cells. Using modeling, quantitative imaging and in vitro killing assays we conclude that this difference in vivo likely reflects different migratory patterns of targets within the spleen and a heterogeneous distribution of CTL, with no detectable difference in the intrinsic susceptibilities of the two populations to lysis. Modeling of the stages involved in the detection and killing of peptide-pulsed targets in vitro revealed that peptide dose influenced the ability of CTL to form conjugates with targets but had no detectable effect on the probability that conjugation resulted in lysis, and that T cell targets took longer to lyse than B cells. We also infer that incomplete killing in vivo of cells pulsed with low doses of peptide may be due to a combination of heterogeneity in peptide uptake and the dissociation, but not internalisation, of peptide-MHC complexes. Our analyses demonstrate how population-averaged parameters in models of immune responses can be dissected to account for both spatial and cellular heterogeneity. PMID:25233372

  6. Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase

    PubMed Central

    Harris, Karen S.; Durek, Thomas; Kaas, Quentin; Poth, Aaron G.; Gilding, Edward K.; Conlan, Brendon F.; Saska, Ivana; Daly, Norelle L.; van der Weerden, Nicole L.; Craik, David J.; Anderson, Marilyn A.

    2015-01-01

    Cyclotides are diverse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Backbone cyclization is a key enzyme-mediated step of cyclotide biosynthesis and confers a measure of stability on the resultant cyclotide. Furthermore, cyclization would be desirable for engineered peptides. Here we report the identification of four asparaginyl endopeptidases (AEPs), proteases implicated in cyclization, from the cyclotide-producing plant Oldenlandia affinis. We recombinantly express OaAEP1b and find it functions preferably as a cyclase by coupling C-terminal cleavage of propeptide substrates with backbone cyclization. Interestingly, OaAEP1b cannot cleave at the N-terminal site of O. affinis cyclotide precursors, implicating additional proteases in cyclotide biosynthesis. Finally, we demonstrate the broad utility of this enzyme by cyclization of peptides unrelated to cyclotides. We propose that recombinant OaAEP1b is a powerful tool for use in peptide engineering applications where increased stability of peptide products is desired. PMID:26680698

  7. Efficient Subcellular Targeting to the Cell Nucleus of Quantum Dots Densely Decorated with a Nuclear Localization Sequence Peptide.

    PubMed

    Maity, Amit Ranjan; Stepensky, David

    2016-01-27

    Organelle-targeted drug delivery can enhance the efficiency of the intracellularly acting drugs and reduce their toxicity. We generated core-shell type CdSe-ZnS quantum dots (QDs) densely decorated with NLS peptidic targeting residues using a 3-stage decoration approach and investigated their endocytosis and nuclear targeting efficiencies. The diameter of the generated QDs increased following the individual decoration stages (16.3, 18.9, and 21.9 nm), the ζ-potential became less negative (-33.2, -17.5, and -11.9 mV), and characteristic changes appeared in the FTIR spectra following decoration with the linker and NLS peptides. Quantitative analysis of the last decoration stage revealed that 37.9% and 33.2% of the alkyne-modified NLS groups that were added to the reaction mix became covalently attached or adsorbed to the QDs surface, respectively. These numbers correspond to 63.6 and 55.7 peptides conjugated or adsorbed to a single QD (the surface density of 42 and 37 conjugated and adsorbed peptides per 1000 nm(2) of the QDs surface), which is higher than in the majority of previous studies that reported decoration efficiencies of formulations intended for nuclear-targeted drug delivery. QDs decorated with NLS peptides undergo more efficient endocytosis, as compared to other investigated QDs formulations, and accumulated to a higher extent in the cell nucleus or in close vicinity to it (11.9%, 14.6%, and 56.1% of the QDs endocytosed by an average cell for the QD-COOH, QD-azide, and QD-NLS formulations, respectively). We conclude that dense decoration of QDs with NLS residues increased their endocytosis and led to their nuclear targeting (preferential accumulation in the cells nuclei or in close vicinity to them). The experimental system and research tools that were used in this study allow quantitative investigation of the mechanisms that govern the QDs nuclear targeting and their dependence on the formulation properties. These findings will contribute to the

  8. Nuclear and perinuclear targeting efficiency of quantum dots depends on density of peptidic targeting residues on their surface.

    PubMed

    Maity, Amit Ranjan; Stepensky, David

    2016-12-29

    Targeted delivery to the cell nucleus can enhance the efficiency of drugs with nuclear site of action (some anti-cancer agents, DNA drugs, etc.), and can reduce their toxicity. Such targeting can be attained using nano-drug delivery systems (nano-DDSs) decorated with nuclear targeting sequences (such as nuclear localization sequence peptides, NLS). Several types of nano-DDSs decorated with NLS peptides were designed, but their investigation usually did not include quantitate analysis of the decoration efficiency and its correlation with the nano-DDSs intracellular localization. Thus, the major mechanisms and limiting factors of the nano-DDSs nuclear targeting are largely unknown yet. In this study, we report quantitative data for specific nano-formulation (CdSe-ZnS quantum dots) that include the efficiencies of its decoration with NLS residues and of its nuclear and perinuclear targeting, and demonstrate correlation between these parameters. For instance, QDs decorated with 83, 246, and 265 NLS peptides accumulated efficiently in the nucleus of HeLa cells or its vicinity (an average of 30.4%, 43.3%, and 49.0% of the intracellular QDs, respectively). On the other hand, QDs decorated with 63, 231, and 308 scrambled peptides accumulated in the nucleus of HeLa cells or its vicinity to a much lower extent (an average of 17.3%, 21.1%, and 25.5% of the intracellular QDs, respectively). Thus, results of our study provide important insights into the structure-activity correlations (i.e., the relationships between the formulation properties and the intracellular fate of nano-DDSs) of nuclear-targeted drug delivery. We plan to apply the research tools that were developed in the course of this and our previous studies to investigate the nuclear and perinuclear targeting activities of different NLS sequences, and to investigate the effects of nano-DDSs size, charge, shape, decoration efficiency with nuclear targeting sequences, and other structural factors on nuclear and

  9. High-Efficiency Synthesis of Human α-Endorphin and Magainin in the Erythrocytes of Transgenic Mice: A Production System for Therapeutic Peptides

    NASA Astrophysics Data System (ADS)

    Sharma, Ajay; Khoury-Christianson, Anastasia M.; White, Steven P.; Dhanjal, Nirpal K.; Huang, Wen; Paulhiac, Clara; Friedman, Eric J.; Manjula, Belur N.; Kumar, Ramesh

    1994-09-01

    Chemical synthesis of peptides, though feasible, is hindered by considerations of cost, purity, and efficiency of synthesizing longer chains. Here we describe a transgenic system for producing peptides of therapeutic interest as fusion proteins at low cost and high purity. Transgenic hemoglobin expression technology using the locus control region was employed to produce fusion hemoglobins in the erythrocytes of mice. The fusion hemoglobin contains the desired peptide as an extension at the C end of human α-globin. A protein cleavage site is inserted between the C end of the α-globin chain and the N-terminal residue of the desired peptide. The peptide is recovered after cleavage of the fusion protein with enzymes that recognize this cleavage signal as their substrate. Due to the selective compartmentalization of hemoglobin in the erythrocytes, purification of the fusion hemoglobin is easy and efficient. Because of its compact and highly ordered structure, the internal sites of hemoglobin are resistant to protease digestion and the desired peptide is efficiently released and recovered. The applicability of this approach was established by producing a 16-mer α-endorphin peptide and a 26-mer magainin peptide in transgenic mice. Transgenic animals and their progeny expressing these fusion proteins remain healthy, even when the fusion protein is expressed at >25% of the total hemoglobin in the erythrocytes. Additional applications and potential improvements of this methodology are discussed.

  10. Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide

    PubMed Central

    Huang, Li-Fen; Tan, Chia-Chun; Yeh, Ju-Fang; Liu, Hsin-Yi; Liu, Yu-Kuo; Ho, Shin-Lon; Lu, Chung-An

    2015-01-01

    Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%–92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp. PMID:26473722

  11. Efficient identification of murine M2 macrophage peptide targeting ligands by phage display and next-generation sequencing

    PubMed Central

    Liu, Gary W.; Livesay, Brynn R.; Kacherovsky, Nataly A.; Cieslewicz, Maryelise; Lutz, Emi; Waalkes, Adam; Jensen, Michael C.; Salipante, Stephen J.; Pun, Suzie H.

    2015-01-01

    Peptide ligands are used to increase the specificity of drug carriers to their target cells and to facilitate intracellular delivery. One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of interest. However, conventional methods for identifying target binders in a library by Sanger sequencing are low-throughput, labor-intensive, and provide a limited perspective (< 0.01%) of the complete sequence space. Moreover, the small sample space can be dominated by non-specific, preferentially amplifying “parasitic sequences” and plastic-binding sequences, which may lead to the identification of false positives or exclude the identification of target-binding sequences. To overcome these challenges, we employed next-generation Illumina sequencing to couple high-throughput screening and high-throughput sequencing, enabling more comprehensive access to the phage display library sequence space. In this work, we define the hallmarks of binding sequences in next-generation sequencing data, and develop a method that identifies several target-binding phage clones for murine, alternatively-activated (M2) macrophages with a high (100%) success rate: sequences and binding motifs were reproducibly present across biological replicates; binding motifs were identified across multiple unique sequences; and an unselected, amplified library accurately filtered out parasitic sequences. In addition, we validate the Multiple Em for Motif Elicitation tool as an efficient and principled means of discovering binding sequences. PMID:26161996

  12. Seneca Valley Virus 3Cpro Substrate Optimization Yields Efficient Substrates for Use in Peptide-Prodrug Therapy.

    PubMed

    Miles, Linde A; Brennen, W Nathaniel; Rudin, Charles M; Poirier, John T

    2015-01-01

    The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3Cpro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3Cpro, we measured cleavage kinetics of predicted SVV-001 3Cpro substrates. An efficient substrate, L/VP4 (kcat/KM = 1932 ± 183 M(-1)s(-1)), was further optimized by a P2' N→P substitution yielding L/VP4.1 (kcat/KM = 17446 ± 2203 M(-1)s(-1)). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3Cpro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3Cpro, with direct implications for anti-cancer therapeutic development.

  13. Seneca Valley Virus 3Cpro Substrate Optimization Yields Efficient Substrates for Use in Peptide-Prodrug Therapy

    PubMed Central

    Miles, Linde A.; Brennen, W. Nathaniel; Rudin, Charles M.; Poirier, John T.

    2015-01-01

    The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3Cpro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3Cpro, we measured cleavage kinetics of predicted SVV-001 3Cpro substrates. An efficient substrate, L/VP4 (kcat/KM = 1932 ± 183 M-1s-1), was further optimized by a P2’ N→P substitution yielding L/VP4.1 (kcat/KM = 17446 ± 2203 M-1s-1). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3Cpro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3Cpro, with direct implications for anti-cancer therapeutic development. PMID:26069962

  14. DNA Subtraction of In Vivo Selected Phage Repertoires for Efficient Peptide Pathology Biomarker Identification in Neuroinflammation Multiple Sclerosis Model

    PubMed Central

    Vargas-Sanchez, Karina; Vekris, Antonios; Petry, Klaus G.

    2016-01-01

    To streamline in vivo biomarker discovery, we developed a suppression subtractive DNA hybridization technique adapted for phage-displayed combinatorial libraries of 12 amino acid peptides (PhiSSH). Physical DNA subtraction is performed in a one-tube-all-reactions format by sequential addition of reagents, producing the enrichment of specific clones of one repertoire. High-complexity phage repertoires produced by in vivo selections in the multiple sclerosis rat model (experimental autoimmune encephalomyelitis, EAE) and matched healthy control rats were used to evaluate the technique. The healthy repertoire served as a physical DNA subtractor from the EAE repertoire to produce the subtraction repertoire. Full next-generation sequencing (NGS) of the three repertoires was performed to evaluate the efficiency of the subtraction technique. More than 96% of the clones common to the EAE and healthy repertoires were absent from the subtraction repertoire, increasing the probability of randomly selecting various specific peptides for EAE pathology to about 70%. Histopathology experiments were performed to confirm the quality of the subtraction repertoire clones, producing distinct labeling of the blood–brain barrier (BBB) affected by inflammation among healthy nervous tissue or the preferential binding to IL1-challenged vs. resting human BBB model. Combining PhiSSH with NGS will be useful for controlled in vivo screening of small peptide combinatorial libraries to discover biomarkers of specific molecular alterations interspersed within healthy tissues. PMID:26917946

  15. Systematic amino acid substitutions improved efficiency of GD2-peptide mimotope vaccination against neuroblastoma.

    PubMed

    Bleeke, Matthias; Fest, Stefan; Huebener, Nicole; Landgraf, Christiane; Schraven, Burkhart; Gaedicke, Gerhard; Volkmer, Rudolf; Lode, Holger N

    2009-11-01

    The likelihood of identifying peptides of sufficient quality for the development of effective cancer vaccines by screening of phage display libraries is low. Here, we introduce the sequential application of systematic amino acid substitution by SPOT synthesis. After the substitution of two amino acids within the sequence of a phage display-derived mimotope of disialoganglioside GD2 (mimotope MA), the novel mimotope C3 showed improved GD2 mimicry in vitro. Peptide vaccination with the C3 mimotope induced an 18-fold increased anti-GD2 serum response associated with reduction of primary tumour growth and spontaneous metastasis in contrast to MA mimotope controls in a syngeneic neuroblastoma model. In summary, SPOT provides an ideal optimisation tool for the development of phage display-derived cancer vaccines.

  16. Dual Receptor Recognizing Cell Penetrating Peptide for Selective Targeting, Efficient Intratumoral Diffusion and Synthesized Anti-Glioma Therapy

    PubMed Central

    Liu, Yayuan; Mei, Ling; Xu, Chaoqun; Yu, Qianwen; Shi, Kairong; Zhang, Li; Wang, Yang; Zhang, Qianyu; Gao, Huile; Zhang, Zhirong; He, Qin

    2016-01-01

    Cell penetrating peptides (CPPs) were widely used for drug delivery to tumor. However, the nonselective in vivo penetration greatly limited the application of CPPs-mediated drug delivery systems. And the treatment of malignant tumors is usually followed by poor prognosis and relapse due to the existence of extravascular core regions of tumor. Thus it is important to endue selective targeting and stronger intratumoral diffusion abilities to CPPs. In this study, an RGD reverse sequence dGR was conjugated to a CPP octa-arginine to form a CendR (R/KXXR/K) motif contained tandem peptide R8-dGR (RRRRRRRRdGR) which could bind to both integrin αvβ3 and neuropilin-1 receptors. The dual receptor recognizing peptide R8-dGR displayed increased cellular uptake and efficient penetration ability into glioma spheroids in vitro. The following in vivo studies indicated the active targeting and intratumoral diffusion capabilities of R8-dGR modified liposomes. When paclitaxel was loaded in the liposomes, PTX-R8-dGR-Lip induced the strongest anti-proliferation effect on both tumor cells and cancer stem cells, and inhibited the formation of vasculogenic mimicry channels in vitro. Finally, the R8-dGR liposomal drug delivery system prolonged the medium survival time of intracranial C6 bearing mice by 2.1-fold compared to the untreated group, and achieved an exhaustive anti-glioma therapy including anti-tumor cells, anti-vasculogenic mimicry and anti-brain cancer stem cells. To sum up, all the results demonstrated that R8-dGR was an ideal dual receptor recognizing CPP with selective glioma targeting and efficient intratumoral diffusion, which could be further used to equip drug delivery system for effective glioma therapy. PMID:26877777

  17. Highly Efficient and Safe Delivery of VEGF siRNA by Bioreducible Fluorinated Peptide Dendrimers for Cancer Therapy.

    PubMed

    Cai, Xiaojun; Zhu, Haofang; Zhang, Yanmei; Gu, Zhongwei

    2017-03-22

    RNA interference (RNAi) has a great promise in treating various acquired and hereditary diseases. However, it remains highly desirable to develop new delivery system to circumvent complex extra- and intracellular barriers for successful clinical translation. Here, we report on a versatile polymeric vector, bioreducible fluorinated peptide dendrimers (BFPD), for efficient and safe small interfering RNA (siRNA) delivery. In virtue of skillfully integrating all of the unique advantages of reversible cross-linking, fluorination, and peptide dendrimers, this novel vector can surmount almost all extra- and intracellular barriers associated with local siRNA delivery through highly improved physiological stability and serum resistance, significantly increased intratumoral enrichment, cellular internalization, successful facilitation of endosomal escape, and cytosolic siRNA release. BFPD polyplexes, carrying small interfering vascular endothelial growth factor (siVEGF), demonstrated excellent VEGF silencing efficacy (∼65%) and a strong capability for inhibiting HeLa cell proliferation. More importantly, these polyplexes showed superior performance in long-term enrichment in the tumor sites and had a high level of tumor growth inhibition. Furthermore, these polyplexes not only exhibited excellent in vivo antitumor efficacy but also demonstrated superior biocompatibility, compared with LPF2000, both in vivo and in vitro. These findings indicate that BFPD is an efficient and safe siRNA delivery system and has remarkable potential for RNAi-based cancer treatment.

  18. Degradable and biocompatible nanoparticles decorated with cyclic RGD peptide for efficient drug delivery to hepatoma cells in vitro.

    PubMed

    Loyer, Pascal; Bedhouche, Wahib; Huang, Zhi Wei; Cammas-Marion, Sandrine

    2013-10-01

    Amphiphilic derivatives of poly(benzyl malate) were synthesized and characterized with the aim of being used as degradable and biocompatible building blocks for the design of functional nanoparticles (NPs). An anti-cancer model drug, doxorubicin, has been successfully encapsulated into the prepared NPs and its release profile has been evaluated in water and in culture medium. NPs bearing biotin molecules were prepared either for site-specific drug delivery via the targeting of biotin receptors overexpressed on the surface of several cancer cells, or for grafting biotinylated cyclic RGD peptide onto their surface using the strong and highly specific interactions between biotin and the streptavidin protein. We have shown that this binding did not affect dramatically the physico-chemical properties of the corresponding NPs. Cyclic RGD grafted fluorescent NPs were more efficiently uptaken by the HepaRG hepatoma cells than biotinylated fluorescent NPs. Furthermore, the targeting of HepaRG hepatoma cells with NPs bearing cyclic RGD was very efficient and much weaker for HeLa and HT29 cell lines confirming that cyclic RGD is a suitable targeting agent for liver cells. Our results also provide a new mean for rapid screening of short hepatotropic peptides in order to design NPs showing specific liver targeting properties.

  19. Synthetic peptides for efficient discrimination of anti-enterovirus antibodies at the serotype level.

    PubMed

    Routsias, John G; Mavrouli, Maria D; Antonaki, Georgia; Spanakis, Nikolaos; Tsakris, Athanassios

    2014-08-01

    Enteroviruses are important human pathogens, causing a broad spectrum of diseases from minor common colds to fatal myocarditis. However, certain disease syndromes are caused by one or few serotypes. Serotype identification is difficult due to the laborious neutralization tests that lack of sensitivity, while in commercial ELISAs homotypic antibodies' activities are largely masked by the recognition of genera-specific epitopes by heterotypic antibodies. In the present study homotypic assays were developed with the ability to discriminate different enterovirus serotypes. Seventy-three children sera, positive for IgM antibodies against enterovirus genus and 49 healthy children were examined for the presence of antibodies against 14 synthetic peptides derived from a non-conserved region of the VP1 protein of coxsackieviruses B2, B3, B4, B5, A9, A16, A24, echoviruses 6, 7, 9, 11, 30, enterovirus 71 and parechovirus 1. 50% of the anti-enterovirus IgM positive sera (>150 BU) reacted with the peptides with the majority of them to preferentially recognize one of them, supporting the homotypic nature of our assay. Inhibition studies yielded homologous inhibition rates 67-95% suggesting that specific peptide recognition actually occurred. The diagnostic value of our assay was tested in blood samples drawn over a 1.5-year period from a 5-year old patient. The anti-enterovirus reactivity was clearly attributed to echovirus serotype 11. The IgM/IgG antibody ratio was reversed 4 months later and subsequently IgM antibodies dropped below the cutoff point. In this paper we demonstrate that our assay can be used to discriminate between antibodies targeting different enterovirus serotypes.

  20. A Biodegradable Polyethylenimine-Based Vector Modified by Trifunctional Peptide R18 for Enhancing Gene Transfection Efficiency In Vivo.

    PubMed

    Hu, Jing; Zhu, Manman; Liu, Kehai; Fan, Hua; Zhao, Wenfang; Mao, Yuan; Zhang, Yaguang

    2016-01-01

    Lack of capacity to cross the nucleus membrane seems to be one of the main reasons for the lower transfection efficiency of gene vectors observed in vivo study than in vitro. To solve this problem, a new non-viral gene vector was designed. First, a degradable polyethylenimine (PEI) derivate was synthesized by crosslinking low-molecular-weight (LMW) PEI with N-octyl-N-quaternary chitosan (OTMCS), and then adopting a designed trifunctional peptide (RGDC-TAT-NLS) with good tumor targeting, cell uptake and nucleus transport capabilities to modify OTMCS-PEI. The new gene vector was termed as OTMCS-PEI-R18 and characterized in terms of its chemical structure and biophysical parameters. Gene transfection efficiency and nucleus transport mechanism of this vector were also evaluated. The polymer showed controlled degradation and remarkable buffer capabilities with the particle size around 100-300 nm and the zeta potential ranged from 5 mV to 40 mV. Agraose gel electrophoresis showed that OTMCS-PEI-R18 could effectively condensed plasmid DNA at a ratio of 1.0. Besides, the polymer was stable in the presence of sodium heparin and could resist digestion by DNase I at a concentration of 63U DNase I/DNA. OTMCS-PEI-R18 also showed much lower cytotoxicity and better transfection rates compared to polymers OTMCS-PEI-R13, OTMCS-PEI and PEI 25 KDa in vitro and in vivo. Furthermore, OTMCS-PEI-R18/DNA complexes could accumulate in the nucleus well soon and not rely on mitosis absolutely due to the newly incorporated ligand peptide NLS with the specific nuclear delivery pathway indicating that the gene delivery system OTMCS-PEI-R18 could reinforce gene transfection efficiency in vivo.

  1. A Biodegradable Polyethylenimine-Based Vector Modified by Trifunctional Peptide R18 for Enhancing Gene Transfection Efficiency In Vivo

    PubMed Central

    Hu, Jing; Zhu, Manman; Fan, Hua; Zhao, Wenfang; Mao, Yuan; Zhang, Yaguang

    2016-01-01

    Lack of capacity to cross the nucleus membrane seems to be one of the main reasons for the lower transfection efficiency of gene vectors observed in vivo study than in vitro. To solve this problem, a new non-viral gene vector was designed. First, a degradable polyethylenimine (PEI) derivate was synthesized by crosslinking low-molecular-weight (LMW) PEI with N-octyl-N-quaternary chitosan (OTMCS), and then adopting a designed trifunctional peptide (RGDC-TAT-NLS) with good tumor targeting, cell uptake and nucleus transport capabilities to modify OTMCS-PEI. The new gene vector was termed as OTMCS-PEI-R18 and characterized in terms of its chemical structure and biophysical parameters. Gene transfection efficiency and nucleus transport mechanism of this vector were also evaluated. The polymer showed controlled degradation and remarkable buffer capabilities with the particle size around 100–300 nm and the zeta potential ranged from 5 mV to 40 mV. Agraose gel electrophoresis showed that OTMCS-PEI-R18 could effectively condensed plasmid DNA at a ratio of 1.0. Besides, the polymer was stable in the presence of sodium heparin and could resist digestion by DNase I at a concentration of 63U DNase I/DNA. OTMCS-PEI-R18 also showed much lower cytotoxicity and better transfection rates compared to polymers OTMCS-PEI-R13, OTMCS-PEI and PEI 25 KDa in vitro and in vivo. Furthermore, OTMCS-PEI-R18/DNA complexes could accumulate in the nucleus well soon and not rely on mitosis absolutely due to the newly incorporated ligand peptide NLS with the specific nuclear delivery pathway indicating that the gene delivery system OTMCS-PEI-R18 could reinforce gene transfection efficiency in vivo. PMID:27935984

  2. A specific aptamer-cell penetrating peptides complex delivered siRNA efficiently and suppressed prostate tumor growth in vivo

    PubMed Central

    Diao, Yanjun; Liu, Jiayun; Ma, Yueyun; Su, Mingquan; Zhang, Hongyi; Hao, Xiaoke

    2016-01-01

    ABSTRACT Specific and efficient delivery of siRNA into intended tumor cells remains as a challenge, even though RNAi has been exploited as a new strategy for prostate cancer therapy. This work aims to address both specificity and efficiency of SURVIVIN-siRNA delivery by constructing a therapeutic complex using combinatorial strategies. A fusion protein STD was first expressed to serve as a backbone, consisting of streptavidin, a cell-penetrating peptide called Trans-Activator of Transcription (TAT) and a double-stranded RNA binding domain. A biotinylated Prostate Specific Membrane Antigen (PSMA) specific aptamer A10 and SURVIVIN-siRNA were then linked to STD protein to form the therapeutic complex. This complex could specifically targeted PSMA+ tumor cells. Compared to lipofectamine and A10-siRNA chimera, it demonstrated higher efficiency in delivering siRNA into target cells by 19.2% and 59.9%, and increased apoptosis by 16.8% and 26.1% respectively. Upon systemic administration, this complex also showed significant efficacy in suppressing tumor growth in athymic mice (p <0.001). We conclude that this therapeutic complex could specifically and efficiently deliver SURVIVIN-siRNA to target cells and suppressed tumor growth in vivo, which indicates its potential to be used as a new strategy in prostate cancer therapy PMID:26954374

  3. Highly efficient peptide formation from N-acetylaminoacyl-AMP anhydride and free amino acid

    NASA Technical Reports Server (NTRS)

    Mullins, D. W., Jr.; Lacey, J. C., Jr.

    1983-01-01

    The kinetics of formation of the N-blocked dipeptide, N-acetylglycylglycine, from N-acetylglycyl adenylate anhydride and glycine in aqueous solution at 25 C, and at various PH's are reported. The reaction is of interest in that over a physiologically relevant pH range (6-8), peptide synthesis proceeds more rapidly than hydrolysis, even at those pH's at which this compound becomes increasingly susceptible to base-catalyzed hydrolysis. Under similar conditions, the corresponding unblocked aminoacyl adenylate anhydrides are considerably more unstable, and undergo appreciable hydrlysis in the presence of free amino acid. Because N-blocked aminoacyl adenylate anhydrides serve as model compounds of peptidyl adenylate anhydrides, these results suggest that primitive amino acid polymerization systems may have operated by cyclic reactivation of the peptidyl carboxyl group, rather than that of the incoming amino acid.

  4. Facile synthesis of yolk-shell magnetic mesoporous carbon microspheres for efficient enrichment of low abundance peptides

    NASA Astrophysics Data System (ADS)

    Wan, Hao; Qin, Hongqiang; Xiong, Zhichao; Zhang, Weibing; Zou, Hanfa

    2013-10-01

    Magnetic mesoporous carbon microspheres with a yolk-shell structure (YSMMCS) have been prepared via a new in situ carbon source strategy. The material was fabricated by two shells coated onto the Fe3O4 particles; the inner dense and thick silica shell could protect the magnetic core from harsh acidic solvents as well as induce the void between the core and the outer shell for the yolk-shell structure, while the outer organosilica shell was used as the template and carbon source for in situ preparation of a carbon shell with mesoporous structure. A C18-alkyl chain was incorporated in situ as the carbon precursor efficiently, avoiding the conventional infiltration step, which was very difficult to manipulate and time-consuming with the possibility of losing the carbon precursor. The resulting yolk-shell magnetic mesoporous carbon microspheres exhibited a high surface area (273.15 m2 g-1), a large pore volume (0.31 cm3 g-1), and a strong magnetic response (a saturation magnetization value of 34.57 emu g-1). As a result of the void between the core and the outer shell and the π-π stacking effect, adsorption capacity reached 191.64 mg g-1 by using Rhodamine B as a standard analyte, indicating the great potential application of the material as drug carriers. Owing to the inherent hydrophobicity and high surface area, the composite material showed better performance in the enrichment of peptides than a magnetic mesoporous silica material (Fe2O3@nSiO2@mSiO2). According to the LC-MS/MS results, about 51 and 29 nonredundant peptides were identified from tryptic digests of 5 nM BSA. Additionally, taking advantage of the mesoporous structure and strong magnetic response, the material was utilized to selectively extract low abundance endogenous peptides from human serum in the presence of high abundance proteins. Based on the LC-MS/MS results, 962 endogenous peptides were obtained by 2.5 mg YSMMCS relative to 539 endogenous peptides by 5 mg Fe2O3@nSiO2@mSiO2, confirming the

  5. Low-density lipoprotein peptide-combined DNA nanocomplex as an efficient anticancer drug delivery vehicle.

    PubMed

    Zhang, Nan; Tao, Jun; Hua, Haiying; Sun, Pengchao; Zhao, Yongxing

    2015-08-01

    DNA is a type of potential biomaterials for drug delivery due to its nanoscale geometry, loading capacity of therapeutics, biocompatibility, and biodegradability. Unfortunately, DNA is easily degraded by DNases in the body circulation and has low intracellular uptake. In the present study, we selected three cationic polymers polyethylenimine (PEI), hexadecyl trimethyl ammonium bromide (CTAB), and low-density lipoprotein (LDL) receptor targeted peptide (RLT), to modify DNA and improve the issues. A potent anti-tumor anthracycline-doxorubicin (DOX) was intercalated into DNA non-covalently and the DOX/DNA was then combined with PEI, CTAB, and RLT, respectively. Compact nanocomplexes were formed by electrostatic interaction and could potentially protect DNA from DNases. More importantly, RLT had the potential to enhance intracellular uptake by LDL receptor mediated endocytosis. In a series of in vitro experiments, RLT complexed DNA enhanced intracellular delivery of DOX, increased tumor cell death and intracellular ROS production, and reduced intracellular elimination of DOX. All results suggested that the easily prepared and targeted RLT/DNA nanocomplexes had great potential to be developed into a formulation for doxorubicin with enhanced anti-tumor activity.

  6. Peptide-Mediated Liposomal Doxorubicin Enhances Drug Delivery Efficiency and Therapeutic Efficacy in Animal Models

    PubMed Central

    Chang, De-Kuan; Li, Pi-Chun; Lu, Ruei-Min; Jane, Wann-Neng; Wu, Han-Chung

    2013-01-01

    Lung cancer ranks among the most common malignancies, and is the leading cause of cancer-related mortality worldwide. Chemotherapy for lung cancer can be made more specific to tumor cells, and less toxic to normal tissues, through the use of ligand-mediated drug delivery systems. In this study, we investigated the targeting mechanism of the ligand-mediated drug delivery system using a peptide, SP5-2, which specifically binds to non-small cell lung cancer (NSCLC) cells. Conjugation of SP5-2 to liposomes enhanced the amount of drug delivered directly into NSCLC cells, through receptor-mediated endocytosis. Functional SP5-2 improved the therapeutic index of Lipo-Dox by enhancing therapeutic efficacy, reducing side effects, and increasing the survival rate of tumor-bearing mice in syngenic, metastatic and orthotopic animal models. Accumulation of SP5-2-conjugated liposomal doxorubicin (SP5-2-LD) in tumor tissues was 11.2-fold higher than that of free doxorubicin, and the area under the concentration-time curve (AUC0–72 hours) was increased 159.2-fold. Furthermore, the experiment of bioavailability was assessed to confirm that SP5-2 elevates the uptake of the liposomal drugs by the tumor cells in vivo. In conclusion, the use of SP5-2-conjugated liposomes enhances pharmacokinetic properties, improves efficacy and safety profiles, and allows for controlled biodistribution and drug release. PMID:24386166

  7. Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization.

    PubMed

    Wei, Xuetuan; Zhou, Yinhua; Chen, Jingbang; Cai, Dongbo; Wang, Dan; Qi, Gaofu; Chen, Shouwen

    2015-02-01

    Nattokinase (NK) possesses the potential for prevention and treatment of thrombus-related diseases. In this study, high-level expression of nattokinase was achieved in Bacillus licheniformis WX-02 via host strain construction and signal peptides optimization. First, ten genes (mpr, vpr, aprX, epr, bpr, wprA, aprE, bprA, hag, amyl) encoding for eight extracellular proteases, a flagellin and an amylase were deleted to obtain B. licheniformis BL10, which showed no extracellular proteases activity in gelatin zymography. Second, the gene fragments of P43 promoter, Svpr, nattokinase and TamyL were combined into pHY300PLK to form the expression vector pP43SNT. In BL10 (pP43SNT), the fermentation activity and product activity per unit of biomass of nattokinase reached 14.33 FU/mL and 2,187.71 FU/g respectively, which increased by 39 and 156 % compared to WX-02 (pP43SNT). Last, Svpr was replaced with SsacC and SbprA, and the maximum fermentation activity (33.83 FU/mL) was achieved using SsacC, which was 229 % higher than that of WX-02 (pP43SNT). The maximum NK fermentation activity in this study reaches the commercial production level of solid state fermentation, and this study provides a promising engineered strain for industrial production of nattokinase, as well as a potential platform host for expression of other target proteins.

  8. Dual-function synthetic peptide derived from BMP4 for highly efficient tumor targeting and antiangiogenesis

    PubMed Central

    Choi, Suk Hyun; Lee, Jue Yeon; Suh, Jin Sook; Park, Yoon Shin; Chung, Chong Pyoung; Park, Yoon Jeong

    2016-01-01

    Angiogenesis plays a critical role in the growth and metastasis of cancer, and growth factors released from cancer promote blood-vessel formation in the tumor microenvironment. The angiogenesis is accelerated via interactions of growth factors with the high-affinity receptors on cancer cells. In particular, heparan sulfate proteoglycans (HSPGs) on the surface of cancer cells have been shown to be important in many aspects of determining a tumor’s phenotype and development. Specifically, the regulation of the interactions between HSPGs and growth factors results in changes in tumor progression. A peptide with heparin-binding (HBP) activity has been developed and synthesized to inhibit tumor growth via the prevention of angiogenesis. We hypothesized that HBP could inhibit the interaction of growth factors and HSPGs on the surface of cancer cells, decrease paracrine signaling in endothelial cells (ECs), and finally decrease angiogenesis in the tumor microenvironment. In this study, we found that HBP had antiangiogenic effects in vitro and in vivo. The conditioned media obtained from a breast cancer cell line treated with HBP were used to culture human umbilical vein ECs (HUVECs) to evaluate the antiangiogenic effect of HBP on ECs. HBP effectively inhibited the migration, invasion, and tube formation of HUVECs in vitro. In addition, the expressions of angiogenesis-mediating factors, including ERK, FAK, and Akt, were considerably decreased. HBP also decreased the levels of invasive factors, including MMP2 and MMP9, secreted by the HUVECs. We demonstrated significant suppression of tumor growth in a breast cancer xenograft model and enhanced distribution of HBP at the site of tumors. Taken together, our results show that HBP has antiangiogenic effects on ECs, and suggest that it may serve as a potential antitumor agent through control of the tumor microenvironment. PMID:27695323

  9. WT1 peptide vaccination combined with BCG-CWS is more efficient for tumor eradication than WT1 peptide vaccination alone.

    PubMed

    Nakajima, Hiroko; Kawasaki, Kotomi; Oka, Yoshihiro; Tsuboi, Akihiro; Kawakami, Manabu; Ikegame, Kazuhiro; Hoshida, Yoshihiko; Fujiki, Fumihiro; Nakano, Akiko; Masuda, Tomoki; Wu, Fei; Taniguchi, Yuki; Yoshihara, Satoshi; Elisseeva, Olga A; Oji, Yusuke; Ogawa, Hiroyasu; Azuma, Ichiro; Kawase, Ichiro; Aozasa, Katsuyuki; Sugiyama, Haruo

    2004-07-01

    A Wilms' tumor gene WT1 is expressed at high levels not only in most types of leukemia but also in various types of solid tumors, including lung and breast cancer. WT1 protein has been reported to serve as a target antigen for tumor-specific immunotherapy both in vitro in human systems and in vivo in murine models. We have shown that mice immunized with WT1 peptide or WT1 cDNA could reject a challenge from WT1-expressing tumor cells (a "prophylactic" model). However, it was not examined whether WT1 peptide vaccination had the potency to reject tumor cells in a "therapeutic" setting. In the present study, we demonstrated for the first time that WT1 peptide vaccination combined with Mycobacterium bovis bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) was more effective for eradication of WT1-expressing tumor cells that had been implanted into mice before vaccination (a "therapeutic" model) compared with WT1 peptide vaccination alone. An intradermal injection of BCG-CWS into mice, followed by that of WT1 peptide at the same site on the next day, generated WT1-specific cytotoxic T lymphocytes (CTLs) and led to rejection of WT1-expressing leukemia or lung cancer cells. These results showed that BCG-CWS, which was well known to enhance innate immunity, could enhance WT1-specific immune responses (acquired immunity) in combination with WT1 peptide vaccination. Therefore, WT1 peptide vaccination combined with BCG-CWS may be applied to cancer immunotherapy in clinical settings.

  10. RGD peptide-modified dendrimer-entrapped gold nanoparticles enable highly efficient and specific gene delivery to stem cells.

    PubMed

    Kong, Lingdan; Alves, Carla S; Hou, Wenxiu; Qiu, Jieru; Möhwald, Helmuth; Tomás, Helena; Shi, Xiangyang

    2015-03-04

    We report the use of arginine-glycine-aspartic (Arg-Gly-Asp, RGD) peptide-modified dendrimer-entrapped gold nanoparticles (Au DENPs) for highly efficient and specific gene delivery to stem cells. In this study, generation 5 poly(amidoamine) dendrimers modified with RGD via a poly(ethylene glycol) (PEG) spacer and with PEG monomethyl ether were used as templates to entrap gold nanoparticles (AuNPs). The native and the RGD-modified PEGylated dendrimers and the respective well characterized Au DENPs were used as vectors to transfect human mesenchymal stem cells (hMSCs) with plasmid DNA (pDNA) carrying both the enhanced green fluorescent protein and the luciferase (pEGFPLuc) reporter genes, as well as pDNA encoding the human bone morphogenetic protein-2 (hBMP-2) gene. We show that all vectors are capable of transfecting the hMSCs with both pDNAs. Gene transfection using pEGFPLuc was demonstrated by quantitative Luc activity assay and qualitative evaluation by fluorescence microscopy. For the transfection with hBMP-2, the gene delivery efficiency was evaluated by monitoring the hBMP-2 concentration and the level of osteogenic differentiation of the hMSCs via alkaline phosphatase activity, osteocalcin secretion, calcium deposition, and von Kossa staining assays. Our results reveal that the stem cell gene delivery efficiency is largely dependent on the composition and the surface functionality of the dendrimer-based vectors. The coexistence of RGD and AuNPs rendered the designed dendrimeric vector with specific stem cell binding ability likely via binding of integrin receptor on the cell surface and improved three-dimensional conformation of dendrimers, which is beneficial for highly efficient and specific stem cell gene delivery applications.

  11. Efficient routes to carbon-silicon bond formation for the synthesis of silicon-containing peptides and azasilaheterocycles.

    PubMed

    Min, Geanna K; Hernández, Dácil; Skrydstrup, Troels

    2013-02-19

    product. We demonstrated that a variety of Ar(2)SiH(2) compounds with methyl substituents on the aromatic core could effectively undergo hydrosilylation and reductive lithiation with a soluble reducing agent, lithium naphthalenide. The electron-rich aromatic groups were more acid labile and, depending on the conditions, could produce either the silane diol or the silanol. In an alternative strategy, we used a highly regioselective Rh-catalyzed sequential double hydrosilylation to form the two C-Si bonds with a single catalyst. This approach is a more efficient, atom economical way to synthesize a wider range of highly functionalized organosilanes with the added possibility of extending this method into an asymmetric protocol. By this method, various functional groups that were not previously tolerated in the lithiation protocol, including OBn, OAc, furyl, and thiophenes, could now be incorporated. Hydrosilylation of a terminal olefin and a peptide functionalized with an enamide at the C-terminus achieved the desired silane in high yields in a one pot reaction without compromising the stereochemical integrity of the peptide. As an extension of this work, we used these methods to efficiently generate a variety of chiral azasilaheterocycles, including silapiperidines and silaindolizidines.

  12. Highly efficient delivery of functional cargoes by the synergistic effect of GAG binding motifs and cell-penetrating peptides

    PubMed Central

    Dixon, James E.; Osman, Gizem; Morris, Gavin E.; Markides, Hareklea; Rotherham, Michael; Bayoussef, Zahia; El Haj, Alicia J.; Denning, Chris; Shakesheff, Kevin M.

    2016-01-01

    Protein transduction domains (PTDs) are powerful nongenetic tools that allow intracellular delivery of conjugated cargoes to modify cell behavior. Their use in biomedicine has been hampered by inefficient delivery to nuclear and cytoplasmic targets. Here we overcame this deficiency by developing a series of novel fusion proteins that couple a membrane-docking peptide to heparan sulfate glycosaminoglycans (GAGs) with a PTD. We showed that this GET (GAG-binding enhanced transduction) system could deliver enzymes (Cre, neomycin phosphotransferase), transcription factors (NANOG, MYOD), antibodies, native proteins (cytochrome C), magnetic nanoparticles (MNPs), and nucleic acids [plasmid (p)DNA, modified (mod)RNA, and small inhibitory RNA] at efficiencies of up to two orders of magnitude higher than previously reported in cell types considered hard to transduce, such as mouse embryonic stem cells (mESCs), human ESCs (hESCs), and induced pluripotent stem cells (hiPSCs). This technology represents an efficient strategy for controlling cell labeling and directing cell fate or behavior that has broad applicability for basic research, disease modeling, and clinical application. PMID:26733682

  13. A highly efficient modified human serum albumin signal peptide to secrete proteins in cells derived from different mammalian species.

    PubMed

    Attallah, Carolina; Etcheverrigaray, Marina; Kratje, Ricardo; Oggero, Marcos

    2017-01-10

    Signal peptides (SPs) are key elements in the production of recombinant proteins; however, little information is available concerning different SP in mammalian cells other than CHO. In order to study the efficiency of different SPs to direct the traffic along the secretory pathway of the green fluorescence protein (GFP) and a scFv-Fc fusion protein; CHO-K1, HEK293 and NS0 cell lines were transfected in a transient and stable way. SP of human azurocidin (AZ), modified human albumin (mSA), modified Cricetulus griseus Ig kappa chain V III region MOPC 63 like (mIgκ C) and modified human Ig kappa chain V III region VG (mIgκ H) were evaluated. The efficiency of SPs to translocate a propeptide across the ER membrane was evaluated by fluorescence microscopy and flow cytometry for the GFP inside the secretory pathway, and by antigen-specific indirect ELISA for the scFv-Fc outside the cell. The mSA SP was successful in directing the secretion of the active proteins in these different types of mammalian cells, regardless of the transgene copy number. The goal of this work was to demonstrate that a modified version of SA SP might be used in different mammalian cells employing the same expression vector.

  14. Peptide Self-Assembled Biofilm with Unique Electron Transfer Flexibility for Highly Efficient Visible-Light-Driven Photocatalysis.

    PubMed

    Pan, Yun-Xiang; Cong, Huai-Ping; Men, Yu-Long; Xin, Sen; Sun, Zheng-Qing; Liu, Chang-Jun; Yu, Shu-Hong

    2015-11-24

    Inspired by natural photosynthesis, biomaterial-based catalysts are being confirmed to be excellent for visible-light-driven photocatalysis, but are far less well explored. Herein, an ultrathin and uniform biofilm fabricated from cold-plasma-assisted peptide self-assembly was employed to support Eosin Y (EY) and Pt nanoparticles to form an EY/Pt/Film catalyst for photocatalytic water splitting to H2 and photocatalytic CO2 reduction with water to CO, under irradiation of visible light. The H2 evolution rate on EY/Pt/Film is 62.1 μmol h(-1), which is about 5 times higher than that on Pt/EY and 1.5 times higher than that on the EY/Pt/TiO2 catalyst. EY/Pt/Film exhibits an enhanced CO evolution rate (19.4 μmol h(-1)), as compared with Pt/EY (2.8 μmol h(-1)) and EY/Pt/TiO2 (6.1 μmol h(-1)). The outstanding activity of EY/Pt/Film results from the unique flexibility of the biofilm for an efficient transfer of the photoinduced electrons. The present work is helpful for designing efficient biomaterial-based catalysts for visible-light-driven photocatalysis and for imitating natural photosynthesis.

  15. A highly efficient route to enantiomerically pure l-N-Bz-Pmp(t-Bu)2-OH and incorporation into a peptide-based protein tyrosine phosphatase inhibitor.

    PubMed

    Hubbard, Caitlin E; Barrios, Amy M

    2008-01-15

    Phosphonomethyl phenylalanine (Pmp), a nonhydrolyzable mimic of phosphotyrosine, is an important building block in the development of peptide-based PTP inhibitors. We have designed a novel, efficient synthesis of N-Bz-Pmp(t-Bu)2-OH. A Pmp-containing peptide based on a known biological substrate of the tyrosine phosphatase CD45 (Ac-TEGQ-Pmp-QPQP-NH2) inhibits CD45 with an IC50 value of approximately 100 microM with virtually no inhibition of TCPTP up to concentrations of 120 microM.

  16. Efficient sequence-directed psoralen targeting using pseudocomplementary Peptide nucleic acids.

    PubMed

    Kim, Ki-Hyun; Fan, Xue-Jun; Nielsen, Peter E

    2007-01-01

    A pair of decameric pseudocomplementary PNAs which bind to their mixed purine-pyrimidine sequence target in duplex DNA by double duplex invasion has been synthesized with a derivative of 8-methoxypsoralen conjugated to one of the PNAs. It is shown that this pair of psoralen-conjugated pseudocomplementary PNA oligomers, which target a site in the pBluescriptKS+ vector, upon irradiation with long-wavelength UV light (UVA) with high efficiency and specificity form photoadducts to an adjacent 5'-TA site, and more than 50% of these adducts are DNA interstrand cross-links. Transcription elongation by T7 or T3 RNA polymerase is specifically arrested at the psoralen cross-linking site, yielding more than 90% arrested product. These results emphasize the potential of pseudocomplementary PNA oligomers for highly specific gene targeting, in particular, with respect to sequence-directed psoralen photomodification of double-stranded DNA. Thus, such psoralen-PNA conjugates could be very useful in a range of biology and drug discovery applications.

  17. MRUniNovo: an efficient tool for de novo peptide sequencing utilizing the hadoop distributed computing framework.

    PubMed

    Li, Chuang; Chen, Tao; He, Qiang; Zhu, Yunping; Li, Kenli

    2016-12-19

    Tandem mass spectrometry-based de novo peptide sequencing is a complex and time-consuming process. The current algorithms for de novo peptide sequencing cannot rapidly and thoroughly process large mass spectrometry datasets. In this paper, we propose MRUniNovo, a novel tool for parallel de novo peptide sequencing. MRUniNovo parallelizes UniNovo based on the Hadoop compute platform. Our experimental results demonstrate that MRUniNovo significantly reduces the computation time of de novo peptide sequencing without sacrificing the correctness and accuracy of the results, and thus can process very large datasets that UniNovo cannot.

  18. Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo.

    PubMed

    Chen, Joy; Peterson, Kenneth R; Iancu-Rubin, Camelia; Bieker, James J

    2010-09-28

    Pharmacological treatments designed to reactivate fetal γ-globin can lead to an effective and successful clinical outcome in patients with hemoglobinopathies. However, new approaches remain highly desired because such treatments are not equally effective for all patients, and toxicity issues remain. We have taken a systematic approach to develop an embedded chimeric peptide nucleic acid (PNA) that effectively enters the cell and the nucleus, binds to its target site at the human fetal γ-globin promoter, and reactivates this transcript in adult transgenic mouse bone marrow and human primary peripheral blood cells. In vitro and in vivo DNA-binding assays in conjunction with live-cell imaging have been used to establish and optimize chimeric PNA design parameters that lead to successful gene activation. Our final molecule contains a specific γ-promoter-binding PNA sequence embedded within two amino acid motifs: one leads to efficient cell/nuclear entry, and the other generates transcriptional reactivation of the target. These embedded PNAs overcome previous limitations and are generally applicable to the design of in vivo transcriptional activation reagents that can be directed to any promoter region of interest and are of direct relevance to clinical applications that would benefit from such a need.

  19. A method for concentrating lipid peptide DNA and siRNA nanocomplexes that retains their structure and transfection efficiency

    PubMed Central

    Tagalakis, Aristides D; Castellaro, Sara; Zhou, Haiyan; Bienemann, Alison; Munye, Mustafa M; McCarthy, David; White, Edward A; Hart, Stephen L

    2015-01-01

    Nonviral gene and small interfering RNA (siRNA) delivery formulations are extensively used for biological and therapeutic research in cell culture experiments, but less so in in vivo and clinical research. Difficulties with formulating the nanoparticles for uniformity and stability at concentrations required for in vivo and clinical use are limiting their progression in these areas. Here, we report a simple but effective method of formulating monodisperse nanocomplexes from a ternary formulation of lipids, targeting peptides, and nucleic acids at a low starting concentration of 0.2 mg/mL of DNA, and we then increase their concentration up to 4.5 mg/mL by reverse dialysis against a concentrated polymer solution at room temperature. The nanocomplexes did not aggregate and they had maintained their biophysical properties, but, importantly, they also mediated DNA transfection and siRNA silencing in cultured cells. Moreover, concentrated anionic nanocomplexes administered by convection-enhanced delivery in the striatum showed efficient silencing of the β-secretase gene BACE1. This method of preparing nanocomplexes could probably be used to concentrate other nonviral formulations and may enable more widespread use of nanoparticles in vivo. PMID:25878500

  20. Which one among aspartyl protease, metallopeptidase, and artificial metallopeptidase is the most efficient catalyst in peptide hydrolysis?

    PubMed

    Bora, Ram Prasad; Barman, Arghya; Zhu, Xiaoxia; Ozbil, Mehmet; Prabhakar, Rajeev

    2010-08-26

    In this comparative DFT study, the hydrolysis of a peptide bond (Phe1-Phe2) by the following three types of catalysts has been studied: (1) beta-secretase (BACE2), (2) matrix metalloproteinase (MMP) and insulin degrading enzyme (IDE), and (3) [Pd(H(2)O)(4)](2+) (I(MPC)) and [Pd(2)(mu-OH)([18]aneN(6))](3+) (I(DPC)). The computed energetics predict that among these catalysts, the Zn(2+) metal center containing MMP is the most efficient in catalyzing this reaction. The two active site aspartate residues containing BACE2 catalyze this reaction with 5.0 kcal/mol higher barrier than MMP. The substitution of a His ligand with Glu in the metal center of MMP generates the active site of IDE that catalyzes the reaction with a 6.9 kcal/mol higher barrier than MMP. Both artificial peptidases I(MPC) and I(DPC) catalyze this reaction with significantly high barriers of 35.4 and 31.0 kcal/mol, respectively. The computed energetics of all the catalysts are in line with the available experimental and theoretical data.

  1. Improving chemotherapeutic efficiency in acute myeloid leukemia treatments by chemically synthesized peptide interfering with CXCR4/CXCL12 axis

    PubMed Central

    Li, Xiaojin; Guo, Hua; Duan, Hongyang; Yang, Yanlian; Meng, Jie; Liu, Jian; Wang, Chen; Xu, Haiyan

    2015-01-01

    Bone marrow stroma can protect acute myeloid leukemia (AML) cells against chemotherapeutic agents and provide anti-apoptosis and chemoresistance signals through secreting chemokine CXCL12 to activate its receptor CXCR4 on AML cells, resulting in minimal residual leukemia and relapse. Therefore disrupting the CXCR4/CXCL12 axis with antagonists is of great significance for improving chemosensitivity and decreasing relapse rate. In a previous study, we reported a novel synthetic peptide E5 with its remarkable effect on inhibiting CXCR4/CXCL12-mediated adhesion and migration of AML cells. Here we presented E5’s capacity of enhancing the therapeutic efficiency of various chemotherapeutics on AML in vitro and in vivo. Results showed that E5 can diminish bone marrow stromal cell-provided protection to leukemia cells, significantly increasing the apoptosis induced by various chemotherapeutics in multiple AML cell lines. In an AML mouse xenograft model, E5 induced 1.84-fold increase of circulating AML cells out of protective stroma niche. Combined with vincristine or cyclophosphamide, E5 inhibited infiltration of AML cells into bone marrow, liver and spleen, as well as prolonged the lifespan of AML mice compared with mice treated with chemotherapy alone. In addition, E5 presented no toxicity in vivo according to the histological analysis and routine clinical parameters of serum analysis. PMID:26538086

  2. pH-Responsive Triblock Copolymeric Micelles Decorated with a Cell-Penetrating Peptide Provide Efficient Doxorubicin Delivery

    NASA Astrophysics Data System (ADS)

    Ng, Khen Eng; Amin, Mohd Cairul Iqbal Mohd; Katas, Haliza; Amjad, Muhammad Wahab; Butt, Adeel Masood; Kesharwani, Prashant; Iyer, Arun K.

    2016-12-01

    This study developed novel triblock pH-responsive polymeric micelles (PMs) using cholic acid-polyethyleneimine-poly- l-arginine (CA-PEI-pArg) copolymers. PEI provided pH sensitivity, while the hydrophilic cell-penetrating pArg peptide promoted cellular PM internalization. The copolymers self-assembled into PMs in aqueous solution at above the critical micelle concentration (2.98 × 10-7 M) and encapsulated doxorubicin in the core region, with a 34.2% ( w/ w) entrapment efficiency. PMs showed pH-dependent swelling, increasing in size by almost sevenfold from pH 7.4 to 5.0. Doxorubicin release was pH-dependent, with about 65% released at pH 5.0, and 32% at pH 7.4. Cellular uptake, assessed by confocal microscopy and flow cytometry, was enhanced by using doxorubicin-loaded CA-PEI-pArg PMs, as compared to free doxorubicin and DOX-loaded CA-PEI PMs. Moreover, 24-h incubation of these PMs with a human breast cancer cell line produced greater cytotoxicity than free doxorubicin. These results indicate that pH-responsive CA-PEI-pArg micelles could provide a versatile delivery system for targeted cancer therapy using hydrophobic drugs.

  3. Development of Nanoparticles Incorporating a Novel Liposomal Membrane Destabilization Peptide for Efficient Release of Cargos into Cancer Cells

    PubMed Central

    Ohgita, Takashi; Kogure, Kentaro

    2014-01-01

    In anti-cancer therapy mediated by a nanoparticle-based drug delivery system (DDS), overall efficacy depends on the release efficiency of cargos from the nanoparticles in the cancer cells as well as the specificity of delivery to tumor tissue. However, conventional liposome-based DDS have no mechanism for specifically releasing the encapsulated cargos inside the cancer cells. To overcome this barrier, we developed nanoparticles containing a novel liposomal membrane destabilization peptide (LMDP) that can destabilize membranes by cleavage with intramembranous proteases on/in cancer cells. Calcein encapsulated in liposomes modified with LMDP (LMDP-lipo) was effectively released in the presence of a membrane fraction containing an LMDP-cleavable protease. The release was inhibited by a protease inhibitor, suggesting that LMDP-lipo could effectively release its cargo into cells in response to a cancer-specific protease. Moreover, when LMDP-lipo contained fusogenic lipids, the release of cargo was accelerated, suggesting that the fusion of LMDP-lipo with cellular membranes was the initial step in the intracellular delivery. Time-lapse microscopic observations showed that the release of cargo from LMDP-lipo occurred immediately after association of LMDP-lipo with target cells. Consequently, LMDP-lipo could be a useful nanoparticle capable of effective release of cargos specifically into targeted cancer cells. PMID:25343714

  4. Efficient help for autoreactive B-cell activation requires CD4+ T-cell recognition of an agonist peptide at the effector stage.

    PubMed

    Hondowicz, Brian D; Batheja, Amrita O; Metzgar, Michele H; Pagán, Antonio J; Perng, Olivia A; Willms, Simone; Caton, Andrew J; Erikson, Jan

    2009-09-01

    T-cell recognition of peptide/MHC complexes is flexible and can lead to differential activation, but how interactions with agonist (full activation) or partial agonist (suboptimal activation) peptides can shape immune responses in vivo is not well characterized. We investigated the effect of stimulation by agonist or partial agonist ligands during initial CD4(+) T-cell priming, and subsequent T-B-cell cognate interactions, on antibody production by anti-chromatin B cells. We found that autoantibody production required TCR recognition of an agonist peptide at the effector stage of B-cell activation. However, interaction with a weak agonist ligand at this effector stage failed to promote efficient autoantibody production, even if the CD4(+) T cells were fully primed by an agonist peptide. These studies suggest that the reactivity of the TCR for a target self-peptide during CD4(+) T-B-cell interaction can be a critical determinant in restraining anti-chromatin autoantibody production.

  5. Construction of a highly active secretory expression system via an engineered dual promoter and a highly efficient signal peptide in Bacillus subtilis.

    PubMed

    Guan, Chengran; Cui, Wenjing; Cheng, Jintao; Liu, Rui; Liu, Zhongmei; Zhou, Li; Zhou, Zhemin

    2016-05-25

    A strong promoter and highly efficient signal peptides are essential for the secretory overproduction of recombinant proteins in Bacillus subtilis. To enhance the limited overexpression capability of natural promoters, various strategies for promoter engineering have been developed and used to construct gene expression systems in B. subtilis and other hosts. By applying a semi-rational approach for promoter engineering, a series of expression plasmids containing single and dual promoters were constructed using aminopeptidase (AP) with an intrinsic signal peptide as the reporter protein. Of the single and dual promoters investigated, the dual promoter PgsiB-PHpaII gave the best performance. To optimize secretion efficiency, the signal peptide YncM was selected after screening a library containing 19 different Sec-type signal peptides. The AP activity detected in the supernatants of a recombinant strain containing the plasmid pBSG24-YncM was as high as 88.86U/mL. The capacity of the expression plasmid pBSG24-YncM was also evaluated with batch fermentation in a 5-L fermentor. Increased production of AP (205U/mL, equal to 1.7g/L) was achieved after 45h of fermentation. These results suggest that this expression system can be used for high-level protein expression in B. subtilis.

  6. Statically Adsorbed Coatings for High Separation Efficiency and Resolution in CE-MS Peptide Analysis: Strategies and Implementation.

    PubMed

    Pattky, Martin; Barkovits, Katalin; Marcus, Katrin; Weiergräber, Oliver H; Huhn, Carolin

    2016-01-01

    Coatings are necessary to prevent protein and peptide adsorption to the capillary surface and obtain high intermediate precision. In this protocol, we first present our basic strategy to address peptide separation using three different coatings: one neutral and two cationic coatings, the latter largely differing in their induced electroosmotic mobility. In detail, we will describe how we apply the statically adsorbed coatings to obtain very high plate numbers and high repeatability.With some model examples, we clearly describe the scope of the method for the analysis of peptide samples: tryptic digests are addressed as well as small glycoproteins and glycopeptides largely differing in their effective electrophoretic mobility. We also show that the method is suitable for a fast screening of peptide samples despite a high matrix load comprising of up to 500 mmol/L sodium chloride. We demonstrate that this basic CE-MS method is rather independent of the polarity of the analytes with a very fast near-baseline separation of very hydrophobic Aβ peptides related to the onset of Alzheimer's disease. These examples will give an impression, which coating is most suitable for a specific analytical application.Special attention is paid to difficult aspects of the coating procedure and the CE-MS method, e.g., the potential of cross-contamination when changing the coatings.

  7. A Robust and Efficient Production and Purification Procedure of Recombinant Alzheimers Disease Methionine-Modified Amyloid-β Peptides

    PubMed Central

    Hoarau, Marie; Hureau, Christelle; Faller, Peter; Gras, Emmanuel; André, Isabelle; Remaud-Siméon, Magali

    2016-01-01

    An improved production and purification method for Alzheimer’s disease related methionine-modified amyloid-β 1–40 and 1–42 peptides is proposed, taking advantage of the formation of inclusion body in Escherichia coli. A Thioflavin-S assay was set-up to evaluate inclusion body formation during growth and optimize culture conditions for amyloid-β peptides production. A simple and fast purification protocol including first the isolation of the inclusion bodies and second, two cycles of high pH denaturation/ neutralization combined with an ultrafiltration step on 30-kDa cut-off membrane was established. Special attention was paid to purity monitoring based on a rational combination of UV spectrophotometry and SDS-PAGE analyses at the various stages of the process. It revealed that this chromatography-free protocol affords good yield of high quality peptides in term of purity. The resulting peptides were fully characterized and are appropriate models for highly reproducible in vitro aggregation studies. PMID:27532547

  8. Impact of Thermostats on Folding and Aggregation Properties of Peptides Using the Optimized Potential for Efficient Structure Prediction Coarse-Grained Model.

    PubMed

    Spill, Yannick G; Pasquali, Samuela; Derreumaux, Philippe

    2011-05-10

    The simulation of amyloid fibril formation is impossible if one takes into account all chemical details of the amino acids and their detailed interactions with the solvent. We investigate the folding and aggregation of two model peptides using the optimized potential for efficient structure prediction (OPEP) coarse-grained model and replica exchange molecular dynamics (REMD) simulations coupled with either the Langevin or the Berendsen thermostat. For both the monomer of blocked penta-alanine and the trimer of the 25-35 fragment of the Alzheimer's amyloid β protein, we find little variations in the equilibrium structures and heat capacity curves using the two thermostats. Despite this high similarity, we detect significant differences in the populations of the dominant conformations at low temperatures, whereas the configurational distributions remain the same in proximity of the melting temperature. Aβ25-35 trimers at 300 K have an averaged β-sheet content of 12% and are primarily characterized by fully disordered peptides or a small curved two-stranded β-sheet stabilized by a disordered peptide. In addition, OPEP molecular dynamics simulations of Aβ25-35 hexamers at 300 K with a small curved six-stranded antiparallel β-sheet do not show any extension of the β-sheet content. These data support the idea that the mechanism of Aβ25-35 amyloid formation does not result from a high fraction of extended β-sheet-rich trimers and hexamers.

  9. Conjugation with RGD Peptides and Incorporation of Vascular Endothelial Growth Factor Are Equally Efficient for Biofunctionalization of Tissue-Engineered Vascular Grafts

    PubMed Central

    Antonova, Larisa V.; Seifalian, Alexander M.; Kutikhin, Anton G.; Sevostyanova, Victoria V.; Matveeva, Vera G.; Velikanova, Elena A.; Mironov, Andrey V.; Shabaev, Amin R.; Glushkova, Tatiana V.; Senokosova, Evgeniya A.; Vasyukov, Georgiy Yu.; Krivkina, Evgeniya O.; Burago, Andrey Yu.; Kudryavtseva, Yuliya A.; Barbarash, Olga L.; Barbarash, Leonid S.

    2016-01-01

    The blend of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) has recently been considered promising for vascular tissue engineering. However, it was shown that PHBV/PCL grafts require biofunctionalization to achieve high primary patency rate. Here we compared immobilization of arginine–glycine–aspartic acid (RGD)-containing peptides and the incorporation of vascular endothelial growth factor (VEGF) as two widely established biofunctionalization approaches. Electrospun PHBV/PCL small-diameter grafts with either RGD peptides or VEGF, as well as unmodified grafts were implanted into rat abdominal aortas for 1, 3, 6, and 12 months following histological and immunofluorescence assessment. We detected CD31+/CD34+/vWF+ cells 1 and 3 months postimplantation at the luminal surface of PHBV/PCL/RGD and PHBV/PCL/VEGF, but not in unmodified grafts, with the further observation of CD31+CD34−vWF+ phenotype. These cells were considered as endothelial and produced a collagen-positive layer resembling a basement membrane. Detection of CD31+/CD34+ cells at the early stages with subsequent loss of CD34 indicated cell adhesion from the bloodstream. Therefore, either conjugation with RGD peptides or the incorporation of VEGF promoted the formation of a functional endothelial cell layer. Furthermore, both modifications increased primary patency rate three-fold. In conclusion, both of these biofunctionalization approaches can be considered as equally efficient for the modification of tissue-engineered vascular grafts. PMID:27854352

  10. Efficient molecular mechanics simulations of the folding, orientation, and assembly of peptides in lipid bilayers using an implicit atomic solvation model

    NASA Astrophysics Data System (ADS)

    Bordner, Andrew J.; Zorman, Barry; Abagyan, Ruben

    2011-10-01

    Membrane proteins comprise a significant fraction of the proteomes of sequenced organisms and are the targets of approximately half of marketed drugs. However, in spite of their prevalence and biomedical importance, relatively few experimental structures are available due to technical challenges. Computational simulations can potentially address this deficit by providing structural models of membrane proteins. Solvation within the spatially heterogeneous membrane/solvent environment provides a major component of the energetics driving protein folding and association within the membrane. We have developed an implicit solvation model for membranes that is both computationally efficient and accurate enough to enable molecular mechanics predictions for the folding and association of peptides within the membrane. We derived the new atomic solvation model parameters using an unbiased fitting procedure to experimental data and have applied it to diverse problems in order to test its accuracy and to gain insight into membrane protein folding. First, we predicted the positions and orientations of peptides and complexes within the lipid bilayer and compared the simulation results with solid-state NMR structures. Additionally, we performed folding simulations for a series of host-guest peptides with varying propensities to form alpha helices in a hydrophobic environment and compared the structures with experimental measurements. We were also able to successfully predict the structures of amphipathic peptides as well as the structures for dimeric complexes of short hexapeptides that have experimentally characterized propensities to form beta sheets within the membrane. Finally, we compared calculated relative transfer energies with data from experiments measuring the effects of mutations on the free energies of translocon-mediated insertion of proteins into lipid bilayers and of combined folding and membrane insertion of a beta barrel protein.

  11. Two interdependent mechanisms of antimicrobial activity allow for efficient killing in nylon-3-based polymeric mimics of innate immunity peptides.

    PubMed

    Lee, Michelle W; Chakraborty, Saswata; Schmidt, Nathan W; Murgai, Rajan; Gellman, Samuel H; Wong, Gerard C L

    2014-09-01

    Novel synthetic mimics of antimicrobial peptides have been developed to exhibit structural properties and antimicrobial activity similar to those of natural antimicrobial peptides (AMPs) of the innate immune system. These molecules have a number of potential advantages over conventional antibiotics, including reduced bacterial resistance, cost-effective preparation, and customizable designs. In this study, we investigate a family of nylon-3 polymer-based antimicrobials. By combining vesicle dye leakage, bacterial permeation, and bactericidal assays with small-angle X-ray scattering (SAXS), we find that these polymers are capable of two interdependent mechanisms of action: permeation of bacterial membranes and binding to intracellular targets such as DNA, with the latter necessarily dependent on the former. We systemically examine polymer-induced membrane deformation modes across a range of lipid compositions that mimic both bacteria and mammalian cell membranes. The results show that the polymers' ability to generate negative Gaussian curvature (NGC), a topological requirement for membrane permeation and cellular entry, in model Escherichia coli membranes correlates with their ability to permeate membranes without complete membrane disruption and kill E. coli cells. Our findings suggest that these polymers operate with a concentration-dependent mechanism of action: at low concentrations permeation and DNA binding occur without membrane disruption, while at high concentrations complete disruption of the membrane occurs. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

  12. Bio-inspired supramolecular hybrid dendrimers self-assembled from low-generation peptide dendrons for highly efficient gene delivery and biological tracking.

    PubMed

    Xu, Xianghui; Jian, Yeting; Li, Yunkun; Zhang, Xiao; Tu, Zhaoxu; Gu, Zhongwei

    2014-09-23

    Currently, supramolecular self-assembly of dendrons and dendrimers emerges as a powerful and challenging strategy for developing sophisticated nanostructures with excellent performances. Here we report a supramolecular hybrid strategy to fabricate a bio-inspired dendritic system as a versatile delivery nanoplatform. With a rational design, dual-functionalized low-generation peptide dendrons (PDs) self-assemble onto inorganic nanoparticles via coordination interactions to generate multifunctional supramolecular hybrid dendrimers (SHDs). These SHDs exhibit well-defined nanostructure, arginine-rich peptide corona, and fluorescent signaling properties. As expected, our bio-inspired supramolecular hybrid strategy largely enhances the gene transfection efficiency of SHDs approximately 50 000-fold as compared to single PDs at the same R/P ratio. Meanwhile the bio-inspired SHDs also (i) provide low cytotoxicity and serum resistance in gene delivery; (ii) provide inherent fluorescence for tracking intracellular pathways including cellular uptake, endosomal escape, and gene release; and (iii) work as an alternative reference for monitoring desired protein expression. More importantly, in vivo animal experiments demonstrate that SHDs offer considerable gene transfection efficiency (in muscular tissue and in HepG2 tumor xenografts) and real-time bioimaging capabilities. These SHDs will likely stimulate studies on bio-inspired supramolecular hybrid dendritic systems for biomedical applications both in vitro and in vivo.

  13. Coupling of a bifunctional peptide R13 to OTMCS-PEI copolymer as a gene vector increases transfection efficiency and tumor targeting

    PubMed Central

    Lv, Hui; Zhu, Qing; Liu, Kewu; Zhu, Manman; Zhao, Wenfang; Mao, Yuan; Liu, Kehai

    2014-01-01

    Background A degradable polyethylenimine (PEI) derivative coupled to a bifunctional peptide R13 was developed to solve the transfection efficiency versus cytotoxicity and tumor-targeting problems of PEI when used as a gene vector. Methods We crossed-linked low molecular weight PEI with N-octyl-N-quaternary chitosan (OTMCS) to synthesize a degradable PEI derivative (OTMCS-PEI), and then used a bifunctional peptide, RGDC-TAT (49–57) called R13 to modify OTMCS-PEI so as to prepare a new gene vector, OTMCS-PEI-R13. This new gene vector was characterized by various physicochemical methods. Its cytotoxicity and gene transfection efficiency were also determined both in vitro and in vivo. Results The vector showed controlled degradation and excellent buffering capacity. The particle size of the OTMCS-PEI-R13/DNA complexes was around 150–250 nm and the zeta potential ranged from 10 mV to 30 mV. The polymer could protect plasmid DNA from being digested by DNase I at a concentration of 23.5 U DNase I/μg DNA. Further, the polymer was resistant to dissociation induced by 50% fetal bovine serum and 400 μg/mL sodium heparin. Compared with PEI 25 kDa, the OTMCS-PEI-R13/DNA complexes showed higher transfection efficiency both in vitro and in vivo. Further, compared with OTMCS-PEI, distribution of OTMCS-PEI-R13 at tumor sites was markedly enhanced, indicating the tumor-targeting specificity of R13. Conclusion OTMCS-PEI-R13 could be a potential candidate as a safe and efficient gene delivery carrier for gene therapy. PMID:24648730

  14. An efficient PEGylated liposomal nanocarrier containing cell-penetrating peptide and pH-sensitive hydrazone bond for enhancing tumor-targeted drug delivery

    PubMed Central

    Ding, Yuan; Sun, Dan; Wang, Gui-Ling; Yang, Hong-Ge; Xu, Hai-Feng; Chen, Jian-Hua; Xie, Ying; Wang, Zhi-Qiang

    2015-01-01

    Cell-penetrating peptides (CPPs) as small molecular transporters with abilities of cell penetrating, internalization, and endosomal escape have potential prospect in drug delivery systems. However, a bottleneck hampering their application is the poor specificity for cells. By utilizing the function of hydration shell of polyethylene glycol (PEG) and acid sensitivity of hydrazone bond, we constructed a kind of CPP-modified pH-sensitive PEGylated liposomes (CPPL) to improve the selectivity of these peptides for tumor targeting. In CPPL, CPP was directly attached to liposome surfaces via coupling with stearate (STR) to avoid the hindrance of PEG as a linker on the penetrating efficiency of CPP. A PEG derivative by conjugating PEG with STR via acid-degradable hydrazone bond (PEG2000-Hz-STR, PHS) was synthesized. High-performance liquid chromatography and flow cytometry demonstrated that PHS was stable at normal neutral conditions and PEG could be completely cleaved from liposome surface to expose CPP under acidic environments in tumor. An optimal CPP density on liposomes was screened to guaranty a maximum targeting efficiency on tumor cells as well as not being captured by normal cells that consequently lead to a long circulation in blood. In vitro and in vivo studies indicated, in 4 mol% CPP of lipid modified system, that CPP exerted higher efficiency on internalizing the liposomes into targeted subcellular compartments while remaining inactive and free from opsonins at a maximum extent in systemic circulation. The 4% CPPL as a drug delivery system will have great potential in the clinical application of anticancer drugs in future. PMID:26491292

  15. Effects of the peptide mycotoxin destruxin E on insect haemocytes and on dynamics and efficiency of the multicellular immune reaction.

    PubMed

    Vey, Alain; Matha, Vladimir; Dumas, Christiane

    2002-07-01

    Destruxins (DTXs) are cyclic peptide toxins secreted by the entomopathogenic fungus Metarhizium anisopliae var. anisopliae. The effects of DTX E, the most active compound of this family on haemocytes, the immunocompetent insect cells, and on the dynamics and efficacy of the multicellular defense of insect hosts have been investigated. Ultrastructural alterations have been observed in circulating plasmatocytes and granular haemocytes, and in attached haemocytes of Galleria mellonella larvae treated with a toxic dose of DTX E (LC50). These changes appear as a consequence of disturbances induced in the cellular calcium balance. An effect on the cell surface of granulocytes was also noted in cells incubated with the toxin and FITC-Con A, even when the concentration of DTX was as low as 0.005 microg/ml. Morphological studies of haemocytic capsules formed in vivo revealed disturbances of the multicellular defense mechanism after toxin treatment However, an attempt to establish if these changes were significant was unsuccessful. In contrast, comparative assays regarding the effect of toxin treatment on the efficacy of the antifungal effect of encapsulation has given conclusive results. The germination of injected Aspergillus niger spores became slightly but significantly increased, and when the granuloma were incubated the fungus escaped more easily from the haemocytic envelope. These results are discussed in terms of significance of the contribution of DTXs to the fungal infection process. It is suggested that the fungal peptides may intervene during the disease by a true immune-inhibitory effect occurring at doses which do not cause paralysis or any general sign of toxicity (e.g., 0.8 microg/g of body weight).

  16. Development of a novel efficient method to construct an adenovirus library displaying random peptides on the fiber knob

    PubMed Central

    Yamamoto, Yuki; Goto, Naoko; Miura, Kazuki; Narumi, Kenta; Ohnami, Shumpei; Uchida, Hiroaki; Miura, Yoshiaki; Yamamoto, Masato; Aoki, Kazunori

    2014-01-01

    Redirection of adenovirus vectors by engineering the capsid-coding region has shown limited success because proper targeting ligands are generally unknown. To overcome this limitation, we constructed an adenovirus library displaying random peptides on the fiber knob, and its screening led to successful selections of several particular targeted vectors. In the previous library construction method, the full length of an adenoviral genome was generated by a Cre-lox mediated in vitro recombination between a fiber-modified plasmid library and the enzyme-digested adenoviral DNA/terminal protein complex (DNA-TPC) before transfection to the producer cells. In this system, the procedures were complicated and time-consuming, and approximately 30% of the vectors in the library were defective with no displaying peptide. These may hinder further extensive exploration of cancer-targeting vectors. To resolve these problems, in this study, we developed a novel method with the transfection of a fiber-modified plasmid library and a fiberless adenoviral DNA-TPC in Cre-expressing 293 cells. The use of in-cell Cre recombination and fiberless adenovirus greatly simplified the library-making steps. The fiberless adenovirus was useful in suppressing the expansion of unnecessary adenovirus vectors. In addition, the complexity of the library was more than a 104 level in one well in a 6-well dish, which was 10-fold higher than that of the original method. The results demonstrated that this novel method is useful in producing a high quality live adenovirus library, which could facilitate the development of targeted adenovirus vectors for a variety of applications in medicine. PMID:24380399

  17. Antiplatelet Aggregation and Antithrombosis Efficiency of Peptides in the Snake Venom of Deinagkistrodon acutus: Isolation, Identification, and Evaluation.

    PubMed

    Ding, Bin; Xu, Zhenghong; Qian, Chaodong; Jiang, Fusheng; Ding, Xinghong; Ruan, Yeping; Ding, Zhishan; Fan, Yongsheng

    2015-01-01

    Two peptides of Pt-A (Glu-Asn-Trp 429 Da) and Pt-B (Glu-Gln-Trp 443 Da) were isolated from venom liquor of Deinagkistrodon acutus. Their antiplatelet aggregation effects were evaluated with platelet-rich human plasma in vitro; the respective IC50 of Pt-A and Pt-B was 66 μM and 203 μM. Both peptides exhibited protection effects on ADP-induced paralysis in mice. After ADP administration, the paralysis time of different concentration of Pt-A and Pt-B lasted as the following: 80 mg/kg Pt-B (152.8 ± 57.8 s) < 40 mg/kg Pt-A (163.5 ± 59.8 s) < 20 mg/kg Pt-A (253.5 ± 74.5 s) < 4 mg/kg clopidogrel (a positive control, 254.5 ± 41.97 s) < 40 mg/kg Pt-B (400.8 ± 35.9 s) < 10 mg/kg Pt-A (422.8 ± 55.4 s), all of which were statistically shorter than the saline treatment (666 ± 28 s). Pulmonary tissue biopsy confirmed that Pt-A and Pt-B prevented the formation of thrombi in the lung. Unlike ADP injection alone, which caused significant reduction of peripheral platelet count, Pt-A treatment prevented the drop of peripheral platelet counts; interestingly, Pt-B could not, even though the same amount of Pt-B also showed protection effects on ADP-induced paralysis and thrombosis. More importantly, intravenous injection of Pt-A and Pt-B did not significantly increase the hemorrhage risks as clopidogrel.

  18. Potential of prodendronic polyamines with modulated segmental charge density as novel coating for fast and efficient analysis of peptides and basic proteins by CE and CE-MS.

    PubMed

    Acunha, Tanize; Ibáñez, Clara; Pascual Reguera, María Isabel; Sarò, Mariagiovanna; Navarro, Rodrigo; Alfonso Redondo, Juan; Reinecke, Helmut; Gallardo, Alberto; Simó, Carolina; Cifuentes, Alejandro

    2015-07-01

    In this work, the suitability of a new polymer family has been investigated as capillary coatings for the analysis of peptides and basic proteins by CE. This polymer family has been designed to minimize or completely prevent protein-capillary wall interactions and to modify the EOF. These coating materials are linear polymeric chains bearing as side cationizable moiety a dentronic triamine derived from N,N,N',N'-tetraethyldiethylenetriamine (TEDETA), which is linked to the backbone through a spacer (unit labeled as TEDETAMA). Four different polymers have been prepared and evaluated: a homopolymer which comprised only of those cationizable repetitive units of TEDETAMA, and three copolymers that randomly incorporate TEDETAMA together with neutral hydrosoluble units of N-(2-hydroxypropyl) methacrylamide (HPMA) at different molar percentages (25:75, 50:50 and 75:25). It has been demonstrated that the composition of the copolymers influences the EOF and therefore the separation of the investigated biopolymers. Among the novel polymers studied, poly-(TEDETAMA-co-HPMA) 50:50 copolymer was successfully applied as coating material of the inner capillary surface in CE-UV and CE-MS, providing EOF reversing together with fast and efficient baseline separation of peptides and basic proteins. Finally, the feasibility of the polymer-coated capillary was shown through the analysis of lysozyme in a cheese sample.

  19. Selective encapsulation of cesium ions using the cyclic peptide moiety of surfactin: Highly efficient removal based on an aqueous giant micellar system.

    PubMed

    Taira, Toshiaki; Yanagisawa, Satohiro; Nagano, Takuto; Zhu, Yanbei; Kuroiwa, Takayoshi; Koumura, Nagatoshi; Kitamoto, Dai; Imura, Tomohiro

    2015-10-01

    Cyclic peptide of surfactin (SF) is one of the promising environment-friendly biosurfactants abundantly produced by microorganisms such as Bacillus subtilis. SF is also known to act as an ionophore, wherein alkali metal ions can be trapped in the cyclic peptide. Especially, SF is expected to show high affinity for Cs(+) because of the distinctive cavity size and coordination number. In this study, we reported the specific interaction between SF and Cs(+) and succeeded in the highly efficient removal of Cs(+) from water using giant SF micelles as a natural sorbent. The specific interaction between SF and Cs(+) to form their inclusion complex was revealed by nuclear magnetic resonance (NMR) and Fourier transform infrared (FT-IR) spectroscopy. We found that SF micelles selectively encapsulate Cs(+), which was suggested by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). A highly effective separation of Cs(+) immobilized on the surface of the SF micelles was also achieved through facile centrifugal ultrafiltration in 91% even in coexisting with other alkali metal ions such as Na(+) and K(+). Thus, the use of the giant micellar system of SF with its high Cs(+) affinity and distinctive assembling properties would be a new approach for the treatment of contaminated soil and water.

  20. Efficient Production of a Bioactive Bevacizumab Monoclonal Antibody Using the 2A Self-cleavage Peptide in Transgenic Rice Callus

    PubMed Central

    Chen, Lei; Yang, Xiaoyu; Luo, Da; Yu, Weichang

    2016-01-01

    Bevacizumab, a humanized monoclonal antibody (mAb) targeting to the vascular endothelial growth factor (VEGF), has been widely used in clinical practice for the treatment of multiple cancers. Bevacizumab was mostly produced by the mammalian cell expression system. We here reported the first plant-derived Bevacizumab by using transgenic rice callus as an alternative gene expression system. Codon-optimized Bevacizumab light chain (BLC) and Bevacizumab heavy chain (BHC) genes were designed, synthesized as a polyprotein with a 2A self-cleavage linker peptide from the Foot-and-mouth disease virus, cloned into a plant binary vector under a constitutive maize ubiquitin promoter, and transformed into rice nuclear genome through Agrobacterium-mediated transformation. Southern blot and western blot analyses confirmed the integration and expression of BLC and BHC genes in transgenic rice callus. Enzyme-linked immunosorbent assay (ELISA) analysis indicated that the rice-derived Bevacizumab mAb was biologically active and the recombinant mAb was expressed at high levels (160.7–242.8 mg/Kg) in transgenic rice callus. The mAb was purified by using protein A affinity chromatography and the purified antibody was tested for its binding affinity with its target human VEGF (hVEGF) antigen by ELISA. Rice callus produced Bevacizumab and a commercial Bevacizumab (Avastin) were shown to have similar binding affinity to hVEGF. These results indicated that rice callus produced Bevacizumab could have similar biological activity and might potentially be used as a cost-effective biosimilar molecule in future cancer treatment. PMID:27555853

  1. Enhanced Digestion Efficiency, Peptide Ionization Efficiency, and Sequence Resolution for Protein Hydrogen/Deuterium Exchange Monitored by FT-ICR Mass Spectrometry

    PubMed Central

    Zhang, Hui-Min; Kazazic, Saša; Schaub, Tanner M.; Tipton, Jeremiah D.; Emmett, Mark R.; Marshall, Alan G.

    2009-01-01

    Solution-phase hydrogen/deuterium exchange (HDX) monitored by high-resolution FT-ICR mass spectrometry offers a rapid method to study protein conformations and protein-protein interactions. Pepsin is usually used to digest proteins in HDX and is known as lack of cleavage specificity. To improve digestion efficiency and specificity, we have optimized digestion conditions and cleavage preferences for pepsin and protease type XIII from Aspergillus saitoi. A dilution series of the proteases was used to determine the digestion efficiency for several test proteins. Protease type XIII prefers to cleave on the C-terminal end of basic amino acids and produced the highest number of fragments and the best sequence coverage compared to pepsin or protease type XVIII from Rhizhopus. Furthermore, protease type XIII exhibited much less self-digestion than pepsin, and thus is superior for HDX experiments. Many highly overlapped segments from protease type XIII and pepsin digestion, combined with high-resolution FT-ICR mass spectrometry, provide high sequence resolution (to as few as one or two amino acids) for the assignment of amide hydrogen exchange rate. Our H/D exchange results correlate well with the secondary and tertiary structure of myoglobin. Such assignments of highly overlapped fragments promise to greatly enhance the accuracy and sequence resolution for determining conformational differences resulting from ligand binding or protein-protein interactions. PMID:19551977

  2. Efficiency of Peptide Nucleic Acid-Directed PCR Clamping and Its Application in the Investigation of Natural Diets of the Japanese Eel Leptocephali

    PubMed Central

    Terahara, Takeshi; Chow, Seinen; Kurogi, Hiroaki; Lee, Sun-Hee; Tsukamoto, Katsumi; Mochioka, Noritaka; Tanaka, Hideki; Takeyama, Haruko

    2011-01-01

    Polymerase chain reaction (PCR)-clamping using blocking primer and DNA-analogs, such as peptide nucleotide acid (PNA), may be used to selectively amplify target DNA for molecular diet analysis. We investigated PCR-clamping efficiency by studying PNA position and mismatch with complementary DNA by designing PNAs at five different positions on the nuclear rDNA internal transcribed spacer 1 of the Japanese eel Anguilla japonica in association with intra-specific nucleotide substitutions. All five PNAs were observed to efficiently inhibit amplification of a fully complementary DNA template. One mismatch between PNA and template DNA inhibited amplification of the template DNA, while two or more mismatches did not. DNA samples extracted from dorsal muscle and intestine of eight wild-caught leptochephalus larvae were subjected to this analysis, followed by cloning, nucleotide sequence analysis, and database homology search. Among 12 sequence types obtained from the intestine sample, six were identified as fungi. No sequence similarities were found in the database for the remaining six types, which were not related to one another. These results, in conjunction with our laboratory observations on larval feeding, suggest that eel leptocephali may not be dependent upon living plankton for their food source. PMID:22069444

  3. Efficient Eradication of Mature Pseudomonas aeruginosa Biofilm via Controlled Delivery of Nitric Oxide Combined with Antimicrobial Peptide and Antibiotics.

    PubMed

    Ren, Hang; Wu, Jianfeng; Colletta, Alessandro; Meyerhoff, Mark E; Xi, Chuanwu

    2016-01-01

    Fast eradication of mature biofilms is the 'holy grail' in the clinical management of device-related infections. Endogenous nitric oxide (NO) produced by macrophages plays an important role in host defense against intracellular pathogens, and NO is a promising agent in preventing biofilms formation in vitro. However, the rate of delivery of NO by various NO donors (e.g., diazeniumdiolates, S-nitrosothiols, etc.) is difficult to control, which hinders fundamental studies aimed at understanding the role of NO in biofilm control. In this study, by using a novel precisely controlled electrochemical NO releasing catheter device, we examine the effect of physiological levels of NO on eradicating mature Pseudomonas aeruginosa biofilm (7 days), as well as the potential application of the combination of NO with antimicrobial agents. It is shown that physiological levels of NO exhibit mixed effects of killing bacteria and dispersing ambient biofilm. The overall biofilm-eradicating effect of NO is quite efficient in a dose-dependent manner over a 3 h period of NO treatment. Moreover, NO also greatly enhances the efficacy of antimicrobial agents, including human beta-defensin 2 (BD-2) and several antibiotics, in eradicating biofilm and its detached cells, which otherwise exhibited high recalcitrance to these antimicrobial agents. The electrochemical NO release technology offers a powerful tool in evaluating the role of NO in biofilm control as well as a promising approach when combined with antimicrobial agents to treat biofilm-associated infections in hospital settings, especially infections resulting from intravascular catheters.

  4. A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

    SciTech Connect

    Han, Hui; Peng, Ji-Run; Chen, Peng-Cheng; Gong, Lei; Qiao, Shi-Shi; Wang, Wen-Zhen; Cui, Zhu-Qingqing; Yu, Xin; Wei, Yu-Hua; Leng, Xi-Sheng

    2011-08-05

    Highlights: {yields} Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. {yields} An ideal artificial APCs system was successfully prepared in vivo. {yields} Controlled release of IL-2 leads to much more T-cell expansion. {yields} This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

  5. Efficient Eradication of Mature Pseudomonas aeruginosa Biofilm via Controlled Delivery of Nitric Oxide Combined with Antimicrobial Peptide and Antibiotics

    PubMed Central

    Ren, Hang; Wu, Jianfeng; Colletta, Alessandro; Meyerhoff, Mark E.; Xi, Chuanwu

    2016-01-01

    Fast eradication of mature biofilms is the ‘holy grail’ in the clinical management of device-related infections. Endogenous nitric oxide (NO) produced by macrophages plays an important role in host defense against intracellular pathogens, and NO is a promising agent in preventing biofilms formation in vitro. However, the rate of delivery of NO by various NO donors (e.g., diazeniumdiolates, S-nitrosothiols, etc.) is difficult to control, which hinders fundamental studies aimed at understanding the role of NO in biofilm control. In this study, by using a novel precisely controlled electrochemical NO releasing catheter device, we examine the effect of physiological levels of NO on eradicating mature Pseudomonas aeruginosa biofilm (7 days), as well as the potential application of the combination of NO with antimicrobial agents. It is shown that physiological levels of NO exhibit mixed effects of killing bacteria and dispersing ambient biofilm. The overall biofilm-eradicating effect of NO is quite efficient in a dose-dependent manner over a 3 h period of NO treatment. Moreover, NO also greatly enhances the efficacy of antimicrobial agents, including human beta-defensin 2 (BD-2) and several antibiotics, in eradicating biofilm and its detached cells, which otherwise exhibited high recalcitrance to these antimicrobial agents. The electrochemical NO release technology offers a powerful tool in evaluating the role of NO in biofilm control as well as a promising approach when combined with antimicrobial agents to treat biofilm-associated infections in hospital settings, especially infections resulting from intravascular catheters. PMID:27582732

  6. Efficient production of recombinant cystatin C using a peptide-tag, 4AaCter, that facilitates formation of insoluble protein inclusion bodies in Escherichia coli.

    PubMed

    Hayashi, Masahiro; Iwamoto, Shigehisa; Sato, Shinya; Sudo, Shigeo; Takagi, Mari; Sakai, Hiroshi; Hayakawa, Tohru

    2013-04-01

    Cystatin C is a cysteine protease inhibitor produced by a variety of human tissues. The blood concentration of cystatin C depends on the glomerular filtration rate and is an endogenous marker of renal dysfunction. Recombinant cystatin C protein with high immunogenicity is therefore in demand for the diagnostic market. In this study, to establish an efficient production system, a synthetic cystatin C gene was designed and synthesized in accordance with the codon preference of Escherichia coli genes. Recombinant cystatin C was expressed as a fusion with a peptide-tag, 4AaCter, which facilitates formation of protein inclusion bodies in E. coli cells. Fusion with 4AaCter-tag dramatically increased the production level of cystatin C, and highly purified protein was obtained without the need for complicated purification steps. The purity and yield of the final product was estimated as 87 ± 5% and 7.1 ± 1.1 mg/l culture, respectively. The recombinant cystatin C prepared by our method was as reactive against anti-cystatin C antibodies as native human cystatin C. Our results suggest that protein production systems using 4AaCter-tag could be a powerful means of preparing significant amounts of antigen protein.

  7. Designing an efficient multi-epitope peptide vaccine against Vibrio cholerae via combined immunoinformatics and protein interaction based approaches.

    PubMed

    Nezafat, Navid; Karimi, Zeinab; Eslami, Mahboobeh; Mohkam, Milad; Zandian, Sanam; Ghasemi, Younes

    2016-06-01

    Cholera continues to be a major global health concern. Among different Vibrio cholerae strains, only O1 and O139 cause acute diarrheal diseases that are related to epidemic and pandemic outbreaks. The currently available cholera vaccines are mainly lived and attenuated vaccines consisting of V. cholerae virulence factors such as toxin-coregulated pili (TCP), outer membrane proteins (Omps), and nontoxic cholera toxin B subunit (CTB). Nowadays, there is a great interest in designing an efficient epitope vaccine against cholera. Epitope vaccines consisting of immunodominant epitopes and adjuvant molecules enhance the possibility of inciting potent protective immunity. In this study, V. cholerae protective antigens (OmpW, OmpU, TcpA and TcpF) and the CTB, which is broadly used as an immunostimulatory adjuvant, were analyzed using different bioinformatics and immunoinformatics tools. The common regions between promiscuous epitopes, binding to various HLA-II supertype alleles, and B-cell epitopes were defined based upon the aforementioned protective antigens. The ultimately selected epitopes and CTB adjuvant were fused together using proper GPGPG linkers to enhance vaccine immunogenicity. A three-dimensional model of the thus constructed vaccine was generated using I-TASSER. The model was structurally validated using the ProSA-web error-detection software and the Ramachandran plot. The validation results indicated that the initial 3D model needed refinement. Subsequently, a high-quality model obtained after various refinement cycles was used for defining conformational B-cell epitopes. Several linear and conformational B-cell epitopes were determined within the epitope vaccine, suggesting likely antibody triggering features of our designed vaccine. Next, molecular docking was performed between the 3D vaccine model and the tertiary structure of the toll like receptor 2 (TLR2). To gain further insight into the interaction between vaccine and TLR2, molecular dynamics

  8. Combination of inverse electron-demand Diels-Alder reaction with highly efficient oxime ligation expands the toolbox of site-selective peptide conjugations.

    PubMed

    Hörner, S; Uth, C; Avrutina, O; Frauendorf, H; Wiessler, M; Kolmar, H

    2015-07-14

    A modular approach combining inverse electron-demand Diels-Alder coupling (DARinv) and oxime ligation expands the toolbox of bioorthogonal peptide chemistry. Applicability of versatile site-specific bifunctional building blocks is demonstrated by generation of defined conjugates comprising linear, cystine-bridged and multi-disulfide functional peptides as well as their conjugation with hybrid silsesquioxane nanoparticles.

  9. Intracellular translocation and differential accumulation of cell-penetrating peptides in bovine spermatozoa: evaluation of efficient delivery vectors that do not compromise human sperm motility

    PubMed Central

    Jones, Sarah; Lukanowska, Monika; Suhorutsenko, Julia; Oxenham, Senga; Barratt, Christopher; Publicover, Steven; Copolovici, Dana Maria; Langel, Ülo; Howl, John

    2013-01-01

    STUDY QUESTION Do cell penetrating peptides (CPPs) translocate into spermatozoa and, if so, could they be utilized to deliver a much larger protein cargo? SUMMARY ANSWER Chemically diverse polycationic CPPs rapidly and efficiently translocate into spermatozoa. They exhibit differential accumulation within intracellular compartments without detrimental influences upon cellular viability or motility but they are relatively ineffective in transporting larger proteins. WHAT IS ALREADY KNOWN Endocytosis, the prevalent route of protein internalization into eukaryotic cells, is severely compromised in mature spermatozoa. Thus, the translocation of many bioactive agents into sperm is relatively inefficient. However, the delivery of bioactive moieties into mature spermatozoa could be significantly improved by the identification and utility of an efficient and inert vectorial delivery technology. STUDY DESIGN CPP translocation efficacies, their subsequent differential intracellular distribution and the influence of peptides upon viability were determined in bovine spermatozoa. Temporal analyses of sperm motility in the presence of exogenously CPPs utilized normozoospermic human donor samples. MATERIALS AND METHODS CPPs were prepared by manual, automated and microwave-enhanced solid phase synthesis. Confocal fluorescence microscopy determined the intracellular distribution of rhodamine-conjugated CPPs in spermatozoa. Quantitative uptake and kinetic analyses compared the translocation efficacies of chemically diverse CPPs and conjugates of biotinylated CPPs and avidin. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) conversion assays were employed to analyse the influence of CPPs upon sperm cell viability and sperm class assays determined the impact of CPPs on motility in capacitated and non-capacitated human samples. MAIN RESULTS Chemically heterogeneous CPPs readily translocated into sperm to accumulate within

  10. Discovery of a novel splice variant of Fcar (CD89) unravels sequence segments necessary for efficient secretion: A story of bad signal peptides and good ones that nevertheless do not make it

    PubMed Central

    Lua, Wai-Heng; Ling, Wei-Li; Su, Chinh Tran-To; Yeo, Joshua Yi

    2017-01-01

    ABSTRACT The IgA receptor, Fcar (CD89) consists of 5 sequence segments: 2 segments (S1, S2) forming the potential signal peptide, 2 extracellular EC domains that include the IgA binding site, and the transmembrane and cytoplasmic tail (TM/C) region. Numerous Fcar splice variants have been reported with various combinations of the sequence segments mentioned above. Here, we report a novel splice variant termed variant APD isolated from a healthy volunteer that lacks only the IgA-binding EC1 domain. Despite possessing the complete signal peptide S1+S2, the variant APD is only found in the intracellular space whereas the wild-type variant 1 is efficiently secreted and variant 4 leaks to the extracellular space. Further mutational experiments involving signal peptide replacements, cleavage site modifications, and studies on alternative isoforms demonstrate that despite the completeness of the signal peptide motif, the presence of the EC1 domain is essential for efficient extracellular export. PMID:28103138

  11. Efficient model chemistries for peptides. I. General framework and a study of the heterolevel approximation in RHF and MP2 with Pople split-valence basis sets.

    PubMed

    Echenique, Pablo; Alonso, José Luis

    2008-07-15

    We present an exhaustive study of more than 250 ab initio potential energy surfaces (PESs) of the model dipeptide HCO-L-Ala-NH(2). The model chemistries (MCs) investigated are constructed as homo- and heterolevels involving possibly different RHF and MP2 calculations for the geometry and the energy. The basis sets used belong to a sample of 39 representants from Pople's split-valence families, ranging from the small 3-21G to the large 6-311++G(2df,2pd). The reference PES to which the rest are compared is the MP2/6-311++G(2df,2pd) homolevel, which, as far as we are aware, is the most accurate PES in the literature. All data sets have been analyzed according to a general framework, which can be extended to other complex problems and which captures the nearness concept in the space of MCs. The great number of MCs evaluated has allowed us to significantly explore this space and show that the correlation between accuracy and computational cost of the methods is imperfect, thus justifying a systematic search for the combination of features in a MC that is optimal to deal with peptides. Regarding the particular MCs studied, the most important conclusion is that the potentially very cost-saving heterolevel approximation is a very efficient one to describe the whole PES of HCO-L-Ala-NH(2). Finally, we show that, although RHF may be used to calculate the geometry if a MP2 single-point energy calculation follows, pure RHF//RHF homolevels are not recommendable for this problem.

  12. Understanding the highly efficient catalysis of prokaryotic peptide deformylases by shedding light on the determinants specifying the low activity of the human counterpart.

    PubMed

    Fieulaine, Sonia; Desmadril, Michel; Meinnel, Thierry; Giglione, Carmela

    2014-02-01

    Peptide deformylases (PDFs), which are essential and ubiquitous enzymes involved in the removal of the N-formyl group from nascent chains, are classified into four subtypes based on the structural and sequence similarity of specific conserved domains. All PDFs share a similar three-dimensional structure, are functionally interchangeable in vivo and display similar properties in vitro, indicating that their molecular mechanism has been conserved during evolution. The human mitochondrial PDF is the only exception as despite its conserved fold it reveals a unique substrate-binding pocket together with an unusual kinetic behaviour. Unlike human PDF, the closely related mitochondrial PDF1As from plants have catalytic efficiencies and enzymatic parameters that are similar to those of other classes of PDFs. Here, the aim was to identify the structural basis underlying the properties of human PDF compared with all other PDFs by focusing on plant mitochondrial PDF1A. The construction of a chimaera composed of plant PDF1A with the nonrandom substitutions found in a conserved motif of its human homologue converted it into an enzyme with properties similar to the human enzyme, indicating the crucial role of these positions. The crystal structure of this human-like plant PDF revealed that substitution of two residues leads to a reduction in the volume of the ligand-binding site together with the introduction of negative charges, unravelling the origin of the weak affinity of human PDF for its substrate. In addition, the substitution of the two residues of human PDF modifies the transition state of the reaction through alteration of the network of interactions between the catalytic residues and the substrate, leading to an overall reduced reaction rate.

  13. Rational design of DKK3 structure-based small peptides as antagonists of Wnt signaling pathway and in silico evaluation of their efficiency

    PubMed Central

    Poorebrahim, Mansour; Sadeghi, Solmaz; Rahimi, Hamzeh; Karimipoor, Morteza; Azadmanesh, Kayhan; Mazlomi, Mohammad Ali; Teimoori-Toolabi, Ladan

    2017-01-01

    Dysregulated Wnt signaling pathway is highly associated with the pathogenesis of several human cancers. Dickkopf proteins (DKKs) are thought to inhibit Wnt signaling pathway through binding to lipoprotein receptor-related protein (LRP) 5/6. In this study, based on the 3-dimensional (3D) structure of DKK3 Cys-rich domain 2 (CRD2), we have designed and developed several peptide inhibitors of Wnt signaling pathway. Modeller 9.15 package was used to predict 3D structure of CRD2 based on the Homology modeling (HM) protocol. After refinement and minimization with GalaxyRefine and NOMAD-REF servers, the quality of selected models was evaluated utilizing VADAR, SAVES and ProSA servers. Molecular docking studies as well as literature-based information revealed two distinct boxes located at CRD2 which are actively involved in the DKK3-LRP5/6 interaction. A peptide library was constructed conducting the backrub sequence tolerance scanning protocol in Rosetta3.5 according to the DKK3-LRP5/6 binding sites. Seven tolerated peptides were chosen and their binding affinity and stability were improved by some logical amino acid substitutions. Molecular dynamics (MD) simulations of peptide-LRP5/6 complexes were carried out using GROMACS package. After evaluation of binding free energies, stability, electrostatic potential and some physicochemical properties utilizing computational approaches, three peptides (PEP-I1, PEP-I3 and PEP-II2) demonstrated desirable features. However, all seven improved peptides could sufficiently block the Wnt-binding site of LRP6 in silico. In conclusion, we have designed and improved several small peptides based on the LRP6-binding site of CRD2 of DKK3. These peptides are highly capable of binding to LRP6 in silico, and may prevent the formation of active Wnt-LRP6-Fz complex. PMID:28234935

  14. The life span of major histocompatibility complex-peptide complexes influences the efficiency of presentation and immunogenicity of two class I-restricted cytotoxic T lymphocyte epitopes in the Epstein-Barr virus nuclear antigen 4

    PubMed Central

    1996-01-01

    We have investigated the reactivity to two human histocompatibility leukocyte antigen (HLA) A11-restricted cytotoxic T lymphocyte (CTL) epitopes derived from amino acids 416-424 (IVTDFSVIK, designated IVT) and 399-408 (AVFDRKSVAK, designated AVF) of the Epstein-Barr virus (EBV) nuclear antigen (EBNA) 4. A strong predominance of CTL clones specific for the IVT epitope was demonstrated in polyclonal cultures generated by stimulation of lymphocytes from the EBV-seropositive donor BK with the autologous B95.8 virus-transformed lymphoblastoid cell line (LCL). This was not due to intrinsic differences of CTL efficiency since clones specific for the two epitopes lysed equally well A11- positive phytohemagglutinin blasts and LCLs pulsed with the relevant synthetic peptide. Irrespective of the endogenous levels of EBNA4 expression, untreated LCLs were lysed more efficiently by the IVT- specific effectors, suggesting that a higher density of A11-IVT complexes is presented at the cell surface. In accordance, 10-50-fold higher amounts of IVT peptides were found in high-performance liquid chromatography fractions of acid extracts corresponding to an abundance of about 350-12,800 IVT and 8-760 AVF molecules per cell. Peptide- mediated competition of CTL sensitization, transport assays in streptolysin-O permeabilized cells, and induction of A11 expression in the transporter associated with antigen presentation-deficient T2/A11 transfectant demonstrated that the IVT and AVF peptides bind with similar affinities to A11, are translocated with equal efficiency to the endoplasmic reticulum, and form complexes of comparable stability over a wide range of temperature and pH conditions. A rapid surface turnover of A11 molecules containing the AVF peptide was demonstrated in metabolically active T2/A11 cells corresponding to a half-life of approximately 3.5 as compared to approximately 2 h for molecules induced at 26 degrees C in the absence of exogenous peptides and >12 h for IVT

  15. Metabolism of Peptides by Rumen Microorganisms

    PubMed Central

    Wright, D. E.

    1967-01-01

    Rumen microorganisms utilize tryptic peptides from Chlorella protein, forming carbon dioxide, volatile fatty acids, and bacterial protein. Peptide carbon is more efficiently converted into bacterial protein than is amino acid carbon. A progressive degradation of the peptides was demonstrated by use of columns of Sephadex G-25. PMID:6035045

  16. Gaining efficiency by parallel quantification and identification of iTRAQ-labeled peptides using HCD and decision tree guided CID/ETD on an LTQ Orbitrap.

    PubMed

    Mischerikow, Nikolai; van Nierop, Pim; Li, Ka Wan; Bernstein, Hans-Gert; Smit, August B; Heck, Albert J R; Altelaar, A F Maarten

    2010-10-01

    Isobaric stable isotope labeling of peptides using iTRAQ is an important method for MS based quantitative proteomics. Traditionally, quantitative analysis of iTRAQ labeled peptides has been confined to beam-type instruments because of the weak detection capabilities of ion traps for low mass ions. Recent technical advances in fragmentation techniques on linear ion traps and the hybrid linear ion trap-orbitrap allow circumventing this limitation. Namely, PQD and HCD facilitate iTRAQ analysis on these instrument types. Here we report a method for iTRAQ-based relative quantification on the ETD enabled LTQ Orbitrap XL, which is based on parallel peptide quantification and peptide identification. iTRAQ reporter ion generation is performed by HCD, while CID and ETD provide peptide identification data in parallel in the LTQ ion trap. This approach circumvents problems accompanying iTRAQ reporter ion generation with ETD and allows quantitative, decision tree-based CID/ETD experiments. Furthermore, the use of HCD solely for iTRAQ reporter ion read out significantly reduces the number of ions needed to obtain informative spectra, which significantly reduces the analysis time. Finally, we show that integration of this method, both with existing CID and ETD methods as well as with existing iTRAQ data analysis workflows, is simple to realize. By applying our approach to the analysis of the synapse proteome from human brain biopsies, we demonstrate that it outperforms a latest generation MALDI TOF/TOF instrument, with improvements in both peptide and protein identification and quantification. Conclusively, our work shows how HCD, CID and ETD can be beneficially combined to enable iTRAQ-based quantification on an ETD-enabled LTQ Orbitrap XL.

  17. Exploration of the Medicinal Peptide Space.

    PubMed

    Gevaert, Bert; Stalmans, Sofie; Wynendaele, Evelien; Taevernier, Lien; Bracke, Nathalie; D'Hondt, Matthias; De Spiegeleer, Bart

    2016-01-01

    The chemical properties of peptide medicines, known as the 'medicinal peptide space' is considered a multi-dimensional subset of the global peptide space, where each dimension represents a chemical descriptor. These descriptors can be linked to biofunctional, medicinal properties to varying degrees. Knowledge of this space can increase the efficiency of the peptide-drug discovery and development process, as well as advance our understanding and classification of peptide medicines. For 245 peptide drugs, already available on the market or in clinical development, multivariate dataexploration was performed using peptide relevant physicochemical descriptors, their specific peptidedrug target and their clinical use. Our retrospective analysis indicates that clusters in the medicinal peptide space are located in a relatively narrow range of the physicochemical space: dense and empty regions were found, which can be explored for the discovery of novel peptide drugs.

  18. Encapsulation of bioactive whey peptides in soy lecithin-derived nanoliposomes: Influence of peptide molecular weight.

    PubMed

    Mohan, Aishwarya; McClements, David Julian; Udenigwe, Chibuike C

    2016-12-15

    Encapsulation of peptides can be used to enhance their stability, delivery and bioavailability. This study focused on the effect of the molecular weight range of whey peptides on their encapsulation within soy lecithin-derived nanoliposomes. Peptide molecular weight did not have a major impact on encapsulation efficiency or liposome size. However, it influenced peptide distribution amongst the surface, core, and bilayer regions of the liposomes, as determined by electrical charge (ζ-potential) and FTIR analysis. The liposome ζ-potential depended on peptide molecular weight, suggesting that the peptide charged groups were in different locations relative to the liposome surfaces. FTIR analysis indicated that the least hydrophobic peptide fractions interacted more strongly with choline on the liposome surfaces. The results suggested that the peptides were unequally distributed within the liposomes, even at the same encapsulation efficiency. These findings are important for designing delivery systems for commercial production of encapsulated peptides with improved functional attributes.

  19. Sustainable efficient way for opioid peptide LVV-h7 preparation from enzymatic proteolysis in a microfluidic-based reaction-extraction process with solvent recycling.

    PubMed

    Elagli, Adil; Belhacene, Kalim; Dhulster, Pascal; Froidevaux, Renato

    2016-05-01

    LVV-h7 (LVVYPWTQFR) is a bioactive peptide that can be obtained from blood as waste of food industry, more precisely from hemoglobin hydrolysis by pepsin. This opioid peptide belongs to the hemorphins family and have strong physiological effects that bring its use in pharmaceutics and various therapeutic treatments attractive, in particular for substituting its costly chemically synthetized analogous. Hemoglobin hydrolysis by pepsin generates a huge variety of peptides among whose LVV-h7 can be purified by liquid-liquid extraction (LLE). Herein, selective preparation of this peptide is proposed by a microfluidic-based continuous reaction-separation process. Hemoglobin hydrolysis in microreactor was firstly coupled to LVV-h7 LLE in octan-1-ol and then coupled to LVV-h7 back LLE in acidic water. This continuous process allowed to prepare pure LVV-h7, as confirmed by liquid chromatography and mass spectrometry. The microfluidic circuit also allowed octan-1-ol recycling in a closed loop, making this method more sustainable than similar biphasic batch process.

  20. Peptide identification

    DOEpatents

    Jarman, Kristin H [Richland, WA; Cannon, William R [Richland, WA; Jarman, Kenneth D [Richland, WA; Heredia-Langner, Alejandro [Richland, WA

    2011-07-12

    Peptides are identified from a list of candidates using collision-induced dissociation tandem mass spectrometry data. A probabilistic model for the occurrence of spectral peaks corresponding to frequently observed partial peptide fragment ions is applied. As part of the identification procedure, a probability score is produced that indicates the likelihood of any given candidate being the correct match. The statistical significance of the score is known without necessarily having reference to the actual identity of the peptide. In one form of the invention, a genetic algorithm is applied to candidate peptides using an objective function that takes into account the number of shifted peaks appearing in the candidate spectrum relative to the test spectrum.

  1. A genital tract peptide epitope vaccine targeting TLR-2 efficiently induces local and systemic CD8 + T cells and protects against herpes simplex virus type 2 challenge

    PubMed Central

    Dasgupta, G; Nesburn, AB; Wu, M; Zhu, X; Carpenter, D; Wechsler, SL; You, S; BenMohamed, L

    2015-01-01

    The next generation of needle-free mucosal vaccines is being rationally designed according to rules that govern the way in which the epitopes are recognized by and stimulate the genital mucosal immune system. We hypothesized that synthetic peptide epitopes extended with an agonist of Toll-like receptor 2 (TLR-2), that are abundantly expressed by dendritic and epithelial cells of the vaginal mucosa, would lead to induction of protective immunity against genital herpes. To test this hypothesis, we intravaginally (IVAG) immunized wild-type B6, TLR-2 (TLR2 −/−) or myeloid differentiation factor 88 deficient (MyD88 −/−) mice with a herpes simplex virus type 2 (HSV-2) CD8 + T-cell peptide epitope extended by a palmitic acid moiety (a TLR-2 agonist). IVAG delivery of the lipopeptide generated HSV-2-specific memory CD8 + cytotoxic T cells both locally in the genital tract draining lymph nodes and systemically in the spleen. Moreover, lipopeptide-immunized TLR2 −/− and MyD88 −/− mice developed significantly less HSV-specific CD8 + T-cell response, earlier death, faster disease progression, and higher vaginal HSV-2 titers compared to lipopeptide-immunized wild-type B6 mice. IVAG immunization with self-adjuvanting lipid-tailed peptides appears to be a novel mucosal vaccine approach, which has attractive practical and immunological features. PMID:19129756

  2. An efficient bioorthogonal strategy using CuAAC click chemistry for radiofluorinations of SNEW peptides and the role of copper depletion.

    PubMed

    Pretze, Marc; Kuchar, Manuela; Bergmann, Ralf; Steinbach, Jörg; Pietzsch, Jens; Mamat, Constantin

    2013-06-01

    The EphB2 receptor is known to be overexpressed in various types of cancer and is therefore a promising target for tumor cell imaging by positron emission tomography (PET). In this regard, imaging could facilitate the early detection of EphB2-overexpressing tumors, monitoring responses to therapy directed toward EphB2, and thus improvement in patient outcomes. We report the synthesis and evaluation of several fluorine-18-labeled peptides containing the SNEW amino acid motif, with high affinity for the EphB2 receptor, for their potential as radiotracers in the non-invasive imaging of cancer using PET. For the purposes of radiofluorination, EphB2-antagonistic SNEW peptides were varied at the C terminus by the introduction of L-cysteine, and further by alkyne- or azide-modified amino acids. In addition, two novel bifunctional and bioorthogonal labeling building blocks [(18)F]AFP and [(18)F]BFP were applied, and their capacity to introduce fluorine-18 was compared with that of the established building block [(18)F]FBAM. Copper-assisted Huisgen 1,3-dipolar cycloaddition, which belongs to the set of bioorthogonal click chemistry reactions, was used to introduce both novel building blocks into azide- or alkyne-modified SNEW peptides under mild conditions. Finally, the depletion of copper immediately after radiolabeling is a highly important step of this novel methodology.

  3. Two interdependent mechanisms of antimicrobial activity allow for efficient killing in nylon-3-based polymeric mimics of innate immunity peptides

    PubMed Central

    Lee, Michelle W.; Chakraborty, Saswata; Schmidt, Nathan W.; Murgai, Rajan; Gellman, Samuel H.; Wong, Gerard C.L.

    2015-01-01

    Novel synthetic mimics of antimicrobial peptides have been developed to exhibit structural properties and antimicrobial activity similar to those of natural antimicrobial peptides (AMPs) of the innate immune system. These molecules have a number of potential advantages over conventional antibiotics, including reduced bacterial resistance, cost-effective preparation, and customizable designs. In this study, we investigate a family of nylon-3 polymer-based antimicrobials. By combining vesicle dye leakage, bacterial permeation, and bactericidal assays with small-angle X-ray scattering (SAXS), we find that these polymers are capable of two interdependent mechanisms of action: permeation of bacterial membranes and binding to intracellular targets such as DNA, with the latter necessarily dependent on the former. We systemically examine polymer-induced membrane deformation modes across a range of lipid compositions that mimic both bacteria and mammalian cell membranes. The results show that the polymers' ability to generate negative Gaussian curvature (NGC), a topological requirement for membrane permeation and cellular entry, in model Escherichia coli membranes correlates with their ability to permeate membranes without complete membrane disruption and kill E. coli cells. Our findings suggest that these polymers operate with a concentration dependent mechanism of action: at low concentrations permeation and DNA binding occur without membrane disruption, while at high concentrations complete disruption of the membrane occurs. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. PMID:24743021

  4. Borinic acid catalysed peptide synthesis.

    PubMed

    El Dine, Tharwat Mohy; Rouden, Jacques; Blanchet, Jérôme

    2015-11-18

    The catalytic synthesis of peptides is a major challenge in the modern organic chemistry hindered by the well-established use of stoichiometric coupling reagents. Herein, we describe for the first time that borinic acid is able to catalyse this reaction under mild conditions with an improved activity compared to our recently developed thiophene-based boronic acid. This catalyst is particularly efficient for peptide bond synthesis affording dipeptides in good yields without detectable racemization.

  5. High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris.

    PubMed

    Laroche, Y; Storme, V; De Meutter, J; Messens, J; Lauwereys, M

    1994-11-01

    Tick anticoagulant peptide (TAP) is a potent and specific inhibitor of the blood coagulation protease Factor Xa. We designed and assembled a synthetic TAP-encoding gene (tapo) based on codons preferentially observed in the highly expressed Pichia pastoris alcohol oxidase 1 gene (AOX1), and fused it to a novel hybrid secretory prepro leader sequence. Expression from this gene yielded biologically active rTAP, which was correctly processed at the amino-terminal fusion site, and accumulated in the medium to approximately 1.7 g/l. This corresponds to a molar concentration of 0.24 mM, and is the highest yet described for a recombinant product secreted from P. pastoris. It also represents a seven-fold improvement in productivity compared to rTAP secretion from Saccharomyces cerevisiae, making P. pastoris an attractive host for the industrial-scale production of this potential therapeutic agent. This system was also used to prepare 21 mg 15N-rTAP, 11 mg 13C-rTAP and 27 mg 15N/13C-rTAP, with isotope incorporation levels higher than 98%, and purities sufficient to allow their use in determining the solution structure of the tick anticoagulant peptide using high field NMR.

  6. Antimicrobial Peptides

    PubMed Central

    Bahar, Ali Adem; Ren, Dacheng

    2013-01-01

    The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. The failure of the most potent antibiotics to kill “superbugs” emphasizes the urgent need to develop other control agents. Here we review the history and new development of antimicrobial peptides (AMPs), a growing class of natural and synthetic peptides with a wide spectrum of targets including viruses, bacteria, fungi, and parasites. We summarize the major types of AMPs, their modes of action, and the common mechanisms of AMP resistance. In addition, we discuss the principles for designing effective AMPs and the potential of using AMPs to control biofilms (multicellular structures of bacteria embedded in extracellular matrixes) and persister cells (dormant phenotypic variants of bacterial cells that are highly tolerant to antibiotics). PMID:24287494

  7. Methyl 2-((succinimidooxy)carbonyl)benzoate (MSB): a new, efficient reagent for N-phthaloylation of amino acid and peptide derivatives.

    PubMed

    Casimir, J Richard; Guichard, Gilles; Briand, Jean-Paul

    2002-05-31

    A new, efficient, and readily available reagent, methyl 2-((succinimidooxy)carbonyl)benzoate (MSB), for N-phthaloylation of amino acids and amino acid derivatives is described. The phthaloylation procedure is simple and racemization-free and gives excellent results with alpha-amino acids, alpha-amino alcohols, dipeptides, alpha-amino carboxamides, and alpha-amino esters.

  8. C-Peptide Test

    MedlinePlus

    ... vital for the body to use its main energy source, glucose . Since C-peptide and insulin are produced ... these cases, C-peptide measurement is a useful alternative to testing for insulin. C-peptide measurements can ...

  9. Predicting protein-peptide interactions from scratch

    NASA Astrophysics Data System (ADS)

    Yan, Chengfei; Xu, Xianjin; Zou, Xiaoqin; Zou lab Team

    Protein-peptide interactions play an important role in many cellular processes. The ability to predict protein-peptide complex structures is valuable for mechanistic investigation and therapeutic development. Due to the high flexibility of peptides and lack of templates for homologous modeling, predicting protein-peptide complex structures is extremely challenging. Recently, we have developed a novel docking framework for protein-peptide structure prediction. Specifically, given the sequence of a peptide and a 3D structure of the protein, initial conformations of the peptide are built through protein threading. Then, the peptide is globally and flexibly docked onto the protein using a novel iterative approach. Finally, the sampled modes are scored and ranked by a statistical potential-based energy scoring function that was derived for protein-peptide interactions from statistical mechanics principles. Our docking methodology has been tested on the Peptidb database and compared with other protein-peptide docking methods. Systematic analysis shows significantly improved results compared to the performances of the existing methods. Our method is computationally efficient and suitable for large-scale applications. Nsf CAREER Award 0953839 (XZ) NIH R01GM109980 (XZ).

  10. Peptide regulation of aging: 35-year research experience.

    PubMed

    Khavinson, V Kh; Anisimov, V N

    2009-07-01

    The authors sum up the results of many-year studies of mechanisms of aging and efficiency of peptide bioregulators in the prevention of age-specific diseases. Data on the effects of peptides, evaluated by the up-to-date methods, are presented. A molecular model of complementary interactions between short peptides and gene promotor sites, underlying the initiation of protein synthesis, is proposed. Prospects of peptide bioregulators in prevention of early aging are discussed.

  11. Antimicrobial Peptides from Marine Proteobacteria

    PubMed Central

    Desriac, Florie; Jégou, Camille; Balnois, Eric; Brillet, Benjamin; Le Chevalier, Patrick; Fleury, Yannick

    2013-01-01

    After years of inadequate use and the emergence of multidrug resistant (MDR) strains, the efficiency of “classical” antibiotics has decreased significantly. New drugs to fight MDR strains are urgently needed. Bacteria hold much promise as a source of unusual bioactive metabolites. However, the potential of marine bacteria, except for Actinomycetes and Cyanobacteria, has been largely underexplored. In the past two decades, the structures of several antimicrobial compounds have been elucidated in marine Proteobacteria. Of these compounds, polyketides (PKs), synthesised by condensation of malonyl-coenzyme A and/or acetyl-coenzyme A, and non-ribosomal peptides (NRPs), obtained through the linkage of (unusual) amino acids, have recently generated particular interest. NRPs are good examples of naturally modified peptides. Here, we review and compile the data on the antimicrobial peptides isolated from marine Proteobacteria, especially NRPs. PMID:24084784

  12. [SYNTHETIC PEPTIDE VACCINES].

    PubMed

    Sergeyev, O V; Barinsky, I F

    2016-01-01

    An update on the development and trials of synthetic peptide vaccines is reviewed. The review considers the successful examples of specific protection as a result of immunization with synthetic peptides using various protocols. The importance of conformation for the immunogenicity of the peptide is pointed out. An alternative strategy of the protection of the organism against the infection using synthetic peptides is suggested.

  13. Combinatorial discovery of tumor targeting peptides using phage display.

    PubMed

    Landon, Linda A; Deutscher, Susan L

    2003-10-15

    Peptides possess appropriate pharmacokinetic properties to serve as cancer imaging or therapeutic targeting agents. Currently, only a small number of rationally-derived, labeled peptide analogues that target only a limited subset of antigens are available. Thus, finding new cancer targeting peptides is a central goal in the field of molecular targeting. Novel tumor-avid peptides can be efficiently identified via affinity selections using complex random peptide libraries containing millions of peptides that are displayed on bacteriophage. In vitro and in situ affinity selections may be used to identify peptides with high affinity for the target antigen in vitro. Unfortunately, it has been found that peptides selected in vitro or in situ may not effectively target tumors in vivo due to poor peptide stability and other problems. To improve in vivo targeting, methodological combinatorial chemistry innovations allow selections to be conducted in the environment of the whole animal. Thus, new targeting peptides with optimal in vivo properties can be selected in vivo in tumor-bearing animals. In vivo selections have been proven successful in identifying peptides that target the vasculature of specific organs. In addition, in vivo selections have identified peptides that bind specifically to the surface of or are internalized into tumor cells. In the future, direct selection of peptides for cancer imaging may be expedited using genetically engineered bacteriophage libraries that encode peptides with intrinsic radiometal-chelation or fluorescent sequences.

  14. A Cell-Penetrating Peptide with a Guanidinylethyl Amine Structure Directed to Gene Delivery

    PubMed Central

    Oba, Makoto; Kato, Takuma; Furukawa, Kaori; Tanaka, Masakazu

    2016-01-01

    A peptide composed of lysine with a guanidinylethyl (GEt) amine structure in the side chain [Lys(GEt)] was developed as a cell-penetrating peptide directed to plasmid DNA (pDNA) delivery. The GEt amine adopted a diprotonated form at neutral pH, which may have led to the more efficient cellular uptake of a Lys(GEt)-peptide than an arginine-peptide at a low concentration. Lys(GEt)-peptide/pDNA complexes showed the highest transfection efficiency due to efficient endosomal escape without any cytotoxicity. Lys(GEt)-peptide may be a promising candidate as a gene delivery carrier. PMID:26814673

  15. Copper-Aβ Peptides and Oxidation of Catecholic Substrates: Reactivity and Endogenous Peptide Damage.

    PubMed

    Pirota, Valentina; Dell'Acqua, Simone; Monzani, Enrico; Nicolis, Stefania; Casella, Luigi

    2016-11-14

    The oxidative reactivity of copper complexes with Aβ peptides 1-16 and 1-28 (Aβ16 and Aβ28) against dopamine and related catechols under physiological conditions has been investigated in parallel with the competitive oxidative modification undergone by the peptides. It was found that both Aβ16 and Aβ28 markedly increase the oxidative reactivity of copper(II) towards the catechol compounds, up to a molar ratio of about 4:1 of peptide/copper(II). Copper redox cycling during the catalytic activity induces the competitive modification of the peptide at selected amino acid residues. The main modifications consist of oxidation of His13/14 to 2-oxohistidine and Phe19/20 to ortho-tyrosine, and the formation of a covalent His6-catechol adduct. Competition by the endogenous peptide is rather efficient, as approximately one peptide molecule is oxidized every 10 molecules of 4-methylcatechol.

  16. Nanotechnology for delivery of peptide nucleic acids (PNAs).

    PubMed

    Gupta, Anisha; Bahal, Raman; Gupta, Meera; Glazer, Peter M; Saltzman, W Mark

    2016-10-28

    Over the past three decades, peptide nucleic acids have been employed in numerous chemical and biological applications. Peptide nucleic acids possess enormous potential because of their superior biophysical properties, compared to other oligonucleotide chemistries. However, for therapeutic applications, intracellular delivery of peptide nucleic acids remains a challenge. In this review, we summarize the progress that has been made in delivering peptide nucleic acids to intracellular targets. In addition, we emphasize recent nanoparticle-based strategies for efficient delivery of conventional and chemically-modified peptides nucleic acids.

  17. Peptide Antimicrobial Agents

    PubMed Central

    Jenssen, Håvard; Hamill, Pamela; Hancock, Robert E. W.

    2006-01-01

    Antimicrobial host defense peptides are produced by all complex organisms as well as some microbes and have diverse and complex antimicrobial activities. Collectively these peptides demonstrate a broad range of antiviral and antibacterial activities and modes of action, and it is important to distinguish between direct microbicidal and indirect activities against such pathogens. The structural requirements of peptides for antiviral and antibacterial activities are evaluated in light of the diverse set of primary and secondary structures described for host defense peptides. Peptides with antifungal and antiparasitic activities are discussed in less detail, although the broad-spectrum activities of such peptides indicate that they are important host defense molecules. Knowledge regarding the relationship between peptide structure and function as well as their mechanism of action is being applied in the design of antimicrobial peptide variants as potential novel therapeutic agents. PMID:16847082

  18. Antimicrobial Peptides in 2014

    PubMed Central

    Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

    2015-01-01

    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. PMID:25806720

  19. PH dependent adhesive peptides

    SciTech Connect

    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. Preparation of Peptide p-Nitroanilides using an Aryl Hydrazine Solid Support

    SciTech Connect

    Kwon, Y; Welsh, K; Mitchell, A R; Camarero, J A

    2004-08-05

    Peptide p-nitroanilides are useful compounds for studying protease activity, however the poor nucleophilicity of p-nitroaniline makes their preparation difficult. We describe a new efficient approach for the Fmoc-based synthesis of peptide p-nitroanilides using an aryl hydrazine resin. Mild oxidation of the peptide hydrazide resin yields a highly reactive acyl diazene, which efficiently reacts with weak nucleophiles. We have prepared several peptide p-nitroanilides, including substrates for the Lethal Factor protease from B. anthracis.

  1. Short peptides allowing preferential detection of Candida albicans hyphae.

    PubMed

    Kaba, Hani E J; Pölderl, Antonia; Bilitewski, Ursula

    2015-09-01

    Whereas the detection of pathogens via recognition of surface structures by specific antibodies and various types of antibody mimics is frequently described, the applicability of short linear peptides as sensor molecules or diagnostic tools is less well-known. We selected peptides which were previously reported to bind to recombinant S. cerevisiae cells, expressing members of the C. albicans Agglutinin-Like-Sequence (ALS) cell wall protein family. We slightly modified amino acid sequences to evaluate peptide sequence properties influencing binding to C. albicans cells. Among the selected peptides, decamer peptides with an "AP"-N-terminus were superior to shorter peptides. The new decamer peptide FBP4 stained viable C. albicans cells more efficiently in their mature hyphal form than in their yeast form. Moreover, it allowed distinction of C. albicans from other related Candida spp. and could thus be the basis for the development of a useful tool for the diagnosis of invasive candidiasis.

  2. Characterization of a possible uptake mechanism of selective antibacterial peptides.

    PubMed

    Polanco, Carlos; Samaniego, José Lino; Castañón-González, Jorge Alberto; Buhse, Thomas; Sordo, Marili Leopold

    2013-01-01

    Selective antibacterial peptides containing less than 30 amino acid residues, cationic, with amphipathic properties, have been the subject of several studies due to their active participation and beneficial effects in strengthening the immune system of all living organisms. This manuscript reports the results of a comparison between the group of selective antibacterial peptides and another group called "cell penetrating peptides". An important number of the selective antibacterial peptides are cell penetrating peptides, suggesting that their toxicity is related to their uptake mechanism. The verification of this observation also includes the adaptation of a method previously published, called Polarity index, which reproduces and confirms the action of this new set of peptides. The efficiency of this method was verified based on four different databases, yielding a high score. The verification was based exclusively on the peptides already reported in the databases which have been experimentally verified.

  3. Peptides homing to tumor vasculature: imaging and therapeutics for cancer.

    PubMed

    Liu, Zhiguo; Wu, Kaichun

    2008-11-01

    A major obstacle to advances in anti-vascular therapy is the lack of molecule candidates that are effective in selectively targeting cancer tissues while sparing normal ones. Phage display peptide library greatly eases the discovery of peptides with specific homing capacity. Many novel peptides homing to angiogenic vessels were isolated recently. Notably, many such peptides showed relatively specific affinity with particular tumor types. These peptides appear to be able to accumulate in the target vascular site of tumor, making them particularly efficient to deliver drugs or other therapeutic and imaging agents. Some homing peptides could not only target to the desired location, but also be internalized into targeted cells, or even induce destruction in desired cells all by the same peptide sequence itself. Accumulating evidence has shown that by tumor specific targeting delivery, improved local effect can be achieved with well tolerated side effects. In the current review, recent literatures and patents in this field have been summarized.

  4. Naturally processed peptides spanning the HPA-1a polymorphism are efficiently generated and displayed from platelet glycoprotein by HLA-DRB3*0101-positive antigen-presenting cells.

    PubMed

    Anani Sarab, Gholamreza; Moss, Michael; Barker, Robert N; Urbaniak, Stanislaw J

    2009-08-27

    In neonatal alloimmune thrombocytopenia, almost all human platelet antigen (HPA)-1b1b mothers who produce anti-HPA-1a antibody through carrying an HPA-1a fetus are human histocompatibility leukocyte antigen (HLA)-DRB3*0101 positive. It is predicted that the HPA-1a Leu(33) polymorphism forms part of an HLA-DRB3*0101-restricted T-helper epitope, and acts as an anchor residue for binding this class II molecule. However, it is not known whether any corresponding peptides are naturally processed and presented from platelet glycoprotein. In this study, peptides displayed by a homozygous HLA-DRB3*0101 antigen-presenting cell line were identified after pulsing with recombinant HPA-1a (Leu(33) plexin-semaphorin-integrin domain). The peptides were eluted from HLA-DR molecules, fractionated by high performance liquid chromatography, and analyzed by tandem mass spectrometry. A "nested set" of naturally presented HPA-1a-derived peptides, each containing the Trp(25)-Leu(33) core epitope, was identified, with the most abundant member being the 16-mer Met(22)-Arg(37). These peptides may provide the basis for novel treatments to tolerize the corresponding T-helper response in women at risk of neonatal alloimmune thrombocytopenia.

  5. A switchable stapled peptide.

    PubMed

    Kalistratova, Aleksandra; Legrand, Baptiste; Verdié, Pascal; Naydenova, Emilia; Amblard, Muriel; Martinez, Jean; Subra, Gilles

    2016-03-01

    The O-N acyl transfer reaction has gained significant popularity in peptide and medicinal chemistry. This reaction has been successfully applied to the synthesis of difficult sequence-containing peptides, cyclic peptides, epimerization-free fragment coupling and more recently, to switchable peptide polymers. Herein, we describe a related strategy to facilitate the synthesis and purification of a hydrophobic stapled peptide. The staple consists of a serine linked through an amide bond formed from its carboxylic acid function and the side chain amino group of diaminopropionic acid and through an ester bond formed from its amino group and the side chain carboxylic acid function of aspartic acid. The α-amino group of serine was protonated during purification. Interestingly, when the peptide was placed at physiological pH, the free amino group initiated the O-N shift reducing the staple length by one atom, leading to a more hydrophobic stapled peptide.

  6. Multiplex De Novo Sequencing of Peptide Antibiotics

    NASA Astrophysics Data System (ADS)

    Mohimani, Hosein; Liu, Wei-Ting; Yang, Yu-Liang; Gaudêncio, Susana P.; Fenical, William; Dorrestein, Pieter C.; Pevzner, Pavel A.

    Proliferation of drug-resistant diseases raises the challenge of searching for new, more efficient antibiotics. Currently, some of the most effective antibiotics (i.e., Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. The isolation and sequencing of cyclic peptide antibiotics, unlike the same activity with linear peptides, is time-consuming and error-prone. The dominant technique for sequencing cyclic peptides is NMR-based and requires large amounts (milligrams) of purified materials that, for most compounds, are not possible to obtain. Given these facts, there is a need for new tools to sequence cyclic NRPs using picograms of material. Since nearly all cyclic NRPs are produced along with related analogs, we develop a mass spectrometry approach for sequencing all related peptides at once (in contrast to the existing approach that analyzes individual peptides). Our results suggest that instead of attempting to isolate and NMR-sequence the most abundant compound, one should acquire spectra of many related compounds and sequence all of them simultaneously using tandem mass spectrometry. We illustrate applications of this approach by sequencing new variants of cyclic peptide antibiotics from Bacillus brevis, as well as sequencing a previously unknown familiy of cyclic NRPs produced by marine bacteria.

  7. Peptide consensus sequence determination for the enhancement of the antimicrobial activity and selectivity of antimicrobial peptides

    PubMed Central

    Almaaytah, Ammar; Ajingi, Ya’u; Abualhaijaa, Ahmad; Tarazi, Shadi; Alshar’i, Nizar; Al-Balas, Qosay

    2017-01-01

    The rise of multidrug-resistant bacteria is causing a serious threat to the world’s human population. Recent reports have identified bacterial strains displaying pan drug resistance against antibiotics and generating fears among medical health specialists that humanity is on the dawn of entering a post-antibiotics era. Global research is currently focused on expanding the lifetime of current antibiotics and the development of new antimicrobial agents to tackle the problem of antimicrobial resistance. In the present study, we designed a novel consensus peptide named “Pepcon” through peptide consensus sequence determination among members of a highly homologous group of scorpion antimicrobial peptides. Members of this group were found to possess moderate antimicrobial activity with significant toxicity against mammalian cells. The aim of our design method was to generate a novel peptide with an enhanced antimicrobial potency and selectivity against microbial rather than mammalian cells. The results of our study revealed that the consensus peptide displayed potent antibacterial activities against a broad range of Gram-positive and Gram-negative bacteria. Our membrane permeation studies displayed that the peptide efficiently induced membrane damage and consequently led to cell death through the process of cell lysis. The microbial DNA binding assay of the peptide was found to be very weak suggesting that the peptide is not targeting the microbial DNA. Pepcon induced minimal cytotoxicity at the antimicrobial concentrations as the hemolytic activity was found to be zero at the minimal inhibitory concentrations (MICs). The results of our study demonstrate that the consensus peptide design strategy is efficient in generating peptides. PMID:28096686

  8. Polymer-Peptide Nanoparticles: Synthesis and Characterization

    NASA Astrophysics Data System (ADS)

    Dong, He; Shu, Jessica Y.; Xu, Ting

    2010-03-01

    Conjugation of synthetic polymers to peptides offers an efficient way to produce novel supramolecular structures. Herein, we report an attempt to prepare synthetic micellar nanoparticles using amphiphilic peptide-polymer conjugates as molecular building blocks. Spherical nanoparticles were formed upon dissolution of peptides in PBS buffer through the segregation of hydrophobic and hydrophilic segments. Both molecular and nano- structures were thoroughly investigated by a variety of biophysical techniques, including circular dichroism (CD), dynamic light scattering (DLS), size exclusion chromatography (SEC), transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS). The results demonstrate that structural properties of these biohybrid materials depend on both the geometry of the hydrophobic domain and the size of synthetic polymers. Given the diversity of functional peptide sequences, hydrophilic polymers and hydrophobic moieties, these materials would be expected to self-assemble into various types of nanostructures to cover a wide range of biological applications.

  9. Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

    PubMed

    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.

  10. Plant peptide hormone signalling.

    PubMed

    Motomitsu, Ayane; Sawa, Shinichiro; Ishida, Takashi

    2015-01-01

    The ligand-receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone-receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions.

  11. Stabilization of exosome-targeting peptides via engineered glycosylation.

    PubMed

    Hung, Michelle E; Leonard, Joshua N

    2015-03-27

    Exosomes are secreted extracellular vesicles that mediate intercellular transfer of cellular contents and are attractive vehicles for therapeutic delivery of bimolecular cargo such as nucleic acids, proteins, and even drugs. Efficient exosome-mediated delivery in vivo requires targeting vesicles for uptake by specific recipient cells. Although exosomes have been successfully targeted to several cellular receptors by displaying peptides on the surface of the exosomes, identifying effective exosome-targeting peptides for other receptors has proven challenging. Furthermore, the biophysical rules governing targeting peptide success remain poorly understood. To evaluate one factor potentially limiting exosome delivery, we investigated whether peptides displayed on the exosome surface are degraded during exosome biogenesis, for example by endosomal proteases. Indeed, peptides fused to the N terminus of exosome-associated transmembrane protein Lamp2b were cleaved in samples derived from both cells and exosomes. To suppress peptide loss, we engineered targeting peptide-Lamp2b fusion proteins to include a glycosylation motif at various positions. Introduction of this glycosylation motif both protected the peptide from degradation and led to an increase in overall Lamp2b fusion protein expression in both cells and exosomes. Moreover, glycosylation-stabilized peptides enhanced targeted delivery of exosomes to neuroblastoma cells, demonstrating that such glycosylation does not ablate peptide-target interactions. Thus, we have identified a strategy for achieving robust display of targeting peptides on the surface of exosomes, which should facilitate the evaluation and development of new exosome-based therapeutics.

  12. Characterization of reducible peptide oligomers as carriers for gene delivery.

    PubMed

    Kiselev, Anton; Egorova, Anna; Laukkanen, Antti; Baranov, Vladislav; Urtti, Arto

    2013-01-30

    The stability of DNA-polyplexes and intracellular DNA release are important features of gene delivery systems. To study these features, we have evaluated reducible cysteine-flanked linear lysine and arginine-rich peptides, modified with histidine residues. The reducible disulfide bonds in cysteine flanked peptides and histidine residues should augment DNA release from the peptide-DNA complexes upon disintegration of the reducible bonds. Template polymerization and oxidative polycondensation were applied to obtain peptide oligomers used for DNA-polyplex preparation. The peptides and DNA-peptide complexes were investigated with physical, chemical and transfection measurements. Physicochemical and transfection properties of DNA-polyplexes depended on the amino acid sequence of the peptidic polymers and type of the polymerization. MALDI-TOF analysis of oxidatively polycondensed products revealed several forms of peptide oligomers corresponding to 5-8 amino acid monomers. DNA-peptide particles based on template-polymerized complexes were more resistant to relaxation by negatively charged heparan sulfate than polyplexes formed with oxidatively condensed peptides. Complexes of DNA with the polycations prepared by oxidative polycondensation exhibited a 100-1000-fold higher level of gene expression compared to DNA/template-polymerized peptide complexes. The most efficient transgene expression was shown with arginine-rich polyplexes. Transfection efficacy of the arginine-rich polyplexes was even 10-fold better than that of DNA/PEI complexes. On average, polyplexes based on cysteine-flanked peptide oligomers showed lower cytotoxicity than non-reducible high molecular weight polylysine/DNA particles. We conclude that reducible peptide oligomers provide efficient DNA transfection and have the potential as vehicles for gene delivery.

  13. Selective enrichment and desalting of hydrophilic peptides using graphene oxide.

    PubMed

    Jiang, Miao; Qi, Linyu; Liu, Peiru; Wang, Zijun; Duan, Zhigui; Wang, Ying; Liu, Zhonghua; Chen, Ping

    2016-08-01

    The wide variety and low abundance of peptides in tissue brought great difficulties to the separation and identification of peptides, which is not in favor of the development of peptidomics. RP-HPLC, which could purify small molecules based on their hydrophobicity, has been widely used in the separation and enrichment of peptide due to its fast, good reproducibility and high resolution. However, RP-HPLC requires the instrument and expensive C18 column and its sample capacity is also limited. Recently, graphene oxide has been applied to the adsorption of amino acids. However, the enrichment efficiency and selectivity of graphene oxide for peptides remain unclear. In this study, the adsorption efficiency and selectivity of graphene oxide and RP-C18 matrix were compared on trypsinized α-actin and also on tissue extracts from pituitary gland and hippocampus. For α-actin, there exhibit similar elution peaks for total trypsinized products and those adsorpted by GO and C18 matrix. But peptides adsorbed by GO showed the higher hydrophilic peaks than which adsorbed by C18 matrix. The resulted RP-HPLC profile showed that most of peptides enriched by graphene oxide were eluted at low concentration of organic solvent, while peptides adsorbed by RP-C18 matrix were mostly eluted at relatively high concentration. Moreover, mass spectrometry analysis suggested that, in pituitary sample, there were 495 peptides enriched by graphene oxide, 447 peptides enriched by RP-C18 matrix while in hippocampus sample 333 and 243 peptides respectively. The GRAVY value analysis suggested that the graphene oxide has a stronger adsorption for highly hydrophilic peptides compared to the RP-C18 matrix. Furthermore, the combination of these two methods could notably increase the number of identification peptides but also the number of predicted protein precursors. Our study provided a new thought to the role of graphene oxide during the enrichment of peptides from tissue which should be useful for

  14. Polycyclic peptide therapeutics.

    PubMed

    Baeriswyl, Vanessa; Heinis, Christian

    2013-03-01

    Owing to their excellent binding properties, high stability, and low off-target toxicity, polycyclic peptides are an attractive molecule format for the development of therapeutics. Currently, only a handful of polycyclic peptides are used in the clinic; examples include the antibiotic vancomycin, the anticancer drugs actinomycin D and romidepsin, and the analgesic agent ziconotide. All clinically used polycyclic peptide drugs are derived from natural sources, such as soil bacteria in the case of vancomycin, actinomycin D and romidepsin, or the venom of a fish-hunting coil snail in the case of ziconotide. Unfortunately, nature provides peptide macrocyclic ligands for only a small fraction of therapeutic targets. For the generation of ligands of targets of choice, researchers have inserted artificial binding sites into natural polycyclic peptide scaffolds, such as cystine knot proteins, using rational design or directed evolution approaches. More recently, large combinatorial libraries of genetically encoded bicyclic peptides have been generated de novo and screened by phage display. In this Minireview, the properties of existing polycyclic peptide drugs are discussed and related to their interesting molecular architectures. Furthermore, technologies that allow the development of unnatural polycyclic peptide ligands are discussed. Recent application of these technologies has generated promising results, suggesting that polycyclic peptide therapeutics could potentially be developed for a broad range of diseases.

  15. Antimicrobial Peptides in Reptiles

    PubMed Central

    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

  16. The natriuretic peptides.

    PubMed

    Baxter, Gary F

    2004-03-01

    The natriuretic peptides are a family of widely distributed, but evolutionarily conserved, polypeptide mediators that exert a range of actions throughout the body. In cardiovascular homeostasis, the endocrine roles of the cardiac-derived atrial and B-type natriuretic peptide (ANP and BNP) in regulating central fluid volume and blood pressure have been recognised for two decades. However, there is a growing realisation that natriuretic peptide actions go far beyond their volume regulating effects. These pleiotropic actions include local (autocrine/paracrine) regulatory actions of ANP and BNP within the heart, and of another natriuretic peptide, CNP, within the vessel wall. Effects on function and growth of the local tissue environment are likely to be of great importance, especially in disease states where tissue and circulating levels of ANP and BNP rise markedly. At present, the relevance of other natriuretic peptides (notably uroguanylin and DNP) to human physiology and pathology remain uncertain. Other articles in this issue of Basic Research in Cardiology review the molecular physiology of natriuretic peptide signalling, with a particular emphasis on the lessons from genetically targetted mice; the vascular activity of natriuretic peptides; the regulation and roles of natriuretic peptides in ischaemic myocardium; and the diagnostic, prognostic and therapeutic roles of natriuretic peptides in heart failure.

  17. Enzyme-mediated ligation technologies for peptides and proteins.

    PubMed

    Schmidt, Marcel; Toplak, Ana; Quaedflieg, Peter Jlm; Nuijens, Timo

    2017-02-18

    With the steadily increasing complexity and quantity requirements for peptides in industry and academia, the efficient and site-selective ligation of peptides and proteins represents a highly desirable goal. Within this context, enzyme-mediated ligation technologies for peptides and proteins have attracted great interest in recent years as they represent an extremely powerful extension to the scope of chemical methodologies (e.g. native chemical ligation) in basic and applied research. Compared to chemical ligation methods, enzymatic strategies using ligases such as sortase, butelase, peptiligase or omniligase generally feature excellent chemoselectivity, therefore making them valuable tools for protein and peptide chemists.

  18. Functions of Cationic Host Defense Peptides in Immunity

    PubMed Central

    Hemshekhar, Mahadevappa; Anaparti, Vidyanand; Mookherjee, Neeloffer

    2016-01-01

    Cationic host defense peptides are a widely distributed family of immunomodulatory molecules with antimicrobial properties. The biological functions of these peptides include the ability to influence innate and adaptive immunity for efficient resolution of infections and simultaneous modulation of inflammatory responses. This unique dual bioactivity of controlling infections and inflammation has gained substantial attention in the last three decades and consequent interest in the development of these peptide mimics as immunomodulatory therapeutic candidates. In this review, we summarize the current literature on the wide range of functions of cationic host defense peptides in the context of the mammalian immune system. PMID:27384571

  19. Porcine parvovirus removal using trimer and biased hexamer peptides

    PubMed Central

    Heldt, Caryn L.; Gurgel, Patrick V.; Jaykus, Lee-Ann; Carbonell, Ruben G.

    2014-01-01

    Assuring the microbiological safety of biological therapeutics remains an important concern. Our group has recently reported small trimeric peptides that have the ability to bind and remove a model non-enveloped virus, porcine parvovirus (PPV), from complex solutions containing human blood plasma. In an effort to improve the removal efficiency of these small peptides, we created a biased library of hexamer peptides that contain two previously reported trimeric peptides designated WRW and KYY. This library was screened and several hexamer peptides were discovered that also removed PPV from solution, but there was no marked improvement in removal efficiency when compared to the trimeric peptides. Based on simulated docking experiments, it appeared that hexamer peptide binding is dictated more by secondary structure, whereas the binding of trimeric peptides is dominated by charge and hydrophobicity. This study demonstrates that trimeric and hexameric peptides may have different, matrix-specific roles to play in virus removal applications. In general, the hexamer ligand may perform better for binding of specific viruses, whereas the trimer ligand may have more broadly reactive virus-binding properties. PMID:21751387

  20. Protein quantification using a cleavable reporter peptide.

    PubMed

    Duriez, Elodie; Trevisiol, Stephane; Domon, Bruno

    2015-02-06

    Peptide and protein quantification based on isotope dilution and mass spectrometry analysis are widely employed for the measurement of biomarkers and in system biology applications. The accuracy and reliability of such quantitative assays depend on the quality of the stable-isotope labeled standards. Although the quantification using stable-isotope labeled peptides is precise, the accuracy of the results can be severely biased by the purity of the internal standards, their stability and formulation, and the determination of their concentration. Here we describe a rapid and cost-efficient method to recalibrate stable isotope labeled peptides in a single LC-MS analysis. The method is based on the equimolar release of a protein reference peptide (used as surrogate for the protein of interest) and a universal reporter peptide during the trypsinization of a concatenated polypeptide standard. The quality and accuracy of data generated with such concatenated polypeptide standards are highlighted by the quantification of two clinically important proteins in urine samples and compared with results obtained with conventional stable isotope labeled reference peptides. Furthermore, the application of the UCRP standards in complex samples is described.

  1. Biomathematical Description of Synthetic Peptide Libraries

    PubMed Central

    Trepel, Martin

    2015-01-01

    Libraries of randomised peptides displayed on phages or viral particles are essential tools in a wide spectrum of applications. However, there is only limited understanding of a library's fundamental dynamics and the influences of encoding schemes and sizes on their quality. Numeric properties of libraries, such as the expected number of different peptides and the library's coverage, have long been in use as measures of a library's quality. Here, we present a graphical framework of these measures together with a library's relative efficiency to help to describe libraries in enough detail for researchers to plan new experiments in a more informed manner. In particular, these values allow us to answer-in a probabilistic fashion-the question of whether a specific library does indeed contain one of the "best" possible peptides. The framework is implemented in a web-interface based on two packages, discreteRV and peptider, to the statistical software environment R. We further provide a user-friendly web-interface called PeLiCa (Peptide Library Calculator, http://www.pelica.org), allowing scientists to plan and analyse their peptide libraries. PMID:26042419

  2. Ubiquitin fusion technology: bioprocessing of peptides.

    PubMed

    Pilon, A; Yost, P; Chase, T E; Lohnas, G; Burkett, T; Roberts, S; Bentley, W E

    1997-01-01

    Ubiquitin fusion technology represents an emerging method for economically producing peptides and small proteins in the bacterium Escherichia coli. Our focus is on peptide production where the need for cost-effective, scaleable processes has recently been highlighted by Kelley (1996). There are two principal features: (1) the expression system consists of a suitable E. coli host strain paired with a plasmid that encodes the ubiquitin fusion and (2) an ubiquitin-specific protease, UCH-L3, which cleaves only C-terminal extensions from ubiquitin. In this work, multigram yields were obtained of four ubiquitin fusions derived from cell paste generated in single 10-L fermentations. All were expressed intracellularly and remained soluble at extremely high levels of expression. Bacterial freeze--thaw lysates contained over 95% pure ubiquitin fusion protein. All four fusions were efficiently cleaved to ubiquitin and the peptide products. In one case, the final yield of peptide was 1.08 g from 3 L of low cell density bacterial culture. The combination of exceptional overexpression of the ubiquitin--peptide fusion proteins and a robust and specific protease are unique advantages contributing to a cost-effective, scaleable, and generic bioprocess for peptide production.

  3. Characterization of Selective Antibacterial Peptides by Polarity Index

    PubMed Central

    Polanco, C.; Samaniego, J. L.; Buhse, T.; Mosqueira, F. G.; Negron-Mendoza, A.; Ramos-Bernal, S.; Castanon-Gonzalez, J. A.

    2012-01-01

    In the recent decades, antibacterial peptides have occupied a strategic position for pharmaceutical drug applications and became subject of intense research activities since they are used to strengthen the immune system of all living organisms by protecting them from pathogenic bacteria. This work proposes a simple and easy statistical/computational method through a peptide polarity index measure by which an antibacterial peptide subgroup can be efficiently identified, that is, characterized by a high toxicity to bacterial membranes but presents a low toxicity to mammal cells. These peptides also have the feature not to adopt to an alpha-helicoidal structure in aqueous solution. The double-blind test carried out to the whole Antimicrobial Peptide Database (November 2011) showed an accuracy of 90% applying the polarity index method for the identification of such antibacterial peptide groups. PMID:22611416

  4. Rapid profiling of peptide stability in proteolytic environments.

    PubMed

    Gorris, Hans H; Bade, Steffen; Röckendorf, Niels; Albers, Eike; Schmidt, M Alexander; Fránek, Milan; Frey, Andreas

    2009-02-15

    The notorious degradation susceptibility of peptides is a major obstacle to their use as medicinal drugs. Assays with which the stability of peptides in complex proteolytic environments can be determined are thus indispensable for peptide drug development. Herein, we describe a new peptide proteolysis assay that meets that demand. It unites the high-throughput capacity of heterogeneous with the well-defined kinetic characteristics of homogeneous assay formats and operates on the cleavage-caused loss of a detection handle. We have confirmed the assay's accuracy with well-defined model interactions and proved its high versatility and robustness with a representative application where we determined the half-lives of 375 different peptides in a crude intestinal protease preparation. With this reliable, reproducible, and efficient assay the enzyme kinetics of any peptide-protease interaction is accessible even for complex protease solutions.

  5. Structure of HLA-A*1101 in complex with a hepatitis B peptide homologue

    PubMed Central

    Blicher, Thomas; Kastrup, Jette Sandholm; Pedersen, Lars Østergaard; Buus, Søren; Gajhede, Michael

    2006-01-01

    A high-resolution structure of the human MHC-I molecule HLA-A*1101 is presented in which it forms a complex with a sequence homologue of a peptide that occurs naturally in hepatitis B virus DNA polymerase. The sequence of the bound peptide is AIMPARFYPK, while that of the corresponding natural peptide is LIMPARFYPK. The peptide does not make efficient use of the middle E pocket for binding, which leads to a rather superficial and exposed binding mode for the central peptide residues. Despite this, the peptide binds with high affinity (IC50 of 31 nM). PMID:17142892

  6. Structure of HLA-A*1101 in complex with a hepatitis B peptide homologue.

    PubMed

    Blicher, Thomas; Kastrup, Jette Sandholm; Pedersen, Lars Østergaard; Buus, Søren; Gajhede, Michael

    2006-12-01

    A high-resolution structure of the human MHC-I molecule HLA-A*1101 is presented in which it forms a complex with a sequence homologue of a peptide that occurs naturally in hepatitis B virus DNA polymerase. The sequence of the bound peptide is AIMPARFYPK, while that of the corresponding natural peptide is LIMPARFYPK. The peptide does not make efficient use of the middle E pocket for binding, which leads to a rather superficial and exposed binding mode for the central peptide residues. Despite this, the peptide binds with high affinity (IC50 of 31 nM).

  7. Ribosomal synthesis of dehydroalanine-containing peptides.

    PubMed

    Seebeck, Florian P; Szostak, Jack W

    2006-06-07

    Dehydroalanine is a nonproteinogenic amino acid, but it is a component of a wide variety of natural products with therapeutic activities. Indeed, this alpha,beta-unsaturated residue is a highly versatile building block due to its rigidifying effect on peptide backbones and its electrophilicity which allows site-specific thiol ligations of peptides with small molecules or proteins. To harness such versatility in genetically encoded, combinatorial peptide libraries, we report a simple and robust method for the ribosomal synthesis of dehydroalanine-containing peptides. Selenalysine, a selenium-containing lysine analogue, was recruited as a masked dehydroalanine equivalent. This residue is efficiently incorporated by a reconstituted Escherichia coli translation system at high fidelity and efficiency despite the presence of low levels of lysine. Mild oxidative conditions were used to convert selenalysine into dehydroalanine post-translationally. Using this method, we demonstrate the preparation of polyunsaturated and highly decorated peptides. This report is an important step toward the preparation and selection of large libraries of protein-reactive compounds with potential use as novel drugs or as analytical tools.

  8. Peptide bioregulators inhibit apoptosis.

    PubMed

    Khavinson, V K; Kvetnoii, I M

    2000-12-01

    The effects of peptide bioregulators epithalon and vilon on the dynamics of irradiation-induced apoptotic death of spleen lymphocytes in rats indicate that these agents inhibit physiologically programmed cell death. The antiapoptotic effect of vilon was more pronounced, which corroborates the concept on tissue-specific effect of peptide bioregulators.

  9. Bacteriocin Inducer Peptides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel peptides produced by bacteriocin-producing bacteria stimulate the production of bacteriocins in vitro. The producer bacteria are cultured in the presence of a novel inducer bacteria and a peptide having a carboxy terminal sequence of VKGLT in order to achieve an increase in bacteriocin produc...

  10. The Specificity of Peptide Chain Extension by N-Carboxyanhydrides

    NASA Astrophysics Data System (ADS)

    Wen, Ke; Orgel, Leslie E.

    2001-06-01

    We have used amino acids activated by carbonyldiimidazole to study the enantiospecificity of peptide elongation in aqueoussolution. Peptide `primers' Glu10 and Ala3Glu10were elongated with the enantiomers of arginine, glutamic acid,asparagine, phenylalanine, serine and valine. The homochiral addition was always the more efficient reaction; the enantiospecificity was large in some cases but very small in others. In every case Ala3Glu10 was elongated more efficiently than Glu10.

  11. Polar characterization of antifungal peptides from APD2 Database.

    PubMed

    Polanco, Carlos; Samaniego-Mendoza, José Lino; Buhse, Thomas; Castañón-González, Jorge Alberto; Leopold-Sordo, Marili

    2014-11-01

    The increase in the number of pathogens due to fungi that are tolerant to therapies does not grow at the same speed than the advance on new antifungal drugs. In this sense, it is imperative to find anti-fungi peptides that are not detrimental to mammalian cells and have an effective toxicity to fungi. In this work, we use a method called polarity index, to identify anti-fungi peptides with an efficiency of 70 %. This method already published, initially identified selective antibacterial peptides from APD2 Database, and was characterized by developing a comprehensive analysis of the polar dynamics of a peptide from its linear sequence. Discriminating tests showed that in addition to being efficient in this identification, it was also good at rejecting other classifications of peptides found in that same database.

  12. Peptide synthesis on glass substrate using acoustic droplet ejector.

    PubMed

    Youngki Choe; Shih-Jui Chen; Eun Sok Kim

    2014-03-01

    This paper describes the synthesis of a 9-mers-long peptide ladder structure of glycine on a modified glass surface using a nanoliter droplet ejector. To synthesize peptide on a glass substrate, SPOT peptide synthesis protocol was followed with a nozzleless acoustic droplet ejector being used to eject about 300 droplets of preactivated amino acid solution to dispense 60 nL of the solution per mer. The coupling efficiency of each mer was measured with FITC fluorescent tag to be 96%, resulting in net 70% efficiency for the whole 9-mer-long peptide of glycine. Usage of a nanoliter droplet ejector for SPOT peptide synthesis increases the density of protein array on a chip.

  13. Antimicrobial Peptides from Fish

    PubMed Central

    Masso-Silva, Jorge A.; Diamond, Gill

    2014-01-01

    Antimicrobial peptides (AMPs) are found widely distributed through Nature, and participate in the innate host defense of each species. Fish are a great source of these peptides, as they express all of the major classes of AMPs, including defensins, cathelicidins, hepcidins, histone-derived peptides, and a fish-specific class of the cecropin family, called piscidins. As with other species, the fish peptides exhibit broad-spectrum antimicrobial activity, killing both fish and human pathogens. They are also immunomodulatory, and their genes are highly responsive to microbes and innate immuno-stimulatory molecules. Recent research has demonstrated that some of the unique properties of fish peptides, including their ability to act even in very high salt concentrations, make them good potential targets for development as therapeutic antimicrobials. Further, the stimulation of their gene expression by exogenous factors could be useful in preventing pathogenic microbes in aquaculture. PMID:24594555

  14. HLA-DM mediates peptide exchange by interacting transiently and repeatedly with HLA-DR1

    PubMed Central

    Narayan, Kedar; Su, Katherine W.; Chou, Chih-Ling; Khoruzhenko, Stanislav

    2009-01-01

    The peptide editor HLA-DM (DM) catalyzes the exchange of peptides bound to MHC class II molecules within antigen presenting cells by generating a “peptide-receptive” MHC class II conformation (MHCreceptive) to which peptides readily bind and rapidly unbind. While recent work has uncovered the determinants of DM recognition and effector functions, the nature of MHCreceptive and its interaction with DM remains unclear. Here, we show that DM induces but does not stabilize MHCreceptive in the absence of peptides. We demonstrate that DM is out-competed by certain superantigens, and increasing solvent viscosity inhibits DM-induced peptide association. We suggest that DM mediates peptide exchange by interacting transiently and repeatedly with MHC class II molecules, continually generating MHCreceptive. The simultaneous presence of peptide and DM in the milieu is thus crucial for the efficient generation of specific peptide-MHC class II complexes over time. PMID:19647320

  15. Review: Formation of Peptide Radical Ions Through Dissociative Electron Transfer in Ternary Metal-Ligand-Peptide Complexes

    SciTech Connect

    Chu, Ivan K.; Laskin, Julia

    2011-12-31

    The formation and fragmentation of odd-electron ions of peptides and proteins is of interest to applications in biological mass spectrometry. Gas-phase redox chemistry occurring during collision-induced dissociation of ternary metal-ligand-peptide complexes enables the formation of a variety of peptide radicals including the canonical radical cations, M{sup +{sm_bullet}}, radical dications, [M{sup +}H]{sup 2+{sm_bullet}}, radical anions, [M-2H]{sup -{sm_bullet}}. In addition, odd-electron peptide ions with well-defined initial location of the radical site are produced through side chain losses from the radical ions. Subsequent fragmentation of these species provides information on the role of charge and the location of the radical site on the competition between radical-induced and proton-driven fragmentation of odd-electron peptide ions. This account summarizes current understanding of the factors that control the efficiency of the intramolecular electron transfer (ET) in ternary metal-ligand-peptide complexes resulting in formation of odd-electron peptide ions. Specifically, we discuss the effect of the metal center, the ligand and the peptide structure on the competition between the ET, proton transfer (PT), and loss of neutral peptide and neutral peptide fragments from the complex. Fundamental studies of the structures, stabilities, and the energetics and dynamics of fragmentation of such complexes are also important for detailed molecular-level understanding of photosynthesis and respiration in biological systems.

  16. Gold Ion-Angiotensin Peptide Interaction by Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Lee, Jenny; Jayathilaka, Lasanthi P.; Gupta, Shalini; Huang, Jin-Sheng; Lee, Bao-Shiang

    2012-05-01

    Stimulated by the interest in developing gold compounds for treating cancer, gold ion-angiotensin peptide interactions are investigated by mass spectrometry. Under the experimental conditions used, the majority of gold ion-angiotensin peptide complexes contain gold in the oxidation states I and III. Both ESI-MS and MALDI-TOF MS detect singly/multiply charged ions for mononuclear/multinuclear gold-attached peptides, which are represented as [peptide + a Au(I) + b Au(III) + (e - a -3b) H]e+, where a,b ≥ 0 and e is charge. ESI-MS data shows singly/multiply charged ions of Au(I)-peptide and Au(III)-peptide complexes. This study reveals that MALDI-TOF MS mainly detects singly charged Au(I)-peptide complexes, presumably due to the ionization process. The electrons in the MALDI plume seem to efficiently reduce Au(III) to Au(I). MALDI also tends to enhance the higher polymeric forms of gold-peptide complexes regardless of the laser power used. Collision-induced dissociation experiments of the mononuclear and dinuclear gold-attached peptide ions for angiotensin peptides show that the gold ion (a soft acid) binding sites are in the vicinity of Cys (a soft ligand), His (a major anchor of peptide for metal ion chelation), and the basic residue Arg. Data also suggests that the abundance of gold-attached peptides increases with higher gold concentration until saturation, after which an increase in gold ion concentration leads to the aggregation and/or precipitation of gold-bound peptides.

  17. Phage-displayed peptide library screening for preferred human substrate peptide sequences for transglutaminase 7.

    PubMed

    Kuramoto, Katsuma; Yamasaki, Risa; Shimizu, Yoshitaka; Tatsukawa, Hideki; Hitomi, Kiyotaka

    2013-09-01

    Transglutaminases are a family of enzymes that catalyze cross-linking reactions between proteins. Among the members, there is currently no information regarding the substrate preferences of transglutaminase 7 (TG7), that would clarify its physiological significance. We previously obtained several highly reactive substrate peptide sequences of transglutaminases from a random peptide library. In this study, we screened for preferred substrate sequences for TG7 from a phage-displayed 12-mer peptide library. The most preferred sequence was selected based on reactivity and isozyme specificity. We firstly exhibited the tendency for the preference of substrate sequence for TG7. Then, using the most efficient peptide, Z3S, we established an in vitro assay system to assess enzymatic activity of TG7.

  18. Postdiffusion of oligo-peptide within exponential growth multilayer films for localized peptide delivery.

    PubMed

    Wang, Xuefei; Ji, Jian

    2009-10-06

    efficient than the equivalent amount of free TAT peptide in the TAT uptake test. The postdiffusion of oligo-peptide within an exponential growth multilayer can serve as an effective approach for localized and sustained peptide delivery.

  19. Cyclic Opioid Peptides.

    PubMed

    Remesic, Michael; Lee, Yeon Sun; Hruby, Victor J

    2016-01-01

    For decades the opioid receptors have been an attractive therapeutic target for the treatment of pain. Since the first discovery of enkephalin, approximately a dozen endogenous opioid peptides have been known to produce opioid activity and analgesia, but their therapeutics have been limited mainly due to low blood brain barrier penetration and poor resistance to proteolytic degradation. One versatile approach to overcome these drawbacks is the cyclization of linear peptides to cyclic peptides with constrained topographical structure. Compared to their linear parents, cyclic analogs exhibit better metabolic stability, lower offtarget toxicity, and improved bioavailability. Extensive structure-activity relationship studies have uncovered promising compounds for the treatment of pain as well as further elucidate structural elements required for selective opioid receptor activity. The benefits that come with employing cyclization can be further enhanced through the generation of polycyclic derivatives. Opioid ligands generally have a short peptide chain and thus the realm of polycyclic peptides has yet to be explored. In this review, a brief history of designing ligands for the opioid receptors, including classic linear and cyclic ligands, is discussed along with recent approaches and successes of cyclic peptide ligands for the receptors. Various scaffolds and approaches to improve bioavailability are elaborated and concluded with a discourse towards polycyclic peptides.

  20. Analytical model of peptide mass cluster centres with applications

    PubMed Central

    Wolski, Witold E; Farrow, Malcolm; Emde, Anne-Katrin; Lehrach, Hans; Lalowski, Maciej; Reinert, Knut

    2006-01-01

    Background The elemental composition of peptides results in formation of distinct, equidistantly spaced clusters across the mass range. The property of peptide mass clustering is used to calibrate peptide mass lists, to identify and remove non-peptide peaks and for data reduction. Results We developed an analytical model of the peptide mass cluster centres. Inputs to the model included, the amino acid frequencies in the sequence database, the average length of the proteins in the database, the cleavage specificity of the proteolytic enzyme used and the cleavage probability. We examined the accuracy of our model by comparing it with the model based on an in silico sequence database digest. To identify the crucial parameters we analysed how the cluster centre location depends on the inputs. The distance to the nearest cluster was used to calibrate mass spectrometric peptide peak-lists and to identify non-peptide peaks. Conclusion The model introduced here enables us to predict the location of the peptide mass cluster centres. It explains how the location of the cluster centres depends on the input parameters. Fast and efficient calibration and filtering of non-peptide peaks is achieved by a distance measure suggested by Wool and Smilansky. PMID:16995952

  1. Anti-antimicrobial Peptides

    PubMed Central

    Ryan, Lloyd; Lamarre, Baptiste; Diu, Ting; Ravi, Jascindra; Judge, Peter J.; Temple, Adam; Carr, Matthew; Cerasoli, Eleonora; Su, Bo; Jenkinson, Howard F.; Martyna, Glenn; Crain, Jason; Watts, Anthony; Ryadnov, Maxim G.

    2013-01-01

    Antimicrobial or host defense peptides are innate immune regulators found in all multicellular organisms. Many of them fold into membrane-bound α-helices and function by causing cell wall disruption in microorganisms. Herein we probe the possibility and functional implications of antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and de novo designed antagonists: anti-antimicrobial peptides. Using sequences from native helical families such as cathelicidins, cecropins, and magainins we demonstrate that designed antagonists can co-fold with antimicrobial peptides into functionally inert helical oligomers. The properties and function of the resulting assemblies were studied in solution, membrane environments, and in bacterial culture by a combination of chiroptical and solid-state NMR spectroscopies, microscopy, bioassays, and molecular dynamics simulations. The findings offer a molecular rationale for anti-antimicrobial responses with potential implications for antimicrobial resistance. PMID:23737519

  2. A viral peptide for intracellular delivery

    NASA Astrophysics Data System (ADS)

    Falanga, Annarita; Tarallo, Rossella; Cantisani, Marco; Della Pepa, Maria Elena; Galdiero, Massimiliano; Galdiero, Stefania

    2012-10-01

    Biological membranes represent a critical hindrance for administering active molecules which are often unable to reach their designated intracellular target sites. In order to overcome this barrier-like behavior not easily circumvented by many pharmacologically-active molecules, synthetic transporters have been exploited to promote cellular uptake. Linking or complexing therapeutic molecules to peptides that can translocate through the cellular membranes could enhance their internal delivery, and consequently, a higher amount of active compound would reach the site of action. Use of cell penetrating peptides (CPPs) is one of the most promising strategy to efficiently translocate macromolecules through the plasma membrane, and have attracted a lot of attention. New translocating peptides are continuously described and in the present review, we will focus on viral derived peptides, and in particular a peptide (gH625) derived from the herpes simplex virus type 1 (HSV-1) glycoprotein H (gH) that has proved to be a useful delivery vehicle due to its intrinsic properties of inducing membrane perturbation.

  3. Melanins from opioid peptides.

    PubMed

    Rosei, M A

    1996-12-01

    Opioid peptides and other Tyr-NH2-terminal peptides are substrates in vitro for mushroom and sepia tyrosine, giving rise to synthetic melanins retaining the peptide moiety (opiomelanins). The melanopeptides are characterized by a total solubility in hydrophylic solvents at neutral and basic pH. Opioid peptides (enkephalins, endorphins, and esorphins), if oxidized by tyrosinase in the presence of Dopa, are easily incorporated into Dopa-melanin, producing mixed-type pigments that can also be solubilized in hydrophylic solvents. Melanins derived from opioid peptides exhibit paramagnetism, as evidenced by an EPR spectrum identical to that of Dopa-melanin. However, the presence of the linked peptide chain is able to influence dramatically the electron transfer properties and the oxidizing behaviour of the melanopeptides, so that whereas Tyr-Gly-melanin appears to behave as Dopa-melanin, Enk-melanin does not exhibit any oxidizing activity. Opiomelanins are characterized by a peculiar UV-VIS spectrum; that is, by the presence of a distinct peak (330 nm) that disappears upon chemical treatment by acid hydrolysis. Opiomelanins are stable pigments at neutral and basic pH in the dark, whereas the addition of H2O2 leads to a 15% degradation. Under stimulated solar illumination, opiomelanins are more easily destroyed with respect to Dopa-melanin, with increasing degradation when exposed to increased hydrogen peroxide concentrations and more alkaline pH. Some speculations on the possible existence and role of opiomelanins have been outlined.

  4. Peptide Optical waveguides.

    PubMed

    Handelman, Amir; Apter, Boris; Shostak, Tamar; Rosenman, Gil

    2017-02-01

    Small-scale optical devices, designed and fabricated onto one dielectric substrate, create integrated optical chip like their microelectronic analogues. These photonic circuits, based on diverse physical phenomena such as light-matter interaction, propagation of electromagnetic waves in a thin dielectric material, nonlinear and electro-optical effects, allow transmission, distribution, modulation, and processing of optical signals in optical communication systems, chemical and biological sensors, and more. The key component of these optical circuits providing both optical processing and photonic interconnections is light waveguides. Optical confinement and transmitting of the optical waves inside the waveguide material are possible due to the higher refractive index of the waveguides in comparison with their surroundings. In this work, we propose a novel field of bionanophotonics based on a new concept of optical waveguiding in synthetic elongated peptide nanostructures composed of ordered peptide dipole biomolecules. New technology of controllable deposition of peptide optical waveguiding structures by nanofountain pen technique is developed. Experimental studies of refractive index, optical transparency, and linear and nonlinear waveguiding in out-of-plane and in-plane diphenylalanine peptide nanotubes have been conducted. Optical waveguiding phenomena in peptide structures are simulated by the finite difference time domain method. The advantages of this new class of bio-optical waveguides are high refractive index contrast, wide spectral range of optical transparency, large optical nonlinearity, and electro-optical effect, making them promising for new applications in integrated multifunctional photonic circuits. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  5. Diversity-Oriented Peptide Stapling: A Third Generation Copper-Catalysed Azide-Alkyne Cycloaddition Stapling and Functionalisation Strategy.

    PubMed

    Tran, Phuong Thu; Larsen, Christian Ørnbøl; Røndbjerg, Tobias; De Foresta, Martina; Kunze, Micha B A; Marek, Ales; Løper, Jacob Hartvig; Boyhus, Lotte-Emilie; Knuhtsen, Astrid; Lindorff-Larsen, Kresten; Pedersen, Daniel Sejer

    2017-01-20

    The introduction of macrocyclic constraints in peptides (peptide stapling) is an important tool within peptide medicinal chemistry for stabilising and pre-organising peptides in a desired conformation. In recent years, the copper-catalysed azide-alkyne cycloaddition (CuAAC) has emerged 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 incorporating two azide-modified amino acids with 1,3,5-triethynylbenzene efficiently provides (i, i+7)- and (i, i+9)-stapled peptides with a single free alkyne positioned on the staple, which can be further conjugated or dimerised. A unique feature of the present method is that it provides easy access to radiolabelled stapled peptides by catalytic tritiation of the alkyne positioned on the staple.

  6. Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis.

    PubMed

    Nguyen, Giang K T; Wang, Shujing; Qiu, Yibo; Hemu, Xinya; Lian, Yilong; Tam, James P

    2014-09-01

    Proteases are ubiquitous in nature, whereas naturally occurring peptide ligases, enzymes catalyzing the reverse reactions of proteases, are rare occurrences. Here we describe the discovery of butelase 1, to our knowledge the first asparagine/aspartate (Asx) peptide ligase to be reported. This highly efficient enzyme was isolated from Clitoria ternatea, a cyclic peptide-producing medicinal plant. Butelase 1 shares 71% sequence identity and the same catalytic triad with legumain proteases but does not hydrolyze the protease substrate of legumain. Instead, butelase 1 cyclizes various peptides of plant and animal origin with yields greater than 95%. With Kcat values of up to 17 s(-1) and catalytic efficiencies as high as 542,000 M(-1) s(-1), butelase 1 is the fastest peptide ligase known. Notably, butelase 1 also displays broad specificity for the N-terminal amino acids of the peptide substrate, thus providing a new tool for C terminus-specific intermolecular peptide ligations.

  7. The Use of Aryl Hydrazide Linkers for the Solid Phase Synthesis of Chemically Modified Peptides

    SciTech Connect

    Woo, Y; Mitchell, A R; Camarero, J A

    2006-11-03

    Since Merrifield introduced the concept of solid phase synthesis in 1963 for the rapid preparation of peptides, a large variety of different supports and resin-linkers have been developed that improve the efficiency of peptide assembly and expand the myriad of synthetically feasible peptides. The aryl hydrazide is one of the most useful resin-linkers for the synthesis of chemically modified peptides. This linker is completely stable during Boc- and Fmoc-based solid phase synthesis and yet it can be cleaved under very mild oxidative conditions. The present article reviews the use of this valuable linker for the rapid and efficient synthesis of C-terminal modified peptides, head-to-tail cyclic peptides and lipidated peptides.

  8. Methods for peptide identification by spectral comparison

    PubMed Central

    Liu, Jian; Bell, Alexander W; Bergeron, John JM; Yanofsky, Corey M; Carrillo, Brian; Beaudrie, Christian EH; Kearney, Robert E

    2007-01-01

    Background Tandem mass spectrometry followed by database search is currently the predominant technology for peptide sequencing in shotgun proteomics experiments. Most methods compare experimentally observed spectra to the theoretical spectra predicted from the sequences in protein databases. There is a growing interest, however, in comparing unknown experimental spectra to a library of previously identified spectra. This approach has the advantage of taking into account instrument-dependent factors and peptide-specific differences in fragmentation probabilities. It is also computationally more efficient for high-throughput proteomics studies. Results This paper investigates computational issues related to this spectral comparison approach. Different methods have been empirically evaluated over several large sets of spectra. First, we illustrate that the peak intensities follow a Poisson distribution. This implies that applying a square root transform will optimally stabilize the peak intensity variance. Our results show that the square root did indeed outperform other transforms, resulting in improved accuracy of spectral matching. Second, different measures of spectral similarity were compared, and the results illustrated that the correlation coefficient was most robust. Finally, we examine how to assemble multiple spectra associated with the same peptide to generate a synthetic reference spectrum. Ensemble averaging is shown to provide the best combination of accuracy and efficiency. Conclusion Our results demonstrate that when combined, these methods can boost the sensitivity and specificity of spectral comparison. Therefore they are capable of enhancing and complementing existing tools for consistent and accurate peptide identification. PMID:17227583

  9. CXCR4-antagonist Peptide R-liposomes for combined therapy against lung metastasis.

    PubMed

    Ieranò, Caterina; Portella, Luigi; Lusa, Sara; Salzano, Giuseppina; D'Alterio, Crescenzo; Napolitano, Maria; Buoncervello, Maria; Macchia, Daniele; Spada, Massimo; Barbieri, Antonio; Luciano, Antonio; Barone, Maria Vittoria; Gabriele, Lucia; Caraglia, Michele; Arra, Claudio; De Rosa, Giuseppe; Scala, Stefania

    2016-04-14

    The chemokine CXCL12 activates CXCR4, initiating multiple pathways that control immune cell trafficking, angiogenesis and embryogenesis; CXCR4 is also overexpressed in multiple tumors affecting metastatic dissemination. While there has been great enthusiasm for exploiting the CXCR4-CXCL12 axis as a target in cancer therapy, to date the promise has yet to be fulfilled. A new class of CXCR4-antagonist cyclic peptides was recently developed and the compound named Peptide R was identified as the most active. With the intent to improve the efficacy and biodistribution of Peptide R, stealth liposomes decorated with Peptide R were developed (PL-Peptide R). In vitro PL-Peptide R efficiently inhibited CXCR4-dependent migration and in vivo it significantly reduced lung metastases and increased overall survival in B16-CXCR4 injected C57BL/6 mice. To evaluate if PL-Peptide R could also be a drug delivery system for CXCR4 expressing tumors, the PL-Peptide R was loaded with doxorubicin (DOX) (PL-Peptide R-DOX). PL-Peptide R-DOX efficiently delivered DOX to CXCR4 expressing cell lines with a consequent decrease in the DOX IC50 efficient dose. In vivo, B16-CXCR4 injected C57BL/6 mice treated with PL-Peptide R-DOX developed fewer lung metastases compared to PL-DOX treated mice. This work provides the proof-of-concept to prevent metastasis by using combined nanomedicine.

  10. Macrocyclic peptides self-assemble into robust vesicles with molecular recognition capabilities.

    PubMed

    Jeong, Woo-jin; Lim, Yong-beom

    2014-11-19

    In this study, we developed macrocyclic peptide building blocks that formed self-assembled peptide vesicles with molecular recognition capabilities. Macrocyclic peptides were significantly different from conventional amphiphiles, in that they could self-assemble into vesicles at very high hydrophilic-to-total mass ratios. The flexibility of the hydrophobic self-assembly segment was critical for vesicle formation. The unique features of this peptide vesicle system include a homogeneous size distribution, unusually small size, and robust structural and thermal stability. The peptide vesicles successfully entrapped a hydrophilic model drug, released the payload very slowly, and were internalized by cells in a highly efficient manner. Moreover, the peptide vesicles exhibited molecular recognition capabilities, in that they selectively bound to target RNA through surface-displayed peptides. This study demonstrates that self-assembled peptide vesicles can be used as strong intracellular delivery vehicles that recognize specific biomacromolecular targets.

  11. Chemical posttranslational modification of phage-displayed peptides.

    PubMed

    Ng, Simon; Tjhung, Katrina F; Paschal, Beth M; Noren, Christopher J; Derda, Ratmir

    2015-01-01

    Phage-displayed peptide library has fueled the discovery of novel ligands for diverse targets. A new type of phage libraries that displays not only linear and disulfide-constrained cyclic peptides but moieties that cannot be encoded genetically or incorporated easily by bacterial genetic machinery has emerged recently. Chemical posttranslational modification of phage library is one of the simplest approaches to encode nonnatural moieties. It confers the library with new functionality and makes it possible to select and evolve molecules with properties not found in the peptides, for instance, glycopeptides recognized by carbohydrate-binding protein and peptides with photoswitching capability. To this end, we describe the newly emerging techniques to chemically modify the phage library and quantify the efficiency of the reaction with a biotin-capture assay. Finally, we provide the methods to construct N-terminal Ser peptide library that allows site-selective modification of phage.

  12. Peptide synthesis in neat organic solvents with novel thermostable proteases.

    PubMed

    Toplak, Ana; Nuijens, Timo; Quaedflieg, Peter J L M; Wu, Bian; Janssen, Dick B

    2015-06-01

    Biocatalytic peptide synthesis will benefit from enzymes that are active at low water levels in organic solvent compositions that allow good substrate and product solubility. To explore the use of proteases from thermophiles for peptide synthesis under such conditions, putative protease genes of the subtilase class were cloned from Thermus aquaticus and Deinococcus geothermalis and expressed in Escherichia coli. The purified enzymes were highly thermostable and catalyzed efficient peptide bond synthesis at 80°C and 60°C in neat acetonitrile with excellent conversion (>90%). The enzymes tolerated high levels of N,N-dimethylformamide (DMF) as a cosolvent (40-50% v/v), which improved substrate solubility and gave good conversion in 5+3 peptide condensation reactions. The results suggest that proteases from thermophiles can be used for peptide synthesis under harsh reaction conditions.

  13. Low-Cost Peptide Microarrays for Mapping Continuous Antibody Epitopes.

    PubMed

    McBride, Ryan; Head, Steven R; Ordoukhanian, Phillip; Law, Mansun

    2016-01-01

    With the increasing need for understanding antibody specificity in antibody and vaccine research, pepscan assays provide a rapid method for mapping and profiling antibody responses to continuous epitopes. We have developed a relatively low-cost method to generate peptide microarray slides for studying antibody binding. Using a setup of an IntavisAG MultiPep RS peptide synthesizer, a Digilab MicroGrid II 600 microarray printer robot, and an InnoScan 1100 AL scanner, the method allows the interrogation of up to 1536 overlapping, alanine-scanning, and mutant peptides derived from the target antigens. Each peptide is tagged with a polyethylene glycol aminooxy terminus to improve peptide solubility, orientation, and conjugation efficiency to the slide surface.

  14. Peptide specific expansion of CD8(+) T cells by recombinant plate bound MHC/peptide complexes.

    PubMed

    Schmidt, Esben G W; Buus, Søren; Thorn, Mette; Stryhn, Anette; Leisner, Christian; Claesson, Mogens H

    2009-01-01

    Development of methods for efficient in vitro stimulation and expansion of peptide specific CD8(+) T cells is compelling not only with respect to adoptive T cell therapy but also regarding analysis of T cell responses and search for new immunogenic peptides. In the present study, a new approach to in vitro T cell stimulation was investigated. By use of an antigenic peptide derived from the cytomegalovirus (CMVp) we tested the stimulatory efficacy of recombinant plate bound MHC molecules (PB-MHC), being immobilized in culture plates. A single stimulation of non-adherent peripheral blood mononuclear cells (NA-PBMCs) with PB-MHC/CMVp resulted in significant expansion of CMVp specific CD8(+) T cells, which was comparable to that achieved by CMVp pulsed mature dendritic cells (DCs). By repeated exposure of NA-PBMCs to PB-MHC/CMVp more than 60% CMVp specific CD8(+) T cells, representing a 240-fold expansion, were reached after only two stimulations. Although stimulation with PB-MHC/CMVp clearly demonstrated efficient peptide specific expansion of CD8(+) T cells, there was a tendency to proliferative exhaustion of the cells after 3-4 stimulations. Thus, it will be of interest to examine the effect of new stimulatory cocktails, e.g. cytokines and co-stimulatory molecules, by use of the present rapid and easy-to-use method of expanding peptide specific T cells.

  15. Antimicrobial Peptides from Plants

    PubMed Central

    Tam, James P.; Wang, Shujing; Wong, Ka H.; Tan, Wei Liang

    2015-01-01

    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. PMID:26580629

  16. Cyclization in opioid peptides.

    PubMed

    Piekielna, Justyna; Perlikowska, Renata; Gach, Katarzyna; Janecka, Anna

    2013-06-01

    Endogenous opioid peptides have been studied extensively as potential therapeutics for the treatment of pain. The major problems of using natural opioid peptides as drug candidates are their poor receptor specificity, metabolic instability and inability to reach the brain after systemic administration. A lot of synthetic efforts have been made to opioid analogs with improved pharmacological properties. One important structural modification leading to such analogs is cyclization of linear sequences. Intramolecular cyclization has been shown to improve biological properties of various bioactive peptides. Cyclization reduces conformational freedom responsible for the simultaneous activation of two or more receptors, increases metabolic stability and lipophilicity which may result in a longer half-life and easier penetration across biological membranes. This review deals with various strategies that have been employed to synthesize cyclic analogs of opioid peptides. Discussed are such bridging bonds as amide and amine linkages, sulfur-containing bonds, including monosulfide, disulfide and dithioether bridges, bismethylene bonds, monosulfide bridges of lanthionine and, finally, carbonyl and guanidine linkages. Opioid affinities and activities of cyclic analogs are given and compared with linear opioid peptides. Analgesic activities of analogs evaluated in the in vivo pain tests are also discussed.

  17. Cullin3 - BTB Interface: A Novel Target for Stapled Peptides

    PubMed Central

    Palmieri, Maddalena; Balasco, Nicole; Esposito, Luciana; Russo, Luigi; Mazzà, Daniela; Di Marcotullio, Lucia; Di Gaetano, Sonia; Malgieri, Gaetano; Vitagliano, Luigi; Pedone, Emilia; Zaccaro, Laura

    2015-01-01

    Cullin3 (Cul3), a key factor of protein ubiquitination, is able to interact with dozens of different proteins containing a BTB (Bric-a-brac, Tramtrack and Broad Complex) domain. We here targeted the Cul3–BTB interface by using the intriguing approach of stabilizing the α-helical conformation of Cul3-based peptides through the “stapling” with a hydrocarbon cross-linker. In particular, by combining theoretical and experimental techniques, we designed and characterized stapled Cul3-based peptides embedding the helix 2 of the protein (residues 49–68). Intriguingly, CD and NMR experiments demonstrate that these stapled peptides were able to adopt the helical structure that the fragment assumes in the parent protein. We also show that some of these peptides were able to bind to the BTB of the tetrameric KCTD11, a substrate adaptor involved in HDAC1 degradation, with high affinity (~ 300–600 nM). Cul3-derived staple peptides are also able to bind the BTB of the pentameric KCTD5. Interestingly, the affinity of these peptides is of the same order of magnitude of that reported for the interaction of full-length Cul3 with some BTB containing proteins. Moreover, present data indicate that stapling endows these peptides with an increased serum stability. Altogether, these findings indicate that the designed stapled peptides can efficiently mimic protein-protein interactions and are potentially able to modulate fundamental biological processes involving Cul3. PMID:25848797

  18. A Highly Scalable Peptide-Based Assay System for Proteomics

    PubMed Central

    Kozlov, Igor A.; Thomsen, Elliot R.; Munchel, Sarah E.; Villegas, Patricia; Capek, Petr; Gower, Austin J.; K. Pond, Stephanie J.; Chudin, Eugene; Chee, Mark S.

    2012-01-01

    We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays. PMID:22701568

  19. Cullin3-BTB interface: a novel target for stapled peptides.

    PubMed

    de Paola, Ivan; Pirone, Luciano; Palmieri, Maddalena; Balasco, Nicole; Esposito, Luciana; Russo, Luigi; Mazzà, Daniela; Di Marcotullio, Lucia; Di Gaetano, Sonia; Malgieri, Gaetano; Vitagliano, Luigi; Pedone, Emilia; Zaccaro, Laura

    2015-01-01

    Cullin3 (Cul3), a key factor of protein ubiquitination, is able to interact with dozens of different proteins containing a BTB (Bric-a-brac, Tramtrack and Broad Complex) domain. We here targeted the Cul3-BTB interface by using the intriguing approach of stabilizing the α-helical conformation of Cul3-based peptides through the "stapling" with a hydrocarbon cross-linker. In particular, by combining theoretical and experimental techniques, we designed and characterized stapled Cul3-based peptides embedding the helix 2 of the protein (residues 49-68). Intriguingly, CD and NMR experiments demonstrate that these stapled peptides were able to adopt the helical structure that the fragment assumes in the parent protein. We also show that some of these peptides were able to bind to the BTB of the tetrameric KCTD11, a substrate adaptor involved in HDAC1 degradation, with high affinity (~ 300-600 nM). Cul3-derived staple peptides are also able to bind the BTB of the pentameric KCTD5. Interestingly, the affinity of these peptides is of the same order of magnitude of that reported for the interaction of full-length Cul3 with some BTB containing proteins. Moreover, present data indicate that stapling endows these peptides with an increased serum stability. Altogether, these findings indicate that the designed stapled peptides can efficiently mimic protein-protein interactions and are potentially able to modulate fundamental biological processes involving Cul3.

  20. Phage Selection of Chemically Stabilized α-Helical Peptide Ligands.

    PubMed

    Diderich, Philippe; Bertoldo, Davide; Dessen, Pierre; Khan, Maola M; Pizzitola, Irene; Held, Werner; Huelsken, Joerg; Heinis, Christian

    2016-05-20

    Short α-helical peptides stabilized by linkages between constituent amino acids offer an attractive format for ligand development. In recent years, a range of excellent ligands based on stabilized α-helices were generated by rational design using α-helical peptides of natural proteins as templates. Herein, we developed a method to engineer chemically stabilized α-helical ligands in a combinatorial fashion. In brief, peptides containing cysteines in position i and i + 4 are genetically encoded by phage display, the cysteines are modified with chemical bridges to impose α-helical conformations, and binders are isolated by affinity selection. We applied the strategy to affinity mature an α-helical peptide binding β-catenin. We succeeded in developing ligands with Kd's as low as 5.2 nM, having >200-fold improved affinity. The strategy is generally applicable for affinity maturation of any α-helical peptide. Compared to hydrocarbon stapled peptides, the herein evolved thioether-bridged peptide ligands can be synthesized more easily, as no unnatural amino acids are required and the cyclization reaction is more efficient and yields no stereoisomers. A further advantage of the thioether-bridged peptide ligands is that they can be expressed recombinantly as fusion proteins.

  1. Detection of selective antibacterial peptides by the Polarity Profile method.

    PubMed

    Polanco, Carlos; Buhse, Thomas; Samaniego, José Lino; Castañón-González, Jorge Alberto

    2013-01-01

    Antimicrobial peptides occupy a prominent place in the production of pharmaceuticals, because of their effective contribution to the protection of the immune system against almost all types of pathogens. These peptides are thoroughly studied by computational methods designed to shed light on their main functions. In this paper, we propose a computational approach, named the Polarity Profile method that represents an improvement to the former Polarity Index method. The Polarity Profile method is very effective in detecting the subgroup of antibacterial peptides called selective cationic amphipathic antibacterial peptides (SCAAP) that show high toxicity towards bacterial membranes and exhibit almost zero toxicity towards mammalian cells. Our study was restricted to the peptides listed in the antimicrobial peptides database (APD2) of December 19, 2012. Performance of the Polarity Profile method is demonstrated through a comparison to the former Polarity Index method by using the same sets of peptides. The efficiency of the Polarity Profile method exceeds 85% taking into account the false positive and/or false negative peptides.

  2. Antimicrobial peptides in the centipede Scolopendra subspinipes mutilans.

    PubMed

    Yoo, Won Gi; Lee, Joon Ha; Shin, Younhee; Shim, Jae-Young; Jung, Myunghee; Kang, Byeong-Chul; Oh, Jaedon; Seong, Jiyeon; Lee, Hak Kyo; Kong, Hong Sik; Song, Ki-Duk; Yun, Eun-Young; Kim, In-Woo; Kwon, Young-Nam; Lee, Dong Gun; Hwang, Ui-Wook; Park, Junhyung; Hwang, Jae Sam

    2014-06-01

    The centipede Scolopendra subspinipes mutilans is an environmentally beneficial and medically important arthropod species. Although this species is increasingly applied as a reliable source of new antimicrobial peptides, the transcriptome of this species is a prerequisite for more rational selection of antimicrobial peptides. In this report, we isolated total RNA from the whole body of adult centipedes, S. subspinipes mutilans, that were nonimmunized and immunized against Escherichia coli, and we generated a total of 77,063 pooled contigs and singletons using high-throughput sequencing. To screen putative antimicrobial peptides, in silico analyses of the S. subspinipes mutilans transcriptome were performed based on the physicochemical evidence of length, charge, isoelectric point, and in vitro and in vivo aggregation scores together with the existence of continuous antimicrobial peptide stretches. Moreover, we excluded some transcripts that showed similarity with both previously known antimicrobial peptides and the human proteome, had a proteolytic cleavage site, and had downregulated expression compared with the nonimmunized sample. As a result, we selected 17 transcripts and tested their antimicrobial activity with a radial diffusion assay. Among them, ten synthetic peptides experimentally showed antimicrobial activity against microbes and no toxicity to mouse erythrocytes. Our results provide not only a useful set of antimicrobial peptide candidates and an efficient strategy for novel antimicrobial peptide development but also the transcriptome data of a big centipede as a valuable resource.

  3. Formyl peptide receptor chimeras define domains involved in ligand binding.

    PubMed

    Perez, H D; Holmes, R; Vilander, L R; Adams, R R; Manzana, W; Jolley, D; Andrews, W H

    1993-02-05

    We have begun to study the structural requirements for the binding of formyl peptides to their specific receptors. As an initial approach, we constructed C5a-formyl peptide receptor chimeras. Unique (and identical) restriction sites were introduced within the transmembrane domains of these receptors that allowed for the exchange of specific areas. Four types of chimeric receptors were generated. 1) The C5a receptor was progressively substituted by the formyl peptide receptor. 2) The formyl peptide receptor was progressively substituted by the C5a receptor. 3) Specific domains of the C5a receptor were substituted by the corresponding domain of the formyl peptide receptor. 4) Specific domains of the formyl peptide receptor were replaced by the same corresponding domain of the C5a receptor. Wild type and chimeric receptors were transfected into COS 7 cells and their ability to bind formyl peptide determined, taking into account efficiency of transfection and expression of chimeric protein. Based on these results, a ligand binding model is presented in which the second, third, and fourth extracellular (and/or their transmembrane) domains together with the first transmembrane domain form a ligand binding pocket for formyl peptides. It is proposed that the amino-terminal domain plays a role by presumably providing a "lid" to the pocket. The carboxyl-terminal cytoplasmic tail appears to modulate ligand binding by regulating receptor affinity.

  4. Peptides in oral diseases.

    PubMed

    Lucchese, Alberta; Guida, Agostino; Petruzzi, Massimo; Capone, Giovanni; Laino, Luigi; Serpico, Rosario

    2012-01-01

    The oral cavity is home to numerous viruses and micro-organisms recognized as having a role in various oral diseases as well as in infections in other parts of the body. Indeed, in general a microbial infection underlies or is believed to underlie the ample spectrum of oral diseases, from tooth enamel decay to periodontal lesions, from candidiasis to virus-induced oral squamous cell carcinomas, and bullous autoimmune oral disorders. This clinico-pathological context stresses the need of targeted therapies to specifically kill infectious agents in a complex environment such as the oral cavity, and explains the current interest in exploring peptide-based therapeutic approaches in oral and dental research. Here, we review the therapeutic potential of antimicrobial peptides such as LL-37, beta defensins, adrenomedullin, histatins, and of various peptides modulating gene expression and immuno-biological interaction(s) in oral diseases.

  5. Molecular modeling of peptides.

    PubMed

    Kuczera, Krzysztof

    2015-01-01

    This article presents a review of the field of molecular modeling of peptides. The main focus is on atomistic modeling with molecular mechanics potentials. The description of peptide conformations and solvation through potentials is discussed. Several important computer simulation methods are briefly introduced, including molecular dynamics, accelerated sampling approaches such as replica-exchange and metadynamics, free energy simulations and kinetic network models like Milestoning. Examples of recent applications for predictions of structure, kinetics, and interactions of peptides with complex environments are described. The reliability of current simulation methods is analyzed by comparison of computational predictions obtained using different models with each other and with experimental data. A brief discussion of coarse-grained modeling and future directions is also presented.

  6. Monolithic capillary columns based on pentaerythritol tetraacrylate for peptide analysis

    NASA Astrophysics Data System (ADS)

    Kucherenko, E. V.; Melnik, D. M.; Korolev, A. A.; Kanateva, A. Yu.; Pirogov, A. V.; Kurganov, A. A.

    2015-09-01

    Monolythic medium-polar capillary columns based on pentaerythritol tetraacrylate were optimized for separation of peptides. The synthesis temperature and time, the fraction of monomer in the initial polymerization mixture, and the nature of alcohol contained in the complex porogen were chosen as optimization parameters. The highest efficiency was attained for columns obtained with 33 and 34% monomer at a polymerization time of 75 min and a temperature of 75°C. The columns with the optimum structure were effective in separation of a model mixture of five peptides. The sensitivity of the method was 200 ng of peptide per column.

  7. Peptide promiscuity: an evolutionary concept for plant defense.

    PubMed

    Franco, Octavio Luiz

    2011-04-06

    The phenomenon of protein promiscuity, in which multiple functions are associated with a single peptide structure, has gained attention in several research fields, including the plant defense field. With this in mind, this report intends to link various plant defense peptides with common scaffolds (defensins, cyclotides and 2S albumins), and multiple activities with the processes of promiscuity generation and protein evolvability. This link seems to create an efficient system of plant defense against insect pests and pathogens, and is thus essential to plant survival and evolution. This review also identifies future possibilities for the use of peptide promiscuity in designing novel drugs and synthetic biotechnological products.

  8. Peptides with Dual Antimicrobial and Anticancer Activities

    PubMed Central

    Felício, Mário R.; Silva, Osmar N.; Gonçalves, Sônia; Santos, Nuno C.; Franco, Octávio L.

    2017-01-01

    In recent years, the number of people suffering from cancer and multi-resistant infections has increased, such that both diseases are already seen as current and future major causes of death. Moreover, chronic infections are one of the main causes of cancer, due to the instability in the immune system that allows cancer cells to proliferate. Likewise, the physical debility associated with cancer or with anticancer therapy itself often paves the way for opportunistic infections. It is urgent to develop new therapeutic methods, with higher efficiency and lower side effects. Antimicrobial peptides (AMPs) are found in the innate immune system of a wide range of organisms. Identified as the most promising alternative to conventional molecules used nowadays against infections, some of them have been shown to have dual activity, both as antimicrobial and anticancer peptides (ACPs). Highly cationic and amphipathic, they have demonstrated efficacy against both conditions, with the number of nature-driven or synthetically designed peptides increasing year by year. With similar properties, AMPs that can also act as ACPs are viewed as future chemotherapeutic drugs, with the advantage of low propensity to resistance, which started this paradigm in the pharmaceutical market. These peptides have already been described as molecules presenting killing mechanisms at the membrane level, but also acting toward intracellular targets, which increases their success compartively to one-target specific drugs. This review will approach the desirable characteristics of small peptides that demonstrated dual activity against microbial infections and cancer, as well as the peptides engaged in clinical trials. PMID:28271058

  9. Novel peptides functionally targeting in vivo human lung cancer discovered by in vivo peptide displayed phage screening.

    PubMed

    Lee, Kyoung Jin; Lee, Jae Hee; Chung, Hye Kyung; Choi, Jinhyang; Park, Jaesook; Park, Seok Soon; Ju, Eun Jin; Park, Jin; Shin, Seol Hwa; Park, Hye Ji; Ko, Eun Jung; Suh, Nayoung; Kim, InKi; Hwang, Jung Jin; Song, Si Yeol; Jeong, Seong-Yun; Choi, Eun Kyung

    2015-02-01

    Discovery of the cancer-specific peptidic ligands have been emphasized for active targeting drug delivery system and non-invasive imaging. For the discovery of useful and applicable peptidic ligands, in vivo peptide-displayed phage screening has been performed in this study using a xenograft mouse model as a mimic microenvironment to tumor. To seek human lung cancer-specific peptides, M13 phage library displaying 2.9 × 10(9) random peptides was intravenously injected into mouse model bearing A549-derived xenograft tumor through the tail vein. Then the phages emerged from a course of four rounds of biopanning in the xenograft tumor tissue. Novel peptides were categorized into four groups according to a sequence-homology phylogenicity, and in vivo tumor-targeting capacity of these peptides was validated by whole body imaging with Cy5.5-labeled phages in various cancer types. The result revealed that novel peptides accumulated only in adenocarcinoma lung cancer cell-derived xenograft tissue. For further confirmation of the specific targeting ability, in vitro cell-binding assay and immunohistochemistry in vivo tumor tissue were performed with a selected peptide. The peptide was found to bind intensely to lung cancer cells both in vitro and in vivo, which was efficiently compromised with unlabeled phages in an in vitro competition assay. In conclusion, the peptides specifically targeting human lung cancer were discovered in this study, which is warranted to provide substantive feasibilities for drug delivery and imaging in terms of a novel targeted therapeutics and diagnostics.

  10. Epitope peptides and immunotherapy.

    PubMed

    Tanabe, Soichi

    2007-02-01

    Allergic diseases affect atopic individuals, who synthesize specific Immunoglobulins E (IgE) to environmental allergens, usually proteins or glycoproteins. These allergens include grass and tree pollens, indoor allergens such as house dust mites and animal dander, and various foods. Because allergen-specific IgE antibodies are the main effector molecules in the immune response to allergens, many studies have focused on the identification of IgE-binding epitopes (called B cell epitopes), specific and minimum regions of allergen molecules that binds to IgE. Our initial studies have provided evidence that only four to five amino acid residues are enough to comprise an epitope, since pentapeptide QQQPP in wheat glutenin is minimally required for IgE binding. Afterwards, various kinds of B cell epitope structures have been clarified. Such information contributes greatly not only to the elucidation of the etiology of allergy, but also to the development of strategies for the treatment and prevention of allergy. Allergen-specific T cells also play an important role in allergy and are obvious targets for intervention in the disease. Currently, the principle approach is to modify B cell epitopes to prevent IgE binding while preserving T cell epitopes to retain the capacity for immunotherapy. There is mounting evidence that the administration of peptide(s) containing immunodominant T cell epitopes from an allergen can induce T cell nonresponsiveness (immunotherapy). There have been clinical studies of peptide immunotherapy performed, the most promising being for bee venom sensitivity. Clinical trials of immunotherapy for cat allergen peptide have also received attention. An alternative strategy for the generation of an effective but hypoallergenic preparation for immunotherapy is to modify T cell epitope peptides by, for example, single amino acid substitution. In this article, I will present an overview of epitopes related to allergic disease, particularly stress on

  11. Quantitative evaluation of peptide-extraction methods by HPLC-triple-quad MS-MS.

    PubMed

    Du, Yan; Wu, Dapeng; Wu, Qian; Guan, Yafeng

    2015-02-01

    In this study, the efficiency of five peptide-extraction methods—acetonitrile (ACN) precipitation, ultrafiltration, C18 solid-phase extraction (SPE), dispersed SPE with mesoporous carbon CMK-3, and mesoporous silica MCM-41—was quantitatively investigated. With 28 tryptic peptides as target analytes, these methods were evaluated on the basis of recovery and reproducibility by using high-performance liquid chromatography-triple-quad tandem mass spectrometry in selected-reaction-monitoring mode. Because of the distinct extraction mechanisms of the methods, their preferences for extracting peptides of different properties were revealed to be quite different, usually depending on the pI values or hydrophobicity of peptides. When target peptides were spiked in bovine serum albumin (BSA) solution, the extraction efficiency of all the methods except ACN precipitation changed significantly. The binding of BSA with target peptides and nonspecific adsorption on adsorbents were believed to be the ways through which BSA affected the extraction behavior. When spiked in plasma, the performance of all five methods deteriorated substantially, with the number of peptides having recoveries exceeding 70% being 15 for ACN precipitation, and none for the other methods. Finally, the methods were evaluated in terms of the number of identified peptides for extraction of endogenous plasma peptides. Only ultrafiltration and CMK-3 dispersed SPE performed differently from the quantitative results with target peptides, and the wider distribution of the properties of endogenous peptides was believed to be the main reason.

  12. Enthalpy-driven interactions with sulfated glycosaminoglycans promote cell membrane penetration of arginine peptides.

    PubMed

    Takechi-Haraya, Yuki; Nadai, Ryo; Kimura, Hitoshi; Nishitsuji, Kazuchika; Uchimura, Kenji; Sakai-Kato, Kumiko; Kawakami, Kohsaku; Shigenaga, Akira; Kawakami, Toru; Otaka, Akira; Hojo, Hironobu; Sakashita, Naomi; Saito, Hiroyuki

    2016-06-01

    The first step of cell membrane penetration of arginine peptides is thought to occur via electrostatic interactions between positive charges of arginine residues and negative charges of sulfated glycosaminoglycans (GAGs) on the cell surface. However, the molecular interaction of arginine peptides with GAG still remains unclear. Here, we compared the interactions of several arginine peptides of Tat, R8, and Rev and their analogues with heparin in relation to the cell membrane penetration efficiency. The high-affinity binding of arginine peptides to heparin was shown to be driven by large favorable enthalpy contributions, possibly reflecting multidentate hydrogen bondings of arginine residues with sulfate groups of heparin. Interestingly, the lysine peptides in which all arginine residues are substituted with lysine residues exhibited negligible binding enthalpy despite of their considerable binding to heparin. In CHO-K1 cells, arginine peptides exhibited a great cell-penetrating ability whereas their corresponding lysine peptides did not penetrate into cells. The degree of cell penetration of arginine peptides markedly decreased by the chlorate treatment of cells which prevents the sulfation of GAG chains. Significantly, the cell penetration efficiency of arginine peptides was found to be correlated with the favorable enthalpy of binding to heparin. These results suggest that the enthalpy-driven strong interaction with sulfated GAGs such as heparan sulfate plays a critical role in the efficient cell membrane penetration of arginine peptides.

  13. Related impurities in peptide medicines.

    PubMed

    D'Hondt, Matthias; Bracke, Nathalie; Taevernier, Lien; Gevaert, Bert; Verbeke, Frederick; Wynendaele, Evelien; De Spiegeleer, Bart

    2014-12-01

    Peptides are an increasingly important group of pharmaceuticals, positioned between classic small organic molecules and larger bio-molecules such as proteins. Currently, the peptide drug market is growing twice as fast as other drug markets, illustrating the increasing clinical as well as economical impact of this medicine group. Most peptides today are manufactured by solid-phase peptide synthesis (SPPS). This review will provide a structured overview of the most commonly observed peptide-related impurities in peptide medicines, encompassing the active pharmaceutical ingredients (API or drug substance) as well as the finished drug products. Not only is control of these peptide-related impurities and degradants critical for the already approved and clinically used peptide-drugs, these impurities also possess the capability of greatly influencing initial functionality studies during early drug discovery phases, possibly resulting in erroneous conclusions. The first group of peptide-related impurities is SPPS-related: deletion and insertion of amino acids are related to inefficient Fmoc-deprotection and excess use of amino acid reagents, respectively. Fmoc-deprotection can cause racemization of amino acid residues and thus diastereomeric impurities. Inefficient deprotection of amino acid side chains results into peptide-protection adducts. Furthermore, unprotected side chains can react with a variety of reagents used in the synthesis. Oxidation of amino acid side chains and dimeric-to-oligomeric impurities were also observed. Unwanted peptide counter ions such as trifluoroacetate, originating from the SPPS itself or from additional purification treatments, may also be present in the final peptide product. Contamination of the desired peptide product by other unrelated peptides was also seen, pointing out the lack of appropriate GMP. The second impurity group results from typical peptide degradation mechanisms such as β-elimination, diketopiperazine, pyroglutamate

  14. Peptide -- Silica Hybrid Networks

    NASA Astrophysics Data System (ADS)

    Altunbas, Aysegul; Sharma, Nikhil; Nagarkar, Radhika; Schneider, Joel; Pochan, Darrin

    2010-03-01

    In this study, a bio-inspired route was used to fabricate scaffolds that display hierarchical organization of an inorganic layer around an organic self-assembled peptide fibril template. The 20 amino acid peptide used in this study intramolecular folds into a beta-hairpin conformation on addition of a desired solution stimulus. This intramolecular folding is followed by intermolecular self-assembly of the peptides into a three dimensional network of entangled fibrils rich in beta-sheet with a high density of lysine groups exposed on the fibril-surfaces. The lysine-rich surface chemistry was utilized to create a silica shell around the fibrils. The mineralization process of the fibrils results in a rigid, porous silica network that retains the microscale and nanoscale structure of the peptide fibril network. Structural characterization via Transmission Electron Microscopy, cryogenic-Scanning Electron Microscopy, mechanical characterization via oscillatory rheology, Small Angle X-ray and Neutron Scattering of the silicified hydrogels will be presented.

  15. Brain Peptides and Psychopharmacology

    ERIC Educational Resources Information Center

    Arehart-Treichel, Joan

    1976-01-01

    Proteins isolated from the brain and used as drugs can improve and apparently even transfer mental states and behavior. Much of the pioneering work and recent research with humans and animals is reviewed and crucial questions that are being posed about the psychologically active peptides are related. (BT)

  16. Peptide Nanofilament Engineering

    DTIC Science & Technology

    2006-05-31

    This laboratory studied four systems involving molecular self-assembly during this project period. Each system will open a new avenue of research in developing novel applications for use in biomedical engineering and materials science. These systems include self-assembling oligopeptides that form stable beta sheets in water, peptides that form inter- and

  17. Bioinformatic identification of plant peptides.

    PubMed

    Lease, Kevin A; Walker, John C

    2010-01-01

    Plant peptides play a number of important roles in defence, development and many other aspects of plant physiology. Identifying additional peptide sequences provides the starting point to investigate their function using molecular, genetic or biochemical techniques. Due to their small size, identifying peptide sequences may not succeed using the default bioinformatic approaches that work well for average-sized proteins. There are two general scenarios related to bioinformatic identification of peptides to be discussed in this paper. In the first scenario, one already has the sequence of a plant peptide and is trying to find more plant peptides with some sequence similarity to the starting peptide. To do this, the Basic Local Alignment Search Tool (BLAST) is employed, with the parameters adjusted to be more favourable for identifying potential peptide matches. A second scenario involves trying to identify plant peptides without using sequence similarity searches to known plant peptides. In this approach, features such as protein size and the presence of a cleavable amino-terminal signal peptide are used to screen annotated proteins. A variation of this method can be used to screen for unannotated peptides from genomic sequences. Bioinformatic resources related to Arabidopsis thaliana will be used to illustrate these approaches.

  18. Roles of Hydrophobicity and Charge Distribution of Cationic Antimicrobial Peptides in Peptide-Membrane Interactions*

    PubMed Central

    Yin, Lois M.; Edwards, Michelle A.; Li, Jessica; Yip, Christopher M.; Deber, Charles M.

    2012-01-01

    Cationic antimicrobial peptides (CAPs) occur as important innate immunity agents in many organisms, including humans, and offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to membrane lysis and eventually cell death. In this work, we studied the biophysical and microbiological characteristics of designed CAPs varying in hydrophobicity levels and charge distributions by a variety of biophysical and biochemical approaches, including in-tandem atomic force microscopy, attenuated total reflection-FTIR, CD spectroscopy, and SDS-PAGE. Peptide structural properties were correlated with their membrane-disruptive abilities and antimicrobial activities. In bacterial lipid model membranes, a time-dependent increase in aggregated β-strand-type structure in CAPs with relatively high hydrophobicity (such as KKKKKKALFALWLAFLA-NH2) was essentially absent in CAPs with lower hydrophobicity (such as KKKKKKAAFAAWAAFAA-NH2). Redistribution of positive charges by placing three Lys residues at both termini while maintaining identical sequences minimized self-aggregation above the dimer level. Peptides containing four Leu residues were destructive to mammalian model membranes, whereas those with corresponding Ala residues were not. This finding was mirrored in hemolysis studies in human erythrocytes, where Ala-only peptides displayed virtually no hemolysis up to 320 μm, but the four-Leu peptides induced 40–80% hemolysis at the same concentration range. All peptides studied displayed strong antimicrobial activity against Pseudomonas aeruginosa (minimum inhibitory concentrations of 4–32 μm). The overall findings suggest optimum routes to balancing peptide hydrophobicity and charge distribution that allow efficient penetration and disruption of the bacterial membranes without damage to mammalian (host) membranes. PMID:22253439

  19. PASSEL: the PeptideAtlas SRMexperiment library.

    PubMed

    Farrah, Terry; Deutsch, Eric W; Kreisberg, Richard; Sun, Zhi; Campbell, David S; Mendoza, Luis; Kusebauch, Ulrike; Brusniak, Mi-Youn; Hüttenhain, Ruth; Schiess, Ralph; Selevsek, Nathalie; Aebersold, Ruedi; Moritz, Robert L

    2012-04-01

    Public repositories for proteomics data have accelerated proteomics research by enabling more efficient cross-analyses of datasets, supporting the creation of protein and peptide compendia of experimental results, supporting the development and testing of new software tools, and facilitating the manuscript review process. The repositories available to date have been designed to accommodate either shotgun experiments or generic proteomic data files. Here, we describe a new kind of proteomic data repository for the collection and representation of data from selected reaction monitoring (SRM) measurements. The PeptideAtlas SRM Experiment Library (PASSEL) allows researchers to easily submit proteomic data sets generated by SRM. The raw data are automatically processed in a uniform manner and the results are stored in a database, where they may be downloaded or browsed via a web interface that includes a chromatogram viewer. PASSELenables cross-analysis of SRMdata, supports optimization of SRMdata collection, and facilitates the review process of SRMdata. Further, PASSELwill help in the assessment of proteotypic peptide performance in a wide array of samples containing the same peptide, as well as across multiple experimental protocols.

  20. Biochemical functionalization of peptide nanotubes with phage displayed peptides

    NASA Astrophysics Data System (ADS)

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

  1. A Peptide Filtering Relation Quantifies MHC Class I Peptide Optimization

    PubMed Central

    Goldstein, Leonard D.; Howarth, Mark; Cardelli, Luca; Emmott, Stephen; Elliott, Tim; Werner, Joern M.

    2011-01-01

    Major Histocompatibility Complex (MHC) class I molecules enable cytotoxic T lymphocytes to destroy virus-infected or cancerous cells, thereby preventing disease progression. MHC class I molecules provide a snapshot of the contents of a cell by binding to protein fragments arising from intracellular protein turnover and presenting these fragments at the cell surface. Competing fragments (peptides) are selected for cell-surface presentation on the basis of their ability to form a stable complex with MHC class I, by a process known as peptide optimization. A better understanding of the optimization process is important for our understanding of immunodominance, the predominance of some T lymphocyte specificities over others, which can determine the efficacy of an immune response, the danger of immune evasion, and the success of vaccination strategies. In this paper we present a dynamical systems model of peptide optimization by MHC class I. We incorporate the chaperone molecule tapasin, which has been shown to enhance peptide optimization to different extents for different MHC class I alleles. Using a combination of published and novel experimental data to parameterize the model, we arrive at a relation of peptide filtering, which quantifies peptide optimization as a function of peptide supply and peptide unbinding rates. From this relation, we find that tapasin enhances peptide unbinding to improve peptide optimization without significantly delaying the transit of MHC to the cell surface, and differences in peptide optimization across MHC class I alleles can be explained by allele-specific differences in peptide binding. Importantly, our filtering relation may be used to dynamically predict the cell surface abundance of any number of competing peptides by MHC class I alleles, providing a quantitative basis to investigate viral infection or disease at the cellular level. We exemplify this by simulating optimization of the distribution of peptides derived from Human

  2. Optimization for Peptide Sample Preparation for Urine Peptidomics

    SciTech Connect

    Sigdel, Tara K.; Nicora, Carrie D.; Hsieh, Szu-Chuan; Dai, Hong; Qian, Weijun; Camp, David G.; Sarwal, Minnie M.

    2014-02-25

    Analysis of native or endogenous peptides in biofluids can provide valuable insights into disease mechanisms. Furthermore, the detected peptides may also have utility as potential biomarkers for non-invasive monitoring of human diseases. The non-invasive nature of urine collection and the abundance of peptides in the urine makes analysis by high-throughput ‘peptidomics’ methods , an attractive approach for investigating the pathogenesis of renal disease. However, urine peptidomics methodologies can be problematic with regards to difficulties associated with sample preparation. The urine matrix can provide significant background interference in making the analytical measurements that it hampers both the identification of peptides and the depth of the peptidomics read when utilizing LC-MS based peptidome analysis. We report on a novel adaptation of the standard solid phase extraction (SPE) method to a modified SPE (mSPE) approach for improved peptide yield and analysis sensitivity with LC-MS based peptidomics in terms of time, cost, clogging of the LC-MS column, peptide yield, peptide quality, and number of peptides identified by each method. Expense and time requirements were comparable for both SPE and mSPE, but more interfering contaminants from the urine matrix were evident in the SPE preparations (e.g., clogging of the LC-MS columns, yellowish background coloration of prepared samples due to retained urobilin, lower peptide yields) when compared to the mSPE method. When we compared data from technical replicates of 4 runs, the mSPE method provided significantly improved efficiencies for the preparation of samples from urine (e.g., mSPE peptide identification 82% versus 18% with SPE; p = 8.92E-05). Additionally, peptide identifications, when applying the mSPE method, highlighted the biology of differential activation of urine peptidases during acute renal transplant rejection with distinct laddering of specific peptides, which was obscured for most proteins

  3. HER2 Targeting Peptides Screening and Applications in Tumor Imaging and Drug Delivery

    PubMed Central

    Geng, Lingling; Wang, Zihua; Jia, Xiangqian; Han, Qiuju; Xiang, Zhichu; Li, Dan; Yang, Xiaoliang; Zhang, Di; Bu, Xiangli; Wang, Weizhi; Hu, Zhiyuan; Fang, Qiaojun

    2016-01-01

    Herein, computational-aided one-bead-one-compound (OBOC) peptide library design combined with in situ single-bead sequencing microarray methods were successfully applied in screening peptides targeting at human epidermal growth factor receptor-2 (HER2), a biomarker of human breast cancer. As a result, 72 novel peptides clustered into three sequence motifs which are PYL***NP, YYL***NP and PPL***NP were acquired. Particularly one of the peptides, P51, has nanomolar affinity and high specificity for HER2 in ex vivo and in vivo tests. Moreover, doxorubicin (DOX)-loaded liposome nanoparticles were modified with peptide P51 or P25 and demonstrated to improve the targeted delivery against HER2 positive cells. Our study provides an efficient peptide screening method with a combination of techniques and the novel screened peptides with a clear binding site on HER2 can be used as probes for tumor imaging and targeted drug delivery. PMID:27279916

  4. Octaarginine-modified liposomes enhance cross-presentation by promoting the C-terminal trimming of antigen peptide.

    PubMed

    Nakamura, Takashi; Ono, Kouhei; Suzuki, Yoshiteru; Moriguchi, Rumiko; Kogure, Kentaro; Harashima, Hideyoshi

    2014-08-04

    Exogenous antigen proteolysis by proteasomes and amino peptidases is essential for the production of mature major histocompatibility complex class I (MHC-I) peptides to induce cross-presentation. We report here that when liposomes are modified with octaarginine (R8-Lip), a type of cell-penetrating peptide, the production of the mature MHC-I peptide is enhanced by promoting the C-terminal trimming of the antigen peptide. The efficiency of cross-presentation of ovalbumin (OVA) using the R8-Lip was dramatically higher than that by octalysine modified liposomes (K8-Lip) in mouse bone-marrow derived dendritic cells (BMDCs), although the physical characters of both liposomes were comparable. In this study, we investigated the mechanism responsible for the enhancement in cross-presentation by R8-Lip. Although the efficiencies of cellular uptake, endosomal escape, proteolysis of OVA and DC maturation between the two systems were essentially the same, an analysis of peptide trimming to SIINFEKL (mature MHC-I peptide of OVA) by using R8-Lip and K8-Lip encapsulating peptides of various length clearly indicates that the use of R8-Lip enhances the efficiency of the C-terminal cleavage of antigen-derived peptides. This finding provides a new strategy for achieving efficient cross-presentation by using R8 peptide and arginine-rich peptides. Moreover, this result may contribute to the development of a new paradigm regarding the machinery associated with antigen peptide production.

  5. Concepts for Biologically Active Peptides

    PubMed Central

    Kastin, Abba J.; Pan, Weihong

    2012-01-01

    Here we review a unique aspect of CNS research on biologically active peptides that started against a background of prevalent dogmas but ended by exerting considerable influence on the field. During the course of refuting some doctrines, we introduced several concepts that were unconventional and paradigm-shifting at the time. We showed that (1) hypothalamic peptides can act ‘up’ on the brain as well as ‘down’ on the pituitary, (2) peripheral peptides can affect the brain, (3) peptides can cross the blood-brain barrier, (4) the actions of peptides can persist longer than their half-lives in blood, (5) perinatal administration of peptides can exert actions persisting into adulthood, (6) a single peptide can have more than one action, (7) dose-response relationships of peptides need not be linear, (8) the brain produces antiopiate as well as opiate peptides, (9) there is a selective high affinity endogenous peptide ligand for the mu-opiate receptor, (10) a peptide’s name does not restrict its effects, and (11) astrocytes assume an active role in response to metabolic disturbance and hyperleptinemia. The evolving questions in our laboratories reflect the diligent effort of the neuropeptide community to identify the roles of peptides in the CNS. The next decade is expected to see greater progress in the following areas: (a) interactions of peptides with other molecules in the CNS; (b) peptide involvement in cell-cell interactions; and (c) peptides in neuropsychiatric, autoimmune, and neurodegenerative diseases. The development of peptidomics and gene silencing approaches will expedite the formation of many new concepts in a new era. PMID:20726835

  6. Semienzymatic cyclization of disulfide-rich peptides using Sortase A.

    PubMed

    Jia, Xinying; Kwon, Soohyun; Wang, Ching-I Anderson; Huang, Yen-Hua; Chan, Lai Y; Tan, Chia Chia; Rosengren, K Johan; Mulvenna, Jason P; Schroeder, Christina I; Craik, David J

    2014-03-07

    Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, α-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential.

  7. The Specificity of Peptide Chain Extension by N-Carboxyanhydrides

    NASA Technical Reports Server (NTRS)

    Wen, Ke; Orgel, Leslie E.

    2001-01-01

    We have used amino acids activated by carbonyldiimidazole to study the enantiospecificity of peptide elongation in aqueous solution. Peptide primers Glu(sub 10) and Ala3Glulo were elongated with the enantiomers of arginine, glutamic acid, asparagine, phenylalanine, serine and valine. The homochiral addition was always the more efficient reaction; the enantiospecificity was large in some cases but very small in others. In every case Ala(sub 3)Glu(sub l0) was elongated more efficiently than Glu(sub 10).

  8. NisT, the transporter of the lantibiotic nisin, can transport fully modified, dehydrated, and unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides.

    PubMed

    Kuipers, Anneke; de Boef, Esther; Rink, Rick; Fekken, Susan; Kluskens, Leon D; Driessen, Arnold J M; Leenhouts, Kees; Kuipers, Oscar P; Moll, Gert N

    2004-05-21

    Lantibiotics are lanthionine-containing peptide antibiotics. Nisin, encoded by nisA, is a pentacyclic lantibiotic produced by some Lactococcus lactis strains. Its thioether rings are posttranslationally introduced by a membrane-bound enzyme complex. This complex is composed of three enzymes: NisB, which dehydrates serines and threonines; NisC, which couples these dehydrated residues to cysteines, thus forming thioether rings; and the transporter NisT. We followed the activity of various combinations of the nisin enzymes by measuring export of secreted peptides using antibodies against the leader peptide and mass spectroscopy for detection. L. lactis expressing the nisABTC genes efficiently produced fully posttranslationally modified prenisin. Strikingly, L. lactis expressing the nisBT genes could produce dehydrated prenisin without thioether rings and a dehydrated form of a non-lantibiotic peptide. In the absence of the biosynthetic NisBC enzymes, the NisT transporter was capable of excreting unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides. Our data show that NisT specifies a broad spectrum (poly)peptide transporter that can function either in conjunction with or independently from the biosynthetic genes. NisT secretes both unmodified and partially or fully posttranslationally modified forms of prenisin and non-lantibiotic peptides. These results open the way for efficient production of a wide range of peptides with increased stability or novel bioactivities.

  9. Peptide mass fingerprinting.

    PubMed

    Thiede, Bernd; Höhenwarter, Wolfgang; Krah, Alexander; Mattow, Jens; Schmid, Monika; Schmidt, Frank; Jungblut, Peter R

    2005-03-01

    Peptide mass fingerprinting by MALDI-MS and sequencing by tandem mass spectrometry have evolved into the major methods for identification of proteins following separation by two-dimensional gel electrophoresis, SDS-PAGE or liquid chromatography. One main technological goal of proteome analyses beside high sensitivity and automation was the comprehensive analysis of proteins. Therefore, the protein species level with the essential information on co- and post-translational modifications must be achieved. The power of peptide mass fingerprinting for protein identification was described here, as exemplified by the identification of protein species with high molecular masses (spectrin alpha and beta), low molecular masses (elongation factor EF-TU fragments), splice variants (alpha A crystallin), aggregates with disulfide bridges (alkylhydroperoxide reductase), and phosphorylated proteins (heat shock protein 27). Helpful tools for these analyses were the use of the minimal protein identifier concept and the software program MS-Screener to remove mass peaks assignable to contaminants and neighbor spots.

  10. Identification of oligomerizing peptides.

    PubMed

    Dhiman, A; Rodgers, M E; Schleif, R

    2001-06-08

    The AraC DNA binding domain is inactive in a monomeric form but can activate transcription from the arabinose operon promoters upon its dimerization. We used this property to identify plasmids encoding peptide additions to the AraC DNA binding domain that could dimerize the domain. We generated a high diversity library of plasmids by inserting 90-base oligonucleotides of random sequence ahead of DNA coding for the AraC DNA binding domain in an expression vector, transforming, and selecting colonies containing functional oligomeric peptide-AraC DNA binding domain chimeric proteins by their growth on minimal arabinose medium. Six of seven Ara(+) candidates were partially characterized, and one was purified. Equilibrium analytical centrifugation experiments showed that it dimerizes with a dissociation constant of approximately 2 micrometer.

  11. Avian host defense peptides.

    PubMed

    Cuperus, Tryntsje; Coorens, Maarten; van Dijk, Albert; Haagsman, Henk P

    2013-11-01

    Host defense peptides (HDPs) are important effector molecules of the innate immune system of vertebrates. These antimicrobial peptides are also present in invertebrates, plants and fungi. HDPs display broad-spectrum antimicrobial activities and fulfill an important role in the first line of defense of many organisms. It is becoming increasingly clear that in the animal kingdom the functions of HDPs are not confined to direct antimicrobial actions. Research in mammals has indicated that HDPs have many immunomodulatory functions and are also involved in other physiological processes ranging from development to wound healing. During the past five years our knowledge about avian HDPs has increased considerably. This review addresses our current knowledge on the evolution, regulation and biological functions of HDPs of birds.

  12. Preferential labeling of alpha-amino N-terminal groups in peptides by biotin: application to the detection of specific anti-peptide antibodies by enzyme immunoassays.

    PubMed

    Sélo, I; Négroni, L; Créminon, C; Grassi, J; Wal, J M

    1996-12-15

    Experimental conditions (pH 6.5, 24 h reaction, peptide:biotin ratio 1:5) were defined for preferential incorporation of the biotin molecule in the N-terminal alpha-amino group of peptides. This strategy could be helpful in numerous applications when an entire peptide chain must remain accessible for antibody or receptor binding. We illustrate this advantage in a solid-phase enzyme immunoassay designed to detect antibodies specific for bovine beta-lactoglobulin present in rabbit or human sera. This test involves synthetic peptides biotinylated in different positions and immobilized on a solid phase. The use of biotin/streptavidin interactions permitted more efficient detection of specific anti-peptide antibodies than solid phases prepared using conventional passive-adsorption techniques. The highest levels of antibody binding were measured when biotinylation occurred at the N-terminal extremity of immobilized peptides.

  13. BT cationic peptides: small peptides that modulate innate immune responses of chicken heterophils and monocytes.

    PubMed

    Kogut, Michael H; Genovese, Kenneth J; He, Haiqi; Swaggerty, Christina L; Jiang, Yi Wei

    2012-01-15

    Neonatal poultry exhibit a transient susceptibility to infectious diseases during the first week of life that stems from inefficient host defense mechanisms. Yet, the initial host immune response to pathogens is a critical determinant of disease resistance and susceptibility. With this context in mind, novel ways to stimulate or modulate the hosts' natural immune response is emerging as an important area of interest for food animal producers including the poultry industry. Specifically, we have been investigating new modulation strategies tailored around the selective stimulation of the host's immune system, and particularly rapid acting innate immunity, as an alternative to direct targeting of microbial pathogens. One such approach that we have been investigating is the use of a group of cationic peptides produced by a Gram-positive soil bacterium, Brevibacillus texasporus (BT peptides). We have previously shown that, provided as a feed additive, BT peptides significantly induced a concentration-dependent protection against cecal colonization and extraintestinal colonization by Salmonella enterica serovar Enteritidis (SE). This protection is not the result of direct antibacterial activity of the BT peptides on the SE since the concentrations used were below the minimum inhibitory concentration for SE. We also found that BT are not absorbed in the intestine, but still induce a significant up-regulation in the functional efficiency of peripheral blood heterophils and monocytes. The mechanisms of this immune modulation are unknown. Here, using in vitro models for measuring: (1) leukocyte oxidative burst, (2) changes in leukocyte cytokine and chemokines gene expression profiles, and (3) phosphorylation of the mitogen activated protein kinases (MAPKs) in leukocytes, we evaluated the role of BT peptides as priming mediators for heterophil and monocyte responses at the level of cell function, gene transcription/expression, and cell phosphorylation following stimulation

  14. Cell-penetrating peptides: Possible transduction mechanisms and therapeutic applications

    PubMed Central

    GUO, ZHENGRONG; PENG, HUANYAN; KANG, JIWEN; SUN, DIANXING

    2016-01-01

    Cell-penetrating peptides (CPPs), also known as protein transduction domains, are a class of diverse peptides with 5–30 amino acids. CPPs are divided into cationic, amphipathic and hydrophobic CPPs. They are able to carry small molecules, plasmid DNA, small interfering RNA, proteins, viruses, imaging agents and other various nanoparticles across the cellular membrane, resulting in internalization of the intact cargos. However, the mechanisms of CPP internalization remain to be elucidated. Recently, CPPs have received considerable attention due to their high transduction efficiency and low cytotoxicity. These peptides have a significant potential for diagnostic and therapeutic applications, such as delivery of fluorescent or radioactive compounds for imaging, delivery of peptides and proteins for therapeutic application, and delivery of molecules into induced pluripotent stem cells for directing differentiation. The present study reviews the classifications and transduction mechanisms of CPPs, as well as their potential applications. PMID:27123243

  15. A new fingerprint to predict nonribosomal peptides activity

    NASA Astrophysics Data System (ADS)

    Abdo, Ammar; Caboche, Ségolène; Leclère, Valérie; Jacques, Philippe; Pupin, Maude

    2012-10-01

    Bacteria and fungi use a set of enzymes called nonribosomal peptide synthetases to provide a wide range of natural peptides displaying structural and biological diversity. So, nonribosomal peptides (NRPs) are the basis for some efficient drugs. While discovering new NRPs is very desirable, the process of identifying their biological activity to be used as drugs is a challenge. In this paper, we present a novel peptide fingerprint based on monomer composition (MCFP) of NRPs. MCFP is a novel method for obtaining a representative description of NRP structures from their monomer composition in fingerprint form. Experiments with Norine NRPs database and MCFP show high prediction accuracy (>93 %). Also a high recall rate (>82 %) is obtained when MCFP is used for screening NRPs database. From this study it appears that our fingerprint, built from monomer composition, allows an effective screening and prediction of biological activities of NRPs database.

  16. Click chemistry in peptide-based drug design.

    PubMed

    Li, Huiyuan; Aneja, Rachna; Chaiken, Irwin

    2013-08-16

    Click chemistry is an efficient and chemoselective synthetic method for coupling molecular fragments under mild reaction conditions. Since the advent in 2001 of methods to improve stereochemical conservation, the click chemistry approach has been broadly used to construct diverse chemotypes in both chemical and biological fields. In this review, we discuss the application of click chemistry in peptide-based drug design. We highlight how triazoles formed by click reactions have been used for mimicking peptide and disulfide bonds, building secondary structural components of peptides, linking functional groups together, and bioconjugation. The progress made in this field opens the way for synthetic approaches to convert peptides with promising functional leads into structure-minimized and more stable forms.

  17. Click Chemistry in Peptide-Based Drug Design

    PubMed Central

    Li, Huiyuan; Aneja, Rachna; Chaiken, Irwin

    2014-01-01

    Click chemistry is an efficient and chemoselective synthetic method for coupling molecular fragments under mild reaction conditions. Since the advent in 2001 of methods to improve stereochemical conservation, the click chemistry approach has been broadly used to construct diverse chemotypes in both chemical and biological fields. In this review, we discuss the application of click chemistry in peptide-based drug design. We highlight how triazoles formed by click reactions have been used for mimicking peptide and disulfide bonds, building secondary structural components of peptides, linking functional groups together, and bioconjugation. The progress made in this field opens the way for synthetic approaches to convert peptides with promising functional leads into structure-minimized and more stable forms. PMID:23959192

  18. Prediction and interpretation of the lipophilicity of small peptides

    NASA Astrophysics Data System (ADS)

    Visconti, Alessia; Ermondi, Giuseppe; Caron, Giulia; Esposito, Roberto

    2015-04-01

    Peptide-based drug discovery has considerably expanded and solid in silico tools for the prediction of physico-chemical properties of peptides are urgently needed. In this work we tested some combinations of descriptors/algorithms to find the best model to predict log D_{ {oct}} of a series of peptides. To do that we evaluate the models statistical performances but also their skills in providing a reliable deconvolution of the balance of intermolecular forces governing the partitioning phenomenon. Results prove that a PLS model based on VolSurf+ descriptors is the best tool to predict log D_{ {oct}} of neutral and ionised peptides. The mechanistic interpretation also reveals that the inclusion in the chemical structure of a HBD group is more efficient in decreasing lipophilicity than the inclusion of a HBA group.

  19. Antimicrobial Peptides and Colitis

    PubMed Central

    Ho, Samantha; Pothoulakis, Charalabos; Koon, Hon Wai

    2013-01-01

    Antimicrobial peptides (AMPs) are important components of innate immunity. They are often expressed in response to colonic inflammation and infection. Over the last several years, the roles of several antimicrobial peptides have been explored. Gene expression of many AMPs (beta defensin HBD2-4 and cathelicidin) is induced in response to invasion of gut microbes into the mucosal barrier. Some AMPs are expressed in a constitutive manner (alpha defensin HD 5-6 and beta defensin HBD1), while others (defensin and bactericidal/permeability increasing protein BPI) are particularly associated with Inflammatory Bowel Disease (IBD) due to altered defensin expression or development of autoantibodies against Bactericidal/permeability increasing protein (BPI). Various AMPs have different spectrum and strength of antimicrobial effects. Some may play important roles in modulating the colitis (cathelicidin) while others (lactoferrin, hepcidin) may represent biomarkers of disease activity. The use of AMPs for therapeutic purposes is still at an early stage of development. A few natural AMPs were shown to be able to modulate colitis when delivered intravenously or intracolonically (cathelicidin, elafin and SLPI) in mouse colitis models. New AMPs (synthetic or artificial non-human peptides) are being developed and may represent new therapeutic approaches against colitis. This review discusses the latest research developments in the AMP field with emphasis in innate immunity and pathophysiology of colitis. PMID:22950497

  20. Investigation on cellular uptake and pharmacodynamics of DOCK2-inhibitory peptides conjugated with cell-penetrating peptides.

    PubMed

    Adachi, Yusuke; Sakamoto, Kotaro; Umemoto, Tadashi; Fukuda, Yasunori; Tani, Akiyoshi; Asami, Taiji

    2017-04-01

    Protein-protein interaction between dedicator of cytokinesis 2 (DOCK2) and Ras-related C3 botulinum toxin substrate 1 (Rac1) is an attractive intracellular target for transplant rejection and inflammatory diseases. Recently, DOCK2-selective inhibitory peptides have been discovered, and conjugation with oligoarginine cell-penetrating peptide (CPP) improved inhibitory activity in a cell migration assay. Although a number of CPPs have been reported, oligoarginine was only one example introduced to the inhibitory peptides. In this study, we aimed to confirm the feasibility of CPP-conjugation approach for DOCK2-inhibitory peptides, and select preferable sequences as CPP moiety. First, we evaluated cell permeability of thirteen known CPPs and partial sequences of influenza A viral protein PB1-F2 using an internalization assay system based on luciferin-luciferase reaction, and then selected four CPPs with efficient cellular uptake. Among four conjugates of these CPPs and a DOCK2-inhibitory peptide, the inhibitory activity of a novel CPP, PB1-F2 fragment 5 (PF5), conjugate was comparable to oligoarginine conjugate and higher than that of the non-conjugated peptide. Finally, internalization assay revealed that oligoarginine and PF5 increased the cellular uptake of inhibitory peptides to the same extent. Hence, we demonstrated that CPP-conjugation approach is applicable to the development of novel anti-inflammatory drugs based on DOCK2 inhibition by investigating both cellular uptake and bioactivity.

  1. Enzyme-Assisted Discovery of Antioxidant Peptides from Edible Marine Invertebrates: A Review.

    PubMed

    Chai, Tsun-Thai; Law, Yew-Chye; Wong, Fai-Chu; Kim, Se-Kwon

    2017-02-16

    Marine invertebrates, such as oysters, mussels, clams, scallop, jellyfishes, squids, prawns, sea cucumbers and sea squirts, are consumed as foods. These edible marine invertebrates are sources of potent bioactive peptides. The last two decades have seen a surge of interest in the discovery of antioxidant peptides from edible marine invertebrates. Enzymatic hydrolysis is an efficient strategy commonly used for releasing antioxidant peptides from food proteins. A growing number of antioxidant peptide sequences have been identified from the enzymatic hydrolysates of edible marine invertebrates. Antioxidant peptides have potential applications in food, pharmaceuticals and cosmetics. In this review, we first give a brief overview of the current state of progress of antioxidant peptide research, with special attention to marine antioxidant peptides. We then focus on 22 investigations which identified 32 antioxidant peptides from enzymatic hydrolysates of edible marine invertebrates. Strategies adopted by various research groups in the purification and identification of the antioxidant peptides will be summarized. Structural characteristic of the peptide sequences in relation to their antioxidant activities will be reviewed. Potential applications of the peptide sequences and future research prospects will also be discussed.

  2. Enzyme-Assisted Discovery of Antioxidant Peptides from Edible Marine Invertebrates: A Review

    PubMed Central

    Chai, Tsun-Thai; Law, Yew-Chye; Wong, Fai-Chu; Kim, Se-Kwon

    2017-01-01

    Marine invertebrates, such as oysters, mussels, clams, scallop, jellyfishes, squids, prawns, sea cucumbers and sea squirts, are consumed as foods. These edible marine invertebrates are sources of potent bioactive peptides. The last two decades have seen a surge of interest in the discovery of antioxidant peptides from edible marine invertebrates. Enzymatic hydrolysis is an efficient strategy commonly used for releasing antioxidant peptides from food proteins. A growing number of antioxidant peptide sequences have been identified from the enzymatic hydrolysates of edible marine invertebrates. Antioxidant peptides have potential applications in food, pharmaceuticals and cosmetics. In this review, we first give a brief overview of the current state of progress of antioxidant peptide research, with special attention to marine antioxidant peptides. We then focus on 22 investigations which identified 32 antioxidant peptides from enzymatic hydrolysates of edible marine invertebrates. Strategies adopted by various research groups in the purification and identification of the antioxidant peptides will be summarized. Structural characteristic of the peptide sequences in relation to their antioxidant activities will be reviewed. Potential applications of the peptide sequences and future research prospects will also be discussed. PMID:28212329

  3. [Hydrolysis of peptides by immobilized bacterial peptide hydrolases].

    PubMed

    Nekliudov, A D; Deniakina, E K

    2004-01-01

    The feasibility of hydrolysis of a mixture of peptides with an enzyme from the bacterium Xanthomonas rubrilineans, displaying a peptidase activity and immobilized on aluminum oxide, was studied. Kinetic schemes and equations allowing for approaching quantitative description of peptide hydrolysis in complex mixtures containing free amino acids and peptides were obtained. It was demonstrated that as a result of hydrolysis, the content of free amino acids in hydrolysates decreased 2.5- to 3-fold and the molecular weight of the constituent peptides, 2-fold.

  4. T-cell receptors: tugging on the anchor for a tighter hold on the tumor-associated peptide.

    PubMed

    Dyson, Julian

    2015-02-01

    Although it has been shown that human tumor-associated, HLA anchor residue modified "heteroclitic" peptides may induce stronger immune responses than wild-type peptides in cancer vaccine trials, it has also been shown that some T cells primed with these heteroclitic peptides subsequently fail to recognize the natural, tumor-expressed peptide efficiently. This may provide a molecular reason for why clinical trials of these peptides have been thus far unsuccessful. In this issue of the European Journal of Immunology, Madura et al. [Eur. J. Immunol. 2015. 45: 584-591] highlight a novel twist on T-cell receptor (TCR) recognition of HLA-peptide complexes. Tumor-associated peptides often lack canonical anchor residues, which can be substituted for the optimal residue to improve their antigenicity. T-cell cross-reactivity between the natural and modified (heteroclitic) peptides is essential for this approach to work and depends on whether the anchor residue substitution influences peptide conformation. The Melan-A/MART-126-35 peptide epitope is an example where T cells can make this distinction, with the natural peptide stimulating higher affinity CD8(+) T cells than the heteroclitic peptide, despite the heteroclitic peptide's more stable association with HLA-A2. The molecular basis for peptide discrimination is identified through the structure of the TCR bound to the natural peptide; TCR engagement of the natural peptide "lifts" its amino-terminus partly away from the HLA peptide binding groove, forming a higher affinity interface with the TCR than is formed with the anchor residue "optimized" heteroclitic peptide, which cannot be "pulled" from the HLA groove.

  5. Rational Design of a Polymer with Robust Efficacy for Intracellular Protein and Peptide Delivery.

    PubMed

    Chang, Hong; Lv, Jia; Gao, Xin; Wang, Xing; Wang, Hui; Chen, Hui; He, Xu; Li, Lei; Cheng, Yiyun

    2017-03-08

    The efficient delivery of biopharmaceutical drugs such as proteins and peptides into the cytosol of target cells poses substantial challenges owing to their large size and susceptibility to degradation. Current protein delivery vehicles have limitations such as the need for protein modification, insufficient delivery of large-size proteins or small peptides, and loss of protein function after the delivery. Here, we adopted a rational approach to design a polymer with robust efficacy for intracellular protein and peptide delivery. The polymer is composed of a dendrimer scaffold, a hydrophobic membrane-disruptive region, and a multivalent protein binding surface. It allows efficient protein/peptide binding, endocytosis, and endosomal disruption and is capable of efficiently delivering various biomacromolecules including bovine serum albumin, R-phycoerythrin, p53, saporin, β-galactosidase, and peptides into the cytosol of living cells. Transduction of apoptotic proteins and peptides successfully induces apoptosis in cancer cells, suggesting that the activities of proteins and peptides are maintained during the delivery. This technology represents an efficient and useful tool for intracellular protein and peptide delivery and has broad applicability for basic research and clinical applications.

  6. Macrocyclization of Unprotected Peptide Isocyanates.

    PubMed

    Vinogradov, Alexander A; Choo, Zi-Ning; Totaro, Kyle A; Pentelute, Bradley L

    2016-03-18

    A chemistry for the facile two-component macrocyclization of unprotected peptide isocyanates is described. Starting from peptides containing two glutamic acid γ-hydrazide residues, isocyanates can be readily accessed and cyclized with hydrazides of dicarboxylic acids. The choice of a nucleophilic linker allows for the facile modulation of biochemical properties of a macrocyclic peptide. Four cyclic NYAD-1 analogues were synthesized using the described method and displayed a range of biological activities.

  7. Biodiscovery of Aluminum Binding Peptides

    DTIC Science & Technology

    2013-08-01

    display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high...scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the self- sustaining peptide libraries to be rapidly screened for high...removal. An eCPX peptide display library was grown and induced as described in the paragraph above. After rinsing samples briefly in PBS, the aluminum

  8. Improving Peptide Applications Using Nanotechnology.

    PubMed

    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.

  9. Biodiscovery of aluminum binding peptides

    NASA Astrophysics Data System (ADS)

    Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Hurley, Margaret M.; Stratis-Cullum, Dimitra

    2013-05-01

    Cell surface peptide display systems are large and diverse libraries of peptides (7-15 amino acids) which are presented by a display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high affinity binders to a given target of interest, and those binders quickly identified. Peptide display systems have traditionally been utilized in conjunction with organic-based targets, such as protein toxins or carbon nanotubes. However, this technology has been expanded for use with inorganic targets, such as metals, for biofabrication, hybrid material assembly and corrosion prevention. While most current peptide display systems employ viruses to host the display scaffold, we have recently shown that a bacterial host, Escherichia coli, displaying peptides in the ubiquitous, membrane protein scaffold eCPX can also provide specific peptide binders to an organic target. We have, for the first time, extended the use of this bacterial peptide display system for the biodiscovery of aluminum binding 15mer peptides. We will present the process of biopanning with macroscopic inorganic targets, binder enrichment, and binder isolation and discovery.

  10. Peptides that influence membrane topology

    NASA Astrophysics Data System (ADS)

    Wong, Gerard C. L.

    2014-03-01

    We examine the mechanism of a range of polypeptides that influence membrane topology, including antimicrobial peptides, cell penetrating peptides, viral fusion peptides, and apoptosis proteins, and show how a combination of geometry, coordination chemistry, and soft matter physics can be used to approach a unified understanding. We will also show how such peptides can impact biomedical problems such as auto-immune diseases (psoriasis, lupus), infectious diseases (viral and bacterial infections), and mitochondrial pathologies (under-regulated apoptosis leads to neurodegenerative diseases whereas over-regulated apoptosis leads to cancer.)

  11. Antitumor Peptides from Marine Organisms

    PubMed Central

    Zheng, Lan-Hong; Wang, Yue-Jun; Sheng, Jun; Wang, Fang; Zheng, Yuan; Lin, Xiu-Kun; Sun, Mi

    2011-01-01

    The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new antitumor agents in the field of the development of marine bioactive substances. In this review, the progress on studies of antitumor peptides from marine sources is provided. The biological properties and mechanisms of action of different marine peptides are described; information about their molecular diversity is also presented. Novel peptides that induce apoptosis signal pathway, affect the tubulin-microtubule equilibrium and inhibit angiogenesis are presented in association with their pharmacological properties. It is intended to provide useful information for further research in the fields of marine antitumor peptides. PMID:22072999

  12. The PeptideAtlas Project.

    PubMed

    Deutsch, Eric W

    2010-01-01

    PeptideAtlas is a multi-species compendium of peptides observed with tandem mass spectrometry methods. Raw mass spectrometer output files are collected from the community and reprocessed through a uniform analysis and validation pipeline that continues to advance. The results are loaded into a database and the information derived from the raw data is returned to the community via several web-based data exploration tools. The PeptideAtlas resource is useful for experiment planning, improving genome annotation, and other data mining projects. PeptideAtlas has become especially useful for planning targeted proteomics experiments.

  13. Antitumor peptides from marine organisms.

    PubMed

    Zheng, Lan-Hong; Wang, Yue-Jun; Sheng, Jun; Wang, Fang; Zheng, Yuan; Lin, Xiu-Kun; Sun, Mi

    2011-01-01

    The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new antitumor agents in the field of the development of marine bioactive substances. In this review, the progress on studies of antitumor peptides from marine sources is provided. The biological properties and mechanisms of action of different marine peptides are described; information about their molecular diversity is also presented. Novel peptides that induce apoptosis signal pathway, affect the tubulin-microtubule equilibrium and inhibit angiogenesis are presented in association with their pharmacological properties. It is intended to provide useful information for further research in the fields of marine antitumor peptides.

  14. Ligand peptide-grafted PEGylated liposomes using HER2 targeted peptide-lipid derivatives for targeted delivery in breast cancer cells: The effect of serine-glycine repeated peptides as a spacer.

    PubMed

    Suga, Tadaharu; Fuchigami, Yuki; Hagimori, Masayori; Kawakami, Shigeru

    2017-02-22

    Ligand peptide-grafted PEGylated liposomes have been widely studied for targeted drug delivery systems. Because ligand peptides are commonly grafted using PEG as a spacer on the surface of PEGylated liposomes, the interaction between ligand peptides and their corresponding receptors can be interrupted by steric hindrance of the PEG layer. Therefore, we aimed to develop ligand peptide-lipid derivatives to enhance the targeting efficiency of ligand peptide-grafted PEGylated liposomes, and designed a new ligand peptide-lipid derivatives having serine-glycine repeats (SG)n as a spacer based on the peptide length calculated by PyMol (v0.99). We selected KCCYSL (KCC) as the ligand peptide for binding to human epidermal growth factor receptor-2 (HER2). We synthesized new KCC-(SG)n-lipid derivatives (n=3, 5, 7) and evaluated their cellular association in breast cancer cells. KCC-(SG)n/PEGylated liposomes dramatically increased cellular association on HER2-positive breast cancer cells. The results suggest that KCC can be grafted on the surface of KCC-(SG)n/PEGylated liposomes prepared from KCC-(SG)n-lipid derivatives (n=3, 5, 7). In summary, we succeeded in developing KCC-(SG)n-lipid derivatives for the preparation of ligand peptide-grafted PEGylated liposomes.

  15. Stereochemical effects of all-hydrocarbon tethers in i,i+4 stapled peptides.

    PubMed

    Kim, Young-Woo; Verdine, Gregory L

    2009-05-01

    The stereochemical effects of the hydrocarbon crosslink on the conformation and cellular uptake of i,i+4 stapled peptides were studied. Compared to its S,S-configurated counterpart, the crosslink bearing the R,R-configuration provided a significantly diminished helix stabilizing effect and conferred less efficient cellular uptake on the stapled peptides. These results suggest that the vesicular trafficking pathway employed by cells to take up stapled peptides is sensitive to the extent of helical character in the peptide, with greater helicity conferring increased cellular uptake.

  16. Peptide synthesis beyond DMF: THF and ACN as excellent and friendlier alternatives.

    PubMed

    Jad, Yahya E; Acosta, Gerardo A; Khattab, Sherine N; de la Torre, Beatriz G; Govender, Thavendran; Kruger, Hendrik G; El-Faham, Ayman; Albericio, Fernando

    2015-02-28

    To date, DMF has been considered as the only solvent suitable for peptide synthesis. Here we demonstrate the capacity of THF and ACN, which are friendlier solvents than DMF, to yield the product in higher purity than DMF. Using various peptide models, both THF and ACN reduced racemization in solution-phase and solid-phase synthesis when compared with DMF. Moreover, the use of ACN and THF in the solid-phase peptide synthesis of hindered peptides, such as Aib-enkephalin pentapeptide and Aib-ACP decapeptide, in combination with a complete polyethylene glycol resin (ChemMatrix), gave a better coupling efficiency than DMF.

  17. Peptide Assembly-Driven Metal-Organic Framework (MOF) Motors for Micro Electric Generator

    PubMed Central

    Ikezoe, Yasuhiro; Fang, Justin; Wasik, Tomasz L.; Uemura, Takashi; Zheng, Yongtai; Kitagawa, Susumu

    2014-01-01

    Peptide-MOF motors, whose motions are driven by anisotropic surface gradients created via peptide self-assembly around nanopores of MOFs, can rotate microscopic rotors and magnet fast enough to generate electric power of 0.1 µW. To make the peptide-MOF generator recyclable, a new MOF is applied as a host motor engine, which has a more rigid framework with higher H2O affinity so that peptide release occurs more efficiently via guest exchange without the destruction of MOF. PMID:25418936

  18. Peptides and food intake.

    PubMed

    Sobrino Crespo, Carmen; Perianes Cachero, Aránzazu; Puebla Jiménez, Lilian; Barrios, Vicente; Arilla Ferreiro, Eduardo

    2014-01-01

    The mechanisms for controlling food intake involve mainly an interplay between gut, brain, and adipose tissue (AT), among the major organs. Parasympathetic, sympathetic, and other systems are required for communication between the brain satiety center, gut, and AT. These neuronal circuits include a variety of peptides and hormones, being ghrelin the only orexigenic molecule known, whereas the plethora of other factors are inhibitors of appetite, suggesting its physiological relevance in the regulation of food intake and energy homeostasis. Nutrients generated by food digestion have been proposed to activate G-protein-coupled receptors on the luminal side of enteroendocrine cells, e.g., the L-cells. This stimulates the release of gut hormones into the circulation such as glucagon-like peptide-1 (GLP-1), oxyntomodulin, pancreatic polypeptides, peptide tyrosine tyrosine, and cholecystokinin, which inhibit appetite. Ghrelin is a peptide secreted from the stomach and, in contrast to other gut hormones, plasma levels decrease after a meal and potently stimulate food intake. Other circulating factors such as insulin and leptin relay information regarding long-term energy stores. Both hormones circulate at proportional levels to body fat content, enter the CNS proportionally to their plasma levels, and reduce food intake. Circulating hormones can influence the activity of the arcuate nucleus (ARC) neurons of the hypothalamus, after passing across the median eminence. Circulating factors such as gut hormones may also influence the nucleus of the tractus solitarius (NTS) through the adjacent circumventricular organ. On the other hand, gastrointestinal vagal afferents converge in the NTS of the brainstem. Neural projections from the NTS, in turn, carry signals to the hypothalamus. The ARC acts as an integrative center, with two major subpopulations of neurons influencing appetite, one of them coexpressing neuropeptide Y and agouti-related protein (AgRP) that increases food

  19. Peptide Amyloid Surface Display

    PubMed Central

    2015-01-01

    Homomeric self-assembly of peptides into amyloid fibers is a feature of many diseases. A central role has been suggested for the lateral fiber surface affecting gains of toxic function. To investigate this, a protein scaffold that presents a discrete, parallel β-sheet surface for amyloid subdomains up to eight residues in length has been designed. Scaffolds that present the fiber surface of islet amyloid polypeptide (IAPP) were prepared. The designs show sequence-specific surface effects apparent in that they gain the capacity to attenuate rates of IAPP self-assembly in solution and affect IAPP-induced toxicity in insulin-secreting cells. PMID:25541905

  20. Plant antimicrobial peptides.

    PubMed

    Nawrot, Robert; Barylski, Jakub; Nowicki, Grzegorz; Broniarczyk, Justyna; Buchwald, Waldemar; Goździcka-Józefiak, Anna

    2014-05-01

    Plant antimicrobial peptides (AMPs) are a component of barrier defense system of plants. They have been isolated from roots, seeds, flowers, stems, and leaves of a wide variety of species and have activities towards phytopathogens, as well as against bacteria pathogenic to humans. Thus, plant AMPs are considered as promising antibiotic compounds with important biotechnological applications. Plant AMPs are grouped into several families and share general features such as positive charge, the presence of disulfide bonds (which stabilize the structure), and the mechanism of action targeting outer membrane structures.

  1. Peptides and Food Intake

    PubMed Central

    Sobrino Crespo, Carmen; Perianes Cachero, Aránzazu; Puebla Jiménez, Lilian; Barrios, Vicente; Arilla Ferreiro, Eduardo

    2014-01-01

    The mechanisms for controlling food intake involve mainly an interplay between gut, brain, and adipose tissue (AT), among the major organs. Parasympathetic, sympathetic, and other systems are required for communication between the brain satiety center, gut, and AT. These neuronal circuits include a variety of peptides and hormones, being ghrelin the only orexigenic molecule known, whereas the plethora of other factors are inhibitors of appetite, suggesting its physiological relevance in the regulation of food intake and energy homeostasis. Nutrients generated by food digestion have been proposed to activate G-protein-coupled receptors on the luminal side of enteroendocrine cells, e.g., the L-cells. This stimulates the release of gut hormones into the circulation such as glucagon-like peptide-1 (GLP-1), oxyntomodulin, pancreatic polypeptides, peptide tyrosine tyrosine, and cholecystokinin, which inhibit appetite. Ghrelin is a peptide secreted from the stomach and, in contrast to other gut hormones, plasma levels decrease after a meal and potently stimulate food intake. Other circulating factors such as insulin and leptin relay information regarding long-term energy stores. Both hormones circulate at proportional levels to body fat content, enter the CNS proportionally to their plasma levels, and reduce food intake. Circulating hormones can influence the activity of the arcuate nucleus (ARC) neurons of the hypothalamus, after passing across the median eminence. Circulating factors such as gut hormones may also influence the nucleus of the tractus solitarius (NTS) through the adjacent circumventricular organ. On the other hand, gastrointestinal vagal afferents converge in the NTS of the brainstem. Neural projections from the NTS, in turn, carry signals to the hypothalamus. The ARC acts as an integrative center, with two major subpopulations of neurons influencing appetite, one of them coexpressing neuropeptide Y and agouti-related protein (AgRP) that increases food

  2. De Novo Peptide Design and Experimental Validation of Histone Methyltransferase Inhibitors

    PubMed Central

    Smadbeck, James; Peterson, Meghan B.; Zee, Barry M.; Garapaty, Shivani; Mago, Aashna; Lee, Christina; Giannis, Athanassios; Trojer, Patrick; Garcia, Benjamin A.; Floudas, Christodoulos A.

    2014-01-01

    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 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 inhibit EZH2. These inhibitors should

  3. Immunomodulatory effects of anti-microbial peptides.

    PubMed

    Otvos, Laszlo

    2016-09-01

    Anti-microbial peptides (AMPs) were originally thought to exert protecting actions against bacterial infection by disintegrating bacterial membranes. Upon identification of internal bacterial targets, the view changed and moved toward inhibition of prokaryote-specific biochemical processes. However, the level of none of these activities can explain the robust efficacy of some of these peptides in animal models of systemic and cutaneous infections. A rapidly growing panel of reports suggests that AMPs, now called host-defense peptides (HDPs), act through activating the immune system of the host. This includes recruitment and activation of macrophages and mast cells, inducing chemokine production and altering NF-κB signaling processes. As a result, both pro- and anti-inflammatory responses are elevated together with activation of innate and adaptive immunity mechanisms, wound healing, and apoptosis. HDPs sterilize the systemic circulation and local injury sites significantly more efficiently than pure single-endpoint in vitro microbiological or biochemical data would suggest and actively aid recovering from tissue damage after or even without bacterial infections. However, the multiple and, often opposing, immunomodulatory functions of HDPs require exceptional care in therapeutic considerations.

  4. Absolute peptide quantification by lutetium labeling and nanoHPLC-ICPMS with isotope dilution analysis.

    PubMed

    Rappel, Christina; Schaumlöffel, Dirk

    2009-01-01

    The need of analytical methods for absolute quantitative protein analysis spurred research on new developments in recent years. In this work, a novel approach was developed for accurate absolute peptide quantification based on metal labeling with lutetium diethylenetriamine pentaacetic acid (Lu-DTPA) and nanoflow high-performance liquid chromatography-inductively coupled plasma isotope dilution mass spectrometry (nanoHPLC-ICP-IDMS). In a two-step procedure peptides were derivatized at amino groups with diethylenetriamine pentaacetic anhydride (DTPAA) followed by chelation of lutetium. Electrospray ionization mass spectrometry (ESI MS) of the reaction product demonstrated highly specific peptide labeling. Under optimized nanoHPLC conditions the labeled peptides were baseline-separated, and the excess labeling reagent did not interfere. A 176Lu-labeled spike was continuously added to the column effluent for quantification by ICP-IDMS. The recovery of a Lu-DTPA-labeled standard peptide was close to 100% indicating high labeling efficiency and accurate absolute quantification. The precision of the entire method was 4.9%. The detection limit for Lu-DTPA-tagged peptides was 179 amol demonstrating that lutetium-specific peptide quantification was by 4 orders of magnitude more sensitive than detection by natural sulfur atoms present in cysteine or methionine residues. Furthermore, the application to peptides in insulin tryptic digest allowed the identification of interfering reagents decreasing the labeling efficiency. An additional advantage of this novel approach is the analysis of peptides, which do not naturally feature ICPMS-detectable elements.

  5. Development of a lytic peptide derived from BH3-only proteins

    PubMed Central

    Liu, Q; Zhao, H; Jiang, Y; Wu, M; Tian, Y; Wang, D; Lao, Y; Xu, N; Li, Z

    2016-01-01

    Despite great advances in cancer therapy, drug resistance is a difficult hurdle to overcome that requires development of anticancer agents with novel and effective modes of action. In a number of studies, lytic peptides have shown remarkable ability to eliminate cancer cells through a different way from traditional treatments. Lytic peptides are positively charged, amphiphilic, and are efficient at binding and disrupting the negatively charged cell membrane of cancer cells. In this study, we described the anticancer properties of a lytic peptide that was developed on the basis of the alignment of amphiphilic BH3 peptides. Our results demonstrated that the positive charge and conformation constraint were favourable for efficient cancer cell elimination. Artificial BCL-2 homology 3 peptides (ABH3) exhibited effective anticancer effects against a series of cancer cell lines in vitro and in HeLa human cervical tumour xenografts in vivo. ABH3 induced cell death in an apoptosis-independent manner through the lytic properties of the peptide that caused disruption of cell membrane. Our results showed that charge tuning and conformation constraining in a lytic peptide could be applied to optimise the anticancer activity of lytic peptides. These results also suggest that ABH3 may be a promising beginning for the development of additional lytic peptides as anticancer reagents. PMID:27551502

  6. The identification of affinity peptide ligands specific to the variable region of human antibodies.

    PubMed

    Akiyama, Yasuto; Miyata, Haruo; Komiyama, Masaru; Nogami, Masahiro; Ozawa, Kazumichi; Oshita, Chie; Kume, Akiko; Ashizawa, Tadashi; Sakura, Naoki; Mochizuki, Tohru; Yamaguchi, Ken

    2014-01-01

    Of all potential biological therapeutics, monoclonal antibody (mAb)-based therapies are becoming the dominant focus of clinical research. In particular, smaller recombinant antibody fragments such as single-chain variable fragments (scFv) have become the subject of intense focus. However, an efficient affinity ligand for antibody fragment purification has not been developed. In the present study, we designed a consensus sequence for the human antibody heavy or light chain-variable regions (Fv) based on the antibody sequences available in the ImMunoGeneTics information system (IMGT), and synthesized these consensus sequences as template Fv antibodies. We then screened peptide ligands that specifically bind to the repertoire-derived human Fv consensus antibody using a 12-mer-peptide library expressed-phage display method. Subsequently, 1 peptide for the VH template and 8 peptides for the VK template were selected as the candidate ligands after 4 rounds of panning the phage display. Using peptide-bead-based immunoprecipitation, the code-4 and code-13 peptides showed recovery rates of the VH and VK templates that were 20-30% and 40-50%, respectively. Both peptides exhibited better recovery rates for trastuzumab scFv (approximately 40%). If it were possible to identify the best combination of VH and VK-binding peptides among the ligand peptides suitable for the human mAb Fv sequence, the result could be a promising purification tool that might greatly improve the cost efficiencies of the purification process.

  7. Production of bioactive peptides in an in vitro system.

    PubMed

    Ozawa, Akihiko; Cai, Yang; Lindberg, Iris

    2007-07-15

    An in vitro system for the preparation of bioactive peptides is described. This system couples three different posttranslational modification enzymes, prohormone convertases (PCs), carboxypeptidase E, and peptidyl alpha-amidating enzyme, to transform recombinant precursors into bioactive peptides. Three different precursors, mouse proopiomelanocortin (mPOMC), rat proenkephalin (rPE), and human proghrelin, were used as model systems. The conversion of mPOMC and rPE to smaller peptide products was measured by radioimmunoassay. After optimization of the system, excellent efficiency was obtained: about 85% of starting mPOMC was converted to des-acetyl alpha-melanocyte-stimulating hormone (alpha-MSH). For proenkephalin, 75 and 96% yields were obtained for the opioid peptides Met-RGL and Met-enk, respectively. Cell-based assays demonstrated that in-vitro-generated des-acetyl alpha-MSH successfully activated the melanocortin 4 receptor. Proghrelin digestion was used to screen the specificity of PC cleavage and to confirm the cleavage site by mass spectroscopy. Mature ghrelin was produced by human furin, mouse prohormone convertase 1, and human prohormone convertase 7 but not by mouse prohormone convertase 2. These results demonstrate that our in vitro system (1) can produce peptides in quantities sufficient to carry out functional analyses, (2) can be used to determine the specificity of proprotein convertases on recombinant precursors, and (3) has the potential to identify novel peptide functions on both known and orphan G-protein-coupled receptors.

  8. Peptide Directed 3D Assembly of Nanoparticles through Biomolecular Interaction

    NASA Astrophysics Data System (ADS)

    Kaur, Prerna

    The current challenge of the 'bottom up' process is the programmed self-assembly of nanoscale building blocks into complex and larger-scale superstructures with unique properties that can be integrated as components in solar cells, microelectronics, meta materials, catalysis, and sensors. Recent trends in the complexity of device design demand the fabrication of three-dimensional (3D) superstructures from multi-nanomaterial components in precise configurations. Bio mimetic assembly is an emerging technique for building hybrid materials because living organisms are efficient, inexpensive, and environmentally benign material generators, allowing low temperature fabrication. Using this approach, a novel peptide-directed nanomaterial assembly technology based on bio molecular interaction of streptavidin and biotin is presented for assembling nanomaterials with peptides for the construction of 3D peptide-inorganic superlattices with defined 3D shape. We took advantage of robust natural collagen triple-helix peptides and used them as nanowire building blocks for 3D peptide-gold nanoparticles superlattice generation. The type of 3D peptide superlattice assembly with hybrid NP building blocks described herein shows potential for the fabrication of complex functional device which demands precise long-range arrangement and periodicity of NPs.

  9. Recognition of Bacterial Signal Peptides by Mammalian Formyl Peptide Receptors

    PubMed Central

    Bufe, Bernd; Schumann, Timo; Kappl, Reinhard; Bogeski, Ivan; Kummerow, Carsten; Podgórska, Marta; Smola, Sigrun; Hoth, Markus; Zufall, Frank

    2015-01-01

    Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system. PMID:25605714

  10. Western blot analysis of Src kinase assays using peptide substrates ligated to a carrier protein.

    PubMed

    Xu, Jie; Sun, Luo; Ghosh, Inca; Xu, Ming-Qun

    2004-06-01

    We have applied intein-mediated peptide ligation (IPL) to the use of peptide substrates for kinase assays and subsequent Western blot analysis. IPL allows for the efficient ligation of a synthetic peptide with an N-terminal cysteine residue to an intein-generated carrier protein containing a cysteine reactive C-terminal thioester through a native peptide bond. A distinct advantage of this procedure is that each carrier protein molecule ligates only one peptide, ensuring that the ligation product forms a sharp band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrate the effectiveness of this approach by mutational analysis of peptide substrates derived from human cyclin-dependent kinase, Cdc2, which contains a phosphorylation site of human c-Src protein tyrosine kinase.

  11. Histidine-containing peptide catalysts developed by a facile library screening method.

    PubMed

    Akagawa, Kengo; Sakai, Nobutaka; Kudo, Kazuaki

    2015-02-02

    Although peptide catalysts have a high potential for the use as organocatalysts, the optimization of peptide sequences is laborious and time-consuming. To address this issue, a facile screening method for finding efficient aminocatalysts from a peptide library has been developed. In the screening for the Michael addition of a malonate to an enal, a dye-labeled product is immobilized on resin-bound peptides through reductive amination to visualize active catalysts. This procedure allows for the monitoring of the reactivity of entire peptides without modifying the resin beads beforehand. Peptides containing histidine at an appropriate position were identified by this method. A novel function of the histidyl residue, which enhances the binding of a substrate to the catalyst by capturing an iminium intermediate, was indicated.

  12. CXCR4-antagonist Peptide R-liposomes for combined therapy against lung metastasis

    NASA Astrophysics Data System (ADS)

    Ieranò, Caterina; Portella, Luigi; Lusa, Sara; Salzano, Giuseppina; D'Alterio, Crescenzo; Napolitano, Maria; Buoncervello, Maria; Macchia, Daniele; Spada, Massimo; Barbieri, Antonio; Luciano, Antonio; Barone, Maria Vittoria; Gabriele, Lucia; Caraglia, Michele; Arra, Claudio; De Rosa, Giuseppe; Scala, Stefania

    2016-03-01

    The chemokine CXCL12 activates CXCR4, initiating multiple pathways that control immune cell trafficking, angiogenesis and embryogenesis; CXCR4 is also overexpressed in multiple tumors affecting metastatic dissemination. While there has been great enthusiasm for exploiting the CXCR4-CXCL12 axis as a target in cancer therapy, to date the promise has yet to be fulfilled. A new class of CXCR4-antagonist cyclic peptides was recently developed and the compound named Peptide R was identified as the most active. With the intent to improve the efficacy and biodistribution of Peptide R, stealth liposomes decorated with Peptide R were developed (PL-Peptide R). In vitro PL-Peptide R efficiently inhibited CXCR4-dependent migration and in vivo it significantly reduced lung metastases and increased overall survival in B16-CXCR4 injected C57BL/6 mice. To evaluate if PL-Peptide R could also be a drug delivery system for CXCR4 expressing tumors, the PL-Peptide R was loaded with doxorubicin (DOX) (PL-Peptide R-DOX). PL-Peptide R-DOX efficiently delivered DOX to CXCR4 expressing cell lines with a consequent decrease in the DOX IC50 efficient dose. In vivo, B16-CXCR4 injected C57BL/6 mice treated with PL-Peptide R-DOX developed fewer lung metastases compared to PL-DOX treated mice. This work provides the proof-of-concept to prevent metastasis by using combined nanomedicine.The chemokine CXCL12 activates CXCR4, initiating multiple pathways that control immune cell trafficking, angiogenesis and embryogenesis; CXCR4 is also overexpressed in multiple tumors affecting metastatic dissemination. While there has been great enthusiasm for exploiting the CXCR4-CXCL12 axis as a target in cancer therapy, to date the promise has yet to be fulfilled. A new class of CXCR4-antagonist cyclic peptides was recently developed and the compound named Peptide R was identified as the most active. With the intent to improve the efficacy and biodistribution of Peptide R, stealth liposomes decorated with Peptide

  13. Design of a multicomponent peptide-woven nanocomplex for delivery of siRNA.

    PubMed

    Jun, Eunsung; Kim, Soyoun; Kim, Jong-Ho; Cha, Kiweon; So, In-Seop; Son, Hye-Nam; Lee, Byung-Heon; Kim, Kwangmeyung; Kwon, Ick Chan; Kim, Sang Yoon; Kim, In-San

    2015-01-01

    We developed and tested a multicomponent peptide-woven siRNA nanocomplex (PwSN) comprising different peptides designed for efficient cellular targeting, endosomal escape, and release of siRNA. To enhance tumor-specific cellular uptake, we connected an interleukin-4 receptor-targeting peptide (I4R) to a nine-arginine peptide (9r), yielding I4R-9r. To facilitate endosomal escape, we blended endosomolytic peptides into the I4R-9r to form a multicomponent nanocomplex. Lastly, we modified 9r peptides by varying the number and positions of positive charges to obtain efficient release of siRNA from the nanocomplex in the cytosol. Using this step-wise approach for overcoming the biological challenges of siRNA delivery, we obtained an optimized PwSN with significant biological activity in vitro and in vivo. Interestingly, surface plasmon resonance analyses and three-dimensional peptide models demonstrated that our designed peptide adopted a unique structure that was correlated with faster complex disassembly and a better gene-silencing effect. These studies further elucidate the siRNA nanocomplex delivery pathway and demonstrate the applicability of our stepwise strategy to the design of siRNA carriers capable of overcoming multiple challenges and achieving efficient delivery.

  14. Cell-Penetrating Ability of Peptide Hormones: Key Role of Glycosaminoglycans Clustering.

    PubMed

    Neree, Armelle Tchoumi; Nguyen, Phuong Trang; Bourgault, Steve

    2015-11-16

    Over the last two decades, the potential usage of cell-penetrating peptides (CPPs) for the intracellular delivery of various molecules has prompted the identification of novel peptidic identities. However, cytotoxic effects and unpredicted immunological responses have often limited the use of various CPP sequences in the clinic. To overcome these issues, the usage of endogenous peptides appears as an appropriate alternative approach. The hormone pituitary adenylate-cyclase-activating polypeptide (PACAP38) has been recently identified as a novel and very efficient CPP. This 38-residue polycationic peptide is a member of the secretin/glucagon/growth hormone-releasing hormone (GHRH) superfamily, with which PACAP38 shares high structural and conformational homologies. In this study, we evaluated the cell-penetrating ability of cationic peptide hormones in the context of the expression of cell surface glycosaminoglycans (GAGs). Our results indicated that among all peptides evaluated, PACAP38 was unique for its potent efficiency of cellular uptake. Interestingly, the abilities of the peptides to reach the intracellular space did not correlate with their binding affinities to sulfated GAGs, but rather to their capacity to clustered heparin in vitro. This study demonstrates that the uptake efficiency of a given cationic CPP does not necessarily correlate with its affinity to sulfated GAGs and that its ability to cluster GAGs should be considered for the identification of novel peptidic sequences with potent cellular penetrating properties.

  15. Design of a Multicomponent Peptide-Woven Nanocomplex for Delivery of siRNA

    PubMed Central

    Jun, Eunsung; Kim, Soyoun; Kim, Jong-Ho; Cha, Kiweon; So, In-Seop; Son, Hye-Nam; Lee, Byung-Heon; Kim, Kwangmeyung; Kwon, Ick Chan; Kim, Sang Yoon; Kim, In-San

    2015-01-01

    We developed and tested a multicomponent peptide-woven siRNA nanocomplex (PwSN) comprising different peptides designed for efficient cellular targeting, endosomal escape, and release of siRNA. To enhance tumor-specific cellular uptake, we connected an interleukin-4 receptor-targeting peptide (I4R) to a nine-arginine peptide (9r), yielding I4R-9r. To facilitate endosomal escape, we blended endosomolytic peptides into the I4R-9r to form a multicomponent nanocomplex. Lastly, we modified 9r peptides by varying the number and positions of positive charges to obtain efficient release of siRNA from the nanocomplex in the cytosol. Using this step-wise approach for overcoming the biological challenges of siRNA delivery, we obtained an optimized PwSN with significant biological activity in vitro and in vivo. Interestingly, surface plasmon resonance analyses and three-dimensional peptide models demonstrated that our designed peptide adopted a unique structure that was correlated with faster complex disassembly and a better gene-silencing effect. These studies further elucidate the siRNA nanocomplex delivery pathway and demonstrate the applicability of our stepwise strategy to the design of siRNA carriers capable of overcoming multiple challenges and achieving efficient delivery. PMID:25705892

  16. Polar profile of antiviral peptides from AVPpred Database.

    PubMed

    Polanco, Carlos; Samaniego, José Lino; Castañón-González, Jorge Alberto; Buhse, Thomas

    2014-11-01

    Diseases of viral origin in humans are among the most serious threats to health and the global economy. As recent history has shown the virus has a high pandemic potential, among other reasons, due to its ability to spread by air, hence the identification, investigation, containment, and treatment of viral diseases should be considered of paramount importance. In this sense, the bioinformatics research has focused on finding fast and efficient algorithms that can identify highly toxic antiviral peptides and to serve as a first filter, so that trials in the laboratory are substantially reduced. The work presented here contributes to this effort through the use of an algorithm already published by this team, called polarity index method, which identifies with high efficiency antiviral peptides from the exhaustive analysis of the polar profile, using the linear sequence of the peptide. The test carried out included all peptides in APD2 Database and 60 antiviral peptides identified by Kumar and co-workers (Nucleic Acids Res 40:W199-204, 2012), to build its AVPpred algorithm. The validity of the method was focused on its discriminating capacity so we included the 15 sub-classifications of both Databases.

  17. Clinical uses of gut peptides.

    PubMed Central

    Geoghegan, J; Pappas, T N

    1997-01-01

    OBJECTIVE: The authors review clinical applications of gut-derived peptides as diagnostic and therapeutic agents. SUMMARY BACKGROUND DATA: An increasing number of gut peptides have been evaluated for clinical use. Earlier uses as diagnostic agents have been complemented more recently by increasing application of gut peptides as therapeutic agents. METHOD: The authors conducted a literature review. RESULTS: Current experience with clinical use of gut peptides is described. Initial clinical applications focused on using secretomotor effects of gut peptides in diagnostic tests, many of which have now fallen into disuse. More recently, attention has been directed toward harnessing these secretomotor effects for therapeutic use in a variety of disorders, and also using the trophic effects of gut peptides to modulate gut mucosal growth in benign and malignant disease. Gut peptides have been evaluated in a variety of other clinical situations including use as adjuncts to imaging techniques, and modification of behaviors such as feeding and panic disorder. CONCLUSIONS: Gut peptides have been used successfully in an increasing variety of clinical conditions. Further refinements in analogue and antagonist design are likely to lead to even more selective agents that may have important clinical applications. Further studies are needed to identity and evaluate these new agents. PMID:9065291

  18. Urinary Peptides in Rett Syndrome.

    ERIC Educational Resources Information Center

    Solaas, K. M.; Skjeldal, O.; Gardner, M. L. G.; Kase, B. F.; Reichelt, K. L.

    2002-01-01

    A study found a significantly higher level of peptides in the urine of 53 girls with Rett syndrome compared with controls. The elevation was similar to that in 35 girls with infantile autism. Levels of peptides were lower in girls with classic Rett syndrome than those with congenital Rett syndrome. (Contains references.) (Author/CR)

  19. Engineered peptide-based nanobiomaterials for electrochemical cell chip

    NASA Astrophysics Data System (ADS)

    Kafi, Md. Abdul; Cho, Hyeon-Yeol; Choi, Jeong-Woo

    2016-07-01

    Biomaterials having cell adhesion ability are considered to be integral part of a cell chip. A number of researches have been carried out to search for a suitable material for effective immobilization of cell on substrate. Engineered ECM materials or their components like collagen, Poly- l-Lysine (PLL), Arg-Gly-Asp (RGD) peptide have been extensively used for mammalian cell adhesion and proliferation with the aim of tissue regeneration or cell based sensing application. This review focuses on the various approaches for two- and three-dimensionally patterned nanostructures of a short peptide i.e. RGD peptide on chip surfaces together with their effects on cell behaviors and electrochemical measurements. Most of the study concluded with positive remarks on the well-oriented engineered RGD peptide over their homogenous thin film. The engineered RGD peptide not only influences cell adhesion, spreading and proliferation but also their periodic nano-arrays directly influence electrochemical measurements of the chips. The electrochemical signals found to be enhanced when RGD peptides were used in well-defined two-dimensional nano-arrays. The topographic alteration of three-dimensional structure of engineered RGD peptide was reported to be suitably contacted with the integrin receptors of cellular membrane which results indicated the enhanced cell-electrode adhesion and efficient electron exchange phenomenon. This enhanced electrochemical signal increases the sensitivity of the chip against the target analytes. Therefore, development of engineered cellular recognizable peptides and its 3D topological design for fabrication of cell chip will provide the synergetic effect on bio-affinity, sensitivity and accuracy for the in situ real-time monitoring of analytes.

  20. Removal of detergents from proteins and peptides in a spin-column format.

    PubMed

    Antharavally, Babu S

    2012-08-01

    To enable downstream analysis, it is critical to remove unbound detergents from protein and peptide samples. This unit describes the use of a high-performance resin that offers exceptional detergent removal for proteins and peptides. The easy-to-use spin format significantly improves results over the standard drip column and batch methodologies, with >95% removal of 1% to 5% detergents, including SDS, sodium deoxycholate, CHAPS, Triton X-100, Triton X-114, NP-40, Brij-35, octyl glucoside, octyl thioglucoside, and lauryl maltoside, with high recovery of proteins and peptides. Detergent removal efficiency is evaluated using colorimetric methods and mass spectrometry (MS). BSA tryptic peptides have been successfully analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption/ionization (MALDI)-MS for identification of protein, following detergent removal using the resin. Advantages of this method include speed (less than 15 min), efficient detergent removal, and high recovery of proteins and peptides.

  1. Bioactive peptides derived from food.

    PubMed

    Rutherfurd-Markwick, Kay J; Moughan, Paul J

    2005-01-01

    As interest in the ability of functional foods to impact on human health has grown over the past decade, so has the volume of knowledge detailing the beneficial roles of food-derived bioactive peptides. Bioactive peptides from both plant and animal proteins have been discovered, with to date, by far the most being isolated from milk-based products. A wide range of activities has been described, including antimicrobial and antifungal properties, blood pressure-lowering effects, cholesterol-lowering ability, antithrombotic effects, enhancement of mineral absorption, immunomodulatory effects, and localized effects on the gut. Although there is still considerable research to be performed in the area of food-derived bioactive peptides, it is clear that the generation of bioactive peptides from dietary proteins during the normal digestive process is of importance. Therefore, it will become necessary when determining dietary protein quality to consider the potential effects of latent bioactive peptides that are released during digestion of the protein.

  2. Microscopic characterization of peptide nanostructures.

    PubMed

    Mammadov, Rashad; Tekinay, Ayse B; Dana, Aykutlu; Guler, Mustafa O

    2012-02-01

    Peptide-based nanomaterials have been utilized for various applications from regenerative medicine to electronics since they provide several advantages including easy synthesis methods, numerous routes for functionalization and biomimicry of secondary structures of proteins which leads to design of self-assembling peptide molecules to form nanostructures. Microscopic characterization at nanoscale is critical to understand processes directing peptide molecules to self-assemble and identify structure-function relationship of the nanostructures. Here, fundamental studies in microscopic characterization of peptide nanostructures are discussed to provide insights in widely used microscopy tools. In this review, we will encompass characterization studies of peptide nanostructures with modern microscopes, such as TEM, SEM, AFM, and advanced optical microscopy techniques. We will also mention specimen preparation methods and describe interpretation of the images.

  3. Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli

    PubMed Central

    Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Sgambati, Valeria; Di Maro, Antimo; Carpentieri, Andrea; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

    2016-01-01

    Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200–250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15–18 mg of recombinant peptide per liter of culture with 96–98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods. PMID:26808536

  4. Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli.

    PubMed

    Pane, Katia; Durante, Lorenzo; Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Sgambati, Valeria; Di Maro, Antimo; Carpentieri, Andrea; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

    2016-01-01

    Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200-250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15-18 mg of recombinant peptide per liter of culture with 96-98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods.

  5. Asymmetric Peptide Nanoribbons.

    PubMed

    Yu, Zhilin; Tantakitti, Faifan; Palmer, Liam C; Stupp, Samuel I

    2016-11-09

    Asymmetry in chemical structure or shape at molecular, nanoscale, or microscopic levels is essential to a vast number of functionalities in both natural and artificial systems. Bottom-up approaches to create asymmetric supramolecular nanostructures are considered promising but this strategy suffers from the potentially dynamic nature of noncovalent interactions. We report here on supramolecular self-assembly of asymmetric peptide amphiphiles consisting of two different molecularly linked domains. We found that strong noncovalent interactions and a high degree of internal order among the asymmetric amphiphiles lead to nanoribbons with asymmetric faces due to the preferential self-association of the two domains. The capture of gold nanoparticles on only one face of the nanoribbons demonstrates symmetry breaking in these supramolecular structures.

  6. Antimicrobial peptides: therapeutic potentials.

    PubMed

    Kang, Su-Jin; Park, Sung Jean; Mishig-Ochir, Tsogbadrakh; Lee, Bong-Jin

    2014-12-01

    The increasing appearance of multidrug-resistant pathogens has created an urgent need for suitable alternatives to current antibiotics. Antimicrobial peptides (AMPs), which act as defensive weapons against microbes, have received great attention because of broad-spectrum activities, unique action mechanisms and rare antibiotic-resistant variants. Despite desirable characteristics, they have shown limitations in pharmaceutical development due to toxicity, stability and manufacturing costs. Because of these drawbacks, only a few AMPs have been tested in Phase III clinical trials and no AMPs have been approved by the US FDA yet. However, these obstacles could be overcome by well-known methods such as changing physicochemical characteristics and introducing nonnatural amino acids, acetylation or amidation, as well as modern techniques like molecular targeted AMPs, liposomal formulations and drug delivery systems. Thus, the current challenge in this field is to develop therapeutic AMPs at a reasonable cost as well as to overcome the limitations.

  7. Analysis of Intrinsic Peptide Detectability via Integrated Label-Free and SRM-Based Absolute Quantitative Proteomics.

    PubMed

    Jarnuczak, Andrew F; Lee, Dave C H; Lawless, Craig; Holman, Stephen W; Eyers, Claire E; Hubbard, Simon J

    2016-09-02

    Quantitative mass spectrometry-based proteomics of complex biological samples remains challenging in part due to the variability and charge competition arising during electrospray ionization (ESI) of peptides and the subsequent transfer and detection of ions. These issues preclude direct quantification from signal intensity alone in the absence of a standard. A deeper understanding of the governing principles of peptide ionization and exploitation of the inherent ionization and detection parameters of individual peptides is thus of great value. Here, using the yeast proteome as a model system, we establish the concept of peptide F-factor as a measure of detectability, closely related to ionization efficiency. F-factor is calculated by normalizing peptide precursor ion intensity by absolute abundance of the parent protein. We investigated F-factor characteristics in different shotgun proteomics experiments, including across multiple ESI-based LC-MS platforms. We show that F-factors mirror previously observed physicochemical predictors as peptide detectability but demonstrate a nonlinear relationship between hydrophobicity and peptide detectability. Similarly, we use F-factors to show how peptide ion coelution adversely affects detectability and ionization. We suggest that F-factors have great utility for understanding peptide detectability and gas-phase ion chemistry in complex peptide mixtures, selection of surrogate peptides in targeted MS studies, and for calibration of peptide ion signal in label-free workflows. Data are available via ProteomeXchange with identifier PXD003472.

  8. Peptide-formation on cysteine-containing peptide scaffolds

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  9. Cell-penetrating peptides: strategies for anticancer treatment.

    PubMed

    Raucher, Drazen; Ryu, Jung Su

    2015-09-01

    Cell-penetrating peptides (CPP) provide an efficient strategy for the intracellular delivery of bioactive molecules in various biomedical applications. This review focuses on recent advances in the use of CPPs to deliver anticancer therapeutics and imaging reagents to cancer cells, along with CPP contributions to novel tumor-targeting techniques. CPPs are now used extensively to deliver a variety of therapeutics, despite lacking cell specificity and having a short duration of action. Resolution of these shortcomings to enable increased cancer cell and/or tumor specificity could improve CPP-based drug delivery strategies, expand combined drug delivery possibilities, and strengthen future clinical applications of these peptides.

  10. Epimerization in peptide thioester condensation.

    PubMed

    Teruya, Kenta; Tanaka, Takeyuki; Kawakami, Toru; Akaji, Kenichi; Aimoto, Saburo

    2012-11-01

    Peptide segment couplings are now widely utilized in protein chemical synthesis. One of the key structures for the strategy is the peptide thioester. Peptide thioester condensation, in which a C-terminal peptide thioester is selectively activated by silver ions then condensed with an amino component, is a powerful tool. But the amino acid adjacent to the thioester is at risk of epimerization. During the preparation of peptide thioesters by the Boc solid-phase method, no substantial epimerization of the C-terminal amino acid was detected. Epimerization was, however, observed during a thioester-thiol exchange reaction and segment condensation in DMSO in the presence of a base. In contrast, thioester-thiol exchange reactions in aqueous solutions gave no epimerization. The epimerization during segment condensation was significantly suppressed with a less polar solvent that is applicable to segments in thioester peptide condensation. These results were applied to a longer peptide thioester condensation. The epimer content of the coupling product of 89 residues was reduced from 27% to 6% in a condensation between segments of 45 and 44 residues for the thioester and the amino component, respectively.

  11. Enhancement of gene delivery using novel homodimeric tat peptide formed by disulfide bond.

    PubMed

    Lee, Soo-Jin; Yoon, Sung-Hwa; Doh, Kyung-Oh

    2011-08-01

    Cationic liposomes have been actively used as gene delivery vehicle because of their minimal toxicity, but their relatively low efficiency of gene delivery is the major disadvantage of these vectors. Recently, cysteine residue incorporation to HIV-1 Tat peptide increased liposomemediated transfection compared with unmodified Tat peptide. Therefore, we designed a novel modified Tat peptide having a homodimeric (Tat-CTHD, Tat-NTHD) and closed structure (cyclic Tat) simply by using the disulfide bond between cysteines to develop a more efficient and safe nonviral gene delivery system. The mixing of Tat-CTHD and Tat-NTHD with DNA before mixing with lipofectamine increased the transfection efficiency compared with unmodified Tat peptide and lipofectamine only in MCF-7 breast cancer cells and rat vascular smooth muscle cells. However, cyclic Tat did not show any improvement in the transfection efficiency. In the gel retardation assay, Tat-CTHD and Tat-NTHD showed more strong binding with DNA than unmodified Tat and cyclic Tat peptide. This enhancement was only shown when Tat-CTHD and Tat-NTHD were mixed with DNA before mixing with lipofectamine. The effects of Tat- CTHD and Tat-NTHD were also valid in the experiment using DOTAP and DMRIE instead of lipofectamine. We could not find any significant cytotoxicity in the working concentration and more usage of these peptides. In conclusion, we have designed a novel transfection-enhancing peptide by easy homodimerization of Tat peptide, and the simple mix of these novel peptides with DNA increased the gene transfer of cationic lipids more efficiently with no additional cytotoxicity.

  12. Characterization of the triphenylphosphonium derivative of peptides by fast atom bombardment-tandem mass spectrometry, and investigations of the mechanisms of fragmentation of peptides

    SciTech Connect

    Wagner, D.S.

    1992-01-01

    Fast atom bombardment collisionally activated dissociation tandem mass spectrometry is a powerful technique for the determination of the primary structure of peptides. However, there are factors that frequently prevent successful sequence analysis by mass spectrometry. Two such factors are the poor ionization efficiency of some hydrophilic peptides and, for many peptides, ambiguities in interpretation of the spectra when key sequence ions are weak or absent. Novel and simple procedures for preparing ethyl-triphenylphosphonium derivatives of peptides are described. These procedures allow an ethyl-triphenylphosphonium moiety to be selectively attached to either the N- or C-terminus. Modification of peptides by these chemical methods significantly enhances the efficiency of fast atom bombardment ionization. Moreover, upon collisionally activated dissociation, the derivatized peptides generate a predictable series of sequence ions from either the C-terminus or the N-terminus, depending on the location of the ethyl-triphenylphosphonium moiety. The potential utility of the ethyl-triphenylphosphonium derivative in structure elucidation is illustrated by a comparison of the mass spectra of underivatized and derivatized peptides containing up to 20 amino acid residues, or contain an N-terminal blocking group, or contain a phosphate group, or contain a disulfide bond, or contain a backbone modification. When protonated peptide molecules and cationized peptide molecules are subjected to high-energy collisionally activated dissociation, skeletal bonds cleave generating sequence-specific fragment ions. These bond cleavages usually involve H-shifts. The utility of selective deuterium labeling was applied here to elucidate fragmentation mechanisms. Skeletal bond cleavages in the ionized peptide H-VGVAPG-OH were investigated, in which the molecule was analyzed in the protonated form, cationized form, or as the charge-localized ethyl-triphenylphosphonium derivative.

  13. Multidimensional peptide separations in proteomics.

    PubMed

    Link, Andrew J

    2002-12-01

    Multidimensional peptide separation will play an increasingly important role in the drive to identify and quantitate the proteome. By increasing the peak and load capacity, multidimensional approaches increase the number and dynamic range of peptides that can be analyzed in a complex biological organism. Separation methods using different physical properties of peptides have been combined with varying degrees of success. The ultimate goal is a rapid separation strategy that can be coupled with analytical methods, such as mass spectrometry, to provide comprehensive monitoring of the changing concentration, interactions, and structures of proteins in the proteome.

  14. Evidence that transporters associated with antigen processing translocate a major histocompatibility complex class I-binding peptide into the endoplasmic reticulum in an ATP-dependent manner.

    PubMed Central

    Androlewicz, M J; Anderson, K S; Cresswell, P

    1993-01-01

    We have investigated the role of the putative peptide transporters associated with antigen processing (TAP) by using a permeabilized-cell system. The main objective was to determine whether these molecules, which bear homology to the ATP-binding cassette family of transporters, translocate antigenic peptides across the endoplasmic reticulum membrane for assembly with major histocompatibility complex (MHC) class I molecules and beta 2-microglobulin light chain. The pore-forming toxin streptolysin O was used to generate permeabilized cells, and peptide translocation was determined by measuring the amount of added radiolabeled peptide bound to endogenous class I molecules. No radiolabeled peptide was associated with MHC class I glycoproteins from unpermeabilized cells. We found that efficient peptide binding to MHC class I molecules in permeabilized cells is both transporter dependent and ATP dependent. In antigen-processing mutant cells lacking a functional transporter, uptake occurs only through a less-efficient transporter and ATP-independent pathway. In addition, short peptides (8-10 amino acids) known to bind MHC class I molecules compete efficiently with a radiolabeled peptide for TAP-dependent translocation, whereas longer peptides and a peptide derived from an endoplasmic reticulum signal sequence do not compete efficiently. This result indicates that the optimal substrates for TAP possess the characteristics of MHC-binding peptides. Images Fig. 2 Fig. 3 Fig. 4 PMID:8415666

  15. Spontaneous intermolecular amide bond formation between side chains for irreversible peptide targeting.

    PubMed

    Zakeri, Bijan; Howarth, Mark

    2010-04-07

    Peptides and synthetic peptide-like molecules are powerful tools for analysis and control of biological function. One major limitation of peptides is the instability of their interactions with biomolecules, because of the limited accessible surface area for noncovalent interactions and the intrinsic flexibility of peptides. Peptide tags are nonetheless fundamental for protein detection and purification, because their small size minimizes the perturbation to protein function. Here we have designed a 16 amino acid peptide that spontaneously forms an amide bond to a protein partner, via reaction between lysine and asparagine side chains. This depended upon splitting a pilin subunit from a human pathogen, Streptococcus pyogenes, which usually undergoes intramolecular amide bond formation to impart mechanical and proteolytic stability to pili. Reaction of the protein partner was able to proceed to 98% conversion. The amide bond formation was independent of redox state and occurred at pH 5-8. The reaction was efficient in phosphate buffered saline and a wide range of biological buffers. Surprisingly, amide bond formation occurred at a similar rate at 4 and 37 degrees C. Both peptide and protein partners are composed of the regular 20 amino acids and reconstituted efficiently inside living E. coli. Labeling also showed high specificity on the surface of mammalian cells. Irreversible targeting of a peptide tag may have application in bioassembly, in cellular imaging, and to lock together proteins subject to high biological forces.

  16. Highly Efficient Proteolysis Accelerated by Electromagnetic Waves for Peptide Mapping

    PubMed Central

    Chen, Qiwen; Liu, Ting; Chen, Gang

    2011-01-01

    Proteomics will contribute greatly to the understanding of gene functions in the post-genomic era. In proteome research, protein digestion is a key procedure prior to mass spectrometry identification. During the past decade, a variety of electromagnetic waves have been employed to accelerate proteolysis. This review focuses on the recent advances and the key strategies of these novel proteolysis approaches for digesting and identifying proteins. The subjects covered include microwave-accelerated protein digestion, infrared-assisted proteolysis, ultraviolet-enhanced protein digestion, laser-assisted proteolysis, and future prospects. It is expected that these novel proteolysis strategies accelerated by various electromagnetic waves will become powerful tools in proteome research and will find wide applications in high throughput protein digestion and identification. PMID:22379392

  17. Separation of Peptides on HALO 2-Micron Particles.

    PubMed

    Mant, Colin T; Hodges, Robert S

    2016-08-01

    Reversed-phase high-performance liquid chromatography (RP-HPLC) is of fundamental importance to the isolation and separation of peptides, proteins, and other biomolecules. Hence, there is a continuing high demand for the development of RP-HPLC stationary-phase materials with enhanced separation efficiency. HALO packing materials began the revolution in "core-shell" technology with the advantages of faster separations, higher resolution and peak capacity, high temperature stability, and rugged reliable performance compared to traditional HPLC and UHPLC. These materials are characterized by a solid core surrounded by a thin layer of porous material, and represent a technology for the future with continuing refinements. Such refinements are aided via the use of designed synthetic peptide standards during stationary-phase development. Concomitantly, such standards also enable the researcher to monitor RP-HPLC column performance and develop optimized separation protocols for peptides from a wide array of sources. © 2016 by John Wiley & Sons, Inc.

  18. Mechanism Matters: A Taxonomy of Cell Penetrating Peptides

    PubMed Central

    Kauffman, W. Berkeley; Fuselier, Taylor; He, Jing; Wimley, William C.

    2016-01-01

    The permeability barrier imposed by cellular membranes limits the access of exogenous compounds to the interior of cells. Researchers and patients alike would benefit from efficient methods for intracellular delivery of a wide range of membrane-impermeant molecules, including biochemically active small molecules, imaging agents, peptides, peptide nucleic acids, proteins, RNA, DNA, and nanoparticles. There has been a sustained effort to exploit cell penetrating peptides (CPPs) for the delivery of such useful cargoes in vitro and in vivo because of their biocompatibility, ease of synthesis, and controllable physical chemistry. Here, we discuss the many mechanisms by which CPPs can function, and describe a taxonomy of mechanisms that could be help organize future efforts in the field. PMID:26545486

  19. Early peptidic enzymes

    NASA Astrophysics Data System (ADS)

    Brack, André; Barbier, Bernard

    Oligopeptides supposed to be essential to primitive living cells could not be obtained by a prebiotic organic chemistry working mainly at random. Selection pathways were required. Experimental evidence is given for selective condensation of amino-acids in water as well as for selective resistance to degradation. Polycationic polypeptides containing lysyl (or arginyl) and hydrophobic residues strongly accelerate the hydrolysis of oligoribonucleotides. A ionic complex is first formed and the polypeptides are particularly active when they adopt a stable conformation, β-sheet or α-helix, in the complex. Well-defined short peptides were synthesized in order to determine the critical chain-length required for chemical activity. In a contemporary cell, proteins represent about 40 % of the dry weight. They fulfil a structural role and they are particularly helpful as chemical catalysts (enzymes). They can be represented as long chains made of twenty different building blocks, the amino-acids NH2-CHR-COOH, which differ by the side-chain R. Proteins are remarkable in the sense that they use amino-acids having only one carbon atom between the -NH2 and -COOH functions. The central carbon atom has always the same spatial asymmetry (chirality) and always bears a hydrogen atom. When the side-chain R is a hydrocarbon, it is branched. When R contains a chemical function, the side functions do not participate to the peptide bond construction. The protein chain results from the condensation of amino-acids, i.e. water molecules are removed between molecules in a medium which is mainly aqueous (the cell contains 75 % of water). The protein chains adopt rigid asymmetric conformations (α-helices, β-sheet structures) which are essential for the protein functions. Proteins, even the smallest ones, are too sophisticated entities to be considered as the products of an organic chemistry working at random, without any chemical selection. The chemist has therefore to understand, with simple

  20. MULTIVALENT DISPLAY OF PENDANT PRO-APOPTOTIC PEPTIDES INCREASES CYTOTOXIC ACTIVITY

    PubMed Central

    Chu, David S.H.; Bocek, Michael J.; Shi, Julie; Ta, Anh; Ngambenjawong, Chayanon; Rostomily, Robert C.; Pun, Suzie H.

    2015-01-01

    Several cationic antimicrobial peptides have been investigated as potential anti-cancer drugs due to their demonstrated selective toxicity towards cancer cells relative to normal cells. For example, intracellular delivery of KLA, a pro-apoptotic peptide, results in toxicity against a variety of cancer cell lines; however, the relatively low activity and small size leads to rapid renal excretion when applied in vivo, limiting its therapeutic potential. In this work, apoptotic peptide-polymer hybrid materials were developed to increase apoptotic peptide activity via multivalent display. Multivalent peptide materials were prepared with comb-like structure by RAFT copolymerization of peptide macromonomers with N-(2-hydroxypropyl) methacrylamide (HPMA). Polymers displayed a GKRK peptide sequence for targeting p32, a protein often overexpressed on the surface of cancer cells, either fused with or as a comonomer to a KLA macromonomer. In three tested cancer cell lines, apoptotic polymers were significantly more cytotoxic than free peptides as evidenced by an order of magnitude decrease in IC50 values for the polymers compared to free peptide. The uptake efficiency and intracellular trafficking of one polymer construct was determined by radiolabeling and subcellular fractionation. Despite their more potent cytotoxic profile, polymeric KLA constructs have poor cellular uptake efficiency (<1%). A significant fraction (20%) of internalized constructs localize with intact mitochondrial fractions. In an effort to increase cellular uptake, polymer amines were converted to guanidines by reaction with O-methylisourea. Guanidinylated polymers disrupted function of isolated mitochondria more than their lysine-based analogs, but overall toxicity was decreased, likely due to inefficient mitochondrial trafficking. Thus, while multivalent KLA polymers are more potent than KLA peptides, these materials can be substantially improved by designing next generation materials with improved

  1. Fully Blind Docking at the Atomic Level for Protein-Peptide Complex Structure Prediction.

    PubMed

    Yan, Chengfei; Xu, Xianjin; Zou, Xiaoqin

    2016-10-04

    Protein-peptide interactions play an important role in many cellular processes. In silico prediction of protein-peptide complex structure is highly desirable for mechanistic investigation of these processes and for therapeutic design. However, predicting all-atom structures of protein-peptide complexes without any knowledge about the peptide binding site and the bound peptide conformation remains a big challenge. Here, we present a docking-based method for predicting protein-peptide complex structures, referred to as MDockPeP, which starts with the peptide sequence and globally docks the all-atom, flexible peptide onto the protein structure. MDockPeP was tested on the peptiDB benchmarking database using both bound and unbound protein structures. The results show that MDockPeP successfully generated near-native peptide binding modes in 95.0% of the bound docking cases and in 92.2% of the unbound docking cases. The performance is significantly better than other existing docking methods. MDockPeP is computationally efficient and suitable for large-scale applications.

  2. Intracellular delivery of tumor antigenic peptides in biodegradable-polymer adjuvant for enhancing cancer immunotherapy.

    PubMed

    Li, H; Ma, W

    2014-01-01

    Tumor antigenic peptides therapeutics is a promising field for cancer immunotherapy; advantages include convenient synthesis and modification of antigenic peptides, as well as little toxicity associated with its administration. Vaccination of the peptides derived from tumor-associated antigen (TAA) was specifically designed for T cells in the context of MHC molecules. In the past decades, tumor antigenic peptides have been examined in clinic but numbered success has been obtained because of the stability of peptide and delivery approaches, consequently leading to an inefficient antigen presentation and low response rates in cancer patients. Thus, the appropriate and efficient peptide vaccine carrier systems still continue to be a major obstacle. However, both sipuleucel-T vaccine and anti-CTLA-4 antibody have been approved by FDA for the treatment of metastatic prostate cancer and melanoma, respectively. PLGA has been recently used as the adjuvant to elicit enhanced immune responses while delivering tumor antigenic peptides. Intracellular delivery of the peptides derived from TAA into DCs would prolong antigen presentation of APC to T cells. This article aims to describe a new delivery method regarding tumor antigenic peptides and rationales of DCs-based vaccination.

  3. Evaluation of a cell penetrating prenylated peptide lacking an intrinsic fluorophore via in situ click reaction.

    PubMed

    Ochocki, Joshua D; Mullen, Daniel G; Wattenberg, Elizabeth V; Distefano, Mark D

    2011-09-01

    Protein prenylation involves the addition of either a farnesyl (C(15)) or geranylgeranyl (C(20)) isoprenoid moiety onto the C-terminus of many proteins. This natural modification serves to direct a protein to the plasma membrane of the cell. A recently discovered application of prenylated peptides is that they have inherent cell-penetrating ability, and are hence termed cell penetrating prenylated peptides. These peptides are able to efficiently cross the cell membrane in an ATP independent, non-endocytotic manner and it was found that the sequence of the peptide does not affect uptake, so long as the geranylgeranyl group is still present [Wollack, J. W.; Zeliadt, N. A.; Mullen, D. G.; Amundson, G.; Geier, S.; Falkum, S.; Wattenberg, E. V.; Barany, G.; Distefano, M. D. Multifunctional Prenylated Peptides for Live Cell Analysis. J. Am. Chem. Soc.2009, 131, 7293-7303]. The present study investigates the effect of removing the fluorophore from the peptides and investigating the uptake by confocal microscopy and flow cytometry. Our results show that the fluorophore is not necessary for uptake of these peptides. This information is significant because it indicates that the prenyl group is the major determinant in allowing these peptides to enter cells; the hydrophobic fluorophore has little effect. Moreover, these studies demonstrate the utility of the Cu-catalyzed click reaction for monitoring the entry of nonfluorescent peptides into cells.

  4. Synthetic Peptides as Protein Mimics

    PubMed Central

    Groß, Andrea; Hashimoto, Chie; Sticht, Heinrich; Eichler, Jutta

    2016-01-01

    The design and generation of molecules capable of mimicking the binding and/or functional sites of proteins represents a promising strategy for the exploration and modulation of protein function through controlled interference with the underlying molecular interactions. Synthetic peptides have proven an excellent type of molecule for the mimicry of protein sites because such peptides can be generated as exact copies of protein fragments, as well as in diverse chemical modifications, which includes the incorporation of a large range of non-proteinogenic amino acids as well as the modification of the peptide backbone. Apart from extending the chemical and structural diversity presented by peptides, such modifications also increase the proteolytic stability of the molecules, enhancing their utility for biological applications. This article reviews recent advances by this and other laboratories in the use of synthetic protein mimics to modulate protein function, as well as to provide building blocks for synthetic biology. PMID:26835447

  5. Marine Peptides: Bioactivities and Applications

    PubMed Central

    Cheung, Randy Chi Fai; Ng, Tzi Bun; Wong, Jack Ho

    2015-01-01

    Peptides are important bioactive natural products which are present in many marine species. These marine peptides have high potential nutraceutical and medicinal values because of their broad spectra of bioactivities. Their antimicrobial, antiviral, antitumor, antioxidative, cardioprotective (antihypertensive, antiatherosclerotic and anticoagulant), immunomodulatory, analgesic, anxiolytic anti-diabetic, appetite suppressing and neuroprotective activities have attracted the attention of the pharmaceutical industry, which attempts to design them for use in the treatment or prevention of various diseases. Some marine peptides or their derivatives have high commercial values and had reached the pharmaceutical and nutraceutical markets. A large number of them are already in different phases of the clinical and preclinical pipeline. This review highlights the recent research in marine peptides and the trends and prospects for the future, with special emphasis on nutraceutical and pharmaceutical development into marketed products. PMID:26132844

  6. Antimicrobial peptides: properties and applicability.

    PubMed

    van 't Hof, W; Veerman, E C; Helmerhorst, E J; Amerongen, A V

    2001-04-01

    All organisms need protection against microorganisms, e. g. bacteria, viruses and fungi. For many years, attention has been focused on adaptive immunity as the main antimicrobial defense system. However, the adaptive immune system, with its network of humoral and cellular responses is only found in higher animals, while innate immunity is encountered in all living creatures. The turning point in the appreciation of the innate immunity was the discovery of antimicrobial peptides in the early eighties. In general these peptides act by disrupting the structural integrity of the microbial membranes. It has become clear that membrane-active peptides and proteins play a crucial role in both the innate and the adaptive immune system as antimicrobial agents. This review is focused on the functional and structural features of the naturally occurring antimicrobial peptides, and discusses their potential as therapeutics.

  7. Moonlighting Peptides with Emerging Function

    PubMed Central

    Rodríguez Plaza, Jonathan G.; Villalón Rojas, Amanda; Herrera, Sur; Garza-Ramos, Georgina; Torres Larios, Alfredo; Amero, Carlos; Zarraga Granados, Gabriela; Gutiérrez Aguilar, Manuel; Lara Ortiz, María Teresa; Polanco Gonzalez, Carlos; Uribe Carvajal, Salvador; Coria, Roberto; Peña Díaz, Antonio; Bredesen, Dale E.; Castro-Obregon, Susana; del Rio, Gabriel

    2012-01-01

    Hunter-killer peptides combine two activities in a single polypeptide that work in an independent fashion like many other multi-functional, multi-domain proteins. We hypothesize that emergent functions may result from the combination of two or more activities in a single protein domain and that could be a mechanism selected in nature to form moonlighting proteins. We designed moonlighting peptides using the two mechanisms proposed to be involved in the evolution of such molecules (i.e., to mutate non-functional residues and the use of natively unfolded peptides). We observed that our moonlighting peptides exhibited two activities that together rendered a new function that induces cell death in yeast. Thus, we propose that moonlighting in proteins promotes emergent properties providing a further level of complexity in living organisms so far unappreciated. PMID:22808104

  8. Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A*

    PubMed Central

    Jia, Xinying; Kwon, Soohyun; Wang, Ching-I Anderson; Huang, Yen-Hua; Chan, Lai Y.; Tan, Chia Chia; Rosengren, K. Johan; Mulvenna, Jason P.; Schroeder, Christina I.; Craik, David J.

    2014-01-01

    Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, α-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential. PMID:24425873

  9. Biodegradable Peptide-Silica Nanodonuts.

    PubMed

    Maggini, Laura; Travaglini, Leana; Cabrera, Ingrid; Castro-Hartmann, Pablo; De Cola, Luisa

    2016-03-07

    We report hybrid organosilica toroidal particles containing a short peptide sequence as the organic component of the hybrid systems. Once internalised in cancer cells, the presence of the peptide allows for interaction with peptidase enzymes, which attack the nanocarrier effectively triggering its structural breakdown. Moreover, these biodegradable nanovectors are characterised by high cellular uptake and exocytosis, showing great potential as biodegradable drug carriers. To demonstrate this feature, doxorubicin was employed and its delivery in HeLa cells investigated.

  10. Peptides and proteins

    SciTech Connect

    Bachovchin, W.W.; Unkefer, C.J.

    1994-12-01

    Advances in magnetic resonance and vibrational spectroscopy make it possible to derive detailed structural information about biomolecular structures in solution. These techniques are critically dependent on the availability of labeled compounds. For example, NMR techniques used today to derive peptide and protein structures require uniformity {sup 13}C-and {sup 15}N-labeled samples that are derived biosynthetically from (U-6-{sup 13}C) glucose. These experiments are possible now because, during the 1970s, the National Stable Isotope Resource developed algal methods for producing (U-6-{sup 13}C) glucose. If NMR techniques are to be used to study larger proteins, we will need sophisticated labelling patterns in amino acids that employ a combination of {sup 2}H, {sup 13}C, and {sup 15}N labeling. The availability of these specifically labeled amino acids requires a renewed investment in new methods for chemical synthesis of labeled amino acids. The development of new magnetic resonance or vibrational techniques to elucidate biomolecular structure will be seriously impeded if we do not see rapid progress in labeling technology. Investment in labeling chemistry is as important as investment in the development of advanced spectroscopic tools.

  11. Antimicrobial peptides from Phyllomedusa frogs: from biomolecular diversity to potential nanotechnologic medical applications.

    PubMed

    Azevedo Calderon, Leonardo de; Silva, Alexandre de Almeida E; Ciancaglini, Pietro; Stábeli, Rodrigo Guerino

    2011-01-01

    Screening for new bioactive peptides in South American anurans has been pioneered in frogs of the genus Phyllomedusa. All frogs of this genus have venomous skin secretions, i.e., a complex mixture of bioactive peptides against potential predators and pathogens that presumably evolved in a scenario of predator-prey interaction and defense against microbial invasion. For every new anuran species studied new peptides are found, with homologies to hormones, neurotransmitters, antimicrobials, and several other peptides with unknown biological activity. From Vittorio Erspamer findings, this genus has been reported as a "treasure store" of bioactive peptides, and several groups focus their research on these species. From 1966 to 2009, more than 200 peptide sequences from different Phyllomedusa species were deposited in UniProt and other databases. During the last decade, the emergence of high-throughput molecular technologies involving de novo peptide sequencing via tandem mass spectrometry, cDNA cloning, pharmacological screening, and surface plasmon resonance applied to peptide discovery, led to fast structural data acquisition and the generation of peptide molecular libraries. Research groups on bioactive peptides in Brazil using these new technologies, accounted for the exponential increase of new molecules described in the last decade, much higher than in any previous decades. Recently, these secretions were also reported as a rich source of multiple antimicrobial peptides effective against multidrug resistant strains of bacteria, fungi, protozoa, and virus, providing instructive lessons for the development of new and more efficient nanotechnological-based therapies for infectious diseases treatment. Therefore, novel drugs arising from the identification and analysis of bioactive peptides from South American anuran biodiversity have a promising future role on nanobiotechnology.

  12. Low-energy collision-induced dissociation fragmentation analysis of cysteinyl-modified peptides.

    PubMed

    Borisov, Oleg V; Goshe, Michael B; Conrads, Thomas P; Rakov, V Sergey; Veenstra, Timothy D; Smith, Richard D

    2002-05-15

    The development of methods to chemically modify and isolate cysteinyl-residue-containing peptides (Cys-peptides) for LC-MS/MS analysis has generated considerable interest in the field of proteomics. Methods using isotope-coded affinity tags (ICAT) and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (iodoacetyl-PEO-biotin) employ similar Cys-modifying reagents that contain a thiolate-specific biotin group to modify and isolate Cys-containing peptides in conjunction with immobilized avidin. For these strategies to be effective on a proteome-wide level, the presence of the ICAT or acetyl-PEO-biotin tag should not interfere with the efficiency of induced dissociation in MS/MS experiments or with the identification of the modified Cys-peptides by automated database searching algorithms. We have compared the collision-induced dissociation (CID) fragmentation patterns of peptides labeled with iodoacetyl-PEO-biotin and the ICAT reagent to those of the unmodified peptides. CID of Cys-peptides modified with either reagent resulted in the formation of ions attributed to the modified Cys-peptides as well as those unique to the labeling reagent. As demonstrated by analyzing acetyl-PEO-biotin labeled peptides from ribonuclease A and the ICAT-labeled proteome of Deinococcus radiodurans, the presence of these label-specific product ions provides a useful identifier to discern whether a peptide has been modified with the Cys-specific reagent, especially when a number of peptides analyzed using these methods do not contain a modified Cys residue, and to differentiate identical Cys-peptides labeled with either ICAT-d0 or ICAT-d8.

  13. Producing peptide arrays for epitope mapping by intein-mediated protein ligation.

    PubMed

    Sun, Luo; Rush, John; Ghosh, Inca; Maunus, Jeremy R; Xu, Ming-Qun

    2004-09-01

    Peptide arrays are increasingly used to define antibody epitopes and substrate specificities of protein kinases. Their use is hampered, however, by ineffective and variable binding efficiency of peptides, which often results in low sensitivity and inconsistent results. To overcome these limitations, we have developed a novel method for making arrays of synthetic peptides on various membranes after ligating the peptide substrates to an intein-generated carrier protein. We have conducted screening for optimal carrier proteins by immunoreactivity and direct assessment of binding using a peptide derivatized at a lysine sidechain with fluorescein, CDPEK(fluorescein)DS. Ligation of a synthetic peptide antigen to a carrier protein, HhaI methylase, resulted in an improved retention of peptides and an increased sensitivity of up to 10(4)-fold in immunoassay- and epitope-scanning experiments. Denaturing the ligation products with 2% sodium dodecyl sulfate (SDS) or an organic solvent (20% methanol) prior to arraying did not significantly affect the immunoreactivity of the HhaI methylase-peptide product. Because the carrier protein dominates the binding of ligation products and contains one peptide reactive site, the amount of peptide arrayed onto the membranes can be effectively normalized. This technique was utilized in the alanine scanning of hemagglutinin (HA) antigen using two monoclonal antibodies, resulting in distinguishing the different antigen epitope profiles. Furthermore, we show that this method can be used to characterize the antibodies that recognize phosphorylated peptides. This novel approach allows for synthetic peptides to be uniformly arrayed onto membranes, compatible with a variety of applications.

  14. Low-Energy Collision-Induced Dissociation Fragmentation Analysis of Cysteinyl-Modified Peptides

    SciTech Connect

    Borisov, Oleg V.; Goshe, Michael B. ); Conrads, Thomas P. ); Rakov, Vsevolod S. ); Veenstra, Timothy D. ); Smith, Richard D. )

    2002-05-15

    The development of methods to chemically modify and isolate cysteinyl-residue containing peptides (Cys-peptides) for LC-MS/MS analysis has generated considerable interest in the field of proteomics. Methods using isotope-coded affinity tags (ICAT) and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (iodoacetyl-PEO-biotin) employ similar Cys-modifying reagents that contain a thiolate-specific biotin group to modify and isolate Cys-containing peptides in conjunction with immobilized avidin. For these strategies to be effective on a proteome-wide level, the presence of the ICAT or acetyl-PEO-biotin tag should not interfere with the efficiency of induced dissociation in MS/MS experiments or with the identification of the modified Cys-peptides by automated database searching algorithms. We have compared the collision-induced dissociation (CID) fragmentation patterns of peptides labeled with iodoacetyl-PEO-biotin and the ICAT reagent to those of the unmodified peptides. CID of Cys-peptides modified with either reagent resulted in the formation of ions attributed to the modified Cys-peptides as well as those unique to the labeling reagent. As demonstrated by analyzing acetyl-PEO-biotin labeled peptides from ribonuclease A and the ICAT-labeled proteome of D. radiodurans, the presence of these labeled-specific product ions provides a useful identifier to discern whether a peptide has been modified with the Cys-specific reagent, especially when a number of peptides analyzed using these methods do not contain a modified Cys-residue, and to differentiate identical Cys-peptides labeled with either ICAT-D0 or ICAT-D8.

  15. Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation.

    PubMed

    Qvit, Nir; Kornfeld, Opher S

    2016-01-26

    Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is a major effort to target PPIs in basic research and in the pharmaceutical industry. Protein-protein interfaces are usually large, flat, and often lack pockets, complicating the discovery of small molecules that target such sites. Alternative targeting approaches using antibodies have limitations due to poor oral bioavailability, low cell-permeability, and production inefficiency. Using peptides to target PPI interfaces has several advantages. Peptides have higher conformational flexibility, increased selectivity, and are generally inexpensive. However, peptides have their own limitations including poor stability and inefficiency crossing cell membranes. To overcome such limitations, peptide cyclization can be performed. Cyclization has been demonstrated to improve peptide selectivity, metabolic stability, and bioavailability. However, predicting the bioactive conformation of a cyclic peptide is not trivial. To overcome this challenge, one attractive approach it to screen a focused library to screen in which all backbone cyclic peptides have the same primary sequence, but differ in parameters that influence their conformation, such as ring size and position. We describe a detailed protocol for synthesizing a library of backbone cyclic peptides targeting specific parasite PPIs. Using a rational design approach, we developed peptides derived from the scaffold protein Leishmania receptor for activated C-kinase (LACK). We hypothesized that sequences in LACK that are conserved in parasites, but not in the mammalian host homolog, may represent interaction sites for proteins that are critical for the parasites' viability. The cyclic peptides were synthesized using microwave irradiation to reduce reaction times and increase efficiency. Developing a library of backbone cyclic peptides with different ring sizes facilitates a

  16. An FPGA Implementation to Detect Selective Cationic Antibacterial Peptides

    PubMed Central

    Polanco González, Carlos; Nuño Maganda, Marco Aurelio; Arias-Estrada, Miguel; del Rio, Gabriel

    2011-01-01

    Exhaustive prediction of physicochemical properties of peptide sequences is used in different areas of biological research. One example is the identification of selective cationic antibacterial peptides (SCAPs), which may be used in the treatment of different diseases. Due to the discrete nature of peptide sequences, the physicochemical properties calculation is considered a high-performance computing problem. A competitive solution for this class of problems is to embed algorithms into dedicated hardware. In the present work we present the adaptation, design and implementation of an algorithm for SCAPs prediction into a Field Programmable Gate Array (FPGA) platform. Four physicochemical properties codes useful in the identification of peptide sequences with potential selective antibacterial activity were implemented into an FPGA board. The speed-up gained in a single-copy implementation was up to 108 times compared with a single Intel processor cycle for cycle. The inherent scalability of our design allows for replication of this code into multiple FPGA cards and consequently improvements in speed are possible. Our results show the first embedded SCAPs prediction solution described and constitutes the grounds to efficiently perform the exhaustive analysis of the sequence-physicochemical properties relationship of peptides. PMID:21738652

  17. An FPGA implementation to detect selective cationic antibacterial peptides.

    PubMed

    Polanco González, Carlos; Nuño Maganda, Marco Aurelio; Arias-Estrada, Miguel; del Rio, Gabriel

    2011-01-01

    Exhaustive prediction of physicochemical properties of peptide sequences is used in different areas of biological research. One example is the identification of selective cationic antibacterial peptides (SCAPs), which may be used in the treatment of different diseases. Due to the discrete nature of peptide sequences, the physicochemical properties calculation is considered a high-performance computing problem. A competitive solution for this class of problems is to embed algorithms into dedicated hardware. In the present work we present the adaptation, design and implementation of an algorithm for SCAPs prediction into a Field Programmable Gate Array (FPGA) platform. Four physicochemical properties codes useful in the identification of peptide sequences with potential selective antibacterial activity were implemented into an FPGA board. The speed-up gained in a single-copy implementation was up to 108 times compared with a single Intel processor cycle for cycle. The inherent scalability of our design allows for replication of this code into multiple FPGA cards and consequently improvements in speed are possible. Our results show the first embedded SCAPs prediction solution described and constitutes the grounds to efficiently perform the exhaustive analysis of the sequence-physicochemical properties relationship of peptides.

  18. Structural Biology of Non-Ribosomal Peptide Synthetases

    PubMed Central

    Miller, Bradley R.; Gulick, Andrew M.

    2016-01-01

    Summary The non-ribosomal peptide synthetases are modular enzymes that catalyze synthesis of important peptide products from a variety of standard and non-proteinogenic amino acid substrates. Within a single module are multiple catalytic domains that are responsible for incorporation of a single residue. After the amino acid is activated and covalently attached to an integrated carrier protein domain, the substrates and intermediates are delivered to neighboring catalytic domains for peptide bond formation or, in some modules, chemical modification. In the final module, the peptide is delivered to a terminal thioesterase domain that catalyzes release of the peptide product. This multi-domain modular architecture raises questions about the structural features that enable this assembly line synthesis in an efficient manner. The structures of the core component domains have been determined and demonstrate insights into the catalytic activity. More recently, multi-domain structures have been determined and are providing clues to the features of these enzyme systems that govern the functional interaction between multiple domains. This chapter describes the structures of NRPS proteins and the strategies that are being used to assist structural studies of these dynamic proteins, including careful consideration of domain boundaries for generation of truncated proteins and the use of mechanism-based inhibitors that trap interactions between the catalytic and carrier protein domains. PMID:26831698

  19. Novel cationic lipophilic peptides for oligodeoxynucleotide delivery.

    PubMed

    Chan, Enoch; Amon, Michael; Marano, Robert J; Wimmer, Norbert; Kearns, Philip S; Manolios, Nicholas; Rakoczy, P Elizabeth; Toth, Istvan

    2007-06-15

    In search of new oligodeoxynucleotide (ODN) delivery agents, we evaluated novel peptides derived from core peptide H-GLRILLLKV-OH (CP). CP is a fragment designed from the T-cell antigen receptor (TCR) alpha-chain transmembrane sequence. CP was able to enter cells including T-cells and inhibited interleukin-2 (IL-2) production. To examine the effect of increased lipophilicity on cellular uptake and activity of CP, a lipoamino acid (2-aminododecanoic acid) was incorporated into peptide CP resulting in 2-aminodecanoyl-CP (LP). The toxicity of CP and LP was assessed by measuring the haemolytic activity. Neither compound caused any haemolysis of red blood cells. We have also compared the biological activities of the CP and LP. Using a T-cell antigen presentation assay, the more lipophilic LP caused greater inhibition of IL-2 production than the parent CP in the antigen stimulated T-cells. The LP also showed increased permeability than CP in the Caco-2 cell assay. We utilised the enhanced cell permeability property of LP in oligodeoxynucleotide ODN1 delivery. Isothermal titration calorimetry (ITC) suggested that CP and LP complex with ODN1 in a 12:1 (CP:ODN1) and 15:1 (LP:ODN1) ratio. These complexes were then transfected into human retinal pigment epithelial cells. The level of transfection was measured by the decreased production of the protein human vascular endothelial growth factor (hVEGF). The results revealed greater transfection efficiency for both CP and LP (47%, 55% more inhibition) compared to commercially available transfection agent cytofectin GSV. These results suggested that the CP and particularly its lipophilic analogue LP have the potential to be used as oligodeoxynucleotide delivery systems.

  20. An integrated artificial photosynthesis system based on peptide nanotubes

    NASA Astrophysics Data System (ADS)

    Xue, Bin; Li, Ying; Yang, Fan; Zhang, Chunfeng; Qin, Meng; Cao, Yi; Wang, Wei

    2014-06-01

    A peptide nanotube platform that integrates both light-harvesting and catalytic units was successfully engineered for artificial photosynthesis. Peptide nanotubes not only serve as a hub for physically combining both units, but also work as mediators that transfer the energy from photo-excited chromophores to catalytic centers. The direct conversion of NAD+ to NADH upon light illumination was demonstrated. This represents a promising step towards efficient and fully integrated artificial photosynthesis systems.A peptide nanotube platform that integrates both light-harvesting and catalytic units was successfully engineered for artificial photosynthesis. Peptide nanotubes not only serve as a hub for physically combining both units, but also work as mediators that transfer the energy from photo-excited chromophores to catalytic centers. The direct conversion of NAD+ to NADH upon light illumination was demonstrated. This represents a promising step towards efficient and fully integrated artificial photosynthesis systems. Electronic supplementary information (ESI) available: Experimental procedures and supporting figures. See DOI: 10.1039/c4nr00295d

  1. Autocrine-Based Selection of Drugs That Target Ion Channels from Combinatorial Venom Peptide Libraries.

    PubMed

    Zhang, Hongkai; Du, Mingjuan; Xie, Jia; Liu, Xiao; Sun, Jingying; Wang, Wei; Xin, Xiu; Possani, Lourival D; Yea, Kyungmoo; Lerner, Richard A

    2016-08-01

    Animal venoms represent a rich source of pharmacologically active peptides that interact with ion channels. However, a challenge to discovering drugs remains because of the slow pace at which venom peptides are discovered and refined. An efficient autocrine-based high-throughput selection system was developed to discover and refine venom peptides that target ion channels. The utility of this system was demonstrated by the discovery of novel Kv1.3 channel blockers from a natural venom peptide library that was formatted for autocrine-based selection. We also engineered a Kv1.3 blocker peptide (ShK) derived from sea anemone to generate a subtype-selective Kv1.3 blocker with a long half-life in vivo.

  2. Preparation and characterization of coacervate microcapsules for the delivery of antimicrobial oyster peptides.

    PubMed

    Zhang, Li; Liu, Yezhou; Wu, Zhongchen; Chen, Haixu

    2009-03-01

    Oyster peptides-loaded alginate/chitosan/starch microcapsules were prepared using external gelation method and internal emulsion gelation method. The solution of oyster peptides complexes was encapsulated into the microcapsules, which endowed the microcapsules with intestine passive targeting properties. The swelling behavior, encapsulation efficiency, and release behavior of oyster peptides from the microcapsules at different pH values were investigated. The microcapsules exhibited sustained release of the peptides in intestinal medium, and the release rate could be regulated by the pH value: in simulated gastric fluid, the release rate was greatly decreased, and in simulated body fluid and intestinal fluid, the microcapsules exhibited a sustained release in 24 h with different release rates. The microspheres were characterized by Fourier transform infrared. The results suggested that the alginate/chitosan/starch microcapsules could be a suitable copolymeric carrier system for intestinal protein or peptides delivery in the intestine.

  3. Peptide-Modulated Self-Assembly of Chromophores toward Biomimetic Light-Harvesting Nanoarchitectonics.

    PubMed

    Zou, Qianli; Liu, Kai; Abbas, Manzar; Yan, Xuehai

    2016-02-10

    Elegant self-assembling complexes by the combination of proteins/peptides with functional chromophores are decisively responsible for highly efficient light-harvesting and energy transfer in natural photosynthetic systems. Mimicking natural light-harvesting complexes through synthetic peptides is attractive due to their advantanges of programmable primary structure, tunable self-assembly architecture and easy availability in comparison to naturally occuring proteins. Here, an overview of recent progresses in the area of biomimetic light-harvesting nanoarchitectonics based on peptide-modulated self-assembly of chromophores is provided. Adjusting the organization of chromophores, either by creating peptide-chromophore conjugates or by the non-covalent assembly of peptides and chromophores are highlighted. The light-harvesting properties, especially the energy transfer of the biomimetic complexes are critically discussed. The applications of such complexes in the mineralization of inorganic nanoparticles, generation of molecular hydrogen and oxygen, and photosynthesis of bioactive molecules are also included.

  4. Infrared multiphoton dissociation of peptide cations in a dual pressure linear ion trap mass spectrometer.

    PubMed

    Gardner, Myles W; Smith, Suncerae I; Ledvina, Aaron R; Madsen, James A; Coon, Joshua J; Schwartz, Jae C; Stafford, George C; Brodbelt, Jennifer S

    2009-10-01

    A dual pressure linear ion trap mass spectrometer was modified to permit infrared multiphoton dissociation (IRMPD) in each of the two cells-the first a high pressure cell operated at nominally 5 x 10(-3) Torr and the second a low pressure cell operated at nominally 3 x 10(-4) Torr. When IRMPD was performed in the high pressure cell, most peptide ions did not undergo significant photodissociation; however, in the low pressure cell peptide cations were efficiently dissociated with less than 25 ms of IR irradiation regardless of charge state. IRMPD of peptide cations allowed the detection of low m/z product ions including the y(1) fragments and immonium ions which are not typically observed by ion trap collision induced dissociation (CID). Photodissociation efficiencies of approximately 100% and MS/MS (tandem mass spectrometry) efficiencies of greater than 60% were observed for both multiply and singly protonated peptides. In general, higher sequence coverage of peptides was obtained using IRMPD over CID. Further, greater than 90% of the product ion current in the IRMPD mass spectra of doubly charged peptide ions was composed of singly charged product ions compared to the CID mass spectra in which the abundances of the multiply and singly charged product ions were equally divided. Highly charged primary product ions also underwent efficient photodissociation to yield singly charged secondary product ions, thus simplifying the IRMPD product ion mass spectra.

  5. Infrared Multiphoton Dissociation of Peptide Cations in a Dual Pressure Linear Ion Trap Mass Spectrometer

    PubMed Central

    Gardner, Myles W.; Smith, Suncerae I.; Ledvina, Aaron R.; Madsen, James A.; Coon, Joshua J.; Schwartz, Jae C.; Stafford, George C.; Brodbelt, Jennifer S.

    2009-01-01

    A dual pressure linear ion trap mass spectrometer was modified to permit infrared multiphoton dissociation (IRMPD) in each of the two cells - the first a high pressure cell operated at nominally 5 × 10-3 Torr and the second a low pressure cell operated at nominally 3 × 10-4 Torr. When IRMPD was performed in the high pressure cell, most peptide ions did not undergo significant photodissociation; however, in the low pressure cell peptide cations were efficiently dissociated with less than 25 ms of IR irradiation regardless of charge state. IRMPD of peptide cations allowed the detection of low m/z product ions including the y1 fragments and immonium ions which are not typically observed by ion trap collision induced dissociation (CID). Photodissociation efficiencies of ~100% and MS/MS (tandem mass spectrometry) efficiencies of greater than 60% were observed for both multiply and singly protonated peptides. In general, higher sequence coverage of peptides was obtained using IRMPD over CID. Further, greater than 90% of the product ion current in the IRMPD mass spectra of doubly charged peptide ions was composed of singly charged product ions compared to the CID mass spectra in which the abundances of the multiply and singly charged product ions were equally divided. Highly charged primary product ions also underwent efficient photodissociation to yield singly charged secondary product ions, thus simplifying the IRMPD product ion mass spectra. PMID:19739654

  6. Cyclic peptide therapeutics: past, present and future.

    PubMed

    Zorzi, Alessandro; Deyle, Kaycie; Heinis, Christian

    2017-02-26

    Cyclic peptides combine several favorable properties such as good binding affinity, target selectivity and low toxicity that make them an attractive modality for the development of therapeutics. Over 40 cyclic peptide drugs are currently in clinical use and around one new cyclic peptide drug enters the market every year on average. The vast majority of clinically approved cyclic peptides are derived from natural products, such as antimicrobials or human peptide hormones. New powerful techniques based on rational design and in vitro evolution have enabled the de novo development of cyclic peptide ligands to targets for which nature does not offer solutions. A look at the cyclic peptides currently under clinical evaluation shows that several have been developed using such techniques. This new source for cyclic peptide ligands introduces a freshness to the field, and it is likely that de novo developed cyclic peptides will be in clinical use in the near future.

  7. Computational modeling of peptide-aptamer binding.

    PubMed

    Rhinehardt, Kristen L; Mohan, Ram V; Srinivas, Goundla

    2015-01-01

    Evolution is the progressive process that holds each living creature in its grasp. From strands of DNA evolution shapes life with response to our ever-changing environment and time. It is the continued study of this most primitive process that has led to the advancement of modern biology. The success and failure in the reading, processing, replication, and expression of genetic code and its resulting biomolecules keep the delicate balance of life. Investigations into these fundamental processes continue to make headlines as science continues to explore smaller scale interactions with increasing complexity. New applications and advanced understanding of DNA, RNA, peptides, and proteins are pushing technology and science forward and together. Today the addition of computers and advances in science has led to the fields of computational biology and chemistry. Through these computational advances it is now possible not only to quantify the end results but also visualize, analyze, and fully understand mechanisms by gaining deeper insights. The biomolecular motion that exists governing the physical and chemical phenomena can now be analyzed with the advent of computational modeling. Ever-increasing computational power combined with efficient algorithms and components are further expanding the fidelity and scope of such modeling and simulations. This chapter discusses computational methods that apply biological processes, in particular computational modeling of peptide-aptamer binding.

  8. Peptide Conjugation to a Polymer Coating via Native Chemical Ligation of Azlactones for Cell Culture.

    PubMed

    Schmitt, Samantha K; Trebatoski, David J; Krutty, John D; Xie, Angela W; Rollins, Benjamin; Murphy, William L; Gopalan, Padma

    2016-03-14

    Conjugation of biomolecules for stable presentation is an essential step toward reliable chemically defined platforms for cell culture studies. In this work, we describe the formation of a stable and site-specific amide bond via the coupling of a cysteine terminated peptide at low concentration to an azlactone containing copolymer coating. A copolymer of polyethylene glycol methyl ether methacrylate-ran-vinyl azlactone-ran-glycidyl methacrylate P(PEGMEMA-r-VDM-r-GMA) was used to form a thin coating (20-30 nm) on silicon and polycarbonate substrates. The formation and stability of coating-peptide bonds for peptides containing free thiols and amines were quantified by X-ray photoelectron spectroscopy (XPS) after exposure to cell culture conditions. Peptides containing a thiol as the only nucleophile coupled via a thioester bond; however, the bond was labile under cell culture conditions and almost all the bound peptides were displaced from the surface over a period of 2 days. Coupling with N-terminal primary amine peptides resulted in the formation of an amide bond with low efficiency (<20%). In contrast, peptides containing an N-terminal cysteine, which contain both nucleophiles (free thiol and amine) in close proximity, bound with 67% efficiency under neutral pH, and were stable under the same conditions for 2 weeks. Control studies confirm that the stable amide formation was a result of an intramolecular rearrangement through a N-acyl intermediate that resembles native chemical ligation. Through a combination of XPS and cell culture studies, we show that the cysteine terminated peptides undergo a native chemical ligation process at low peptide concentration in aqueous media, short reaction time, and at room temperature resulting in the stable presentation of peptides beyond 2 weeks for cell culture studies.

  9. LL-37-derived peptides eradicate multidrug-resistant Staphylococcus aureus from thermally wounded human skin equivalents.

    PubMed

    Haisma, Elisabeth M; de Breij, Anna; Chan, Heelam; van Dissel, Jaap T; Drijfhout, Jan W; Hiemstra, Pieter S; El Ghalbzouri, Abdoelwaheb; Nibbering, Peter H

    2014-08-01

    Burn wound infections are often difficult to treat due to the presence of multidrug-resistant bacterial strains and biofilms. Currently, mupirocin is used to eradicate methicillin-resistant Staphylococcus aureus (MRSA) from colonized persons; however, mupirocin resistance is also emerging. Since we consider antimicrobial peptides to be promising candidates for the development of novel anti-infective agents, we studied the antibacterial activities of a set of synthetic peptides against different strains of S. aureus, including mupirocin-resistant MRSA strains. The peptides were derived from P60.4Ac, a peptide based on the human cathelicidin LL-37. The results showed that peptide 10 (P10) was the only peptide more efficient than P60.4Ac, which is better than LL-37, in killing MRSA strain LUH14616. All three peptides displayed good antibiofilm activities. However, both P10 and P60.4Ac were more efficient than LL-37 in eliminating biofilm-associated bacteria. No toxic effects of these three peptides on human epidermal models were detected, as observed morphologically and by staining for mitochondrial activity. In addition, P60.4Ac and P10, but not LL-37, eradicated MRSA LUH14616 and the mupirocin-resistant MRSA strain LUH15051 from thermally wounded human skin equivalents (HSE). Interestingly, P60.4Ac and P10, but not mupirocin, eradicated LUH15051 from the HSEs. None of the peptides affected the excretion of interleukin 8 (IL-8) by thermally wounded HSEs upon MRSA exposure. In conclusion, the synthetic peptides P60.4Ac and P10 appear to be attractive candidates for the development of novel local therapies to treat patients with burn wounds infected with multidrug-resistant bacteria.

  10. Definition of an extended MHC class II-peptide binding motif for the autoimmune disease-associated Lewis rat RT1.BL molecule.

    PubMed

    Wauben, M H; van der Kraan, M; Grosfeld-Stulemeyer, M C; Joosten, I

    1997-02-01

    The Lewis rat, an inbred rat strain susceptible to several well-characterized experimental autoimmune diseases, provides a good model to study peptide-mediated immunotherapy. Peptide immunotherapy focussing on the modulation of T cell responses by interfering with TCR-peptide-MHC complex formation requires the elucidation of the molecular basis of TCR-peptide-MHC interactions for an efficient design of modulatory peptides. In the Lewis rat most autoimmune-associated CD4+ T cell responses are MHC class II RT1.BL restricted. In this study, the characteristics of RT1.BL-peptide interactions were explored. A series of substitution analogs of two Lewis rat T cell epitopes was examined in a direct peptide-MHC binding assay on isolated RT1.BL molecules. Furthermore, other autoimmune-related as well as non-disease-related T cell epitopes were tested in the binding assay. This has led to the definition of an extended RT1.BL-peptide binding motif. The RT1.BL-peptide binding motif established in this study is the first described rat MHC-peptide binding motif based on direct MHC-peptide binding experiments. To predict good or intermediate RT1.BL binding peptides, T cell epitope search profiles were deduced from this motif. The motif and search profiles will greatly facilitate the prediction of modulatory peptides based on autoimmune-associated T cell epitopes and the identification of target structures in experimental autoimmune diseases in Lewis rats.

  11. Twilight reloaded: the peptide experience

    PubMed Central

    Weichenberger, Christian X.; Pozharski, Edwin; Rupp, Bernhard

    2017-01-01

    The de facto commoditization of biomolecular crystallography as a result of almost disruptive instrumentation automation and continuing improvement of software allows any sensibly trained structural biologist to conduct crystallo­graphic studies of biomolecules with reasonably valid outcomes: that is, models based on properly interpreted electron density. Robust validation has led to major mistakes in the protein part of structure models becoming rare, but some depositions of protein–peptide complex structure models, which generally carry significant interest to the scientific community, still contain erroneous models of the bound peptide ligand. Here, the protein small-molecule ligand validation tool Twilight is updated to include peptide ligands. (i) The primary technical reasons and potential human factors leading to problems in ligand structure models are presented; (ii) a new method used to score peptide-ligand models is presented; (iii) a few instructive and specific examples, including an electron-density-based analysis of peptide-ligand structures that do not contain any ligands, are discussed in detail; (iv) means to avoid such mistakes and the implications for database integrity are discussed and (v) some suggestions as to how journal editors could help to expunge errors from the Protein Data Bank are provided. PMID:28291756

  12. Peptide Vaccine: Progress and Challenges

    PubMed Central

    Li, Weidang; Joshi, Medha D.; Singhania, Smita; Ramsey, Kyle H.; Murthy, Ashlesh K.

    2014-01-01

    Conventional vaccine strategies have been highly efficacious for several decades in reducing mortality and morbidity due to infectious diseases. The bane of conventional vaccines, such as those that include whole organisms or large proteins, appear to be the inclusion of unnecessary antigenic load that, not only contributes little to the protective immune response, but complicates the situation by inducing allergenic and/or reactogenic responses. Peptide vaccines are an attractive alternative strategy that relies on usage of short peptide fragments to engineer the induction of highly targeted immune responses, consequently avoiding allergenic and/or reactogenic sequences. Conversely, peptide vaccines used in isolation are often weakly immunogenic and require particulate carriers for delivery and adjuvanting. In this article, we discuss the specific advantages and considerations in targeted induction of immune responses by peptide vaccines and progresses in the development of such vaccines against various diseases. Additionally, we also discuss the development of particulate carrier strategies and the inherent challenges with regard to safety when combining such technologies with peptide vaccines. PMID:26344743

  13. Antimicrobial Peptides Under Clinical Trials.

    PubMed

    Greber, Katarzyna E; Dawgul, Małgorzata

    2017-01-01

    Today microbial drug resistance has become a serious problem not only within inpatient setting but also within outpatient setting. Repeated intake and unnecessary usage of antibiotics as well as the transfer of resistance genes are the most important factors that make the microorganisms resistant to conventional antibiotics. A large number of antimicrobials successfully used for prophylaxis and therapeutic purposes have now become ineffective [1, 2]. Therefore, new molecules are being studied to be used in the treatment of various diseases. Some of these molecules are structural compounds based on a combination of peptides, for example, naturally occurring endogenous peptide antibiotics and their synthetic analogues or molecules designed de novo using QSAR (quantitative structureproperty relationships)-based methods [3]. Trying to exploit numerous advantages of antimicrobial peptides such as high potency and selectivity, broad range of targets, potentially low toxicity and low accumulation in tissues, pharmaceutical industry aims to develop them as commercially available drugs and appropriate clinical trials are being conducted [4]. In this paper we define clinical trials steps and describe current status of several antimicrobial peptides under clinical development as well as briefly depict peptide drug formulation.

  14. Mitochondrial targeting functional peptides as potential devices for the mitochondrial delivery of a DF-MITO-Porter.

    PubMed

    Kawamura, Eriko; Yamada, Yuma; Harashima, Hideyoshi

    2013-11-01

    To achieve mitochondrial therapy, we previously reported on the use of an octaarginine (R8) modified Dual Function (DF)-MITO-Porter for delivering molecules to mitochondria in living cells. In this study, using isolated mitochondria, homogenates and living cells, we evaluated the utility of mitochondrial targeting functional peptides as a ligand for delivering carriers. The S2 peptide modified carrier showed a high mitochondrial targeting activity in homogenates and living cells. In addition, the S2 peptide had a lower cell toxicity compared to R8 modified liposomes. The S2 peptide represents a potentially useful moiety for constructing an efficient and safe mitochondrial delivery system.

  15. Key comparison study on peptide purity—synthetic human C-peptide

    NASA Astrophysics Data System (ADS)

    Josephs, R. D.; Li, M.; Song, D.; Westwood, S.; Stoppacher, N.; Daireaux, A.; Choteau, T.; Wielgosz, R.; Xiao, P.; Liu, Y.; Gao, X.; Zhang, C.; Zhang, T.; Mi, W.; Quan, C.; Huang, T.; Li, H.; Flatschart, R.; Borges Oliveira, R.; Melanson, J. E.; Ohlendorf, R.; Henrion, A.; Kinumi, T.; Wong, L.; Liu, Q.; Oztug Senal, M.; Vatansever, B.; Ün, I.; Gören, A. C.; Akgöz, M.; Quaglia, M.; Warren, J.

    2017-01-01

    . More detailed studies on the identification/quantification of peptide related impurities and the hydrolysis efficiency revealed that the integrity of the impurity profile of the related peptide impurities obtained by the participant is crucial for the impact on accuracy of the hCP mass fraction assignment. The assessment of the mass fraction of peptide impurities is based on the assumption that only the most exhaustive and elaborate set of results is taken for the calculation of the KCRVPepImp. The KCRVPepImp for the peptide related impurity mass fractions of the material was 83.3 mg/g with a combined standard uncertainty of 1.5 mg/g. Inspection of the degree of equivalence plots for the mass fraction of peptide impurities and additional information obtained from the peptide related impurity profile indicates that in many cases only a very small number of impurities have been identified and quantified resulting in an underestimation of the peptide related impurity mass fractions. The approach to obtain a KCRVhCP for the mass fraction of hCP is based on a mass balance calculation that takes into account the most exhaustive and elaborate set of results for the peptide related impurities KCRVPepImp, the TFA mass fraction value, water and other minor counter ions obtained by the coordinating laboratories. Differences in the quality of the results obtained for both peptides related impurity mass fractions and hCP mass fractions are better weighted and reflected in smaller uncertainties. The KCRVhCP for CCQM-K115 is 801.8 mg/g with a corresponding combined standard uncertainty of 3.1 mg/g. In general, mass balance approaches show smaller uncertainties than PICAA approaches and the majority of results obtained by the PICAA approach are in agreement because of larger corresponding uncertainties. Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The

  16. Recent advances in the research and development of marine antimicrobial peptides.

    PubMed

    El-Gamal, Mohammed I; Abdel-Maksoud, Mohammed S; Oh, Chang-Hyun

    2013-08-01

    Antimicrobial peptides are a group of natural or semi-synthetic molecules possessing antimicrobial activities against bacteria, fungi, viruses, parasites, etc. They are considered as promising candidates for treatment of microbial infections and suppression of microbial resistance. The increasing emergence of bacterial resistance has required development of new efficient antibiotics that can be added to the antibacterial armamentarium. The marine world provides a rich source of antimicrobial peptides. That world has not been highly explored yet, and much more effort is required in order to discover new efficient antimicrobial peptides. In the present article, we have reviewed the recent progress in the field of marine antimicrobial peptides from 2009 until the mid of 2012.

  17. SITS Derivatization of Peptides to Enhance 266 nm Ultraviolet Photodissociation (UVPD)

    NASA Astrophysics Data System (ADS)

    Quick, M. Montana; Mehaffey, M. Rachel; Johns, Robert W.; Parker, W. Ryan; Brodbelt, Jennifer S.

    2017-03-01

    N-terminal derivatization of peptides with the chromogenic reagent 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS) is demonstrated to enhance the efficiency of 266 nm ultraviolet photodissociation (UVPD). Attachment of the chromophore results in a mass shift of 454 Da and provides significant gains in the number and abundances of diagnostic fragment ions upon UVPD. Activation of SITS-tagged peptides with 266 nm UVPD leads to many fragment ions akin to the a/b/y ions commonly produced by CID, along with other sequence ions (c, x, and z) typically accessed through higher energy pathways. Extreme bias towards C-terminal fragment ions is observed upon activation of SITS-tagged peptides using multiple 266 nm laser pulses. Due to the high reaction efficiency of the isothiocyanate coupling to the N-terminus of peptides, we demonstrate the ability to adapt this strategy to a high-throughput LC-MS/MS workflow with 266 nm UVPD.

  18. Peptides Labeled with Pyridinium Salts for Sensitive Detection and Sequencing by Electrospray Tandem Mass Spectrometry

    PubMed Central

    Waliczek, Mateusz; Kijewska, Monika; Rudowska, Magdalena; Setner, Bartosz; Stefanowicz, Piotr; Szewczuk, Zbigniew

    2016-01-01

    Mass spectrometric analysis of trace amounts of peptides may be problematic due to the insufficient ionization efficiency resulting in limited sensitivity. One of the possible ways to overcome this problem is the application of ionization enhancers. Herein we developed new ionization markers based on 2,4,6-triphenylpyridinium and 2,4,6-trimethylpyridinium salts. Using of inexpensive and commercially available pyrylium salt allows selective derivatization of primary amino groups, especially those sterically unhindered, such as ε-amino group of lysine. The 2,4,6-triphenylpyridinium modified peptides generate in MS/MS experiments an abundant protonated 2,4,6-triphenylpyridinium ion. This fragment is a promising reporter ion for the multiple reactions monitoring (MRM) analysis. In addition, the fixed positive charge of the pyridinium group enhances the ionization efficiency. Other advantages of the proposed ionization enhancers are the simplicity of derivatization of peptides and the possibility of convenient incorporation of isotopic labels into derivatized peptides. PMID:27892962

  19. Natriuretic peptide metabolism, clearance and degradation.

    PubMed

    Potter, Lincoln R

    2011-06-01

    Atrial natriuretic peptide, B-type natriuretic peptide and C-type natriuretic peptide constitute a family of three structurally related, but genetically distinct, signaling molecules that regulate the cardiovascular, skeletal, nervous, reproductive and other systems by activating transmembrane guanylyl cyclases and elevating intracellular cGMP concentrations. This review broadly discusses the general characteristics of natriuretic peptides and their cognate signaling receptors, and then specifically discusses the tissue-specific metabolism of natriuretic peptides and their degradation by neprilysin, insulin-degrading enzyme, and natriuretic peptide receptor-C.

  20. Antifungal Activities of Peptides Derived from Domain 5 of High-Molecular-Weight Kininogen

    PubMed Central

    Sonesson, Andreas; Nordahl, Emma Andersson; Malmsten, Martin; Schmidtchen, Artur

    2011-01-01

    In both immunocompromised and immunocompetent patients, Candida and Malassezia are causing or triggering clinical manifestations such as cutaneous infections and atopic eczema. The innate immune system provides rapid responses to microbial invaders, without requiring prior stimulation, through a sophisticated system of antimicrobial peptides (AMPs). High molecular weight kininogen (HMWK) and components of the contact system have previously been reported to bind to Candida and other pathogens, leading to activation of the contact system. A cutaneous Candida infection is characterized by an accumulation of neutrophils, leading to an inflammatory response and release of enzymatically active substances. In the present study we demonstrate that antifungal peptide fragments are generated through proteolytic degradation of HMWK. The recombinant domain 5 (rD5) of HMWK, D5-derived peptides, as well as hydrophobically modified D5-derived peptides efficiently killed Candida and Malassezia. Furthermore, the antifungal activity of modified peptides was studied at physiological conditions. Binding of a D5-derived peptide, HKH20 (His479-His498), to the fungal cell membrane was visualized by fluorescence microscopy. Our data disclose a novel antifungal activity of D5-derived peptides and also show that proteolytic cleavage of HMWK results in fragments exerting antifungal activity. Of therapeutic interest is that structurally modified peptides show an enhanced antifungal activity. PMID:21941573

  1. A p7 Ion Channel-derived Peptide Inhibits Hepatitis C Virus Infection in Vitro*

    PubMed Central

    Hong, Wei; Lang, Yange; Li, Tian; Zeng, Zhengyang; Song, Yu; Wu, Yingliang; Li, Wenxin; Cao, Zhijian

    2015-01-01

    Viral infection is an early stage of its life cycle and represents a promising target for antiviral drug development. Here we designed and characterized three peptide inhibitors of hepatitis C virus (HCV) infection based on the structural features of the membrane-associated p7 polypeptide of HCV. The three peptides exhibited low toxicity and high stability while potently inhibiting initial HCV infection and suppressed established HCV infection at non-cytotoxic concentrations in vitro. The most efficient peptide (designated H2-3), which is derived from the H2 helical region of HCV p7 ion channel, inhibited HCV infection by inactivating both intracellular and extracellular viral particles. The H2-3 peptide inactivated free HCV with an EC50 (50% effective concentration) of 82.11 nm, which is >1000-fold lower than the CC50 (50% cytotoxic concentration) of Huh7.5.1 cells. H2-3 peptide also bound to cell membrane and protected host cells from viral infection. The peptide H2-3 did not alter the normal electrophysiological profile of the p7 ion channel or block viral release from Huh7.5.1 cells. Our work highlights a new anti-viral peptide design strategy based on ion channel, giving the possibility that ion channels are potential resources to generate antiviral peptides. PMID:26251517

  2. Stitched α-helical peptides via bis ring-closing metathesis.

    PubMed

    Hilinski, Gerard J; Kim, Young-Woo; Hong, Jooyeon; Kutchukian, Peter S; Crenshaw, Charisse M; Berkovitch, Shaunna S; Chang, Andrew; Ham, Sihyun; Verdine, Gregory L

    2014-09-03

    Conformationally stabilized α-helical peptides are capable of inhibiting disease-relevant intracellular or extracellular protein-protein interactions in vivo. We have previously reported that the employment of ring-closing metathesis to introduce a single all-hydrocarbon staple along one face of an α-helical peptide greatly increases α-helical content, binding affinity to a target protein, cell penetration through active transport, and resistance to proteolytic degradation. In an effort to improve upon this technology for stabilizing a peptide in a bioactive α-helical conformation, we report the discovery of an efficient and selective bis ring-closing metathesis reaction leading to peptides bearing multiple contiguous staples connected by a central spiro ring junction. Circular dichroism spectroscopy, NMR, and computational analyses have been used to investigate the conformation of these "stitched" peptides, which are shown to exhibit remarkable thermal stabilities. Likewise, trypsin proteolysis assays confirm the achievement of a structural rigidity unmatched by peptides bearing a single staple. Furthermore, fluorescence-activated cell sorting (FACS) and confocal microscopy assays demonstrate that stitched peptides display superior cell penetrating ability compared to their stapled counterparts, suggesting that this technology may be useful not only in the context of enhancing the drug-like properties of α-helical peptides but also in producing potent agents for the intracellular delivery of proteins and oligonucleotides.

  3. Investigating the lytic activity and structural properties of Staphylococcus aureus phenol soluble modulin (PSM) peptide toxins.

    PubMed

    Laabei, Maisem; Jamieson, W David; Yang, Yi; van den Elsen, Jean; Jenkins, A Toby A

    2014-12-01

    The ubiquitous bacterial pathogen, Staphylococcus aureus, expresses a large arsenal of virulence factors essential for pathogenesis. The phenol-soluble modulins (PSMs) are a family of cytolytic peptide toxins which have multiple roles in staphylococcal virulence. To gain an insight into which specific factors are important in PSM-mediated cell membrane disruption, the lytic activity of individual PSM peptides against phospholipid vesicles and T cells was investigated. Vesicles were most susceptible to lysis by the PSMα subclass of peptides (α1-3 in particular), when containing between 10 and 30mol% cholesterol, which for these vesicles is the mixed solid ordered (so)-liquid ordered (lo) phase. Our results show that the PSMβ class of peptides has little effect on vesicles at concentrations comparable to that of the PSMα class and exhibited no cytotoxicity. Furthermore, within the PSMα class, differences emerged with PSMα4 showing decreased vesicle and cytotoxic activity in comparison to its counterparts, in contrast to previous studies. In order to understand this, peptides were studied using helical wheel projections and circular dichroism measurements. The degree of amphipathicity, alpha-helicity and properties such as charge and hydrophobicity were calculated, allowing a structure-function relationship to be inferred. The degree of alpha-helicity of the peptides was the single most important property of the seven peptides studied in predicting their lytic activity. These results help to redefine this class of peptide toxins and also highlight certain membrane parameters required for efficient lysis.

  4. Recombinant expression of antimicrobial peptides using a novel self-cleaving aggregation tag in Escherichia coli.

    PubMed

    Luan, Chao; Xie, Yong Gang; Pu, Yu Tian; Zhang, Hai Wen; Han, Fei Fei; Feng, Jie; Wang, Yi Zhen

    2014-03-01

    Antimicrobial peptides (AMPs) are part of the innate immune system of complex multicellular organisms. Despite the fact that AMPs show great potential as a novel class of antibiotics, the lack of a cost-effective means for their mass production limits both basic research and clinical use. In this work, we describe a novel expression system for the production of antimicrobial peptides in Escherichia coli by combining ΔI-CM mini-intein with the self-assembling amphipathic peptide 18A to drive the formation of active aggregates. Two AMPs, human β-defensin 2 and LL-37, were fused to the self-cleaving tag and expressed as active protein aggregates. The active aggregates were recovered by centrifugation and the intact antimicrobial peptides were released into solution by an intein-mediated cleavage reaction in cleaving buffer (phosphate-buffered saline supplemented with 40 mmol/L Bis-Tris, 2 mmol/L EDTA, pH 6.2). The peptides were further purified by cation-exchange chromatography. Peptides yields of 0.82 ± 0.24 and 0.59 ± 0.11 mg/L were achieved for human β-defensin 2 and LL-37, respectively, with demonstrated antimicrobial activity. Using our expression system, intact antimicrobial peptides were recovered by simple centrifugation from active protein aggregates after the intein-mediated cleavage reaction. Thus, we provide an economical and efficient way to produce intact antimicrobial peptides in E. coli.

  5. Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands

    PubMed Central

    Shou, Yulin; Schmidt, Emily N.; Paavola, Chad D.; Naranjo, Leslie; Bemdich, Sara; Swanson, Basil I.; Bradbury, Andrew R. M.; Martinez, Jennifer S.

    2016-01-01

    Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The “helper cell” packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands. PMID:27626637

  6. Dynamics of Protonated Peptide Ion Collisions with Organic Surfaces: Consonance of Simulation and Experiment

    SciTech Connect

    Pratihar, Subha; Barnes, George L.; Laskin, Julia; Hase, William L.

    2016-08-18

    In this Perspective mass spectrometry experiments and chemical dynamics simulations are described which have explored the atomistic dynamics of protonated peptide ions, peptide-H+, colliding with organic surfaces. These studies have investigated surface-induced dissociation (SID) for which peptide-H+ fragments upon collision with the surface, peptide-H+ physisorption on the surface, soft landing (SL), and peptide-H+ reaction with the surface, reactive landing (RL). The simulations include QM+MM and QM/MM direct dynamics. For collisions with self-assembled monolayer (SAM) surfaces there is quite good agreement between experiment and simulation in the efficiency of energy transfer to the peptide-H+ ion’s internal degrees of freedom. Both the experiments and simulations show two mechanisms for peptide-H+ fragmentation, i.e. shattering and statistical, RRKM dynamics. Mechanisms for SL are probed in simulations of collisions of protonated dialanine with a perfluorinated SAM surface. RL has been studied experimentally for a number of peptide-H+ + surface systems, and qualitative agreement between simulation and experiment is found for two similar systems.

  7. Mass spectrometric peptide mapping analysis and structural characterization of dihydrodiol dehydrogenase isoenzymes.

    PubMed Central

    Gauss, C; Klein, J; Post, K; Suckau, D; Schneider, K; Thomas, H; Oesch, F; Przybylski, M

    1990-01-01

    The direct molecular weight determination and structural analysis of polypeptides and peptide mixtures have become amenable by the recent development of fast atom bombardment (FABMS) and 252Cf-plasma desorption (PDMS) mass spectrometry. FABMS and PDMS peptide mapping, i.e., the direct analysis of peptide mixtures resulting from proteolytic digestion, have been developed as powerful methods for the structural characterization of epoxide-metabolizing isoenzymes. The major advantage of this approach is provided by the selectivity of the endoproteolytic cleavage, combined with the specific and accurate molecular weight determination of complex digest mixtures containing peptides up to several thousands daltons in size. Furthermore, the mass spectrometric peptide mapping analysis can be combined with a range of protein-chemical modification reactions and with sequential degradation such as by carboxypeptidases. Both FABMS and PDMS peptide mapping have already been successfully applied to the structural differentiation of glutathione transferase and epoxide hydrolase isoenzymes in cases where references sequence data for at least one isoenzyme form was available. In the application described here, for a series of dihydrodiol dehydrogenase (DDH) isoenzymes with hitherto undetermined primary structures, a direct correlation between the structural differentiation from peptide mapping data and differences in their substrate specificities could be demonstrated. The mass spectrometric peptide mapping analysis of isoenzymes proved to be an efficient basis for the elucidation of the structure of one major DDH isoenzyme form; partial sequence data for this protein are reported. PMID:2272334

  8. Evaluation of a Cell Penetrating Prenylated Peptide Lacking an Intrinsic Fluorophore via in situ Click Reaction

    PubMed Central

    Ochocki, Joshua D.; Mullen, Daniel G.; Wattenberg, Elizabeth V.; Distefano, Mark D.

    2011-01-01

    Protein prenylation involves the addition of either a farnesyl (C15) or geranylgeranyl (C20) isoprenoid moiety onto the C-terminus of many proteins. This natural modification serves to direct a protein to the plasma membrane of the cell. A recently discovered application of prenylated peptides is that they have inherent cell-penetrating ability, and are hence termed cell penetrating prenylated peptides. These peptides are able to efficiently cross the cell membrane in an ATP independent, non-endocytotic manner and it was found that the sequence of the peptide does not affect uptake, so long as the geranylgeranyl group is still present. The present study investigates the effect of removing the fluorophore from the peptides and investigating the uptake by confocal microsopy and flow cytometry. Our results show that the fluorophore is not necessary for uptake of these peptides. This information is significant because it indicates that the prenyl group is the major determinant in allowing these peptides to enter cells; the hydrophobic fluorophore has little effect. Moreover, these studies demonstrate the utility of the Cu-catalyzed click reaction for monitoring the entry of nonfluorescent peptides into cells. PMID:21632248

  9. Dietary bioactive peptides: Human studies.

    PubMed

    Bouglé, Dominique; Bouhallab, Saïd

    2017-01-22

    Current opinion strongly links nutrition and health. Among nutrients, proteins, and peptides which are encrypted in their sequences and released during digestion could play a key role in improving health. These peptides have been claimed to be active on a wide spectrum of biological functions or diseases, including blood pressure and metabolic risk factors (coagulation, obesity, lipoprotein metabolism, and peroxidation), gut and neurological functions, immunity, cancer, dental health, and mineral metabolism. A majority of studies involved dairy peptides, but the properties of vegetal, animal, and sea products were also assessed. However, these allegations are mainly based on in vitro and experimental studies which are seldom confirmed in humans. This review focused on molecules which were tested in humans, and on the mechanisms explaining discrepancies between experimental and human studies.

  10. Antiviral active peptide from oyster

    NASA Astrophysics Data System (ADS)

    Zeng, Mingyong; Cui, Wenxuan; Zhao, Yuanhui; Liu, Zunying; Dong, Shiyuan; Guo, Yao

    2008-08-01

    An active peptide against herpes virus was isolated from the enzymic hydrolysate of oyster ( Crassostrea gigas) and purified with the definite direction hydrolysis technique in the order of alcalase and bromelin. The hydrolysate was fractioned into four ranges of molecular weight (>10 kDa, 10 5 kDa, 5 1 kDa and <1 kDa) using ultrafiltration membranes and dialysis. The fraction of 10 5 kDa was purified using consecutive chromatographic methods including DEAE Sephadex A-25 column, Sephadex G-25 column, and high performance liquid chromatogram (HPLC) by activity-guided isolation. The antiviral effect of the obtained peptide on herpetic virus was investigated in Vero cells by observing cytopathic effect (CPE). The result shows that the peptide has high inhibitory activity on herpetic virus.

  11. An Investigation on a Novel Anti-tumor Fusion Peptide of FSH33-53-IIKK

    PubMed Central

    Yang, Runlin; Liu, Ping; Pan, Donghui; zhang, Pengjun; Bai, Zhicheng; Xu, Yuping; Wang, Lizhen; Yan, Junjie; Yan, Yongjun; Liu, Xingdang; Yang, Min

    2016-01-01

    A novel fusion peptide FSH33-53-IIKK was designed and expected to combine the follicle stimulating hormone receptor (FSHR) targeting and tumor toxicity. In vitro and in vivo study showed the anti-tumor activity of FSH33-53-IIKK was enhanced compared to that of IIKK only. FSH33-53-IIKK could inhibit the growth of tumor via apoptosis and autophagy pathways. In summary, combining the tumor marker-target peptide and anti-tumor peptide together may be an efficient way to search for better anti-tumor candidates. PMID:27313792

  12. "Click" immobilization of a VEGF-mimetic peptide on decellularized endothelial extracellular matrix to enhance angiogenesis.

    PubMed

    Wang, Lin; Zhao, Meirong; Li, Siheng; Erasquin, Uriel J; Wang, Hao; Ren, Li; Chen, Changyi; Wang, Yingjun; Cai, Chengzhi

    2014-06-11

    We show that coating of decellularized extracellular matrix (DC-ECM) on substrate surfaces is an efficient way to generate a platform mimicking the native ECM environment. Moreover, the DC-ECM can be modified with a peptide (QK) mimicking vascular endothelial growth factor without apparently compromising its integrity. The modification was achieved through metabolic incorporation of a "clickable" handle to DC-ECM followed by rapid attachment of the QK peptide with an azido tag using copper-catalyzed click reaction. The attachment of the QK peptide on to DC-ECM in this way further enhanced the angiogenic responses (formation of branched tubular networks) of endothelial cells.

  13. Construction of a peptide microarray for auto-anti- body detection.

    PubMed

    Cosandey, Vincent; Debrot, Fabien; Kaeser, Jérémy; Marti, Roger; Passeraub, Philippe; Pétremand, Jannick; Prim, Denis; Pfeifer, Marc E

    2012-01-01

    Peptide and protein microarrays provide a multiplex approach to identification and quantification of protein-protein interactions (PPI), useful to study for instance antigen-antibody properties. Multivariate serology assays detecting multiple tumor auto-antibodies (TAA) is an emerging class of blood tests for cancer detection. Here we describe the efficient coupling of peptide baits derived from the BRCA1-associated RING domain protein 1 (BARD1) to a solid surface and detection of a commercially available anti-BARD1 antibody with this newly designed peptide microarray. Analytical sensitivity and specificity were shown to be comparable to a microtiter plate based enzyme-linked immunosorbent assay (ELISA).

  14. Manufacturing of Peptide Microarrays Based on Catalyst-Free Click Chemistry.

    PubMed

    Prim, Denis; Pfeifer, Marc E

    2016-01-01

    Immobilization of peptides to a solid surface is frequently an important first step before they can be probed with a variety of biological samples in a heterogeneous assay format for research and clinical diagnostic purposes. Peptides can be derivatized in many ways to subsequently covalently attach them to an activated solid surface such as epoxy-functionalized glass slides. Here, we describe a clean, efficient, and reproducible fabrication process based on catalyst-free click chemistry compatible with the construction of low- to high-density peptide microarrays.

  15. Use of the 2-chlorotrityl chloride resin for microwave-assisted solid phase peptide synthesis.

    PubMed

    Ieronymaki, Matthaia; Androutsou, Maria Eleni; Pantelia, Anna; Friligou, Irene; Crisp, Molly; High, Kirsty; Penkman, Kirsty; Gatos, Dimitrios; Tselios, Theodore

    2015-09-01

    A fast and efficient microwave (MW)-assisted solid-phase peptide synthesis protocol using the 2-chlorotrityl chloride resin and the Fmoc/tBu methodology, has been developed. The established protocol combines the advantages of MW irradiation and the acid labile 2-chlorotrityl chloride resin. The effect of temperature during the MW irradiation, the degree of resin substitution during the coupling of the first amino acids and the rate of racemization for each amino acid were evaluated. The suggested solid phase methodology is applicable for orthogonal peptide synthesis and for the synthesis of cyclic peptides.

  16. Synthesis of hybrid hydrazino peptides: protected vs unprotected chiral α-hydrazino acids.

    PubMed

    Suć, Josipa; Jerić, Ivanka

    2015-01-01

    Peptidomimetics based on hydrazino derivatives of α-amino acids represent an important class of peptidic foldamers with promising biological activities, like protease inhibition and antimicrobial activity. However, the lack of straightforward method for the synthesis of optically pure hydrazino acids and efficient incorporation of hydrazino building blocks into peptide sequence hamper wider exploitation of hydrazino peptidomimetics. Here we described the utility of N (α)-benzyl protected and unprotected hydrazino derivatives of natural α-amino acids in synthesis of peptidomimetics. While incorporation of N (α)-benzyl-hydrazino acids into peptide chain and deprotection of benzyl moiety proceeded with difficulties, unprotected hydrazino acids allowed fast and simple construction of hybrid peptidomimetics.

  17. Allylic Amines as Key Building Blocks in the Synthesis of (E)-Alkene Peptide Isosteres

    PubMed Central

    Skoda, Erin M.; Davis, Gary C.

    2012-01-01

    Nucleophilic imine additions with vinyl organometallics have developed into efficient, high yielding, and robust methodologies to generate structurally diverse allylic amines. We have used the hydrozirconation-transmetalation-imine addition protocol in the synthesis of allylic amine intermediates for peptide bond isosteres, phosphatase inhibitors, and mitochondria-targeted peptide mimetics. The gramicidin S-derived XJB-5-131 and JP4-039 and their analogs have been prepared on up to 160 g scale for preclinical studies. These (E)-alkene peptide isosteres adopt type II′ β-turn secondary structures and display impressive biological properties, including selective reactions with reactive oxygen species (ROS) and prevention of apoptosis. PMID:22323894

  18. Use of multiple peptides containing T cell epitopes is a feasible approach for peptide-based immunotherapy in Can f 1 allergy

    PubMed Central

    Immonen, Anu K; Taivainen, Antti H; Närvänen, Ale T O; Kinnunen, Tuure T; Saarelainen, Soili A; Rytkönen-Nissinen, Marja A; Virtanen, Tuomas I

    2007-01-01

    We have previously shown that the major dog allergen Can f 1 contains seven T cell epitope regions, none of which was preferentially recognized. To identify the immune characteristics of Can f 1 epitopes and to verify their suitability for peptide-based allergen immunotherapy, short-term T cell lines were generated with epitope-containing peptides from peripheral blood mononuclear cells of Can f 1 skinprick test-positive allergic and healthy control subjects. The lines were examined for their proliferative capacity and cytokine production upon stimulation with the allergen peptide, a homologous peptide from human tear lipocalin (TL) and Can f 1 and TL proteins. Can f 1 peptides induced proliferation of T cells and gave rise to T cell lines with comparable efficiencies. In particular, the T cell lines of allergic subjects induced with p33–48 and p107–122 favoured the production of interferon-γ and interleukin-10, respectively. A greater number of Can f 1-specific T cell lines were generated from allergic than from healthy individuals. Two p107–122-induced Can f 1-specific T cell lines also reacted to a homologous peptide of human TL. Our results suggest that several T cell epitope-containing peptides should be used in combination for specific immunotherapy in Can f 1 allergy. PMID:17233739

  19. A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry

    PubMed Central

    Keough, T.; Youngquist, R. S.; Lacey, M. P.

    1999-01-01

    A method has been developed for de novo peptide sequencing using matrix-assisted laser desorption ionization mass spectrometry. This method will facilitate biological studies that require rapid determination of peptide or protein sequences, e.g., determination of posttranslational modifications, identification of active compounds isolated from combinatorial peptide libraries, and the selective identification of proteins as part of proteome studies. The method involves fast, one-step addition of a sulfonic acid group to the N terminus of tryptic peptides followed by acquisition of postsource decay (PSD) fragment ion spectra. The derivatives are designed to promote efficient charge site-initiated fragmentation of the backbone amide bonds and to selectively enhance the detection of a single fragment ion series that contains the C terminus of the molecule (y-ions). The overall method has been applied to pmol quantities of peptides. The resulting PSD fragment ion spectra often exhibit uninterrupted sequences of 20 or more amino acid residues. However, fragmentation efficiency decreases considerably at amide bonds on the C-terminal side of Pro. The spectra are simple enough that de novo sequence tagging is routine. The technique has been successfully applied to peptide mixtures, to high-mass peptides (up to 3,600 Da) and to the unambiguous identification of proteins isolated from two-dimensional gel electrophoresis. The PSD spectra of these derivatized peptides often allow far more selective protein sequence database searches than those obtained from the spectra of native peptides. PMID:10377380

  20. Efficient functionalization of alginate biomaterials.

    PubMed

    Dalheim, Marianne Ø; Vanacker, Julie; Najmi, Maryam A; Aachmann, Finn L; Strand, Berit L; Christensen, Bjørn E

    2016-02-01

    Peptide coupled alginates obtained by chemical functionalization of alginates are commonly used as scaffold materials for cells in regenerative medicine and tissue engineering. We here present an alternative to the commonly used carbodiimide chemistry, using partial periodate oxidation followed by reductive amination. High and precise degrees of substitution were obtained with high reproducibility, and without formation of by-products. A protocol was established using l-Tyrosine methyl ester as a model compound and the non-toxic pic-BH3 as the reducing agent. DOSY was used to indirectly verify covalent binding and the structure of the product was further elucidated using NMR spectroscopy. The coupling efficiency was to some extent dependent on alginate composition, being most efficient on mannuronan. Three different bioactive peptide sequences (GRGDYP, GRGDSP and KHIFSDDSSE) were coupled to 8% periodate oxidized alginate resulting in degrees of substitution between 3.9 and 6.9%. Cell adhesion studies of mouse myoblasts (C2C12) and human dental stem cells (RP89) to gels containing various amounts of GRGDSP coupled alginate demonstrated the bioactivity of the material where RP89 cells needed higher peptide concentrations to adhere.

  1. Naturally occurring and synthetic peptides acting on nicotinic acetylcholine receptors.

    PubMed

    Kasheverov, Igor E; Utkin, Yuri N; Tsetlin, Victor I

    2009-01-01

    Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane-bound proteins belonging to the large family of ligand-gated ion channels. nAChRs possess various binding sites which interact with compounds of different chemical nature, including peptides. Historically first peptides found to act on nAChR were synthetic fragments of snake alpha-neurotoxins, competitive receptor antagonists. Later it was shown that fragments of glycoprotein from rabies virus, having homology to alpha-neurotoxins, and polypeptide neurotoxins waglerins from the venom of Wagler's pit viper Trimeresurus (Tropidolaemus) wagleri bind in a similar way, waglerins being efficient blockers of muscle-type nAChRs. Neuropeptide substance P appears to interact with the channel moiety of nAChR. beta-Amyloid, a peptide forming senile plaques in Alzheimer's disease, also can bind to nAChR, although the mode of binding is still unclear. However, the most well-studied peptides interacting with the ligand-binding sites of nAChRs are so-called alpha-conotoxins, peptide neurotoxins from marine snails of Conus genus. First alpha-conotoxins were discovered in the late 1970s, and now it is a rapidly growing family due to isolation of peptides from multiple Conus species, as well as to cloning, and chemical synthesis of new analogues. Because of their unique selectivity towards distinct nAChR subtypes, alpha-conotoxins became valuable tools in nAChR research. Recent X-ray structures of alpha-conotoxin complexes with acetylcholine-binding protein, a model of nAChR ligand-binding domains, revealed the details of the nAChR ligand-binding sites and provided the basis for design of novel ligands.

  2. Membrane peptides and their role in protobiological evolution

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Wilson, Michael A.; Chipot, Christophe

    2003-01-01

    How simple membrane peptides performed such essential protocellular functions as transport of ions and organic matter across membranes separating the interior of the cell from the environment, capture and utilization of energy, and transduction of environmental signals, is a key question in protobiological evolution. On the basis of detailed, molecular-level computer simulations we explain how these peptides fold at water-membrane interfaces, insert into membranes, self-assemble into higher-order structures and acquire functions. We have investigated the interfacial behavior and folding of several peptides built of leucine and glutamine residues and have demonstrated that many of them tend to adopt ordered structures. Further, we have studied the insertion of an alpha-helical peptide containing leucine (L) and serine (S) of the form (LSLLLSL)3 into a model membrane. The transmembrane state is metastable, and approximately 15 kcal mol(-1) is required to insert the peptide into the membrane. Investigations of dimers formed by (LSLLLSL)3 and glycophorin A demonstrate how the favorable free energy of helix association can offset the unfavorable free energy of insertion, leading to self-assembly of peptide helices in the membrane. An example of a self-assembled structure is the tetrameric transmembrane pore of the influenza virus M2 protein, which is an efficient and selective voltage-gated proton channel. Our simulations explain the gating mechanism and provide guidelines how to re-engineer the channel to act as a simple proton pump. In general, emergence of integral membrane proteins appears to be quite feasible and may be easier to envision than the emergence of water-soluble proteins.

  3. Synthesis of cysteine-rich peptides by native chemical ligation without use of exogenous thiols.

    PubMed

    Tsuda, Shugo; Yoshiya, Taku; Mochizuki, Masayoshi; Nishiuchi, Yuji

    2015-04-03

    Native chemical ligation (NCL) performed without resorting to the use of thiol additives was demonstrated to be an efficient and effective procedure for synthesizing Cys-rich peptides. This method using tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent facilitates the ligation reaction even at the Thr-Cys or Ile-Cys site and enables one-pot synthesis of Cys-rich peptides throughout NCL and oxidative folding.

  4. Cholesterol-Peptide Hybrids to Form Liposome-Like Vesicles for Gene Delivery

    PubMed Central

    Tang, Qiong; Cao, Bin; Wu, Haiyan; Cheng, Gang

    2013-01-01

    In this paper, four amphiphilic cholesterol-peptide conjugates (Ch-R5H5, Ch-R3H3, Ch-R5 and Ch-R5) were designed and synthesized, and their properties in gene delivery were evaluated in vitro with an aim of developing more efficient gene delivery carriers. These amphiphilic cholesterol-peptide conjugates are composed of hydrophobic cholesterol and positively charged peptides. They were able to self-assemble into micelles at low concentrations and their critical micelle concentrations in phosphate buffered saline (pH 7.4) are ≤85 µg/mL. Amphiphilic cholesterol-peptide conjugates condensed DNA more efficiently than a hydrophilic cationic oligoarginine (R10) peptide with no hydrophobic segment. Their transfection efficiencies were at least two orders of magnitude greater than that of R10 peptide in HEK-293 cells. Moreover, the introduction of histidine residues in cholesterol-peptide conjugates led to higher gene expression efficiency compared with cholesterol-peptides without histidine (Ch-R5 and Ch-R3), and the luciferase expression level was comparable or even higher than that induced by PEI at its optimal N/P ratio. In particular, Ch-R5H5 condensed DNA into smaller nanoparticles than Ch-R3H3 at higher N/P ratios, and the minimum size of Ch-R5H5/DNA complexes was 180 nm with zeta potential of 23 mV, achieved at the N/P ratio of 30. This liposome-like vesicle may be a promising gene delivery carrier for intravenous therapy. PMID:23382899

  5. A stabilized peptide ligand for multifunctional glioma targeted drug delivery.

    PubMed

    Ying, Man; Shen, Qing; Zhan, Changyou; Wei, Xiaoli; Gao, Jie; Xie, Cao; Yao, Bingxin; Lu, Weiyue

    2016-12-10

    Peptide ligands consisting of l-amino acids are subject to proteolysis in vivo. When modified on the surface of nanocarriers, those peptide ligands would readily degrade and the targeting efficacy is significantly attenuated. It has received increasing scrutiny to design stable peptide ligands for targeted drug delivery. Here, we present the design of a stable peptide ligand by the formation of a head-to-tail amide bond as an example. Even though the linear l-peptide A7R (termed (L)A7R) can bind specifically to vascular endothelial growth factor receptor 2 (VEGFR2) and neuropilin-1 (NRP-1) that are overexpressed on glioma cells, neovasculature and glioma vasculogenic mimicry (VM), the tumor-homing capacity of (L)A7R is greatly impaired in vivo due to proteolysis (e.g. in the serum). A cyclic A7R (cA7R) peptide was identified by computer-aided peptide design and synthesized with high yield by combining solid phase peptide synthesis and native chemical ligation. The binding of cA7R to both receptors was theoretically and experimentally assessed. In our simulated model hydrophobic and ionic interactions dominated the binding of (L)A7R to receptors. It is very interesting that cA7R adopting a different structure from (L)A7R retained high binding affinities to receptors without affecting the hydrophobic and ionic interactions. After head-to-tail cyclization by the formation of an amide bond, cA7R exhibited exceptional stability in mouse serum. Either cA7R or (L)A7R was conjugated on the surface of doxorubicin (DOX) loaded liposomes (cA7R-LS/DOX or (L)A7R-LS/DOX). The results of in vitro cellular assays indicated that cA7R-LS/DOX not only displayed stronger anti-proliferative effect against glioma cells, but also demonstrated to be more efficient in destruction of VM and HUVEC tubes in comparison to (L)A7R-LS/DOX and plain liposomes (LS/DOX, without peptide conjugation). cA7R conjugation could achieve significantly higher accumulation of liposomes in glioma than did (L

  6. Membrane Perturbation Induced by Interfacially Adsorbed Peptides

    PubMed Central

    Zemel, Assaf; Ben-Shaul, Avinoam; May, Sylvio

    2004-01-01

    The structural and energetic characteristics of the interaction between interfacially adsorbed (partially inserted) α-helical, amphipathic peptides and the lipid bilayer substrate are studied using a molecular level theory of lipid chain packing in membranes. The peptides are modeled as “amphipathic cylinders” characterized by a well-defined polar angle. Assuming two-dimensional nematic order of the adsorbed peptides, the membrane perturbation free energy is evaluated using a cell-like model; the peptide axes are parallel to the membrane plane. The elastic and interfacial contributions to the perturbation free energy of the “peptide-dressed” membrane are evaluated as a function of: the peptide penetration depth into the bilayer's hydrophobic core, the membrane thickness, the polar angle, and the lipid/peptide ratio. The structural properties calculated include the shape and extent of the distorted (stretched and bent) lipid chains surrounding the adsorbed peptide, and their orientational (C-H) bond order parameter profiles. The changes in bond order parameters attendant upon peptide adsorption are in good agreement with magnetic resonance measurements. Also consistent with experiment, our model predicts that peptide adsorption results in membrane thinning. Our calculations reveal pronounced, membrane-mediated, attractive interactions between the adsorbed peptides, suggesting a possible mechanism for lateral aggregation of membrane-bound peptides. As a special case of interest, we have also investigated completely hydrophobic peptides, for which we find a strong energetic preference for the transmembrane (inserted) orientation over the horizontal (adsorbed) orientation. PMID:15189858

  7. Tuning Liposome Membrane Permeability by Competitive Peptide Dimerization and Partitioning-Folding Interactions Regulated by Proteolytic Activity

    NASA Astrophysics Data System (ADS)

    Lim, Seng Koon; Sandén, Camilla; Selegård, Robert; Liedberg, Bo; Aili, Daniel

    2016-02-01

    Membrane active peptides are of large interest for development of drug delivery vehicles and therapeutics for treatment of multiple drug resistant infections. Lack of specificity can be detrimental and finding routes to tune specificity and activity of membrane active peptides is vital for improving their therapeutic efficacy and minimize harmful side effects. We describe a de novo designed membrane active peptide that partition into lipid membranes only when specifically and covalently anchored to the membrane, resulting in pore-formation. Dimerization with a complementary peptide efficiently inhibits formation of pores. The effect can be regulated by proteolytic digestion of the inhibitory peptide by the matrix metalloproteinase MMP-7, an enzyme upregulated in many malignant tumors. This system thus provides a precise and specific route for tuning the permeability of lipid membranes and a novel strategy for development of recognition based membrane active peptides and indirect enzymatically controlled release of liposomal cargo.

  8. Orthogonal ring-closing alkyne and olefin metathesis for the synthesis of small GTPase-targeting bicyclic peptides

    PubMed Central

    Cromm, Philipp M.; Schaubach, Sebastian; Spiegel, Jochen; Fürstner, Alois; Grossmann, Tom N.; Waldmann, Herbert

    2016-01-01

    Bicyclic peptides are promising scaffolds for the development of inhibitors of biological targets that proved intractable by typical small molecules. So far, access to bioactive bicyclic peptide architectures is limited due to a lack of appropriate orthogonal ring-closing reactions. Here, we report chemically orthogonal ring-closing olefin (RCM) and alkyne metathesis (RCAM), which enable an efficient chemo- and regioselective synthesis of complex bicyclic peptide scaffolds with variable macrocycle geometries. We also demonstrate that the formed alkyne macrocycle can be functionalized subsequently. The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand. This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far. The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets. PMID:27075966

  9. Sample Limited Characterization of a Novel Disulfide-Rich Venom Peptide Toxin from Terebrid Marine Snail Terebra variegata

    PubMed Central

    Anand, Prachi; Grigoryan, Alexandre; Bhuiyan, Mohammed H.; Ueberheide, Beatrix; Russell, Victoria; Quinoñez, Jose; Moy, Patrick; Chait, Brian T.; Poget, Sébastien F.; Holford, Mandë

    2014-01-01

    Disulfide-rich peptide toxins found in the secretions of venomous organisms such as snakes, spiders, scorpions, leeches, and marine snails are highly efficient and effective tools for novel therapeutic drug development. Venom peptide toxins have been used extensively to characterize ion channels in the nervous system and platelet aggregation in haemostatic systems. A significant hurdle in characterizing disulfide-rich peptide toxins from venomous animals is obtaining significant quantities needed for sequence and structural analyses. Presented here is a strategy for the structural characterization of venom peptide toxins from sample limited (4 ng) specimens via direct mass spectrometry sequencing, chemical synthesis and NMR structure elucidation. Using this integrated approach, venom peptide Tv1 from Terebra variegata was discovered. Tv1 displays a unique fold not witnessed in prior snail neuropeptides. The novel structural features found for Tv1 suggest that the terebrid pool of peptide toxins may target different neuronal agents with varying specificities compared to previously characterized snail neuropeptides. PMID:24713808

  10. NMR chemical shift study of the interaction of selected peptides with liposomal and micellar models of apoptotic cells.

    PubMed

    Van Koninckxloo, Aurore; Henoumont, Céline; Laurent, Sophie; Muller, Robert N; Vander Elst, Luce

    2014-12-01

    The interaction between two peptides previously selected by phage display to target apoptotic cells and phospholipidic models of these cells (liposomes or micelles made of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and/or 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS, phosphatidylserine analog) was studied by the simple analysis of the changes induced on the proton NMR chemical shifts of the peptides. Our approach which does not need healthy and/or apoptotic cells for assessing the affinity of different peptides is fast and efficient and requires small amounts of peptide to determine the association constant, the interacting protons, and the number of interaction sites. The micellar model gave more reliable results than the liposomal one. The preferential interaction of the peptide with DPPS was evidenced by the change of the chemical shifts of specific amino acids of the peptides. Our micellar model is thus well suited to mimic apoptotic cells.

  11. Novel Formulations for Antimicrobial Peptides

    PubMed Central

    Carmona-Ribeiro, Ana Maria; Carrasco, Letícia Dias de Melo

    2014-01-01

    Peptides in general hold much promise as a major ingredient in novel supramolecular assemblies. They may become essential in vaccine design, antimicrobial chemotherapy, cancer immunotherapy, food preservation, organs transplants, design of novel materials for dentistry, formulations against diabetes and other important strategical applications. This review discusses how novel formulations may improve the therapeutic index of antimicrobial peptides by protecting their activity and improving their bioavailability. The diversity of novel formulations using lipids, liposomes, nanoparticles, polymers, micelles, etc., within the limits of nanotechnology may also provide novel applications going beyond antimicrobial chemotherapy. PMID:25302615

  12. Novel formulations for antimicrobial peptides.

    PubMed

    Carmona-Ribeiro, Ana Maria; de Melo Carrasco, Letícia Dias

    2014-10-09

    Peptides in general hold much promise as a major ingredient in novel supramolecular assemblies. They may become essential in vaccine design, antimicrobial chemotherapy, cancer immunotherapy, food preservation, organs transplants, design of novel materials for dentistry, formulations against diabetes and other important strategical applications. This review discusses how novel formulations may improve the therapeutic index of antimicrobial peptides by protecting their activity and improving their bioavailability. The diversity of novel formulations using lipids, liposomes, nanoparticles, polymers, micelles, etc., within the limits of nanotechnology may also provide novel applications going beyond antimicrobial chemotherapy.

  13. Peptides and the new endocrinology

    NASA Astrophysics Data System (ADS)

    Schwyzer, Robert

    1982-01-01

    The discovery of regulatory peptides common to the nervous and the endocrine systems (brain, gut, and skin) has brought about a revolution in our concepts of endocrinology and neurology. We are beginning to understand some of the complex interrelationships between soma and psyche that might, someday, be important for an integrated treatment of diseases. Examples of the actions of certain peptides in the periphery and in the central nervous system are given, and their biosynthesis and molecular anatomy as carriers for information are discussed.

  14. Butelase-mediated cyclization and ligation of peptides and proteins.

    PubMed

    Nguyen, Giang K T; Qiu, Yibo; Cao, Yuan; Hemu, Xinya; Liu, Chuan-Fa; Tam, James P

    2016-10-01

    Enzymes that catalyze efficient macrocyclization or site-specific ligation of peptides and proteins can enable tools for drug design and protein engineering. Here we describe a protocol to use butelase 1, a recently discovered peptide ligase, for high-efficiency cyclization and ligation of peptides and proteins ranging in size from 10 to >200 residues. Butelase 1 is the fastest known ligase and is found in pods of the common medicinal plant Clitoria ternatea (also known as butterfly pea). It has a very simple C-terminal-specific recognition motif that requires Asn/Asp (Asx) at the P1 position and a dipeptide His-Val at the P1' and P2' positions. Substrates for butelase-mediated ligation can be prepared by standard Fmoc (9-fluorenylmethyloxycarbonyl) chemistry or recombinant expression with the minimal addition of this tripeptide Asn-His-Val motif at the C terminus. Butelase 1 achieves cyclizations that are 20,000 times faster than those of sortase A, a commonly used enzyme for backbone cyclization. Unlike sortase A, butelase is traceless, and it can be used for the total synthesis of naturally occurring peptides and proteins. Furthermore, butelase 1 is also useful for intermolecular ligations and synthesis of peptide or protein thioesters, which are versatile activated intermediates necessary for and compatible with many chemical ligation methods. The protocol describes steps for isolation and purification of butelase 1 from plant extract using a four-step chromatography procedure, which takes ∼3 d. We then describe steps for intramolecular cyclization, intermolecular ligation and butelase-mediated synthesis of protein thioesters. Butelase reactions are generally completed within minutes and often achieve excellent yields.

  15. Streptomyces as Overexpression System for Heterologous Production of an Antimicrobial Peptide.

    PubMed

    Roldán-Tapia, Marisol; Anné, Jozef; Reyes, Ana Gisela; Carrasco, Ulises; Millán-Pacheco, Cesar; Barrios-González, Javier; Mejía, Armando

    2017-02-08

    Defense mechanisms of plants against phytopathogens include cationic antimicrobial peptides (CAPs). The broad-spectrum activity of these peptides has been evaluated previously against different phytopathogens. Their lack of toxicity for plants and animals is a promising feature for pest control. Although, some attempts have been made previously in order to increase their heterologous expressions, the employed strategies have so far proven to be ineffective. Low production yield and elevated costs are the obstacles to overcome. In this study, a strategy for CAP overexpression is presented based on the construction of an expression cassette for Streptomyces lividans TK24. This system contains elements that allow the increase of the efficiency of the peptide's expression. The main elements included in this cassette are the sequences of the promoter and signal peptide from a subtilisin inhibitor gene of Streptomyces venezuelae. This allows the efficient secretion of the peptide to the growth medium, thereby simplifying its recovery and avoiding its toxic effect on the producing organism. The results obtained demonstrate the system's efficiency by achieving a peptide concentration of 11.61 mg/ml. This represents at least a 10-fold increase compared to previously established strategies.

  16. Towards rice bran protein utilization: In silico insight on the role of oryzacystatins in biologically-active peptide production.

    PubMed

    Udenigwe, Chibuike C

    2016-01-15

    Rice bran proteins (RBP) have been demonstrated to harbour biologically active peptides, which can be released by proteases and applied in human health promotion. In this study, the roles of rice bran cysteine protease inhibitors, oryzacystatins, were considered for efficient production of bioactive peptides from RBP. In silico evidence demonstrates that aspartate protease (pepsin at pH>2) and metalloproteinase (thermolysin) have strong prospects for use in simultaneously cleaving the QXVXGX motif of oryzacystatins, which can lead to their inactivation, and in releasing bioactive sequences from the protease inhibitors. The cleaved bioactive peptides are known to possess activities that can be applied in the management of hypertension, oxidative stress, type 2 diabetes mellitus and other aberrant cellular processes. Moreover, several potentially bioactive di- and tripeptides were identified in oryzacystatin peptide pools. This study provides an important consideration and a direction that can lead to efficient release of bioactive peptides from rice bran proteins for functional food applications.

  17. Peptide Antibodies: Past, Present, and Future.

    PubMed

    Houen, Gunnar

    2015-01-01

    Peptide antibodies recognize epitopes with amino acid residues adjacent in sequence ("linear" epitopes). Such antibodies can be made to virtually any sequence and have been immensely important in all areas of molecular biology and diagnostics due to their versatility and to the rapid growth in protein sequence information. Today, peptide antibodies can be routinely and rapidly made to large numbers of peptides, including peptides with posttranslationally modified residues, and are used for immunoblotting, immunocytochemistry, immunohistochemistry, and immunoassays. In the future, peptide antibodies will continue to be immensely important for molecular biology, TCR- and MHC-like peptide antibodies may be produced routinely, peptide antibodies with predetermined conformational specificities may be designed, and peptide-based vaccines may become part of vaccination programs.

  18. Computational approach for designing tumor homing peptides

    PubMed Central

    Sharma, Arun; Kapoor, Pallavi; Gautam, Ankur; Chaudhary, Kumardeep; Kumar, Rahul; Chauhan, Jagat Singh; Tyagi, Atul; Raghava, Gajendra P. S.

    2013-01-01

    Tumor homing peptides are small peptides that home specifically to tumor and tumor associated microenvironment i.e. tumor vasculature, after systemic delivery. Keeping in mind the huge therapeutic importance of these peptides, we have made an attempt to analyze and predict tumor homing peptides. It was observed that certain types of residues are preferred in tumor homing peptides. Therefore, we developed support vector machine based models for predicting tumor homing peptides using amino acid composition and binary profiles of peptides. Amino acid composition, dipeptide composition and binary profile-based models achieved a maximum accuracy of 86.56%, 82.03%, and 84.19% respectively. These methods have been implemented in a user-friendly web server, TumorHPD. We anticipate that this method will be helpful to design novel tumor homing peptides. TumorHPD web server is freely accessible at http://crdd.osdd.net/raghava/tumorhpd/. PMID:23558316

  19. Membrane disruption mechanism of antimicrobial peptides

    NASA Astrophysics Data System (ADS)

    Lee, Ka Yee

    2012-04-01

    Largely distributed among living organisms, antimicrobial peptides are a class of small (<100 residues) host defense peptides that induce selective membrane lytic activity against microbial pathogens. The permeabilizing behavior of these diverse peptides has been commonly attributed to the formation of pores, and such pore formation has been categorized as barrel-stave, toroidal, or carpet-like. With the continuing discovery of new peptide species, many are uncharacterized and the exact mechanism is unknown. Through the use of atomic force microscopy, the disruption of supported lipid bilayer patches by protegrin-1 is concentration-dependent. The intercalation of antimicrobial peptide into the bilayer results in structures beyond that of pore formation, but with the formation of worm-like micelles at high peptide concentration. Our results suggest that antimicrobial peptide acts to lower the interfacial energy of the bilayer in a way similar to detergents. Antimicrobial peptides with structural differences, magainin-1 and aurein 1.1, exhibit a mechanistic commonality.

  20. Stapled peptide induces cancer cell death.

    PubMed

    Whelan, Jo

    2004-11-01

    Hydrocarbon stapling could enable peptides from the key domains of natural proteins to be used therapeutically. Using the technique on a peptide involved in apoptosis, researchers have succeeded in destroying cancer cells in a mouse model of leukaemia.

  1. Investigating Endogenous Peptides and Peptidases using Peptidomics

    PubMed Central

    Tinoco, Arthur D.; Saghatelian, Alan

    2012-01-01

    Rather than simply being protein degradation products, peptides have proven to be important bioactive molecules. Bioactive peptides act as hormones, neurotransmitters and antimicrobial agents in vivo. The dysregulation of bioactive peptide signaling is also known to be involved in disease, and targeting peptide hormone pathways has been successful strategy in the development of novel therapeutics. The importance of bioactive peptides in biology has spurred research to elucidate the function and regulation of these molecules. Classical methods for peptide analysis have relied on targeted immunoassays, but certain scientific questions necessitated a broader and more detailed view of the peptidome–all the peptides in a cell, tissue or organism. In this review we discuss how peptidomics has emerged to fill this need through the application of advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods that provide unique insights into peptide activity and regulation. PMID:21786763

  2. Identification of tissue-specific targeting peptide

    NASA Astrophysics Data System (ADS)

    Jung, Eunkyoung; Lee, Nam Kyung; Kang, Sang-Kee; Choi, Seung-Hoon; Kim, Daejin; Park, Kisoo; Choi, Kihang; Choi, Yun-Jaie; Jung, Dong Hyun

    2012-11-01

    Using phage display technique, we identified tissue-targeting peptide sets that recognize specific tissues (bone-marrow dendritic cell, kidney, liver, lung, spleen and visceral adipose tissue). In order to rapidly evaluate tissue-specific targeting peptides, we performed machine learning studies for predicting the tissue-specific targeting activity of peptides on the basis of peptide sequence information using four machine learning models and isolated the groups of peptides capable of mediating selective targeting to specific tissues. As a representative liver-specific targeting sequence, the peptide "DKNLQLH" was selected by the sequence similarity analysis. This peptide has a high degree of homology with protein ligands which can interact with corresponding membrane counterparts. We anticipate that our models will be applicable to the prediction of tissue-specific targeting peptides which can recognize the endothelial markers of target tissues.

  3. Boosting production yield of biomedical peptides

    NASA Technical Reports Server (NTRS)

    Manatt, S. L.

    1978-01-01

    Nuclear magnetic resonance (NMR) technique is employed to monitor synthesis of biomedical peptides. Application of NMR technique may improve production yields of insulin, ACTH, and growth hormones, as well as other synthesized biomedical peptides.

  4. Streptavidin-binding peptides and uses thereof

    NASA Technical Reports Server (NTRS)

    Szostak, Jack W. (Inventor); Wilson, David S. (Inventor); Keefe, Anthony D. (Inventor)

    2006-01-01

    The invention provides peptides with high affinity for streptavidin. These peptides may be expressed as part of fusion proteins to facilitate the detection, quantitation, and purification of proteins of interest.

  5. Streptavidin-binding peptides and uses thereof

    NASA Technical Reports Server (NTRS)

    Szostak, Jack W. (Inventor); Wilson, David S. (Inventor); Keefe, Anthony D. (Inventor)

    2005-01-01

    The invention provides peptides with high affinity for streptavidin. These peptides may be expressed as part of fusion proteins to facilitate the detection, quantitation, and purification of proteins of interest.

  6. Carbohydrates in peptide and protein design.

    PubMed

    Jensen, Knud J; Brask, Jesper

    2005-01-01

    Monosaccharides and amino acids are fundamental building blocks in the assembly of nature's polymers. They have different structural aspects and, to a significant extent, different functional groups. Oligomerization gives rise to oligosaccharides and peptides, respectively. While carbohydrates and peptides can be found conjoined in nature, e.g., in glycopeptides, the aim of this review is the radical redesign of peptide structures using carbohydrates, particularly monosaccharides and cyclic oligosaccharides, to produce novel peptides, peptidomimetics, and abiotic proteins. These hybrid molecules, chimeras, have properties arising largely from the combination of structural characteristics of carbohydrates with the functional group diversity of peptides. This field includes de novo designed synthetic glycopeptides, sugar (carbohydrate) amino acids, carbohydrate scaffolds for nonpeptidal peptidomimetics of cyclic peptides, cyclodextrin functionalized peptides, and carboproteins, i.e., carbohydrate-based proteinmimetics. These successful applications demonstrate the general utility of carbohydrates in peptide and protein architecture.

  7. Digesting New Elements in Peptide Transport.

    PubMed

    Lyons, Joseph A; Nissen, Poul

    2015-10-06

    In this issue of Structure, Beale et al. (2015) define structurally and functionally a large extracellular domain unique to mammalian peptide transporters and its implications for the transport of basic di- and tri-peptides (Beale et al., 2015).

  8. Surface Plasmon Resonance Studies of the Specific Interactions of Hexamer Peptide Ligands with Human Immunoglobulin G

    NASA Astrophysics Data System (ADS)

    Islam, Nafisa

    structure of the peptide contributed to a constant rate adsorption constant, ka, at all concentrations of IgG, regardless of flow rates, when peptides were grafted onto pure or mixed SAMs. However the adsorption rate constant, ka showed partial diffusion-controlled behavior when peptides were grafted onto branched amines on SAMs. This was attributed to the three dimensional matrix of branched amines introducing mass transport limitations when IgG binds to the peptides grafted onto the branched amines. It was also found that theoretical models must incorporate parameters to account for multiple-site binding of IgG on the branched amine-based surfaces. The performances of the biosensing surfaces with peptides grafted onto pure SAMs, onto mixed SAMs and onto branched amines were analyzed in presence of complex mixtures and for their regeneration potentials. Complex mixture IgG binding gave the best results for branched amine-based peptide surfaces for a wide range of IgG concentrations. Regeneration behavior was optimum for the branched amine-based surfaces and peptide activity showed negligible decline after eight cycles of IgG binding and regeneration. Thus, among the peptide-based surfaces studied, the peptides grafted on branched amines were optimal with regards to peptide density, efficiency of amine terminus usage, sensitivity and regeneration potential. The theoretical models developed in this work helped to understand the binding mechanism of IgG to the peptide on various supports. The potential for commercial deployment of peptide-based IgG biosensors has been demonstrated in this research work. (Abstract shortened by UMI.)

  9. The evolution of peptide hormones.

    PubMed

    Niall, H D

    1982-01-01

    Despite limitations in our present knowledge it is already possible to discern the main features of peptide hormone evolution, since the same mechanisms (and indeed the same hormone molecules) function in many different ways. This underlying unity of organization has its basis in the tendency of biochemical networks, once established, to survive and diversify. The most surprising recent findings in endocrinology have been the discovery of vertebrate peptide hormones in multiple sites within the same organism, and the reports, persuasive but requiring confirmation, of vertebrate hormones in primitive unicellular organisms (20, 20a). Perhaps the major challenge for the future is to define the roles and interactions of the many peptide hormones identified in brain (18). The most primitive bacteria and the human brain, though an enormous evolutionary distance apart, may have more in common than we have recognized until now. As Axelrod & Hamilton have pointed out in a recent provocative article, "The Evolution of Cooperation" (1), bacteria, though lacking a brain, are capable of adaptive behavior that can be analysed in terms of game theory. It is clear that we can learn a great deal about the whole evolutionary process from a study of the versatile and durable peptide hormones molecules.

  10. Acyclic peptide inhibitors of amylases.

    PubMed

    Pohl, Nicola

    2005-12-01

    In this issue of Chemistry and Biology, a library screening approach reveals a linear octapeptide inhibitor of alpha-amylases reached by de novo design . The selected molecule shares characteristics with naturally occurring protein inhibitors -- a result that suggests general rules for the design of peptide-based amylase inhibitors may be achievable.

  11. Bi- or multifunctional peptide drugs

    PubMed Central

    Schiller, Peter W.

    2009-01-01

    Strategies for the design of bi- or multifunctional drugs are reviewed. A distinction is made between bifunctional drugs interacting in a monovalent fashion with two targets and ligands containing two distinct pharmacophores binding in a bivalent mode to the two binding sites in a receptor heterodimer. Arguments are presented to indicate that some of the so-called “bivalent” ligands reported in the literature are unlikely to simultaneously interact with two binding sites. Aspects related to the development of bi- or multifunctional drugs are illustrated with examples from the field of opioid analgesics. The drug-like properties of the tetrapeptide Dmt1[DALDA] with triple action as a μ opioid agonist, norepinephrine uptake inhibitor and releaser of endogenous opioid peptides to produce potent spinal analgesia are reviewed. Rationales for the development of opioid peptides with mixed agonist/antagonist profiles as analgesics with reduced side effects are presented. Progress in the development of mixed μ opioid agonist/δ opioid antagonists with low propensity to produce tolerance and physical dependence is reviewed. Efforts to develop bifunctional peptides containing a μ opioid agonist and a cholecystokinin antagonist or an NK1 receptor antagonist as analgesics expected to produce less tolerance and dependence are also reviewed. A strategy to improve the drug-like properties of bifunctional opioid peptide analgesics is presented. PMID:19285088

  12. Free-living nematode peptides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    All nematodes employ a wide array of peptide messengers to control nearly all aspects of the life cycle, including hatching, locomotion, feeding, defense, mating, reproduction, and other behavioral and metabolic events. There are molecular and biological similarities, as well as significant differen...

  13. Toxins and antimicrobial peptides: interactions with membranes

    NASA Astrophysics Data System (ADS)

    Schlamadinger, Diana E.; Gable, Jonathan E.; Kim, Judy E.

    2009-08-01

    The innate immunity to pathogenic invasion of organisms in the plant and animal kingdoms relies upon cationic antimicrobial peptides (AMPs) as the first line of defense. In addition to these natural peptide antibiotics, similar cationic peptides, such as the bee venom toxin melittin, act as nonspecific toxins. Molecular details of AMP and peptide toxin action are not known, but the universal function of these peptides to disrupt cell membranes of pathogenic bacteria (AMPs) or a diverse set of eukaryotes and prokaryotes (melittin) is widely accepted. Here, we have utilized spectroscopic techniques to elucidate peptide-membrane interactions of alpha-helical human and mouse AMPs of the cathelicidin family as well as the peptide toxin melittin. The activity of these natural peptides and their engineered analogs was studied on eukaryotic and prokaryotic membrane mimics consisting of <200-nm bilayer vesicles composed of anionic and neutral lipids as well as cholesterol. Vesicle disruption, or peptide potency, was monitored with a sensitive fluorescence leakage assay. Detailed molecular information on peptidemembrane interactions and peptide structure was further gained through vibrational spectroscopy combined with circular dichroism. Finally, steady-state fluorescence experiments yielded insight into the local environment of native or engineered tryptophan residues in melittin and human cathelicidin embedded in bilayer vesicles. Collectively, our results provide clues to the functional structures of the engineered and toxic peptides and may impact the design of synthetic antibiotic peptides that can be used against the growing number of antibiotic-resistant pathogens.

  14. Identification of multifunctional peptides from human milk.

    PubMed

    Mandal, Santi M; Bharti, Rashmi; Porto, William F; Gauri, Samiran S; Mandal, Mahitosh; Franco, Octavio L; Ghosh, Ananta K

    2014-06-01

    Pharmaceutical industries have renewed interest in screening multifunctional bioactive peptides as a marketable product in health care applications. In this context, several animal and plant peptides with potential bioactivity have been reported. Milk proteins and peptides have received much attention as a source of health-enhancing components to be incorporated into nutraceuticals and functional foods. By using this source, 24 peptides have been fractionated and purified from human milk using RP-HPLC. Multifunctional roles including antimicrobial, antioxidant and growth stimulating activity have been evaluated in all 24 fractions. Nevertheless, only four fractions show multiple combined activities among them. Using a proteomic approach, two of these four peptides have been identified as lactoferrin derived peptide and kappa casein short chain peptide. Lactoferrin derived peptide (f8) is arginine-rich and kappa casein derived (f12) peptide is proline-rich. Both peptides (f8 and f12) showed antimicrobial activities against both Gram-positive and Gram-negative bacteria. Fraction 8 (f8) exhibits growth stimulating activity in 3T3 cell line and f12 shows higher free radical scavenging activity in comparison to other fractions. Finally, both peptides were in silico evaluated and some insights into their mechanism of action were provided. Thus, results indicate that these identified peptides have multiple biological activities which are valuable for the quick development of the neonate and may be considered as potential biotechnological products for nutraceutical industry.

  15. Peptide-functionalized iron oxide magnetic nanoparticle for gold mining

    NASA Astrophysics Data System (ADS)

    Shen, Wei-Zheng; Cetinel, Sibel; Sharma, Kumakshi; Borujeny, Elham Rafie; Montemagno, Carlo

    2017-02-01

    Here, we present our work on preparing a novel nanomaterial composed of inorganic binding peptides and magnetic nanoparticles for inorganic mining. Two previously selected and well-characterized gold-binding peptides from cell surface display, AuBP1 and AuBP2, were exploited. This nanomaterial (AuBP-MNP) was designed to fulfill the following two significant functions: the surface conjugated gold-binding peptide will recognize and selectively bind to gold, while the magnetic nano-sized core will respond and migrate according to the applied external magnetic field. This will allow the smart nanomaterial to mine an individual material (gold) from a pool of mixture, without excessive solvent extraction, filtration, and concentration steps. The working efficiency of AuBP-MNP was determined by showing a dramatic reduction of gold nanoparticle colloid concentration, monitored by spectroscopy. The binding kinetics of AuBP-MNP onto the gold surface was determined using surface plasmon resonance (SPR) spectroscopy, which exhibits around 100 times higher binding kinetics than peptides alone. The binding capacity of AuBP-MNP was demonstrated by a bench-top mining test with gold microparticles.

  16. Molecular biomimetics: nanotechnology and bionanotechnology using genetically engineered peptides.

    PubMed

    Tamerler, Candan; Sarikaya, Mehmet

    2009-05-13

    Nature provides inspiration for designing materials and systems that derive their functions from highly organized structures. Biological hard tissues are hybrid materials having inorganics within a complex organic matrix, the molecular scaffold controlling the inorganic structures. Biocomposites incorporate both biomacromolecules such as proteins, lipids and polysaccharides, and inorganic materials, such as hydroxyapatite, silica, magnetite and calcite. The ordered organization of hierarchical structures in organisms begins via the molecular recognition of inorganics by proteins that control interactions and is followed by the highly efficient self-assembly across scales. Following the molecular biological principle, proteins could also be used in controlling materials formation in practical engineering via self-assembled, hybrid, functional materials structures. In molecular biomimetics, material-specific peptides could be the key in the molecular engineering of biology-inspired materials. With the recent developments of nanoscale engineering in physical sciences and the advances in molecular biology, we now combine genetic tools with synthetic nanoscale constructs to create a novel methodology. We first genetically select and/or design peptides with specific binding to functional solids, tailor their binding and assembly characteristics, develop bifunctional peptide/protein genetic constructs with both material binding and biological activity, and use these as molecular synthesizers, erectors and assemblers. Here, we give an overview of solid-binding peptides as novel molecular agents coupling bio- and nanotechnology.

  17. Resurrecting inactive antimicrobial peptides from the lipopolysaccharide trap.

    PubMed

    Mohanram, Harini; Bhattacharjya, Surajit

    2014-01-01

    Host defense antimicrobial peptides (AMPs) are a promising source of antibiotics for the treatment of multiple-drug-resistant pathogens. Lipopolysaccharide (LPS), the major component of the outer leaflet of the outer membrane of Gram-negative bacteria, functions as a permeability barrier against a variety of molecules, including AMPs. Further, LPS or endotoxin is the causative agent of sepsis killing 100,000 people per year in the United States alone. LPS can restrict the activity of AMPs inducing aggregations at the outer membrane, as observed for frog AMPs, temporins, and also in model AMPs. Aggregated AMPs, "trapped" by the outer membrane, are unable to traverse the cell wall, causing their inactivation. In this work, we show that these inactive AMPs can overcome LPS-induced aggregations while conjugated with a short LPS binding β-boomerang peptide motif and become highly bactericidal. The generated hybrid peptides exhibit activity against Gram-negative and Gram-positive bacteria in high-salt conditions and detoxify endotoxin. Structural and biophysical studies establish the mechanism of action of these peptides in LPS outer membrane. Most importantly, this study provides a new concept for the development of a potent broad-spectrum antibiotic with efficient outer membrane disruption as the mode of action.

  18. Peptide-templating dye-sensitized solar cells.

    PubMed

    Han, Tae Hee; Moon, Hyoung-Seok; Hwang, Jin Ok; Seok, Sang Il; Im, Sang Hyuk; Kim, Sang Ouk

    2010-05-07

    A hollow TiO(2) nanoribbon network electrode for dye-sensitized solar cells (DSSC) was fabricated by a biotemplating process combining peptide self-assembly and atomic layer deposition (ALD). An aromatic peptide of diphenylalanine was assembled into a three-dimensional network consisting of highly entangled nanoribbons. A thin TiO(2) layer was deposited at the surface of the peptide template via the ALD process. After the pyrolysis of the peptide template, a highly entangled nanotubular TiO(2) framework was successfully prepared. Evolution of the crystal phase and crystallite size of the TiO(2) nanostructure was exploited by controlling the calcination temperature. Finally, the hollow TiO(2) nanoribbon network electrode was integrated into DSSC devices and their photochemical performances were investigated. Hollow TiO(2) nanoribbon-based DSSCs exhibited a power conversion efficiency of 3.8%, which is comparable to the conventional TiO(2) nanoparticle-based DSSCs (3.5%). Our approach offers a novel pathway for DSSCs consisting of TiO(2) electrodes via biotemplating.

  19. C-type natriuretic peptide stimulates ovarian follicle development.

    PubMed

    Sato, Yorino; Cheng, Yuan; Kawamura, Kazuhiro; Takae, Seido; Hsueh, Aaron J W

    2012-07-01

    C-type natriuretic peptide (CNP) encoded by the NPPC (Natriuretic Peptide Precursor C) gene expressed in ovarian granulosa cells inhibits oocyte maturation by activating the natriuretic peptide receptor (NPR)B (NPRB) in cumulus cells. RT-PCR analyses indicated increased NPPC and NPRB expression during ovarian development and follicle growth, associated with increases in ovarian CNP peptides in mice. In cultured somatic cells from infantile ovaries and granulosa cells from prepubertal animals, treatment with CNP stimulated cGMP production. Also, treatment of cultured preantral follicles with CNP stimulated follicle growth whereas treatment of cultured ovarian explants from infantile mice with CNP, similar to FSH, increased ovarian weight gain that was associated with the development of primary and early secondary follicles to the late secondary stage. Of interest, treatment with FSH increased levels of NPPC, but not NPRB, transcripts in ovarian explants. In vivo studies further indicated that daily injections of infantile mice with CNP for 4 d promoted ovarian growth, allowing successful ovulation induction by gonadotropins. In prepubertal mice, CNP treatment alone also promoted early antral follicle growth to the preovulatory stage, leading to efficient ovulation induction by LH/human chorionic gonadotropin. Mature oocytes retrieved after CNP treatment could be fertilized in vitro and developed into blastocysts, allowing the delivery of viable offspring. Thus, CNP secreted by growing follicles is capable of stimulating preantral and antral follicle growth. In place of FSH, CNP treatment could provide an alternative therapy for female infertility.

  20. Novor: Real-Time Peptide de Novo Sequencing Software

    NASA Astrophysics Data System (ADS)

    Ma, Bin

    2015-11-01

    De novo sequencing software has been widely used in proteomics to sequence new peptides from tandem mass spectrometry data. This study presents a new software tool, Novor, to greatly improve both the speed and accuracy of today's peptide de novo sequencing analyses. To improve the accuracy, Novor's scoring functions are based on two large decision trees built from a peptide spectral library with more than 300,000 spectra with machine learning. Important knowledge about peptide fragmentation is extracted automatically from the library and incorporated into the scoring functions. The decision tree model also enables efficient score calculation and contributes to the speed improvement. To further improve the speed, a two-stage algorithmic approach, namely dynamic programming and refinement, is used. The software program was also carefully optimized. On the testing datasets, Novor sequenced 7%-37% more correct residues than the state-of-the-art de novo sequencing tool, PEAKS, while being an order of magnitude faster. Novor can de novo sequence more than 300 MS/MS spectra per second on a laptop computer. The speed surpasses the acquisition speed of today's mass spectrometer and, therefore, opens a new possibility to de novo sequence in real time while the spectrometer is acquiring the spectral data.

  1. Combinatorial Approach for Large-scale Identification of Linked Peptides from Tandem Mass Spectrometry Spectra*

    PubMed Central

    Wang, Jian; Anania, Veronica G.; Knott, Jeff; Rush, John; Lill, Jennie R.; Bourne, Philip E.; Bandeira, Nuno

    2014-01-01

    The combination of chemical cross-linking and mass spectrometry has recently been shown to constitute a powerful tool for studying protein–protein interactions and elucidating the structure of large protein complexes. However, computational methods for interpreting the complex MS/MS spectra from linked peptides are still in their infancy, making the high-throughput application of this approach largely impractical. Because of the lack of large annotated datasets, most current approaches do not capture the specific fragmentation patterns of linked peptides and therefore are not optimal for the identification of cross-linked peptides. Here we propose a generic approach to address this problem and demonstrate it using disulfide-bridged peptide libraries to (i) efficiently generate large mass spectral reference data for linked peptides at a low cost and (ii) automatically train an algorithm that can efficiently and accurately identify linked peptides from MS/MS spectra. We show that using this approach we were able to identify thousands of MS/MS spectra from disulfide-bridged peptides through comparison with proteome-scale sequence databases and significantly improve the sensitivity of cross-linked peptide identification. This allowed us to identify 60% more direct pairwise interactions between the protein subunits in the 20S proteasome complex than existing tools on cross-linking studies of the proteasome complexes. The basic framework of this approach and the MS/MS reference dataset generated should be valuable resources for the future development of new tools for the identification of linked peptides. PMID:24493012

  2. Peptide microarray with ligands at high density based on symmetrical carrier landscape phage for detection of cellulase.

    PubMed

    Qi, Huan; Wang, Fei; Petrenko, Valery A; Liu, Aihua

    2014-06-17

    Peptide microarrays evolved recently as a routine analytical implementation in various research areas due to their unique characteristics. However, the immobilization of peptides with high density in each spot during the fabricating process remains a problem, which will affect the performance of the resultant microarray greatly. To respond to this challenge, a novel peptide immobilization method using symmetrical phage carrier was developed in this work. The cellulytic enzyme endoglucanase I (EG I) was used as a model for selection of its specific peptide ligands from the f8/8 landscape library. Three phage monoclones were selected and identified by the specificity array, of which one phage monoclone displaying the fusion peptide EGSDPRMV (phage EGSDPRMV) could bind EG I specifically with highest affinity. Subsequently, the phage EGSDPRMV was used directly to construct peptide microarray. For comparison, major coat protein pVIII fused EG I specific peptide EGSDPRMV (pVIII-fused EGSDPRMV) which was isolated from phage EGSDPRMV was also immobilized by traditional method to fabricate peptide microarray. The fluorescent signal of the phage EGSDPRMV-mediated peptide microarray was more reproducible and about four times higher than the value for pVIII-fused EGSDPRMV-based microarray, suggesting the high efficiency of the proposed phage EGSDPRMV-mediated peptide immobilization method. Further, the phage EGSDPRMV based microarray not only simplified the procedure of microarray construction but also exhibited significantly enhanced sensitivity due to the symmetrical carrier landscape phage, which dramatically increased the density and sterical regularity of immobilized peptides in each spot. Thus, the proposed strategy has the advantages that the immobilizing peptide ligands were not disturbed by their composition and the immobilized peptides were highly regular with free amino-terminal.

  3. A Method for Selective Enrichment and Analysis of Nitrotyrosine-Containing Peptides in Complex Proteome Samples

    SciTech Connect

    Zhang, Qibin; Qian, Weijun; Knyushko, Tanya V.; Clauss, Therese RW; Purvine, Samuel O.; Moore, Ronald J.; Sacksteder, Colette A.; Chin, Mark H.; Smith, Desmond J.; Camp, David G.; Bigelow, Diana J.; Smith, Richard D.

    2007-06-01

    Elevated levels of protein tyrosine nitration have been found in various neurodegenerative diseases and aging related pathologies; however, the lack of an efficient enrichment method has prevented the analysis of this important low level protein modification. We have developed an efficient method for specific enrichment of nitrotyrosine containing peptides that permits nitrotyrosine peptides and specific nitration sites to be unambiguously identified with LC-MS/MS. The method is based on the derivatization of nitrotyrosine into free sulfhydryl groups followed by high efficiency enrichment of sulfhydryl-containing peptides with thiopropyl sepharose beads. The derivatization process starts with acetylation with acetic anhydride to block all primary amines, followed by reduction of nitrotyrosine to aminotyrosine, then derivatization of aminotyrosine with N-Succinimidyl S-Acetylthioacetate (SATA), and finally deprotecting of S-acetyl on SATA to form free sulfhydryl groups. This method was evaluated using nitrotyrosine containing peptides, in-vitro nitrated human histone 1.2, and bovine serum albumin (BSA). 91% and 62% of the identified peptides from enriched histone and BSA samples were nitrotyrosine derivatized peptides, respectively, suggesting relative high specificity of the enrichment method. The application of this method to in-vitro nitrated mouse brain homogenate resulted in 35% of identified peptides containing nitrotyrosine (compared to only 5.9% observed from the global analysis of unenriched sample), and a total of 150 unique nitrated peptides covering 102 proteins were identified with a false discovery rate estimated at 3.3% from duplicate LC-MS/MS analyses of a single enriched sample.

  4. Sustained delivery of VEGF from designer self-assembling peptides improves cardiac function after myocardial infarction

    SciTech Connect

    Guo, Hai-dong; Cui, Guo-hong; Yang, Jia-jun; Wang, Cun; Zhu, Jing; Zhang, Li-sheng; Jiang, Jun; Shao, Shui-jin

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer The designer peptide LRKKLGKA could self-assemble into nanofibers. Black-Right-Pointing-Pointer Injection of LRKKLGKA peptides could promote the sustained delivery of VEGF. Black-Right-Pointing-Pointer Injection of VEGF with LRKKLGKA peptides lead to sufficient angiogenesis. Black-Right-Pointing-Pointer Injection of VEGF with LRKKLGKA peptides improves heart function. -- Abstract: Poor vascularization and insufficient oxygen supply are detrimental to the survival of residual cardiomyocytes or transplanted stem cells after myocardial infarction. To prolong and slow the release of angiogenic factors, which stimulate both angiogenesis and vasculogenesis, we constructed a novel self-assembling peptide by attaching the heparin-binding domain sequence LRKKLGKA to the self-assembling peptide RADA16. This designer self-assembling peptide self-assembled into nanofiber scaffolds under physiological conditions, as observed by atomic force microscopy. The injection of designer self-assembling peptides can efficiently provide the sustained delivery of VEGF for at least 1 month. At 4 weeks after transplantation, cardiac function was improved, and scar size and collagen deposition were markedly reduced in the group receiving VEGF with the LRKKLGKA scaffolds compared with groups receiving VEGF alone, LRKKLGKA scaffolds alone or VEGF with RADA16 scaffolds. The microvessel density in the VEGF with LRKKLGKA group was higher than that in the VEGF with RADA16 group. TUNEL and cleaved caspase-3 expression assays showed that the transplantation of VEGF with LRKKLGKA enhanced cell survival in the infarcted heart. These results present the tailor-made peptide scaffolds as a new generation of sustained-release biomimetic biomaterials and suggest that the use of angiogenic factors along with designer self-assembling peptides can lead to myocardial protection, sufficient angiogenesis, and improvement in cardiac function.

  5. Cu nanocrystal growth on peptide nanotubes by biomineralization: Size control of Cu nanocrystals by tuning peptide conformation

    NASA Astrophysics Data System (ADS)

    Banerjee, Ipsita A.; Yu, Lingtao; Matsui, Hiroshi

    2003-12-01

    With recent interest in seeking new biologically inspired device-fabrication methods in nanotechnology, a new biological approach was examined to fabricate Cu nanotubes by using sequenced histidine-rich peptide nanotubes as templates. The sequenced histidine-rich peptide molecules were assembled as nanotubes, and the biological recognition of the specific sequence toward Cu lead to efficient Cu coating on the nanotubes. Cu nanocrystals were uniformly coated on the histidine-incorporated nanotubes with high packing density. In addition, the diameter of Cu nanocrystal was controlled between 10 and 30 nm on the nanotube by controlling the conformation of histidine-rich peptide by means of pH changes. Those nanotubes showed significant change in electronic structure by varying the nanocrystal diameter; therefore, this system may be developed to a conductivity-tunable building block for microelectronics and biological sensors. This simple biomineralization method can be applied to fabricate various metallic and semiconductor nanotubes with peptides whose sequences are known to mineralize specific ions.

  6. Combinatorial peptide library-based identification of peptide ligands for tumor-reactive cytolytic T lymphocytes of unknown specificity.

    PubMed

    Rubio-Godoy, Verena; Ayyoub, Maha; Dutoit, Valerie; Servis, Catherine; Schink, Amy; Rimoldi, Donata; Romero, Pedro; Cerottini, Jean-Charles; Simon, Richard; Zhao, Yindong; Houghten, Richard A; Pinilla, Clemencia; Valmori, Danila

    2002-08-01

    A novel approach for the identification of tumor antigen-derived sequences recognized by CD8(+) cytolytic T lymphocytes (CTL) consists in using synthetic combinatorial peptide libraries. Here we have screened a library composed of 3.1 x 10(11) nonapeptides arranged in a positional scanning format, in a cytotoxicity assay, to search the antigen recognized by melanoma-reactive CTL of unknown specificity. The results of this analysis enabled the identification of several optimal peptide ligands, as most of the individual nonapeptides deduced from the primary screening were efficiently recognized by the CTL. The results of the library screening were also analyzed with a mathematical approach based on a model of independent and additive contribution of individual amino acids to antigen recognition. This biometrical data analysis enabled the retrieval, in public databases, of the native antigenic peptide SSX-2(41-49), whose sequence is highly homologous to the ones deduced from the library screening, among the ones with the highest stimulatory score. These results underline the high predictive value of positional scanning synthetic combinatorial peptide library analysis and encourage its use for the identification of CTL ligands.

  7. Delivery of siRNA using ternary complexes containing branched cationic peptides: the role of peptide sequence, branching and targeting.

    PubMed

    Kudsiova, Laila; Welser, Katharina; Campbell, Frederick; Mohammadi, Atefeh; Dawson, Natalie; Cui, Lili; Hailes, Helen C; Lawrence, M Jayne; Tabor, Alethea B

    2016-03-01

    Ternary nanocomplexes, composed of bifunctional cationic peptides, lipids and siRNA, as delivery vehicles for siRNA have been investigated. The study is the first to determine the optimal sequence and architecture of the bifunctional cationic peptide used for siRNA packaging and delivery using lipopolyplexes. Specifically three series of cationic peptides of differing sequence, degrees of branching and cell-targeting sequences were co-formulated with siRNA and vesicles prepared from a 1 : 1 molar ratio of the cationic lipid DOTMA and the helper lipid, DOPE. The level of siRNA knockdown achieved in the human alveolar cell line, A549-luc cells, in both reduced serum and in serum supplemented media was evaluated, and the results correlated to the nanocomplex structure (established using a range of physico-chemical tools, namely small angle neutron scattering, transmission electron microscopy, dynamic light scattering and zeta potential measurement); the conformational properties of each component (circular dichroism); the degree of protection of the siRNA in the lipopolyplex (using gel shift assays) and to the cellular uptake, localisation and toxicity of the nanocomplexes (confocal microscopy). Although the size, charge, structure and stability of the various lipopolyplexes were broadly similar, it was clear that lipopolyplexes formulated from branched peptides containing His-Lys sequences perform best as siRNA delivery agents in serum, with protection of the siRNA in serum balanced against efficient release of the siRNA into the cytoplasm of the cell.

  8. Peptide array-based characterization and design of ZnO-high affinity peptides.

    PubMed

    Okochi, Mina; Sugita, Tomoya; Furusawa, Seiji; Umetsu, Mitsuo; Adschiri, Tadafumi; Honda, Hiroyuki

    2010-08-15

    Peptides with both an affinity for ZnO and the ability to generate ZnO nanoparticles have attracted attention for the self-assembly and templating of nanoscale building blocks under ambient conditions with compositional uniformity. In this study, we have analyzed the specific binding sites of the ZnO-binding peptide, EAHVMHKVAPRP, which was identified using a phage display peptide library. The peptide binding assay against ZnO nanoparticles was performed using peptides synthesized on a cellulose membrane using the spot method. Using randomized rotation of amino acids in the ZnO-binding peptide, 125 spot-synthesized peptides were assayed. The peptide binding activity against ZnO nanoparticles varied greatly. This indicates that ZnO binding does not depend on total hydrophobicity or other physical parameters of these peptides, but rather that ZnO recognizes the specific amino acid alignment of these peptides. In addition, several peptides were found to show higher binding ability compared with that of the original peptides. Identification of important binding sites in the EAHVMHKVAPRP peptide was investigated by shortened, stepwise sequence from both termini. Interestingly, two ZnO-binding sites were found as 6-mer peptides: HVMHKV and HKVAPR. The peptides identified by amino acid substitution of HKVAPR were found to show high affinity and specificity for ZnO nanoparticles.

  9. Future directions for peptide therapeutics development.

    PubMed

    Kaspar, Allan A; Reichert, Janice M

    2013-09-01

    The notable expansion of peptide therapeutics development in the late 1990s and the 2000s led to an unprecedented number of marketing approvals in 2012 and has provided a robust pipeline that should deliver numerous approvals during the remainder of the 2010s. To document the current status of the pipeline, we collected data for peptide therapeutics in clinical studies and regulatory review, as well as those recently approved. In this Foundation review, we provide an overview of the pipeline, including therapeutic area and molecular targets, with a focus on glucagon-like peptide 1 receptor agonists. Areas for potential expansion, for example constrained peptides and peptide-drug conjugates, are profiled.

  10. Peptides and Peptidomimetics for Antimicrobial Drug Design

    PubMed Central

    Mojsoska, Biljana; Jenssen, Håvard

    2015-01-01

    The purpose of this paper is to introduce and highlight a few classes of traditional antimicrobial peptides with a focus on structure-activity relationship studies. After first dissecting the important physiochemical properties that influence the antimicrobial and toxic properties of antimicrobial peptides, the contributions of individual amino acids with respect to the peptides antibacterial properties are presented. A brief discussion of the mechanisms of action of different antimicrobials as well as the development of bacterial resistance towards antimicrobial peptides follows. Finally, current efforts on novel design strategies and peptidomimetics are introduced to illustrate the importance of antimicrobial peptide research in the development of future antibiotics. PMID:26184232

  11. Fabrication of Odor Sensor Using Peptide

    NASA Astrophysics Data System (ADS)

    Hotokebuchi, Yuta; Hayashi, Kenshi; Toko, Kiyoshi; Chen, Ronggang; Ikezaki, Hidekazu

    We report fabrication of an odor sensor using peptides. Peptides were designed to acquire the specific reception for a target odor molecule. Au surface of the sensor electrode was coated by the designed peptide using the method of self assembled monolayers (SAMs). Functionalized Au surfaces by the peptides were confirmed by ellipsometry and cyclic voltammetry. The odorants of vanillin, phenethyl alcohol and hexanol were discriminated by QCM sensor with the peptide surface. Moreover, we verified specific interaction between amino acid (Trp) and vanillin by fluorescence assay.

  12. Isoelectric focusing of proteins and peptides

    NASA Technical Reports Server (NTRS)

    Egen, N.

    1979-01-01

    Egg-white solution was chosen as the reference solution in order to assess the effects of operational parameters (voltage, flow rate, ampholine pH range and concentration, and protein concentration) of the RIEF apparatus on protein resolution. Topics of discussion include: (1) comparison of RIEF apparatus to conventional IEF techniques (column and PAG) with respect to resolution and throughput; (2) peptide and protein separation (AHF, Thymosin - Fraction 5, vasoactive peptide, L-asparaginase and ACP); and (3) detection of peptides - dansyl derivatives of amino acids and peptides, post-focusing fluorescent labeling of amino acids, peptides and proteins, and ampholine extraction from focused gels.

  13. The First Salamander Defensin Antimicrobial Peptide

    PubMed Central

    Jiang, Ke; Rong, Mingqiang; Lai, Ren

    2013-01-01

    Antimicrobial peptides have been widely identified from amphibian skins except salamanders. A novel antimicrobial peptide (CFBD) was isolated and characterized from skin secretions of the salamander, Cynops fudingensis. The cDNA encoding CFBD precursor was cloned from the skin cDNA library of C. fudingensis. The precursor was composed of three domains: signal peptide of 17 residues, mature peptide of 41 residues and intervening propeptide of 3 residues. There are six cysteines in the sequence of mature CFBD peptide, which possibly form three disulfide-bridges. CFBD showed antimicrobial activities against Staphylococcus aureus, Bacillus subtilis, Candida albicans and Escherichia coli. This peptide could be classified into family of β-defensin based on its seqeuence similarity with β-defensins from other vertebrates. Evolution analysis indicated that CFBD was close to fish β-defensin. As far as we know, CFBD is the first β-defensin antimicrobial peptide from salamanders. PMID:24386139

  14. Biology of the CAPA peptides in insects.

    PubMed

    Predel, R; Wegener, C

    2006-11-01

    CAPA peptides have been isolated from a broad range of insect species as well as an arachnid, and can be grouped into the periviscerokinin and pyrokinin peptide families. In insects, CAPA peptides are the characteristic and most abundant neuropeptides in the abdominal neurohemal system. In many species, CAPA peptides exert potent myotropic effects on different muscles such as the heart. In others, including blood-sucking insects able to transmit serious diseases, CAPA peptides have strong diuretic or anti-diuretic effects and thus are potentially of medical importance. CAPA peptides undergo cell-type-specific sorting and packaging, and are the first insect neuropeptides shown to be differentially processed. In this review, we discuss the current knowledge on the structure, distribution, receptors and physiological actions of the CAPA peptides.

  15. The first salamander defensin antimicrobial peptide.

    PubMed

    Meng, Ping; Yang, Shilong; Shen, Chuanbin; Jiang, Ke; Rong, Mingqiang; Lai, Ren

    2013-01-01

    Antimicrobial peptides have been widely identified from amphibian skins except salamanders. A novel antimicrobial peptide (CFBD) was isolated and characterized from skin secretions of the salamander, Cynops fudingensis. The cDNA encoding CFBD precursor was cloned from the skin cDNA library of C. fudingensis. The precursor was composed of three domains: signal peptide of 17 residues, mature peptide of 41 residues and intervening propeptide of 3 residues. There are six cysteines in the sequence of mature CFBD peptide, which possibly form three disulfide-bridges. CFBD showed antimicrobial activities against Staphylococcus aureus, Bacillus subtilis, Candida albicans and Escherichia coli. This peptide could be classified into family of β-defensin based on its sequence similarity with β-defensins from other vertebrates. Evolution analysis indicated that CFBD was close to fish β-defensin. As far as we know, CFBD is the first β-defensin antimicrobial peptide from salamanders.

  16. Therapeutic uses of gastrointestinal peptides.

    PubMed

    Redfern, J S; O'Dorisio, T M

    1993-12-01

    The GI tract is one of nature's great pharmacies. Most, if not all, biologically active peptides can be found there, and it is quite likely that others remain to be discovered. Our ability to exploit this resource has expanded considerably over the past two decades. Advances in analytical techniques have allowed investigators to rapidly isolate and purify new compounds from tissue extracts. Sequencing and de novo synthesis of newly discovered peptides are now routine, and the structural modifications required to alter activity and tailor a compound to a particular use are easily made. A number of gastrointestinal peptides or their analogues for use in clinical studies are available from commercial sources (see Table 7). Somatostatin is the first gut peptide to successfully complete development and yield a pharmaceutical compound with a broad range of action. Several of the peptides discussed in this article have similar potential. TRH stands out as a candidate because of its effectiveness in the treatment of experimental spinal cord injury and a variety of shock states. Such a broad range of action in critical fields may justify the intensive development required to yield potent, long-acting, and highly specific analogues. Similarly, the antimetastatic and immunostimulant properties of the enkephalins offer promise for new therapies in the treatment of AIDS, ARC, and cancer. Studies with amylin may lead to new and more precise regimens of blood sugar control in insulin-dependent diabetics and could in turn, prevent some of the worst long-term effects of the disease. The development of effective intranasal forms of GHRH could spare children with GH-GHRH deficiency the distress of repeated injections and help to prevent excessive GH blood levels. Secretin, glucagon, or CGRP might be used one day in cardiovascular emergencies, and VIP or its analogues could prove effective in the treatment of asthma. Although preliminary results with many of these peptides are

  17. Improving gene annotation using peptide mass spectrometry

    PubMed Central

    Tanner, Stephen; Shen, Zhouxin; Ng, Julio; Florea, Liliana; Guigó, Roderic; Briggs, Steven P.; Bafna, Vineet

    2007-01-01

    Annotation of protein-coding genes is a key goal of genome sequencing projects. In spite of tremendous recent advances in computational gene finding, comprehensive annotation remains a challenge. Peptide mass spectrometry is a powerful tool for researching the dynamic proteome and suggests an attractive approach to discover and validate protein-coding genes. We present algorithms to construct and efficiently search spectra against a genomic database, with no prior knowledge of encoded proteins. By searching a corpus of 18.5 million tandem mass spectra (MS/MS) from human proteomic samples, we validate 39,000 exons and 11,000 introns at the level of translation. We present translation-level evidence for novel or extended exons in 16 genes, confirm translation of 224 hypothetical proteins, and discover or confirm over 40 alternative splicing events. Polymorphisms are efficiently encoded in our database, allowing us to observe variant alleles for 308 coding SNPs. Finally, we demonstrate the use of mass spectrometry to improve automated gene prediction, adding 800 correct exons to our predictions using a simple rescoring strategy. Our results demonstrate that proteomic profiling should play a role in any genome sequencing project. PMID:17189379

  18. F5-peptide induces aspermatogenesis by disrupting organization of actin- and microtubule-based cytoskeletons in the testis

    PubMed Central

    Gao, Ying; Mruk, Dolores D.; Lui, Wing-yee; Lee, Will M.; Cheng, C. Yan

    2016-01-01

    During the release of sperm at spermiation, a biologically active F5-peptide, which can disrupt the Sertoli cell tight junction (TJ) permeability barrier, is produced at the site of the degenerating apical ES (ectoplasmic specialization). This peptide coordinates the events of spermiation and blood-testis barrier (BTB) remodeling at stage VIII of the epithelial cycle, creating a local apical ES-BTB axis to coordinate cellular events across the epithelium. The mechanism(s) by which F5-peptide perturbs BTB restructuring, and its involvement in apical ES dynamics remain unknown. F5-peptide, besides perturbing BTB integrity, was shown to induce germ cell release from the epithelium following its efficient in vivo overexpression in the testis. Overexpression of F5-peptide caused disorganization of actin- and microtubule (MT)-based cytoskeletons, mediated by altering the spatiotemporal expression of actin binding/regulatory proteins in the seminiferous epithelium. F5-peptide perturbed the ability of actin microfilaments and/or MTs from converting between their bundled and unbundled/defragmented configuration, thereby perturbing adhesion between spermatids and Sertoli cells. Since apical ES and basal ES/BTB are interconnected through the underlying cytoskeletal networks, this thus provides an efficient and novel mechanism to coordinate different cellular events across the epithelium during spermatogenesis through changes in the organization of actin microfilaments and MTs. These findings also illustrate the potential of F5-peptide being a male contraceptive peptide for men. PMID:27611949

  19. RFamide peptides in agnathans and basal chordates.

    PubMed

    Osugi, Tomohiro; Son, You Lee; Ubuka, Takayoshi; Satake, Honoo; Tsutsui, Kazuyoshi

    2016-02-01

    Since a peptide with a C-terminal Arg-Phe-NH2 (RFamide peptide) was first identified in the ganglia of the venus clam in 1977, RFamide peptides have been found in the nervous system of both invertebrates and vertebrates. In vertebrates, the RFamide peptide family includes gonadotropin-inhibitory hormone (GnIH), neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP), pyroglutamylated RFamide peptide/26RFamide peptide (QRFP/26RFa), and kisspeptins (kiss1 and kiss2). They are involved in important functions such as the release of hormones, regulation of sexual or social behavior, pain transmission, reproduction, and feeding. In contrast to tetrapods and jawed fish, the information available on RFamide peptides in agnathans and basal chordates is limited, thus preventing further insights into the evolution of RFamide peptides in vertebrates. In this review, we focus on the previous research and recent advances in the studies on RFamide peptides in agnathans and basal chordates. In agnathans, the genes encoding GnIH, NPFF, and PrRP precursors and the mature peptides have been identified in lamprey (Petromyzon marinus) and hagfish (Paramyxine atami). Putative kiss1 and kiss2 genes have also been found in the genome database of lamprey. In basal chordates, namely, in amphioxus (Branchiostoma japonicum), a common ancestral form of GnIH and NPFF genes and their mature peptides, as well as the ortholog of the QRFP gene have been identified. The studies revealed that the number of orthologs of vertebrate RFamide peptides present in agnathans and basal chordates is greater than expected, suggesting that the vertebrate RFamide peptides might have emerged and expanded at an early stage of chordate evolution.

  20. Anionic phospholipids modulate peptide insertion into membranes.

    PubMed

    Liu, L P; Deber, C M

    1997-05-06

    While the insertion of a hydrophobic peptide or membrane protein segment into the bilayer can be spontaneous and driven mainly by the hydrophobic effect, anionic lipids, which comprise ca. 20% of biological membranes, provide a source of electrostatic attractions for binding of proteins/peptides into membranes. To unravel the interplay of hydrophobicity and electrostatics in the binding of peptides into membranes, we designed peptides de novo which possess the typical sequence Lys-Lys-Ala-Ala-Ala-X-Ala-Ala-Ala-Ala-Ala-X-Ala-Ala-Trp-Ala-Ala-X-Ala-Al a-Ala-Lys-Lys-Lys-Lys-amide, where X residues correspond to "guest" residues which encompass a range of hydrophobicity (Leu, Ile, Gly, and Ser). Circular dichroism spectra demonstrated that peptides were partially (40-90%) random in aqueous buffer but were promoted to form 100% alpha-helical structures by anionic lipid micelles. In neutral lipid micelles, only the relatively hydrophobic peptides (X = L and I) spontaneously adopted the alpha-helical conformation, but when 25% of negatively charged lipids were mixed in to mimic the content of anionic lipids in biomembranes, the less hydrophobic (X = S and G) peptides then formed alpha-helical conformations. Consistent with these findings, fluorescence quenching by the aqueous-phase quencher iodide indicated that in anionic (dimyristoylphosphatidylglycerol) vesicles, the peptide Trp residue was buried in the lipid vesicle hydrophobic core, while in neutral (dimyristoylphosphatidylcholine) vesicles, only hydrophobic (X = L and I) peptides were shielded from the aqueous solution. Trp emission spectra of peptides in the presence of phospholipids doxyl-labeled at the 5-, 7-, 10-, 12-, and 16-fatty acid positions implied not only a transbilayer orientation for inserted peptides but also that mixed peptide populations (transbilayer + surface-associated) may arise. Overall results suggest that for hydrophobic peptides with segmental threshold hydrophobicity below that which

  1. Cell surface profiling with peptide libraries yields ligand arrays that classify breast tumor subtypes.

    PubMed

    Dane, Karen Y; Gottstein, Claudia; Daugherty, Patrick S

    2009-05-01

    Cancer heterogeneity renders risk stratification and therapy decisions challenging. Thus, genomic and proteomic methodologies have been used in an effort to identify biomarkers that can differentiate tumor subtypes to improve therapeutic outcome. Here, we report a generally applicable strategy to generate tumor type-specific peptide ligand arrays. Peptides that specifically recognize breast tumor-derived cell lines (MDA-MB-231, MCF-7, and T47-D) were identified using cell-displayed peptide libraries carrying an intrinsic fluorescent marker allowing for sorting and characterization with quantitative flow cytometry. Tumor cell specificity was achieved by depleting libraries of ligands binding to normal mammary epithelial cells (HMEC and MCF-10A). Although integrin binding RGD motifs were favored by some cell lines, screening with RGD competitors yielded several novel consensus motifs exhibiting improved tumor specificity. The resultant peptide array contained multiple consensus motifs exhibiting strong similarity to breast tumor-associated proteins. Profiling a panel of breast cancer cell lines with the peptide array revealed receptor expression patterns distinctive for luminal or basal tumor subtypes. In addition, peptide displaying bacteria and peptide functionalized microparticles enabled fluorescent labeling of tumor cells and frozen tumor tissue sections. Our results indicate that cell surface profiling using highly specific breast tumor cell binding ligands may provide an efficient route for tumor subtype classification, biomarker identification, and for the development of targeted diagnostics and therapeutics.

  2. Glucagon‐related peptides and the regulation of food intake in chickens

    PubMed Central

    2016-01-01

    Abstract The regulatory mechanisms underlying food intake in chickens have been a focus of research in recent decades to improve production efficiency when raising chickens. Lines of evidence have revealed that a number of brain‐gut peptides function as a neurotransmitter or peripheral satiety hormone in the regulation of food intake both in mammals and chickens. Glucagon, a 29 amino acid peptide hormone, has long been known to play important roles in maintaining glucose homeostasis in mammals and birds. However, the glucagon gene encodes various peptides that are produced by tissue‐specific proglucagon processing: glucagon is produced in the pancreas, whereas oxyntomodulin (OXM), glucagon‐like peptide (GLP)‐1 and GLP‐2 are produced in the intestine and brain. Better understanding of the roles of these peptides in the regulation of energy homeostasis has led to various physiological roles being proposed in mammals. For example, GLP‐1 functions as an anorexigenic neurotransmitter in the brain and as a postprandial satiety hormone in the peripheral circulation. There is evidence that OXM and GLP‐2 also induce anorexia in mammals. Therefore, it is possible that the brain‐gut peptides OXM, GLP‐1 and GLP‐2 play physiological roles in the regulation of food intake in chickens. More recently, a novel GLP and its specific receptor were identified in the chicken brain. This review summarizes current knowledge about the role of glucagon‐related peptides in the regulation of food intake in chickens. PMID:27150835

  3. Charge Effect on the Quantum Dots-Peptide Self-Assembly Using Fluorescence Coupled Capillary Electrophoresis.

    PubMed

    Wang, Jianhao; Li, Jingyan; Teng, Yiwan; Bi, Yanhua; Hu, Wei; Li, Jinchen; Wang, Cheli; Qiu, Lin; Jiang, Pengju

    2016-04-01

    We present a molecular characterization of metal-affinity driven self-assembly between CdSe-ZnS quantum dots and a series of hexahistidine peptides with different charges. In particular, we uti- lized fluorescence coupled capillary electrophoresis to test the self-assembly process of quantum dots with peptides in solution. Four peptides with different charges can be efficiently separated by fluorescence coupled capillary electrophoresis. The migration time appeared to be influenced by the charges of the peptide. In addition, the kinetics of self-assembly process of quantum dots with one of the peptides manifested a bi-phasic kinetics followed by a saturating stage. This work revealed that there exist two types of binding sites on the surface of quantum dots for peptide 1: one type termed "high priority" binding site and a "low priority" site which is occupied after the first binding sites are fully occupied. The total self-assembly process finishes in solution within 80 s. Our work represents the systematic investigation of the details of self-assembly kinetics utilizing high-resolution fluorescence coupled capillary electrophoresis. The charge effect of peptide coating quantum dots provides a new way of preparing bioprobes.

  4. High-resolution structure of HLA-A*1101 in complex with SARS nucleocapsid peptide.

    PubMed

    Blicher, Thomas; Kastrup, Jette Sandholm; Buus, Søren; Gajhede, Michael

    2005-08-01

    The structure of the human MHC-I molecule HLA-A*1101 in complex with a nonameric peptide (KTFPPTEPK) has been determined by X-ray crystallography to 1.45 A resolution. The peptide is derived from the SARS-CoV nucleocapsid protein positions 362-370 (SNP362-370). It is conserved in all known isolates of SARS-CoV and has been verified by in vitro peptide-binding studies to be a good to intermediate binder to HLA-A*0301 and HLA-A*1101, with IC50 values of 70 and 186 nM, respectively [Sylvester-Hvid et al. (2004), Tissue Antigens, 63, 395-400]. In terms of the residues lining the peptide-binding groove, the HLA-A*1101-SNP362-370 complex is very similar to other known structures of HLA-A*1101 and HLA-A*6801. The SNP362-370 peptide is held in place by 17 hydrogen bonds to the alpha-chain residues and by nine water molecules which are also tightly bound in the peptide-binding groove. Thr6 of the peptide (Thr6p) does not make efficient use of the middle (E) pocket. For vaccine development, there seems to be a potential for optimization targeted at this position. All residues except Thr2p and Lys9p are accessible for T-cell recognition.

  5. SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture

    PubMed Central

    Fierer, Jacob O.; Veggiani, Gianluca; Howarth, Mark

    2014-01-01

    Individual proteins can now often be modified with atomic precision, but there are still major obstacles to connecting proteins into larger assemblies. To direct protein assembly, ideally, peptide tags would be used, providing the minimal perturbation to protein function. However, binding to peptides is generally weak, so assemblies are unstable over time and disassemble with force or harsh conditions. We have recently developed an irreversible protein–peptide interaction (SpyTag/SpyCatcher), based on a protein domain from Streptococcus pyogenes, that locks itself together via spontaneous isopeptide bond formation. Here we develop irreversible peptide–peptide interaction, through redesign of this domain and genetic dissection into three parts: a protein domain termed SpyLigase, which now ligates two peptide tags to each other. All components expressed efficiently in Escherichia coli and peptide tags were reactive at the N terminus, at the C terminus, or at internal sites. Peptide–peptide ligation enabled covalent and site-specific polymerization of affibodies or antibodies against the tumor markers epidermal growth factor receptor (EGFR) and HER2. Magnetic capture of circulating tumor cells (CTCs) is one of the most promising approaches to improve cancer prognosis and management, but CTC capture is limited by inefficient recovery of cells expressing low levels of tumor antigen. SpyLigase-assembled protein polymers made possible the isolation of cancerous cells expressing lower levels of tumor antigen and should have general application in enhancing molecular capture. PMID:24639550

  6. Plasma proteome coverage is increased by unique peptide recovery from sodium deoxycholate precipitate.

    PubMed

    Serra, Aida; Zhu, Hongbin; Gallart-Palau, Xavier; Park, Jung Eun; Ho, Hee Haw; Tam, James P; Sze, Siu Kwan

    2016-03-01

    The ionic detergent sodium deoxycholate (SDC) is compatible with in-solution tryptic digestion and LC-MS/MS-based shotgun proteomics by virtue of being easy to separate from the peptide products via precipitation in acidic buffers. However, it remains unclear whether unique human peptides co-precipitate with SDC during acid treatment of complex biological samples. In this study, we demonstrate for the first time that a large quantity of unique peptides in human blood plasma can be co-precipitated with SDC using an optimized sample preparation method prior to shotgun proteomic analysis. We show that the plasma peptides co-precipitated with SDC can be successfully recovered using a sequential re-solubilization and precipitation procedure, and that this approach is particularly efficient at the extraction of long peptides. Recovery of peptides from the SDC pellet dramatically increased overall proteome coverage (>60 %), thereby improving the identification of low-abundance proteins and enhancing the identification of protein components of membrane-bound organelles. In addition, when we analyzed the physiochemical properties of the co-precipitated peptides, we observed that SDC-based sample preparation improved the identification of mildly hydrophilic/hydrophobic proteins that would otherwise be lost upon discarding the pellet. These data demonstrate that the optimized SDC protocol is superior to sodium dodecyl sulfate (SDS)/urea treatment for identifying plasma biomarkers by shotgun proteomics.

  7. Rice Bran Protein as a Potent Source of Antimelanogenic Peptides with Tyrosinase Inhibitory Activity.

    PubMed

    Ochiai, Akihito; Tanaka, Seiya; Tanaka, Takaaki; Taniguchi, Masayuki

    2016-10-28

    Rice (Oryza sativa) is consumed as a staple food globally, and rice bran, the byproduct, is an unused biomass that is ultimately discarded as waste. Thus, in the present study, a technique for producing tyrosinase inhibitory peptides from rice bran protein (RBP) was developed. Simultaneous treatment of RBP with chymotrypsin and trypsin produced numerous peptides. Subsequently, six tyrosinase inhibitory peptides were isolated from the hydrolysate fractions in a multistep purification protocol, and their amino acid sequences were determined. Three of these peptides had a C-terminal tyrosine residue and exhibited significant inhibitory effects against tyrosinase-mediated monophenolase reactions. Furthermore, peptide CT-2 (Leu-Gln-Pro-Ser-His-Tyr) potently inhibited melanogenesis in mouse B16 melanoma cells without causing cytotoxicity, suggesting the potential of CT-2 as an agent for melanin-related skin disorder treatment. The present data indicate that RBP is a potent source of tyrosinase inhibitory peptides and that simultaneous treatment of RBP with chymotrypsin and trypsin efficiently produces these peptides.

  8. Computer-aided design of peptide near infrared fluorescent probe for tumor diagnosis

    NASA Astrophysics Data System (ADS)

    Zhang, Congying; Gu, Yueqing

    2014-09-01

    Integrin αvβ3 receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth, so they become hot research tagets in cancer diagnosis. Peptides possess several attractive features when compared to protein and small molecule, such as small size and high structural compatibility with target proteins. Efficient design of high-affinity peptide ligands to Integrin αvβ3 receptors has been an important problem. Designed peptides in silico provide a valuable and high-selectivity peptide, meanwhile decrease the time of drug screening. In this study, we design peptide which can bind with integrin αvβ3 via computer, and then synthesis near infrared fluorescent probe. The characterization of this near infrared fluorescent probe was detected by UV. To investigate the tumor cell targeting of this probe, it was labeled with visible fluorescent dye Rhodamine B (RhB) for microscopy. To evaluate the targeting capability of this near infrared fluorescent probe, mice bearing integrin αvβ3 positive tumor xenografts were used. In vitro cellular experiments indicated that this probe have a clear binding affinity to αvβ3-positive tumor cells. In vivo experiments confirmed the receptor binding specificity of this probe. The peptide of computational design can bind with integrin αvβ3. Combined peptide near-infrared fluorescent probe with imaging technology use for clinical and tumor diagnosis have a greater development in future.

  9. Facile preparation of mesoporous carbon-silica-coated graphene for the selective enrichment of endogenous peptides.

    PubMed

    Zhang, Quanqing; Zhang, Qinghe; Xiong, Zhichao; Wan, Hao; Chen, Xiaoting; Li, Hongmei; Zou, Hanfa

    2016-01-01

    A sandwich-like composite composed of ordered mesoporous carbon-silica shell-coated graphene (denoted as graphene@mSiO2-C) was prepared by an in-situ carbonation strategy. A mesoporous silica shell was synthesized by a sol-gel method, and cetyltrimethyl ammonium bromide inside the mesopores were in-situ carbonized as a carbon source to obtain a carbon-silica shell. The resulting mesoporous carbon-silica material with a sandwich structure possesses a high surface area (600 m(2) g(-1)), large pore volume (0.587 cm(3) g(-1)), highly ordered mesoporous pore (3 nm), and high carbon content (30%). This material shows not only high hydrophobicity of graphene and mesoporous carbon but also a hydrophilic silica framework that ensures excellent dispersibility in aqueous solution. The material can capture many more peptides from bovine serum albumin tryptic digests than mesoporous silica shell-coated graphene, demonstrating great enrichment efficiency for peptides. Furthermore, the prepared composite was applied to the enrichment of low-abundance endogenous peptides in human serum. Based on Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry identification, the graphene@mSiO2-C could efficiently size-exclude proteins and enriches the low-abundant peptides on the graphene and mesoporous carbon. And based on the LC-MS/MS results, 892 endogenous peptides were obtained by graphene@mSiO2-C, hinting at its great potential in peptides analysis.

  10. Cellular Uptake of Gold Nanoparticles Directly Cross-linked with Carrier Peptides by Osteosarcoma Cells

    PubMed Central

    Mandal, Deendayal; Maran, Avudaippan; Yaszemski, Michael J.; Bolander, Mark E; Sarkar, Gobinda

    2010-01-01

    Nanoparticles have been extensively used for a variety of biomedical applications and there is a growing need for highly specific and efficient delivery of the nanoparticles into target cells and subcellular location. We attempted to accomplish this goal by modifying gold particles with peptide motif’s that are known to deliver a ‘cargo’ into chosen cellular location specifically, we intended to deliver nanogold particles into cells through chemical cross-linking with different peptides known to carry protein into cells. Our results suggest that specific sequence of such ‘carrier peptides’ can efficiently deliver gold nanoparticles into cells when chemically cross-linked with the metal particles. PMID:18807262

  11. Resonance Energy Transfer Relates the Gas-Phase Structure and Pharmacological Activity of Opioid Peptides.

    PubMed

    Kopysov, Vladimir; Boyarkin, Oleg V

    2016-01-11

    Enkephalins are efficient pain-relief drugs that bind to transmembrane opioid receptors. One key structural parameter that governs the pharmacological activity of these opioid peptides and is typically determined from condensed-phase structures is the distance between the aromatic rings of their Tyr and Phe residues. We use resonance energy transfer, detected by a combination of cold ion spectroscopy and mass spectrometry, to estimate the Tyr-Phe spacing for enkephalins in the gas phase. In contrast to the condensed-phase structures, these distances appear to differ substantially in enkephalins with different pharmacological efficiencies, suggesting that gas-phase structures might be a better pharmacophoric metric for ligand peptides.

  12. Taylor Dispersion Analysis as a promising tool for assessment of peptide-peptide interactions.

    PubMed

    Høgstedt, Ulrich B; Schwach, Grégoire; van de Weert, Marco; Østergaard, Jesper

    2016-10-10

    Protein-protein and peptide-peptide (self-)interactions are of key importance in understanding the physiochemical behavior of proteins and peptides in solution. However, due to the small size of peptide molecules, characterization of these interactions is more challenging than for proteins. In this work, we show that protein-protein and peptide-peptide interactions can advantageously be investigated by measurement of the diffusion coefficient using Taylor Dispersion Analysis. Through comparison to Dynamic Light Scattering it was shown that Taylor Dispersion Analysis is well suited for the characterization of protein-protein interactions of solutions of α-lactalbumin and human serum albumin. The peptide-peptide interactions of three selected peptides were then investigated in a concentration range spanning from 0.5mg/ml up to 80mg/ml using Taylor Dispersion Analysis. The peptide-peptide interactions determination indicated that multibody interactions significantly affect the PPIs at concentration levels above 25mg/ml for the two charged peptides. Relative viscosity measurements, performed using the capillary based setup applied for Taylor Dispersion Analysis, showed that the viscosity of the peptide solutions increased with concentration. Our results indicate that a viscosity difference between run buffer and sample in Taylor Dispersion Analysis may result in overestimation of the measured diffusion coefficient. Thus, Taylor Dispersion Analysis provides a practical, but as yet primarily qualitative, approach to assessment of the colloidal stability of both peptide and protein formulations.

  13. Peptides and methods against diabetes

    DOEpatents

    Albertini, Richard J.; Falta, Michael T.

    2000-01-01

    This invention relates to methods of preventing or reducing the severity of diabetes. In one embodiment, the method involves administering to the individual a peptide having substantially the sequence of a on-conserved region sequence of a T cell receptor present on the surface of T cells mediating diabetes or a fragment thereof, wherein the peptide or fragment is capable of causing an effect on the immune system to regulate the T cells. In particular, the T cell receptor has the V.beta. regional V.beta.6 or V.beta.14. In another embodiment, the method involves gene therapy. The invention also relates to methods of diagnosing diabetes by determining the presence of diabetes predominant T cell receptors.

  14. Antimicrobial Peptides in Human Sepsis

    PubMed Central

    Martin, Lukas; van Meegern, Anne; Doemming, Sabine; Schuerholz, Tobias

    2015-01-01

    Nearly 100 years ago, antimicrobial peptides (AMPs) were identified as an important part of innate immunity. They exist in species from bacteria to mammals and can be isolated in body fluids and on surfaces constitutively or induced by inflammation. Defensins have anti-bacterial effects against Gram-positive and Gram-negative bacteria as well as anti-viral and anti-yeast effects. Human neutrophil peptides (HNP) 1–3 and human beta-defensins (HBDs) 1–3 are some of the most important defensins in humans. Recent studies have demonstrated higher levels of HNP 1–3 and HBD-2 in sepsis. The bactericidal/permeability-increasing protein (BPI) attenuates local inflammatory response and decreases systemic toxicity of endotoxins. Moreover, BPI might reflect the severity of organ dysfunction in sepsis. Elevated plasma lactoferrin is detected in patients with organ failure. HNP 1–3, lactoferrin, BPI, and heparin-binding protein are increased in sepsis. Human lactoferrin peptide 1–11 (hLF 1–11) possesses antimicrobial activity and modulates inflammation. The recombinant form of lactoferrin [talactoferrin alpha (TLF)] has been shown to decrease mortality in critically ill patients. A phase II/III study with TLF in sepsis did not confirm this result. The growing number of multiresistant bacteria is an ongoing problem in sepsis therapy. Furthermore, antibiotics are known to promote the liberation of pro-inflammatory cell components and thus augment the severity of sepsis. Compared to antibiotics, AMPs kill bacteria but also neutralize pathogenic factors such as lipopolysaccharide. The obstacle to applying naturally occurring AMPs is their high nephro- and neurotoxicity. Therefore, the challenge is to develop peptides to treat septic patients effectively without causing harm. This overview focuses on natural and synthetic AMPs in human and experimental sepsis and their potential to provide significant improvements in the treatment of critically ill with severe infections

  15. A novel fuzzy Fisher classifier for signal peptide prediction.

    PubMed

    Gao, Cui-Fang; Qiu, Zi-Xue; Wu, Xiao-Jun; Tian, Feng-Wei; Zhang, Hao; Chen, Wei

    2011-08-01

    Signal peptides recognition by bioinformatics approaches is particularly important for the efficient secretion and production of specific proteins. We concentrate on developing an integrated fuzzy Fisher clustering (IFFC) and designing a novel classifier based on IFFC for predicting secretory proteins. IFFC provides a powerful optimal discriminant vector calculated by fuzzy intra-cluster scatter matrix and fuzzy inter-cluster scatter matrix. Because the training samples and test samples are processed together in IFFC, it is convenient for users to employ their own specific samples of high reliability as training data if necessary. The cross-validation results on some existing datasets indicate that the fuzzy Fisher classifier is quite promising for signal peptide prediction.

  16. Synthesis of lipoic acid-peptide conjugates and their effect on collagen and melanogenesis.

    PubMed

    Lu, Chichong; Kim, Bo Mi; Lee, Duckhee; Lee, Min Hee; Kim, Jin Hwa; Pyo, Hyeong-Bae; Chai, Kyu Yun

    2013-11-01

    We report new examples of lipoic acid (LA)-peptide conjugates, their potential as codrugs having anti-melanogenic and anti-aging properties was evaluated. These multifunctional molecules were prepared by linking lipophilic moiety (LA) to the pentapeptide KTTKS. The inhibitory effect of LA-peptide conjugates on melanin synthesis and tyrosinase activity is stronger than that of LA or the pentapeptide alone. Importantly, the conjugates display no cytotoxicity at a high concentration. LA-KTTKS and LA-PEG-KTTKS also inhibit UV-induced matrix metalloproteinase-1 expression up to 49.5% and 69.5% at 0.5 mM, respectively. LA-peptide conjugates stimulate collagen biosynthesis in fibroblasts more efficiently than their parent molecules do. These data suggest that LA-peptide conjugates may have cosmeceutical application as anti-melanogenic and anti-aging agents.

  17. Reversed-phase high-performance liquid chromatography of protected peptide segments.

    PubMed

    Pedroso, E; Grandas, A; Amor, J C; Giralt, E

    1987-11-13

    There is little evidence that reversed-phase high-performance liquid chromatography can be successfully used in the analysis of protected peptide segments. The use of C18 and CN packings and mobile phases containing water-acetonitrile with or without propionic acid in the separation of complex mixtures of synthetic protected peptides is reported. CN packings show a lower efficiency and exhibit poorer resolution than C18 packings but provide different separations. The addition of propionic acid to the mobile phase increases the retention time of peptides but also provides dramatic and useful changes in selectivity. Retention is not related to the molecular mass of the protected peptides but mainly to their hydrophobicity.

  18. Preorganized Peptide Scaffolds as Mimics of Phosphorylated Proteins Binding Sites with a High Affinity for Uranyl.

    PubMed

    Starck, Matthieu; Sisommay, Nathalie; Laporte, Fanny A; Oros, Stéphane; Lebrun, Colette; Delangle, Pascale

    2015-12-07

    Cyclic peptides with two phosphoserines and two glutamic acids were developed to mimic high-affinity binding sites for uranyl found in proteins such as osteopontin, which is believed to be a privileged target of this ion in vivo. These peptides adopt a β-sheet structure that allows the coordination of the latter amino acid side chains in the equatorial plane of the dioxo uranyl cation. Complementary spectroscopic and analytical methods revealed that these cyclic peptides are efficient uranyl chelating peptides with a large contribution from the phosphorylated residues. The conditional affinity constants were measured by following fluorescence tryptophan quenching and are larger than 10(10) at physiological pH. These compounds are therefore promising models for understanding uranyl chelation by proteins, which is relevant to this actinide ion toxicity.

  19. Peptide Paratope Mimics of the Broadly Neutralizing HIV-1 Antibody b12.

    PubMed

    Haußner, Christina; Damm, Dominik; Nirschl, Sandra; Rohrhofer, Anette; Schmidt, Barbara; Eichler, Jutta

    2017-01-26

    The broadly neutralizing HIV-1 antibody b12 recognizes the CD4 binding site of the HIV-1 envelope glycoprotein gp120 and efficiently neutralizes HIV-1 infections in vitro and in vivo. Based on the 3D structure of a b12⋅gp120 complex, we have designed an assembled peptide (b12-M) that presents the parts of the three heavy-chain complementarity-determining regions (CDRs) of b12, which contain the contact sites of the antibody for gp120. This b12-mimetic peptide, as well as a truncated peptide presenting only two of the three heavy-chain CDRs of b12, were shown to recognize gp120 in a similar manner to b12, as well as to inhibit HIV-1 infection, demonstrating functional mimicry of b12 by the paratope mimetic peptides.

  20. Gene introduction into the mitochondria of Arabidopsis thaliana via peptide-based carriers.

    PubMed

    Chuah, Jo-Ann; Yoshizumi, Takeshi; Kodama, Yutaka; Numata, Keiji

    2015-01-13

    Available methods in plant genetic transformation are nuclear and plastid transformations because similar procedures have not yet been established for the mitochondria. The double membrane and small size of the organelle, in addition to its large population in cells, are major obstacles in mitochondrial transfection. Here we report the intracellular delivery of exogenous DNA localized to the mitochondria of Arabidopsis thaliana using a combination of mitochondria-targeting peptide and cell-penetrating peptide. Low concentrations of peptides were sufficient to deliver DNA into the mitochondria and expression of imported DNA reached detectable levels within a short incubation period (12 h). We found that electrostatic interaction with the cell membrane is not a critical factor for complex internalization, instead, improved intracellular penetration of mitochondria-targeted complexes significantly enhanced gene transfer efficiency. Our results delineate a simple and effective peptide-based method, as a starting point for the development of more sophisticated plant mitochondrial transfection strategies.

  1. Solid-phase synthesis, characterization, and cellular activities of collagen-model nanodiamond-peptide conjugates.

    PubMed

    Knapinska, Anna M; Tokmina-Roszyk, Dorota; Amar, Sabrina; Tokmina-Roszyk, Michal; Mochalin, Vadym N; Gogotsi, Yury; Cosme, Patrick; Terentis, Andrew C; Fields, Gregg B

    2015-05-01

    Nanodiamonds (NDs) have received considerable attention as potential drug delivery vehicles. NDs are small (∼5 nm diameter), can be surface modified in a controllable fashion with a variety of functional groups, and have little observed toxicity in vitro and in vivo. However, most biomedical applications of NDs utilize surface adsorption of biomolecules, as opposed to covalent attachment. Covalent modification provides reliable and reproducible ND-biomolecule ratios, and alleviates concerns over biomolecule desorption prior to delivery. The present study has outlined methods for the efficient solid-phase conjugation of ND to peptides and characterization of ND-peptide conjugates. Utilizing collagen-derived peptides, the ND was found to support or even enhance the cell adhesion and viability activities of the conjugated sequence. Thus, NDs can be incorporated into peptides and proteins in a selective manner, where the presence of the ND could potentially enhance the in vivo activities of the biomolecule it is attached to.

  2. Structure-Activity Relations of Myxinidin, an Antibacterial Peptide Derived from the Epidermal Mucus of Hagfish

    PubMed Central

    Cantisani, Marco; Leone, Marilisa; Mignogna, Eleonora; Kampanaraki, Katerina; Falanga, Annarita; Morelli, Giancarlo

    2013-01-01

    The structure-activity relations of myxinidin, a peptide derived from epidermal mucus of hagfish, Myxine glutinosa L., were investigated. Analysis of key residues allowed us to design new peptides with increased efficiency. Antimicrobial activity of native and modified peptides demonstrated the key role of uncharged residues in the sequence; the loss of these residues reduces almost entirely myxinidin antimicrobial activity, while insertion of arginine at charged and uncharged position increases antimicrobial activity compared with that of native myxinidin. Particularly, we designed a peptide capable of achieving a high inhibitory effect on bacterial growth. Experiments were conducted using both Gram-negative and Gram-positive bacteria. Nuclear magnetic resonance (NMR) studies showed that myxinidin is able to form an amphipathic α-helical structure at the N terminus and a random coil region at the C terminus. PMID:24002100

  3. Solid-Phase Synthesis, Characterization, and Cellular Activities of Collagen-Model Nanodiamond-Peptide Conjugates

    PubMed Central

    Knapinska, Anna M.; Tokmina-Roszyk, Dorota; Amar, Sabrina; Tokmina-Roszyk, Michal; Mochalin, Vadym N.; Gogotsi, Yury; Cosme, Patrick; Terentis, Andrew C.; Fields, Gregg B.

    2015-01-01

    Nanodiamonds (NDs) have received considerable attention as potential drug delivery vehicles. NDs are small (~5 nm diameter), can be surface modified in a controllable fashion with a variety of functional groups, and have little observed toxicity in vitro and in vivo. However, most biomedical applications of NDs utilize surface adsorption of biomolecules, as opposed to covalent attachment. Covalent modification provides reliable and reproducible ND–biomolecule ratios, and alleviates concerns over biomolecule desorption prior to delivery. The present study has outlined methods for the efficient solid-phase conjugation of ND to peptides and characterization of ND–peptide conjugates. Utilizing collagen-derived peptides, the ND was found to support or even enhance the cell adhesion and viability activities of the conjugated sequence. Thus, NDs can be incorporated into peptides and proteins in a selective manner, where the presence of the ND could potentially enhance the in vivo activities of the biomolecule it is attached to. PMID:25753561

  4. Short polyhistidine peptides penetrate effectively into Nicotiana tabacum-cultured cells and Saccharomyces cerevisiae cells.

    PubMed

    Kimura, Sayaka; Kawano, Tsuyoshi; Iwasaki, Takashi

    2017-01-01

    The polyhistidine peptides (PHPs) have been previously reported as novel cell-penetrating peptides and are efficiently internalized into mammal cells; however, penetration of PHPs into other cell types is unknown. In this study, the cellular uptake of PHPs in plant and yeast cells was found to be dependent on the number of histidines, and short PHPs (H6-H10 peptides) showed effective internalization. The H8 peptide showed the highest cell-penetrating capacity and localized to vacuoles in plant and yeast cells. Low-temperature conditions inhibited significantly the cellular uptake of short PHPs by both cells. However, net charge neutralization of PHPs also completely inhibited cellular uptake by plant cells, but not by yeast cells. These results indicate that short PHPs penetrate effectively into plant and yeast cells by similar mechanism with the exception of net charge dependency. The findings show the short PHPs are promising candidates for new delivery tools into plant and yeast cells.

  5. Self-Assembled Peptide- and Protein-Based Nanomaterials for Antitumor Photodynamic and Photothermal Therapy.

    PubMed

    Abbas, Manzar; Zou, Qianli; Li, Shukun; Yan, Xuehai

    2017-03-01

    Tremendous interest in self-assembly of peptides and proteins towards functional nanomaterials has been inspired by naturally evolving self-assembly in biological construction of multiple and sophisticated protein architectures in organisms. Self-assembled peptide and protein nanoarchitectures are excellent promising candidates for facilitating biomedical applications due to their advantages of structural, mechanical, and functional diversity and high biocompability and biodegradability. Here, this review focuses on the self-assembly of peptides and proteins for fabrication of phototherapeutic nanomaterials for antitumor photodynamic and photothermal therapy, with emphasis on building blocks, non-covalent interactions, strategies, and the nanoarchitectures of self-assembly. The exciting antitumor activities achieved by these phototherapeutic nanomaterials are also discussed in-depth, along with the relationships between their specific nanoarchitectures and their unique properties, providing an increased understanding of the role of peptide and protein self-assembly in improving the efficiency of photodynamic and photothermal therapy.

  6. Exploitation of starch industry liquid by-product to produce bioactive peptides from rice hydrolyzed proteins.

    PubMed

    Dei Piu', Lucilla; Tassoni, Annalisa; Serrazanetti, Diana Isabella; Ferri, Maura; Babini, Elena; Tagliazucchi, Davide; Gianotti, Andrea

    2014-07-15

    Small peptides show higher antioxidant capacity than native proteins and may be absorbed in the intestine without further digestion. In our study, a protein by-product from rice starch industry was hydrolyzed with commercial proteolytic enzymes (Alcalase, Neutrase, Flavourzyme) and microbial whole cells of Bacillus spp. and the released peptides were tested for antioxidant activity. Among enzymes, Alcalase was the most performing, while microbial proteolytic activity was less efficient. Conversely, the antioxidant activity was higher in the samples obtained by microbial hydrolysis and particularly with Bacillus pumilus AG1. The sequences of low molecular weight antioxidant peptides were determined and analyzed for aminoacidic composition. The results obtained so far suggest that the hydrolytic treatment of this industrial by-product, with selected enzymes and microbial systems, can allow its exploitation for the production of functional additives and supplements rich in antioxidant peptides, to be used in new food formulas for human consumption.

  7. Rational, computer-enabled peptide drug design: principles, methods, applications and future directions.

    PubMed

    Diller, David J; Swanson, Jon; Bayden, Alexander S; Jarosinski, Mark; Audie, Joseph

    2015-01-01

    Peptides provide promising templates for developing drugs to occupy a middle space between small molecules and antibodies and for targeting 'undruggable' intracellular protein-protein interactions. Importantly, rational or in cerebro design, especially when coupled with validated in silico tools, can be used to efficiently explore chemical space and identify islands of 'drug-like' peptides to satisfy diverse drug discovery program objectives. Here, we consider the underlying principles of and recent advances in rational, computer-enabled peptide drug design. In particular, we consider the impact of basic physicochemical properties, potency and ADME/Tox opportunities and challenges, and recently developed computational tools for enabling rational peptide drug design. Key principles and practices are spotlighted by recent case studies. We close with a hypothetical future case study.

  8. Anticancer Activity of the Antimicrobial Peptide Scolopendrasin VII Derived from the Centipede, Scolopendra subspinipes mutilans.

    PubMed

    Lee, Joon Ha; Kim, In-Woo; Kim, Sang-Hee; Kim, Mi-Ae; Yun, Eun-Young; Nam, Sung-Hee; Ahn, Mi-Young; Kang, Dongchul; Hwang, Jae Sam

    2015-08-01

    Previously, we performed de novo RNA sequencing of Scolopendra subspinipes mutilans using high-throughput sequencing technology and identified several antimicrobial peptide candidates. Among them, a cationic antimicrobial peptide, scolopendrasin VII, was selected based on its physicochemical properties, such as length, charge, and isoelectric point. Here, we assessed the anticancer activities of scolopendrasin VII against U937 and Jurkat leukemia cell lines. The results showed that scolopendrasin VII decreased the viability of the leukemia cells in MTS assays. Furthermore, flow cytometric analysis and acridine orange/ethidium bromide staining revealed that scolopendrasin VII induced necrosis in the leukemia cells. Scolopendrasin VII-induced necrosis was mediated by specific interaction with phosphatidylserine, which is enriched in the membrane of cancer cells. Taken together, these data indicated that scolopendrasin VII induced necrotic cell death in leukemia cells, probably through interaction with phosphatidylserine. The results provide a useful anticancer peptide candidate and an efficient strategy for new anticancer peptide development.

  9. Modulating charge transfer through cyclic D,L-alpha-peptide self-assembly.

    PubMed

    Horne, W Seth; Ashkenasy, Nurit; Ghadiri, M Reza

    2005-02-04

    We describe a concise, solid support-based synthetic method for the preparation of cyclic d,l-alpha-peptides bearing 1,4,5,8-naphthalenetetracarboxylic acid diimide (NDI) side chains. Studies of the structural and photoluminescence properties of these molecules in solution show that the hydrogen bond-directed self-assembly of the cyclic d,l-alpha-peptide backbone promotes intermolecular NDI excimer formation. The efficiency of NDI charge transfer in the resulting supramolecular assemblies is shown to depend on the length of the linker between the NDI and the peptide backbone, the distal NDI substituent, and the number of NDIs incorporated in a given structure. The design rationale and synthetic strategies described here should provide a basic blueprint for a series of self-assembling cyclic d,l-alpha-peptide nanotubes with interesting optical and electronic properties.

  10. Modulating Charge Transfer Through Cyclic D,L α-Peptide Self-Assembly

    PubMed Central

    Horne, W. Seth; Ashkenasy, Nurit; Ghadiri, M. Reza

    2007-01-01

    We describe a concise solid support-based synthetic method for the preparation of cyclic D,L α-peptides bearing 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) side chains. Studies of the structural and photoluminescence properties of these molecules in solution show that the hydrogen bond directed self-assembly of the cyclic D,L α-peptide backbone promotes intermolecular NDI excimer formation. The efficiency of NDI charge transfer in the resulting supramolecular assemblies is shown to depend on the length of the linker between the NDI and the peptide backbone, the distal NDI substituent, and the number of NDIs incorporated in a given structure. The design rationale and synthetic strategies described here should provide a basic blueprint for a series of self-assembling cyclic D,L α-peptide nanotubes with interesting optical and electronic properties. PMID:15624124

  11. Antihypertensive Peptides from Milk Proteins

    PubMed Central

    Jäkälä, Pauliina; Vapaatalo, Heikki

    2010-01-01

    Dietary proteins possess a wide range of nutritional and functional properties. They are used as a source of energy and amino acids, which are needed for growth and development. Many dietary proteins, especially milk proteins, contain physiologically active peptides encrypted in the protein sequence. These peptides may be released during gastrointestinal digestion or food processing and once liberated, cause different physiological functions. Milk-derived bioactive peptides are shown to have antihypertensive, antimicrobial, immunomodulatory, antioxidative and mineral-binding properties. During the fermentation of milk with certain lactobacilli, two interesting tripeptides Ile-Pro-Pro and Val-Pro-Pro are released from casein to the final product. These lactotripeptides have attenuated the development of hypertension in several animal models and lowered blood pressure in clinical studies. They inhibit ACE in vitro at micromolar concentrations, protect endothelial function in vitro and reduce arterial stiffness in humans. Thus, milk as a traditional food product can after certain processing serve as a functional food and carry specific health-promoting effects, providing an option to control blood pressure. PMID:27713251

  12. Recent Advances in Peptide Immunomodulators.

    PubMed

    Zerfas, Breanna L; Gao, Jianmin

    2015-01-01

    With the continued rise in antibiotic-resistant bacteria, there is an immense need for the development of new therapeutic agents. Host-defense peptides (HDPs) offer a unique alternative to many of the current approved antibiotics. By targeting the host rather than the pathogen, HDPs offer several benefits over traditional small molecule drug treatments, such as a slower propensity towards resistance, broad-spectrum activity and lower risk of patients developing sepsis. However, natural peptide structures have many disadvantages as well, including susceptibility to proteolytic degradation, significant costs of synthesis and host toxicity. For this reason, much work has been done to examine peptidomimetic structures, in the hopes of finding a structure with all of the desired qualities of an antibiotic drug. Recently, this research has included synthetic constructs that mimic the behavior of HDPs but have no structural similarity to peptides. This review article focuses on the progression of this field of research, beginning with an analysis of a few prominent examples of natural HDPs and moving on to describe how the information learned by studying them have led to the current design platforms.

  13. Peptide sequences mediating tropism to intact blood-brain barrier: an in vivo biodistribution study using phage display.

    PubMed

    Smith, Mathew W; Al-Jayyoussi, Ghaith; Gumbleton, Mark

    2012-11-01

    Peptide motifs that demonstrate tropism for the blood brain barrier (BBB) are of real translational value in developing innovative delivery strategies for biological brain targeted therapies. In vivo peptide-phage display affords peptide selection against the full complement of biological markers within the correct cellular macro- and micro-environments. Here a stringent in vivo biopanning protocol was employed in the rat aimed at identifying cyclic 7-mer peptide motifs that mediate tropism to brain microvasculature. Five rounds of biopanning identified 349 unique peptide motifs in the brain tissue gray matter compartment (microvasculature and parenchyma). While in general no consensus was evident linking peptide physico-chemical properties and brain tropism, peptides bearing c-SxTSSTx-c or c-xxxSSTx-c motifs were found to be present in high abundance. Based on amino acid frequency distribution of the 349 unique peptides sequences a theoretical 'idealized' peptide pattern, c-PP(S/P)SSST-c, could be derived. For the most abundant experimental peptide sequence found in brain tissue, c-SYTSSTM-c, an in vivo pharmacokinetic and whole body tissue biodistribution study was performed. Based upon tissue exposure data (i.e. tissue AUC((0-infinity))) the sequence c-SYTSSTM-c efficiently retargeted phage virions to the brain providing an approximate 5-fold greater (P<0.05) accumulation in brain over control phage; in all other organs no significant (P>0.05) difference in tissue tropism between c-SYTSSTM-c and control phages were evident. This peptide and more generally the peptide motifs, -SxTSSTx- or -xxxSSTx-, warrant further investigation as agents mediating sequence-dependent tropism to brain microvasculature potentially able to deliver biologic cargo to the CNS.

  14. Peptide mass fingerprinting peak intensity prediction: extracting knowledge from spectra.

    PubMed

    Gay, Steven; Binz, Pierre-Alain; Hochstrasser, Denis F; Appel, Ron D

    2002-10-01

    Matrix-assisted laser desorption/ionization-time of flight mass spectrometry has become a valuable tool in proteomics. With the increasing acquisition rate of mass spectrometers, one of the major issues is the development of accurate, efficient and automatic peptide mass fingerprinting (PMF) identification tools. Current tools are mostly based on counting the number of experimental peptide masses matching with theoretical masses. Almost all of them use additional criteria such as isoelectric point, molecular weight, PTMs, taxonomy or enzymatic cleavage rules to enhance prediction performance. However, these identification tools seldom use peak intensities as parameter as there is currently no model predicting the intensities based on the physicochemical properties of peptides. In this work, we used standard datamining methods such as classification and regression methods to find correlations between peak intensities and the properties of the peptides composing a PMF spectrum. These methods were applied on a dataset comprising a series of PMF experiments involving 157 proteins. We found that the C4.5 method gave the more informative results for the classification task (prediction of the presence or absence of a peptide in a spectra) and M5' for the regression methods (prediction of the normalized intensity of a peptide peak). The C4.5 result correctly classified 88% of the theoretical peaks; whereas the M5' peak intensities had a correlation coefficient of 0.6743 with the experimental peak intensities. These methods enabled us to obtain decision and model trees that can be directly used for prediction and identification of PMF results. The work performed permitted to lay the foundations of a method to analyze factors influencing the peak intensity of PMF spectra. A simple extension of this analysis could lead to improve the accuracy of the results by using a larger dataset. Additional peptide characteristics or even PMF experimental parameters can also be taken into

  15. Determinant selection of major histocompatibility complex class I- restricted antigenic peptides is explained by class I-peptide affinity and is strongly influenced by nondominant anchor residues

    PubMed Central

    1994-01-01

    The contribution of major histocompatibility complex (MHC) class I- peptide affinity to immunodominance of particular peptide antigens (Ags) in the class I-restricted cytotoxic T lymphocyte (CTL) response is not clearly established. Therefore, we have compared the H-2Kb- restricted binding and presentation of the immunodominant ovalbumin (OVA)257-264 (SIINFEKL) determinant to that of a subdominant OVA determinant OVA55-62 (KVVRFDKL). Immunodominance of OVA257-264 was not attributable to the specific T cell repertoire but correlated instead with more efficient Ag presentation. This enhanced Ag presentation could be accounted for by the higher affinity of Kb/OVA257-264 compared with Kb/OVA55-62 despite the presence of a conserved Kb-binding motif in both peptides. Kinetic binding studies using purified soluble H-2Kb molecules (Kbs) and biosensor techniques indicated that the Kon for association of OVA257-264-C6 and Kbs at 25 degrees C was integral of 10- fold faster (5.9 x 10(3) M-1 s-1 versus 6.5 x 10(2) M-1 s-1), and the Koff approximately twofold slower (9.1 x 10(-6) s-1 versus 1.6 x 10(-5) s-1), than the rate constants for interaction of OVA55-62-C6 and Kbs. The association of these peptides with Kb was significantly influenced by multiple residues at presumed nonanchor sites within the peptide sequence. The contribution of each peptide residue to Kb-binding was dependent upon the sequence context and the summed contributions were not additive. Thus the affinity of MHC class I-peptide binding is a critical factor controlling presentation of peptide Ag and immunodominance in the class I-restricted CTL response. PMID:7523572

  16. Stability of peptide drugs in the colon.

    PubMed

    Wang, Jie; Yadav, Vipul; Smart, Alice L; Tajiri, Shinichiro; Basit, Abdul W

    2015-10-12

    This study was the first to investigate the colonic stability of 17 peptide molecules (insulin, calcitonin, glucagon, secretin, somatostatin, desmopressin, oxytocin, Arg-vasopressin, octreotide, ciclosporin, leuprolide, nafarelin, buserelin, histrelin, [D-Ser(4)]-gonadorelin, deslorelin, and goserelin) in a model of the large intestine using mixed human faecal bacteria. Of these, the larger peptides - insulin, calcitonin, somatostatin, glucagon and secretin - were metabolized rapidly, with complete degradation observed within 5 min. In contrast, a number of the smaller peptides - Arg-vasopressin, desmopressin, oxytocin, gonadorelin, goserelin, buserelin, leuprolide, nafarelin and deslorelin - degraded more slowly, while octreotide, histrelin and ciclosporin were seen to be more stable as compared to the other small peptides under the same conditions. Peptide degradation rate was directly correlated to peptide lipophilicity (logP); those peptides with a higher logP were more stable in the colonic model (R(2)=0.94). In the absence of human faecal bacteria, all peptides were stable. This study highlights the impact of the colonic environment - in particular, the gut microbiota - on the metabolism of peptide drugs, and identifies potential peptide candidates for drug delivery to the colon.

  17. Structure-function relationships of antimicrobial peptides.

    PubMed

    Hwang, P M; Vogel, H J

    1998-01-01

    Antimicrobial peptides are ubiquitously produced throughout nature. Many of these relatively short peptides (6-50 residues) are lethal towards bacteria and fungi, yet they display minimal toxicity towards mammalian cells. All of the peptides are highly cationic and hydrophobic. It is widely believed that they act through nonspecific binding to biological membranes, even though the exact nature of these interactions is presently unclear. High-resolution nuclear magnetic resonance (NMR) has contributed greatly to knowledge in this field, providing insight about peptide structure in aqueous solution, in organic cosolvents, and in micellar systems. Solid-state NMR can provide additional information about peptide-membrane binding. Here we review our current knowledge about the structure of antimicrobial peptides. We also discuss studies pertaining to the mechanism of action. Despite the different three-dimensional structural motifs of the various classes, they all have similar amphiphilic surfaces that are well-suited for membrane binding. Many antimicrobial peptides bind in a membrane-parallel orientation, interacting only with one face of the bilayer. This may be sufficient for antimicrobial action. At higher concentrations, peptides and phospholipids translocate to form multimeric transmembrane channels that seem to contribute to the peptide's hemolytic activity. An understanding of the key features of the secondary and tertiary structures of the antimicrobial peptides and their effects on bactericidal and hemolytic activity can aid the rational design of improved analogs for clinical use.

  18. Bioprospecting open reading frames for peptide effectors.

    PubMed

    Xiong, Ling; Scott, Charles

    2014-01-01

    Recent successes in the development of small-molecule antagonists of protein-protein interactions designed based on co-crystal structures of peptides bound to their biological targets confirm that short peptides derived from interacting proteins can be high-value ligands for pharmacologic validation of targets and for identification of druggable sites. Evolved sequence space is likely to be enriched for interacting peptides, but identifying minimal peptide effectors within genomic sequence can be labor intensive. Here we describe the use of incremental truncation to diversify genetic material on the scale of open reading frames into comprehensive libraries of constituent peptides. The approach is capable of generating peptides derived from both continuous and discontinuous sequence elements, and is compatible with the expression of free linear or backbone cyclic peptides, with peptides tethered to amino- or carboxyl-terminal fusion partners or with the expression of peptides displayed within protein scaffolds (peptide aptamers). Incremental truncation affords a valuable source of molecular diversity to interrogate the druggable genome or evaluate the therapeutic potential of candidate genes.

  19. In situ study by polarization modulated Fourier transform infrared spectroscopy of the structure and orientation of lipids and amphipathic peptides at the air-water interface.

    PubMed Central

    Cornut, I; Desbat, B; Turlet, J M; Dufourcq, J

    1996-01-01

    Free amphipathic peptides and peptides bound to dimyristoylphosphatidylcholine (DMPC) were studied directly at the air/water interface using polarization modulation infrared reflection absorption spectroscopy (PMIRRAS). Such differential reflectivity measurements proved to be a sensitive and efficient technique to investigate in situ the respective conformations and orientations of lipid and peptide molecules in pure and mixed films. Data obtained for melittin, a natural hemolytic peptide, are compared to those of L15K7, an ideally amphipathic synthetic peptide constituted by only apolar Leu and polar Lys residues. For pure peptidic films, the intensity, shape, and position of the amide I and II bands indicate that the L15K7 peptide adopts a totally alpha-helical structure, whereas the structure of melittin is mainly alpha-helical and presents some unordered domains. The L15K7 alpha-helix axis is oriented essentially parallel to the air-water interface plane; it differs for melittin. When injected into the subphase, L15K7 and melittin insert into preformed expanded DMPC monolayers and can be detected by PMIRRAS, even at low peptide content (> 50 DMPC molecules per peptide). In such conditions, peptides have the same secondary structure and orientation as in pure peptidic films. PMID:8770206

  20. Identification of novel Lck-derived T helper epitope long peptides applicable for HLA-A2(+) cancer patients as cancer vaccine.

    PubMed

    Matsueda, Satoko; Shichijo, Shigeki; Nagata, Sayaka; Seki, Chieko; Yamada, Akira; Noguchi, Masanori; Itoh, Kyogo

    2015-11-01

    The present study attempted to identify T helper epitope long peptides capable of inducing cytotoxic T lymphocytes (CTL) from Lck antigen (p56(Lck) ), the src family tyrosine kinase, which is known to be aberrantly expressed in metastatic cancers cells, in order to develop a long peptide-based cancer vaccine for HLA-A2(+) cancer patients. Based on the biding motif to the HLA-DR and HLA-A2 alleles, 94 peptides were prepared from the Lck antigen. These peptides were screened for their reactivity to immunoglobulin G (IgG) from plasma of cancer patients, followed by testing of their ability to induce both CD4(+) and CD8(+) T lymphocytes showing not only peptide-specific IFN-γ production but cytotoxicity against HLA-A2(+) cancer cells from peripheral blood mononuclear cells (PBMC) of HLA-A2(+) cancer patients. Among 94 peptides tested, the three T helper epitope long peptides and their inner CTL epitope short peptides with HLA-A2 binding motifs were frequently recognized by IgG of cancer patients, and efficiently induced both CD4(+) IFN-γ(+) and CD8(+) IFN-γ(+) T lymphocytes. Patients' PBMC stimulated with these long peptides showed cytotoxicity against HLA-A2(+) Lck(+) cancer cells in HLA-class I and HLA-class II dependent manners. These three peptides might be useful for long peptide-based vaccines for HLA-A2(+) cancer patients with Lck(+) tumor cells.

  1. Human Antimicrobial Peptides and Proteins

    PubMed Central

    Wang, Guangshun

    2014-01-01

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

  2. Batch efficiency

    NASA Astrophysics Data System (ADS)

    Schwickerath, Ulrich; Silva, Ricardo; Uria, Christian

    2010-04-01

    A frequent source of concern for resource providers is the efficient use of computing resources in their centers. This has a direct impact on requests for new resources. There are two different but strongly correlated aspects to be considered: while users are mostly interested in a good turn-around time for their jobs, resource providers are mostly interested in a high and efficient usage of their available resources. Both things, the box usage and the efficiency of individual user jobs, need to be closely monitored so that the sources of the inefficiencies can be identified. At CERN, the Lemon monitoring system is used for both purposes. Examples of such sources are poorly written user code, inefficient access to mass storage systems, and dedication of resources to specific user groups. As a first step for improvements CERN has launched a project to develop a scheduler add-on that allows careful overloading of worker nodes that run idle jobs.

  3. Characterization of the cell penetrating properties of a human salivary proline-rich peptide.

    PubMed

    Radicioni, Giorgia; Stringaro, Annarita; Molinari, Agnese; Nocca, Giuseppina; Longhi, Renato; Pirolli, Davide; Scarano, Emanuele; Iavarone, Federica; Manconi, Barbara; Cabras, Tiziana; Messana, Irene; Castagnola, Massimo; Vitali, Alberto

    2015-11-01

    Saliva contains hundreds of small proline-rich peptides most of which derive from the post-translational and post-secretory processing of the acidic and basic salivary proline-rich proteins. Among these peptides we found that a 20 residue proline-rich peptide (p1932), commonly present in human saliva and patented for its antiviral activity, was internalized within cells of the oral mucosa. The cell-penetrating properties of p1932 have been studied in a primary gingival fibroblast cell line and in a squamous cancer cell line, and compared to its retro-inverso form. We observed by mass-spectrometry, flow cytometry and confocal microscopy that both peptides were internalized in the two cell lines on a time scale of minutes, being the natural form more efficient than the retro-inverso one. The cytosolic localization was dependent on the cell type: both peptide forms were able to localize within nuclei of tumoral cells, but not in the nuclei of gingival fibroblasts. The uptake was shown to be dependent on the culture conditions used: peptide internalization was indeed effective in a complete medium than in a serum-free one allowing the hypothesis that the internalization could be dependent on the cell cycle. Both peptides were internalized likely by a lipid raft-mediated endocytosis mechanism as suggested by the reduced uptake in the presence of methyl-ß-cyclodextrin. These results suggest that the natural peptide may play a role within the cells of the oral mucosa after its secretion and subsequent internalization. Furthermore, lack of cytotoxicity of both peptide forms highlights their possible application as novel drug delivery agents.

  4. Characterizing the adsorption of peptides to TiO2 in aqueous solutions by liquid chromatography.

    PubMed

    Gertler, Golan; Fleminger, Gideon; Rapaport, Hanna

    2010-05-04

    The interactions between titanium oxide (TiO(2)) and flexible peptides, decorated by amine, carboxyl, and phosphoserine functional groups, were characterized using analytical liquid chromatography with various loading and eluting solutions. This approach enabled discernment of the type of intermolecular interactions generated between the peptides and the metal oxide surfaces in addition to unraveling more subtle effects, specific ions, and oxide phase may have on the adsorption. The peptide presenting Lys residues adsorbed to the oxide surface in the presence of Tris buffer and eluted under conditions that indicated its binding via electrostatic interactions at physiological pH values. Upon adsorption to the oxide in the presence of phosphate buffer, the same peptide exhibited stronger electrostatic interactions with the surface, mediated by the buffer phosphate ions. In Tris-buffered saline (TBS), pH 7.4, as the adsorption medium, the peptide with the phosphoserine residues exhibited affinity indicative of coordinative binding to the titanium oxide, whereas a similar peptide decorated by carboxylate groups failed to adsorb. On the basis of differences in the interactions of these peptides with the TiO(2), the efficient separation of the two peptides was demonstrated. A basic amphiphilic peptide, composed mostly of Lys and Leu residues, was found to strongly adsorb to TiO(2) while in helical conformation only, demonstrating the strong impact the secondary structure may have on adsorption to the surface. The methodology presented in this study allows the elucidation of in situ binding mechanism and relative strengths to titanium oxide surfaces at conditions which resemble biologically relevant environments.

  5. Protein hydrolysates in animal nutrition: Industrial production, bioactive peptides, and functional significance.

    PubMed

    Hou, Yongqing; Wu, Zhenlong; Dai, Zhaolai; Wang, Genhu; Wu, Guoyao

    2017-01-01

    Recent years have witnessed growing interest in the role of peptides in animal nutrition. Chemical, enzymatic, or microbial hydrolysis of proteins in animal by-products or plant-source feedstuffs before feeding is an attractive means of generating high-quality small or large peptides that have both nutritional and physiological or regulatory functions in livestock, poultry and fish. These peptides may also be formed from ingested proteins in the gastrointestinal tract, but the types of resultant peptides can vary greatly with the physiological conditions of the animals and the composition of the diets. In the small intestine, large peptides are hydrolyzed to small peptides, which are absorbed into enterocytes faster than free amino acids (AAs) to provide a more balanced pattern of AAs in the blood circulation. Some peptides of plant or animal sources also have antimicrobial, antioxidant, antihypertensive, and immunomodulatory activities. Those peptides which confer biological functions beyond their nutritional value are called bioactive peptides. They are usually 2-20 AA residues in length but may consist of >20 AA residues. Inclusion of some (e.g. 2-8%) animal-protein hydrolysates (e.g., porcine intestine, porcine mucosa, salmon viscera, or poultry tissue hydrolysates) or soybean protein hydrolysates in practical corn- and soybean meal-based diets can ensure desirable rates of growth performance and feed efficiency in weanling pigs, young calves, post-hatching poultry, and fish. Thus, protein hydrolysates hold promise in optimizing the nutrition of domestic and companion animals, as well as their health (particularly gut health) and well-being.

  6. Antibacterial peptides and mitochondrial presequences affect mitochondrial coupling, respiration and protein import.

    PubMed

    Hugosson, M; Andreu, D; Boman, H G; Glaser, E

    1994-08-01

    Cecropins A and P1, antibacterial peptides from insects and from pig and some related peptides released respiratory control, inhibited protein import and at higher concentrations also inhibited respiration. However, PR-39, an antibacterial peptide from pig intestine, was found to be almost inert towards mitochondria. The concentrations at which the three mitochondrial functions were effected varied for different peptides. Melittin, magainin and Cecropin-A-(1,13)-Melittin(1,13)-NH2, a hybrid between cecropin A and melittin, were most potent, while the two cecropins acted at higher concentrations. The biosynthesis of cecropin A is known and the intermediates are synthesized. We have used four peptides from this pathway to investigate their effects on coupling, respiration and protein import into mitochondria. Mature cecropin A followed by the preproprotein were most aggressive whereas the intermediates were less active or inert. The efficiency of different derivatives of cecropin A as uncouplers correlates well with their capacity to release membrane potential measured as fluorescence quenching of Rhodamine 123. Inhibition of respiration was found to be dependent on membrane potential and was most pronounced with mature cecropin A, less so with its three precursors. We also found that three peptides derived from mitochondrial presequences showed antibacterial activity. It is concluded that, there are similarities in the functions of antibacterial peptides and mitochondrial presequences, uncoupling activity in mitochondria cannot be correlated with the antibacterial activity (contrary to a previous suggestion), the processing of preprocecropin A may have evolved in such a way that there is a minimum of membrane damage from the intermediates in the pathway, and peptides produced for delivery outside of an animal have evolved to be more aggressive against mitochondria than peptides for delivery inside of the animal.

  7. Efficiency Goals

    ERIC Educational Resources Information Center

    Graham, Donald

    2009-01-01

    The lighting of learning environments is an important focus in designing new schools and renovating older schools. Studies long have shown that appropriate lighting levels and daylighting improve learning; now, climbing energy budgets have spurred school administrators to seek more efficient use of lighting. Electricity rates are expected to rise…

  8. Microwave-assisted solid-phase synthesis of side-chain to side-chain lactam-bridge cyclic peptides.

    PubMed

    Tala, Srinivasa R; Schnell, Sathya M; Haskell-Luevano, Carrie

    2015-12-15

    Side-chain to side-chain lactam-bridged cyclic peptides have been utilized as therapeutic agents and biochemical tools. Previous synthetic methods of these peptides need special reaction conditions, form side products and take longer reaction times. Herein, an efficient microwave-assisted synthesis of side-chain to side-chain lactam-bridge cyclic peptides SHU9119 and MTII is reported. The synthesis time and efforts are significantly reduced in the present method, without side product formation. The analytical and pharmacological data of the synthesized cyclic peptides are in accordance with the commercially obtained compounds. This new method could be used to synthesize other side-chain to side-chain lactam-bridge peptides and amenable to automation and extensive SAR compound derivatization.

  9. Site-Directed Glycosylation of Peptide/Protein with Homogeneous O-Linked Eukaryotic N-Glycans.

    PubMed

    Wu, Zhigang; Jiang, Kuan; Zhu, Hailiang; Ma, Cheng; Yu, Zaikuan; Li, Lei; Guan, Wanyi; Liu, Yunpeng; Zhu, He; Chen, Yanyi; Li, Shanshan; Li, Jing; Cheng, Jiansong; Zhang, Lianwen; Wang, Peng George

    2016-09-21

    Here we report a facile and efficient method for site-directed glycosylation of peptide/protein. The method contains two sequential steps: generation of a GlcNAc-O-peptide/protein, and subsequent ligation of a eukaryotic N-glycan to the GlcNAc moiety. A pharmaceutical peptide, glucagon-like peptide-1 (GLP-1), and a model protein, bovine α-Crystallin, were successfully glycosylated using such an approach. It was shown that the GLP-1 with O-linked N-glycan maintained an unchanged secondary structure after glycosylation, suggesting the potential application of this approach for peptide/protein drug production. In summary, the coupled approach provides a general strategy to produce homogeneous glycopeptide/glycoprotein bearing eukaryotic N-glycans.

  10. Anti-angiogenic peptides for cancer therapeutics

    PubMed Central

    Rosca, Elena V.; Koskimaki, Jacob E.; Rivera, Corban G.; Pandey, Niranjan B.; Tamiz, Amir P.; Popel, Aleksander S.

    2011-01-01

    Peptides have emerged as important therapeutics that are being rigorously tested in angiogenesis-dependent diseases due to their low toxicity and high specificity. Since the discovery of endogenous proteins and protein fragments that inhibit microvessel formation (thrombospondin, endostatin) several peptides have shown promise in pre-clinical and clinical studies for cancer. Peptides have been derived from thrombospondin, collagens, chemokines, coagulation cascade proteins, growth factors, and other classes of proteins and target different receptors. Here we survey recent developments for anti-angiogenic peptides with length not exceeding 50 amino acid residues that have shown activity in pre-clinical models of cancer or have been tested in clinical trials; some of the peptides have been modified and optimized, e.g., through L-to-D and non-natural amino acid substitutions. We highlight technological advances in peptide discovery and optimization including computational and bioinformatics tools and novel experimental techniques. PMID:21470139

  11. Formulation strategies to improve oral peptide delivery.

    PubMed

    Maher, Sam; Ryan, Ben; Duffy, Aoife; Brayden, David J

    2014-05-01

    Delivery of peptides by the oral route greatly appeals due to commercial, patient convenience and scientific arguments. While there are over 60 injectable peptides marketed worldwide, and many more in development, most delivery strategies do not yet adequately overcome the barriers to oral delivery. Peptides are sensitive to chemical and enzymatic degradation in the intestine, and are poorly permeable across the intestinal epithelium due to sub-optimal physicochemical properties. A successful oral peptide delivery technology should protect potent peptides from presystemic degradation and improve epithelial permeation to achieve a target oral bioavailability with acceptable intra-subject variability. This review provides a comprehensive up-to-date overview of the current status of oral peptide delivery with an emphasis on patented formulations that are yielding promising clinical data.

  12. Ribosomally encoded cyclic peptide toxins from mushrooms.

    PubMed

    Walton, Jonathan D; Luo, Hong; Hallen-Adams, Heather

    2012-01-01

    The cyclic peptide toxins of poisonous Amanita mushrooms are chemically unique among known natural products. Furthermore, they differ from other fungal cyclic peptides in being synthesized on ribosomes instead of by nonribosomal peptide synthetases. Because of their novel structures and biogenic origins, elucidation of the biosynthetic pathway of the Amanita cyclic peptides presents both challenges and opportunities. In particular, a full understanding of the pathway should lead to the ability to direct synthesis of a large number of novel cyclic peptides based on the Amanita toxin scaffold by genetic engineering of the encoding genes. Here, we highlight some of the principal methods for working with the Amanita cyclic peptides and the known steps in their biosynthesis.

  13. Cryptic Peptides from Collagen: A Critical Review.

    PubMed

    Banerjee, Pradipta; Shanthi, C

    2016-01-01

    Collagen, a predominant structural protein in extracellular matrix (ECM), is now considered to have probable roles in many biological activities and hence, in different forms have found application as nutraceutical or pharmaceutical therapy option. Many of the biological properties are believed to be due to small hidden peptide residues in the collagen molecules, which come into play after the biodegradation or biosorption of the parent molecule. These peptide regions are called cryptic peptides or by some, as cryptides. The proteolytic hydrolysis of the ECM protein releases the cryptic peptides with many novel biological activities not exhibited directly by the parental protein which include angiogenic, antimicrobial, mitogenic and chemotactic properties. The research for understanding the role of these cryptic peptide regions and making use of them in medical field is very active. Such an understanding could lead to the development of peptide supplements for many biomedical applications. The prolific research in this area is reviewed in this paper.

  14. Peptide YY receptors in the brain

    SciTech Connect

    Inui, A.; Oya, M.; Okita, M.; Inoue, T.; Sakatani, N.; Morioka, H.; Shii, K.; Yokono, K.; Mizuno, N.; Baba, S.

    1988-01-15

    Radiolabelled ligand binding studies demonstrated that specific receptors for peptide YY are present in the porcine as well as the canine brains. Peptide YY was bound to brain tissue membranes via high-affinity (dissociation constant, 1.39 X 10(-10)M) and low-affinity (dissociation constant, 3.72 X 10(-8)M) components. The binding sites showed a high specificity for peptide YY and neuropeptide Y, but not for pancreatic polypeptide or structurally unrelated peptides. The specific activity of peptide YY binding was highest in the hippocampus, followed by the pituitary gland, the hypothalamus, and the amygdala of the porcine brain, this pattern being similarly observed in the canine brain. The results suggest that peptide YY and neuropeptide Y may regulate the function of these regions of the brain through interaction with a common receptor site.

  15. Toward structure prediction of cyclic peptides.

    PubMed

    Yu, Hongtao; Lin, Yu-Shan

    2015-02-14

    Cyclic peptides are a promising class of molecules that can be used to target specific protein-protein interactions. A computational method to accurately predict their structures would substantially advance the development of cyclic peptides as modulators of protein-protein interactions. Here, we develop a computational method that integrates bias-exchange metadynamics simulations, a Boltzmann reweighting scheme, dihedral principal component analysis and a modified density peak-based cluster analysis to provide a converged structural description for cyclic peptides. Using this method, we evaluate the performance of a number of popular protein force fields on a model cyclic peptide. All the tested force fields seem to over-stabilize the α-helix and PPII/β regions in the Ramachandran plot, commonly populated by linear peptides and proteins. Our findings suggest that re-parameterization of a force field that well describes the full Ramachandran plot is necessary to accurately model cyclic peptides.

  16. Synthesis of peptide .alpha.-thioesters

    DOEpatents

    Camarero, Julio A.; Mitchell, Alexander R.; De Yoreo, James J.

    2008-08-19

    Disclosed herein is a new method for the solid phase peptide synthesis (SPPS) of C-terminal peptide .alpha. thioesters using Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazine linker, which is totally stable to conditions required for Fmoc-SPPS. When the peptide synthesis has been completed, activation of the linker is achieved by mild oxidation. The oxidation step converts the acyl-hydrazine group into a highly reactive acyl-diazene intermediate which reacts with an .alpha.-amino acid alkylthioester (H-AA-SR) to yield the corresponding peptide .alpha.-thioester in good yield. A variety of peptide thioesters, cyclic peptides and a fully functional Src homology 3 (SH3) protein domain have been successfully prepared.

  17. Anti-angiogenic peptides for cancer therapeutics.

    PubMed

    Rosca, Elena V; Koskimaki, Jacob E; Rivera, Corban G; Pandey, Niranjan B; Tamiz, Amir P; Popel, Aleksander S

    2011-08-01

    Peptides have emerged as important therapeutics that are being rigorously tested in angiogenesis-dependent diseases due to their low toxicity and high specificity. Since the discovery of endogenous proteins and protein fragments that inhibit microvessel formation (thrombospondin, endostatin) several peptides have shown promise in pre-clinical and clinical studies for cancer. Peptides have been derived from thrombospondin, collagens, chemokines, coagulation cascade proteins, growth factors, and other classes of proteins and target different receptors. Here we survey recent developments for anti-angiogenic peptides with length not exceeding 50 amino acid residues that have shown activity in pre-clinical models of cancer or have been tested in clinical trials; some of the peptides have been modified and optimized, e.g., through L-to-D and non-natural amino acid substitutions. We highlight technological advances in peptide discovery and optimization including computational and bioinformatics tools and novel experimental techniques.

  18. Use of Galerina marginata genes and proteins for peptide production

    DOEpatents

    Hallen-Adams, Heather E.; Scott-Craig, John S.; Walton, Jonathan D.; Luo, Hong

    2017-03-21

    The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.

  19. Use of Galerina marginata genes and proteins for peptide production

    DOEpatents

    Hallen-Adams, Heather E.; Scott-Craig, John S.; Walton, Jonathan D.; Luo, Hong

    2016-03-01

    The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides spec