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Sample records for active site peptide

  1. Characterization and sequencing of an active-site cysteine-containing peptide from the xylanase of a thermotolerant Streptomyces.

    PubMed

    Keskar, S S; Rao, M B; Deshpande, V V

    1992-02-01

    The kinetics of chemical modification of the xylanase from a thermotolerant Streptomyces T7 indicated the involvement of 1 mol of cysteine residue/mol of enzyme [Keskar, Srinivasan & Deshpande (1989) Biochem. J. 261, 49-55]. The chromophoric reagent N-(2,4-dinitroanilino)maleimide (DAM) reacts covalently with thiol groups of xylanase with complete inactivation. Protection against inactivation was provided by the substrate (xylan). The purified xylanase that had been modified with DAM was digested with pepsin and the peptides were purified by gel filtration followed by peptide mapping. The active-site peptide was distinguished from the other thiol-containing peptides by comparison of the peptides generated by labelling the enzyme in the presence and in the absence of the substrate. The peptide mapping of the modified enzyme in the absence of xylan showed three yellow peptides, whereas in the presence of xylan only two yellow peptides were detected. The active-site peptide protected by the substrate failed to form the complex with DAM. The modified active-site peptide was isolated and sequenced. Gas-phase sequencing provided the following sequence: Ser-Val-Ile-Met-Xaa-Ile-Asp-His-Ile-Arg-Phe. This is the first report on the isolation and sequencing of the active-site peptide from a xylanase. The comparison of reactive cysteine-containing peptide sequence with the catalytic regions of other glucanases revealed the presence of a conserved aspartic acid residue.

  2. Conversed mutagenesis of an inactive peptide to ASIC3 inhibitor for active sites determination.

    PubMed

    Osmakov, Dmitry I; Koshelev, Sergey G; Andreev, Yaroslav A; Dyachenko, Igor A; Bondarenko, Dmitry A; Murashev, Arkadii N; Grishin, Eugene V; Kozlov, Sergey A

    2016-06-15

    Peptide Ugr9-1 from the venom of sea anemone Urticina grebelnyi selectively inhibits the ASIC3 channel and significantly reverses inflammatory and acid-induced pain in vivo. A close homolog peptide Ugr 9-2 does not have these features. To find the pharmacophore residues and explore structure-activity relationships of Ugr 9-1, we performed site-directed mutagenesis of Ugr 9-2 and replaced several positions by the corresponding residues from Ugr 9-1. Mutant peptides Ugr 9-2 T9F and Ugr 9-2 Y12H were able to inhibit currents of the ASIC3 channels 2.2 times and 1.3 times weaker than Ugr 9-1, respectively. Detailed analysis of the spatial models of Ugr 9-1, Ugr 9-2 and both mutant peptides revealed the presence of the basic-aromatic clusters on opposite sides of the molecule, each of which is responsible for the activity. Additionally, Ugr9-1 mutant with truncated N- and C-termini retained similar with the Ugr9-1 action in vitro and was equally potent in vivo model of thermal hypersensitivity. All together, these results are important for studying the structure-activity relationships of ligand-receptor interaction and for the future development of peptide drugs from animal toxins. PMID:26686983

  3. The thrombin receptor extracellular domain contains sites crucial for peptide ligand-induced activation.

    PubMed Central

    Bahou, W F; Coller, B S; Potter, C L; Norton, K J; Kutok, J L; Goligorsky, M S

    1993-01-01

    A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize alpha-thrombin- and peptide ligand-induced activation responses using two different antibodies: anti-TR34-52 directed against a 20-amino acid peptide spanning the thrombin cleavage site, and anti-TR1-160 generated against the NH2-terminal 160 amino acids of the TR expressed as a chimeric protein in Escherichia coli. Activation-dependent responses to both alpha-thrombin (10 nM) and peptide ligand (20 microM) were studied using fura 2-loaded cells and microspectrofluorimetry. Whereas preincubation of CHO-TR with anti-TR34-52 abolished only alpha-thrombin-induced [Ca2+]i transients, preincubation with anti-TR1-160 abrogated both alpha-thrombin- and peptide ligand-induced responses. This latter inhibitory effect was dose dependent and similar for both agonists, with an EC50 of approximately 90 micrograms/ml. Anti-TR1-160 similarly abolished peptide ligand-induced [Ca2+]i transients in platelets and HUVECs, whereas qualitatively different responses characterized by delayed but sustained elevations in [Ca2+]i transients were evident using alpha-thrombin. Platelet aggregation to low concentrations of both ligands was nearly abolished by anti-TR1-160, although some shape change remained; anti-TR34-52 only inhibited alpha-thrombin-induced aggregation. These data establish that a critical recognition sequence for peptide ligand-mediated receptor activation is contained on the NH2-terminal portion of the receptor, upstream from the first transmembrane domain. Furthermore, alpha

  4. Activation of erythropoietin receptor in the absence of hormone by a peptide that binds to a domain different from the hormone binding site

    PubMed Central

    Naranda, Tatjana; Wong, Kenneth; Kaufman, R. Ilene; Goldstein, Avram; Olsson, Lennart

    1999-01-01

    Applying a homology search method previously described, we identified a sequence in the extracellular dimerization site of the erythropoietin receptor, distant from the hormone binding site. A peptide identical to that sequence was synthesized. Remarkably, it activated receptor signaling in the absence of erythropoietin. Neither the peptide nor the hormone altered the affinity of the other for the receptor; thus, the peptide does not bind to the hormone binding site. The combined activation of signal transduction by hormone and peptide was strongly synergistic. In mice, the peptide acted like the hormone, protecting against the decrease in hematocrit caused by carboplatin. PMID:10377456

  5. Amyloid-β peptide active site: theoretical Cu K-edge XANES study

    NASA Astrophysics Data System (ADS)

    Chaynikov, A. P.; Soldatov, M. A.; Streltsov, V.; Soldatov, A. V.

    2013-04-01

    This article is dedicated to the local atomic structure analysis of the copper binding site in amyloid-β peptide. Here we considered two possible structural models that were previously obtained by means of EXAFS analysis and density functional theory simulations. We present the calculations of Cu K-edge XANES spectra for both models and make comparison of these spectra with experiment.

  6. Neurospora tryptophan synthase: N-terminal analysis and the sequence of the pyridoxal phosphate active site peptide

    SciTech Connect

    Pratt, M.L.; Hsu, P.Y.; DeMoss, J.A.

    1986-05-01

    Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with (/sup 3/H)-NaBH/sub 4/, and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active site peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P.

  7. Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli

    SciTech Connect

    Nicholas, R.A.; Suzuki, H.; Hirota, Y.; Strominger, J.L.

    1985-07-02

    This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with (/sup 14/C)penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed that the serine residue in the middle of the sequence was covalently bonded to the (/sup 14/C)penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.

  8. Active site peptide of beta-lactamase from Shigella flexneri UCSF-129.

    PubMed

    Campos, M; González, H; Bocaz, G; Vásquez, O

    1997-01-01

    The peptide containing the catalytic serine of beta-lactamase from Shigella flexneri was determined as V-D-E-R-F-P-M-M-S*-T-F-K. It is a local pathogenic strain which produces intestinal problems, especially in children. The highly purified enzyme was prepared by affinity chromatography in phenylboronic acid-agarose gels. The peptide was obtained by tryptic hydrolysis, with further purification by Bio-Gel P-4, Sephadex QAE-25 and Sephadex SP-25. The relevance of the serine, lysine and arginine residues was mainly shown by the loss of enzymatic activity after specific chemical modifications. Finally, this enzyme was classified as A, according to the similarity of this peptide with that of class A beta-lactamases such as R-TEM 1 and 2. PMID:9301069

  9. A single mutation in the hepta-peptide active site of Aspergillus niger PhyA phytase leads to myriad of biochemical changes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The active site motif of proteins belonging to ‘Histidine Acid Phosphatase’ (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs has revealed the fourth residue of the hepta-peptide to be E instead of A, which is the case with A. niger phyA phytase. However,...

  10. Correlation between the compact regions predicted by the average distance map (ADM) and the biologically active sites in atrial natriuretic peptide.

    PubMed

    Kikuchi, T

    1992-10-01

    The prediction of the short-range compact regions of human atrial natriuretic peptide (alpha-hANP), one of the biologically active peptides, has been made by means of the Average Distance Map(ADM). We found out that the location of the predicted short-range compact regions is consistent with the structural units determined by the NMR analysis (Kobayashi et al., 1988). Furthermore, the short-range compact regions correspond well to the biologically active areas of atriopeptin (103-125)-amide (which is homologous peptide to alpha-hANP), detected by the glycine substitution technique (Konishi et al., 1987). The results suggest that a predicted short-range compact region can be regarded as a possible active site in a biologically active peptide.

  11. Binding stability of peptides derived from 1ALA residue and 7GLY residues to sites near active center of fluctuating papain

    NASA Astrophysics Data System (ADS)

    Nishiyama, Katsuhiko

    2012-05-01

    We investigated the binding stability of peptides derived from 1ALA residue and 7GLY residues to sites near active center of fluctuating papain via molecular dynamics and docking simulations. Replacing GLY residue in 8GLY with ALA residue had a positive effect on binding stability to the sites in some cases although the replacing had a negative effect on it in other cases. Furthermore the replacing had a negative effect on the chance of binding to the sites. Residue in peptide should be replaced on the basis of systematic exploration of its position.

  12. Femtomolar Zn(II) affinity in a peptide-based ligand designed to model thiolate-rich metalloprotein active sites.

    PubMed

    Petros, Amy K; Reddi, Amit R; Kennedy, Michelle L; Hyslop, Alison G; Gibney, Brian R

    2006-12-11

    Metal-ligand interactions are critical components of metalloprotein assembly, folding, stability, electrochemistry, and catalytic function. Research over the past 3 decades on the interaction of metals with peptide and protein ligands has progressed from the characterization of amino acid-metal and polypeptide-metal complexes to the design of folded protein scaffolds containing multiple metal cofactors. De novo metalloprotein design has emerged as a valuable tool both for the modular synthesis of these complex metalloproteins and for revealing the fundamental tenets of metalloprotein structure-function relationships. Our research has focused on using the coordination chemistry of de novo designed metalloproteins to probe the interactions of metal cofactors with protein ligands relevant to biological phenomena. Herein, we present a detailed thermodynamic analysis of Fe(II), Co(II), Zn(II), and[4Fe-4S]2(+/+) binding to IGA, a 16 amino acid peptide ligand containing four cysteine residues, H2N-KLCEGG-CIGCGAC-GGW-CONH2. These studies were conducted to delineate the inherent metal-ion preferences of this unfolded tetrathiolate peptide ligand as well as to evaluate the role of the solution pH on metal-peptide complex speciation. The [4Fe-4S]2(+/+)-IGA complex is both an excellent peptide-based synthetic analogue for natural ferredoxins and is flexible enough to accommodate mononuclear metal-ion binding. Incorporation of a single ferrous ion provides the FeII-IGA complex, a spectroscopic model of a reduced rubredoxin active site that possesses limited stability in aqueous buffers. As expected based on the Irving-Williams series and hard-soft acid-base theory, the Co(II) and Zn(II) complexes of IGA are significantly more stable than the Fe(II) complex. Direct proton competition experiments, coupled with determinations of the conditional dissociation constants over a range of pH values, fully define the thermodynamic stabilities and speciation of each MII-IGA complex. The

  13. FRET analysis using sperm-activating peptides tagged with fluorescent proteins reveals that ligand-binding sites exist as clusters.

    PubMed

    Arcos-Hernández, César; Romero, Francisco; Sánchez-Guevara, Yoloxochitl; Beltrán, Carmen; Nishigaki, Takuya

    2016-02-01

    Long-range cellular communication between the sperm and egg is critical for external fertilization. Sperm-activating peptides (SAPs) are diffusible components of the outer layer of eggs in echinoderms, and function as chemoattractants for spermatozoa. The decapeptide named speract is the best-characterized sea urchin SAP. Biochemical and physiological actions of speract have been studied with purified or chemically synthesized peptides. In this work, we prepared recombinant speract fused to a fluorescent protein (FP; FP-speract) using three color variants: a cyan (eCFP), a yellow (mVenus) and a large Stokes shift yellow (mAmetrine) FP. Although these fluorescence tags are 20 times larger than speract, competitive binding experiments using mAmetrine-speract revealed that this FP-speract has binding affinity to the receptor that is comparable (7.6-fold less) to that of non-labeled speract. Indeed, 10 nmol l(-1) eCFP-speract induces physiological sperm responses such as membrane potential changes and increases in intracellular pH and Ca(2+) concentrations similar to those triggered by 10 nmol l(-1) speract. Furthermore, FP-speract maintains its fluorescence upon binding to its receptor. Using this property, we performed fluorescence resonance energy transfer (FRET) measurements with eCFP-speract and mVenus-speract as probes and obtained a positive FRET signal upon binding to the receptor, which suggests that the speract receptor exists as an oligomer, at least as a dimer, or alternatively that a single speract receptor protein possesses multiple binding sites. This property could partially account for the positive and/or negative cooperative binding of speract to the receptor.

  14. Active site specificity profiling of the matrix metalloproteinase family: Proteomic identification of 4300 cleavage sites by nine MMPs explored with structural and synthetic peptide cleavage analyses.

    PubMed

    Eckhard, Ulrich; Huesgen, Pitter F; Schilling, Oliver; Bellac, Caroline L; Butler, Georgina S; Cox, Jennifer H; Dufour, Antoine; Goebeler, Verena; Kappelhoff, Reinhild; Keller, Ulrich Auf dem; Klein, Theo; Lange, Philipp F; Marino, Giada; Morrison, Charlotte J; Prudova, Anna; Rodriguez, David; Starr, Amanda E; Wang, Yili; Overall, Christopher M

    2016-01-01

    Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6' in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1' (with a ~32-93% preference for leucine depending on the MMP), and basic and small residues in P2' and P3', respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1' leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1'-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with

  15. Definition of a physiologic aging autoantigen by using synthetic peptides of membrane protein band 3: localization of the active antigenic sites.

    PubMed

    Kay, M M; Marchalonis, J J; Hughes, J; Watanabe, K; Schluter, S F

    1990-08-01

    Senescent cell antigen (SCA), an aging antigen, is a protein that appears on old cells and marks them for removal by the immune system in mammals. It is derived from band 3, a ubiquitous membrane transport protein found in diverse cell types and tissues. We have used synthetic peptides to identify aging antigenic sites on band 3, using a competitive inhibition assay and immunoblotting with IgG directed against the aging antigen on old cells. Results indicate that: (i) the active antigenic sites of the aging antigen reside on membrane protein band 3 residues that are extracellular regions implicated in anion transport (residues 538-554 and 788-827); (ii) a putative ankyrin-binding-region peptide is not involved in SCA activity; and (iii) carbohydrate moieties are not required for the antigenicity or recognition of SCA because synthetic peptides alone abolish binding of senescent cell IgG to erythrocytes. One of the putative transport sites that contributes to the aging antigen is located toward the carboxyl terminus. A model of band 3 is presented. Localization of the active antigenic site on the band 3 molecule facilitates definition of the molecular changes occurring during aging that initiate molecular as well as cellular degeneration. PMID:1696010

  16. Active-site peptide "fingerprinting" of glycosidases in complex mixtures by mass spectrometry. Discovery of a novel retaining beta-1,4-glycanase in Cellulomonas fimi.

    PubMed

    Hekmat, Omid; Kim, Young-Wan; Williams, Spencer J; He, Shouming; Withers, Stephen G

    2005-10-21

    New proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Here, we report the first gel-free, mass spectrometry-based strategy for mechanism-based profiling of retaining beta-endoglycosidases in complex proteomes. Using a biotinylated, cleavable 2-deoxy-2-fluoroxylobioside inactivator, we have isolated and identified the active-site peptides of target retaining beta-1,4-glycanases in systems of increasing complexity: pure enzymes, artificial proteomes, and the secreted proteome of the aerobic mesophilic soil bacterium Cellulomonas fimi. The active-site peptide of a new C. fimi beta-1,4-glycanase was identified in this manner, and the peptide sequence, which includes the catalytic nucleophile, is highly conserved among glycosidase family 10 members. The glycanase gene (GenBank accession number DQ146941) was cloned using inverse PCR techniques, and the protein was found to comprise a catalytic domain that shares approximately 70% sequence identity with those of xylanases from Streptomyces sp. and a family 2b carbohydrate-binding module. The new glycanase hydrolyzes natural and artificial xylo-configured substrates more efficiently than their cello-configured counterparts. It has a pH dependence very similar to that of known C. fimi retaining glycanases. PMID:16085650

  17. X-ray structure at 1.75 resolution of a norovirus 3C protease linked to an active site-directed peptide inhibitor

    SciTech Connect

    Cooper, Jon; Coates, Leighton; Hussey, Robert

    2010-01-01

    Noroviruses are recognized universally as the most important cause of human epidemic non-bacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.75 resolution, following initial MAD phasing with a selenomethionine derivative. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, based on a 3C protease cleavage recognition sequences in the 200kDa polyprotein substrate, reacts covalently through its propenylethylester group (X) with the active site nucleophile, Cys 139. The 3C protease-inhibitor structure permits, for the first time, the identification of substrate recognition and binding groups and provides important new information for the development of antiviral prophylactics.

  18. Degradation and antioxidant activities of peptides and zinc-peptide complexes during in vitro gastrointestinal digestion.

    PubMed

    Wang, Chan; Li, Bo; Wang, Bo; Xie, Ningning

    2015-04-15

    The degradation characteristics of three peptides (Ser-Met, Asn-Cys-Ser, and glutathione) and their zinc-peptide complexes were studied using a two-stage in vitro digestion model. Enzyme-resistant peptides and zinc-peptide complexes, antioxidant activities, and free amino acids released by digestive enzymes, were measured in this study. The results revealed that the three peptides and their zinc-peptide complexes were resistant to pepsin but not to pancreatin. Pancreatin can partly hydrolyse both peptides and zinc-peptide complexes, but more than half of them remaining in their original form after gastrointestinal digestion. The coordination of zinc improved the enzymatic resistance of the peptide due to lower solubility of complexes and affected the hydrolytic site of pepsin and pancreatin. Zinc-Asn-Cys-Ser, which is highly resistant to enzymatic hydrolysis and maintains Zn in a soluble form, may have potential to improve Zn bioavailability.

  19. Antimicrobial activity of polycationic peptides.

    PubMed

    Giacometti, A; Cirioni, O; Barchiesi, F; Del Prete, M S; Scalise, G

    1999-11-01

    The in vitro activity of six polycationic peptides, buforin II, cecropin P1, indolicidin, magainin II, nisin, and ranalexin, were evaluated against several clinical isolates of gram-positive and gram-negative aerobic bacteria, yeasts, Pneumocystis carinii and Cryptosporidium parvum, by using microbroth dilution methods. The peptides exhibited different antibacterial activities and rapid time-dependent killing. The gram-negative organisms were more susceptible to buforin II and cecropin P1, whereas buforin II and ranalexin were the most active compounds against the gram-positive strains. Similarly, ranalexin showed the highest activity against Candida spp., whereas magainin II exerted the highest anticryptococcal activity. Finally, the peptides showed high anti-Pneumocystis activity, whereas no compound had strong inhibitory effect on C. parvum. PMID:10612440

  20. Specific binding sites for muramyl peptides on murine macrophages

    SciTech Connect

    Silverman, D.H.S.; Krueger, J.M.; Karnovsky, M.L.

    1986-03-15

    Two radiolabeled (/sup 125/I) muramyl peptide derivatives of high specific activity were prepared: a tripeptide with an iodinated C-terminal tyrosine methyl ester (Ligand I), and a muramyl tripeptide with a C-terminal lysine derivatized with Bolton-Hunter reagent (Ligand II). These were used to characterize binding of muramyl peptides to monolayers of murine macrophages. Saturable high-affinity binding to resident, caseinate-elicited, and Listeria-activated peritoneal cells was observed with both radioligands. Binding affinities varied with the state of activation of the macrophages, and K/sub D/ values ranged from 48 +/- 33 pM (for resident macrophages, Ligand I) to 1020 +/- 90 pM (for activated macrophages, Ligand II). Specific binding sites were also found on a macrophage-derived cell line. The ability of several unlabeled muramyl peptides to compete with Ligands I and II for their binding sites was tested. Competition was stereospecific and correlated with known biological activities of these compounds (i.e., immunoadjuvanticity, pyrogenicity, and somnogenicity). The sites identified here for Ligands I and II may mediate some of the effects that muramyl peptides have previously been demonstrated to have on macrophages.

  1. Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors.

    PubMed

    Roessler, Claudia; Nowak, Theresa; Pannek, Martin; Gertz, Melanie; Nguyen, Giang T T; Scharfe, Michael; Born, Ilona; Sippl, Wolfgang; Steegborn, Clemens; Schutkowski, Mike

    2014-09-26

    Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5. PMID:25111069

  2. 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.

  3. Non-coding nucleotides and amino acids near the active site regulate peptide deformylase expression and inhibitor susceptibility in Chlamydia trachomatis

    PubMed Central

    Bao, Xiaofeng; Pachikara, Niseema D.; Oey, Christopher B.; Balakrishnan, Amit; Westblade, Lars F.; Tan, Ming; Chase, Theodore; Nickels, Bryce E.

    2011-01-01

    Chlamydia trachomatis, an obligate intracellular bacterium, is a highly prevalent human pathogen. Hydroxamic-acid-based matrix metalloprotease inhibitors can effectively inhibit the pathogen both in vitro and in vivo, and have exhibited therapeutic potential. Here, we provide genome sequencing data indicating that peptide deformylase (PDF) is the sole target of the inhibitors in this organism. We further report molecular mechanisms that control chlamydial PDF (cPDF) expression and inhibition efficiency. In particular, we identify the σ66-dependent promoter that controls cPDF gene expression and demonstrate that point mutations in this promoter lead to resistance by increasing cPDF transcription. Furthermore, we show that substitution of two amino acids near the active site of the enzyme alters enzyme kinetics and protein stability. PMID:21719536

  4. PeptiSite: a structural database of peptide binding sites in 4D.

    PubMed

    Acharya, Chayan; Kufareva, Irina; Ilatovskiy, Andrey V; Abagyan, Ruben

    2014-03-21

    We developed PeptiSite, a comprehensive and reliable database of biologically and structurally characterized peptide-binding sites, in which each site is represented by an ensemble of its complexes with protein, peptide and small molecule partners. The unique features of the database include: (1) the ensemble site representation that provides a fourth dimension to the otherwise three dimensional data, (2) comprehensive characterization of the binding site architecture that may consist of a multimeric protein assembly with cofactors and metal ions and (3) analysis of consensus interaction motifs within the ensembles and identification of conserved determinants of these interactions. Currently the database contains 585 proteins with 650 peptide-binding sites. http://peptisite.ucsd.edu/ link allows searching for the sites of interest and interactive visualization of the ensembles using the ActiveICM web-browser plugin. This structural database for protein-peptide interactions enables understanding of structural principles of these interactions and may assist the development of an efficient peptide docking benchmark. PMID:24406170

  5. Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

    PubMed

    Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

    2015-12-18

    A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

  6. Inactivation of glyceraldehyde-3-phosphate dehydrogenase by a reactive metabolite of acetaminophen and mass spectral characterization of an arylated active site peptide.

    PubMed

    Dietze, E C; Schäfer, A; Omichinski, J G; Nelson, S D

    1997-10-01

    Acetaminophen (4'-hydroxyacetanilide, APAP) is a widely used analgesic and antipyretic drug that can cause hepatic necrosis under some circumstances via cytochrome P450-mediated oxidation to a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Although the mechanism of hepatocellular injury caused by APAP is not fully understood, it is known that NAPQI forms covalent adducts with several hepatocellular proteins. Reported here is the identification of one of these proteins as glyceraldehyde-3-phosphate dehydrogenase [GAPDH, D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12]. Two hours after the administration of hepatotoxic doses of [14C]APAP to mice, at a time prior to overt cell damage, hepatocellular GAPDH activity was significantly decreased concurrent with the formation of a 14C-labeled GAPDH adduct. A nonhepatotoxic regioisomer of APAP, 3'-hydroxyacetanilide (AMAP), was found to decrease GAPDH activity to a lesser extent than APAP, and radiolabel from [14C]AMAP bound to a lesser extent to GAPDH at a time when its overall binding to hepatocellular proteins was almost equivalent to that of APAP. In order to determine the nature of the covalent adduct between GAPDH and APAP, its major reactive and toxic metabolite, NAPQI, was incubated with purified porcine muscle GAPDH. Microsequencing analysis and fast atom bombardment mass spectrometry (FAB-MS) with collision-induced dissociation (CID) were used to characterize one of the adducts as APAP bound to the cysteinyl sulfhydryl group of Cys-149 in the active site peptide of GAPDH. PMID:9348431

  7. Natural and synthetic peptides with antifungal activity.

    PubMed

    Ciociola, Tecla; Giovati, Laura; Conti, Stefania; Magliani, Walter; Santinoli, Claudia; Polonelli, Luciano

    2016-08-01

    In recent years, the increase of invasive fungal infections and the emergence of antifungal resistance stressed the need for new antifungal drugs. Peptides have shown to be good candidates for the development of alternative antimicrobial agents through high-throughput screening, and subsequent optimization according to a rational approach. This review presents a brief overview on antifungal natural peptides of different sources (animals, plants, micro-organisms), peptide fragments derived by proteolytic cleavage of precursor physiological proteins (cryptides), synthetic unnatural peptides and peptide derivatives. Antifungal peptides are schematically reported based on their structure, antifungal spectrum and reported effects. Natural or synthetic peptides and their modified derivatives may represent the basis for new compounds active against fungal infections. PMID:27502155

  8. Design and production of peptides mimicking the active site of serine esterases with covalent binding to the organophosphorous poison soman. Annual report, 1 July 1984-30 June 1985

    SciTech Connect

    Seltzman, H.H.

    1985-12-09

    The objective of this research program is to design, synthesize, and test peptides and peptide mimics that will scavange soman in vivo and thereby provide protection against this CW agent. The test compounds were designed to mimic the active site of serine esterases (AChE), which are the natural targets of soman, enabling them to react with soman and thus protect endogenous AChE. Cyclodextrins derivatized with peptide functional groups and their equivalents such as imidazole, histamine, ethylene diamine, diethylene triamine, catechol, and ethane dithiol were synthesized for testing. The synthesis of precursors to cyclohexapeptides containing histidine, serine, and aspartic acid, which are amino acids that have been implicated in the active site of numerous esterases, were pursued. Testing of the ability of alpha-, beta, and gamma-cyclodextrins to protect AChE frominactivation by soman was carried out in vitro. From this group of compounds, beta-cyclodextrin was observed to preserve the activity of AChE in a dose response manner achieving a 72.1% preservation of activity when present in 200,000 fold excess versus soman after only ten minutes incubation time (beta-cyclodextrin + soman). Neither alpha, nor gamma-cyclodextrin showed any protective effect at the same doses. The test results suggest that beta cyclodextrin is uniquely suited to scavange soman. Improved scavanging might be achieved with the modified cyclodextrins prepared above for testing.

  9. Isolation and sequencing of an active-site peptide from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase after affinity labeling with 2-((Bromoacetyl)amino)pentitol 1,5-bisphosphate

    SciTech Connect

    Fraij, B.; Hartman, F.C.

    1983-01-01

    2-((Bromoacetyl)amino)pentitol 1,5-bisphosphate was reported to be a highly selective affinity label for ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. The enzyme has now been inactivated with a /sup 14/C-labeled reagent in order to identify the target residue at the sequence level. Subsequent to inactivation, the enzyme was carboxymethylated with iodoacetate and then digested with trypsin. The only radioactive peptide in the digest was obtained at a high degree of purity by successive chromatography on DEAE-cellulose, SP-Sephadex, and Sephadex G-25. On the basis of amino acid analysis of the purified peptide, the derivatized residue was a methionyl sulfonium salt. Automated Edman degradation confirmed the purity of the labeled peptide and established its sequence as Leu-Gln-Gly-Ala-Ser-Gly-Ile-His-Thr-Gly-Thr-Met-Gly-Phe-Gly-Lys-Met-Glu-Gly-Glu-Ser-Ser-Asp-Arg. Cleavage of this peptide with cyanogen bromide showed that the reagent moiety was covalently attached to the second methionyl residue. Sequence homology with the carboxylase/oxygenase from spinach indicates that the lysyl residue immediately preceding the alkylated methionine corresponds to Lys-334, a residue previously implicated at the active site. 31 references, 4 figures, 3 tables.

  10. Hydrazide Reactive Peptide Tags for Site-Specific Protein Labeling

    PubMed Central

    Eldridge, Glenn M.; Weiss, Gregory A.

    2011-01-01

    New site-specific protein labeling (SSPL) reactions for targeting specific, short peptides could be useful for the real time detection of proteins inside of living cells. One SSPL approach matches bioorthogonal reagents with complementary peptides. Here, hydrazide reactive peptides were selected from phage-displayed libraries using reaction-based selections. Selection conditions included washes of varying pH and treatment with NaCNBH3 in order to specifically select reactive carbonyl containing peptides. Selected peptides were fused to T4 lysozyme or synthesized on filter paper for colorimetric assays of the peptide-hydrazide interaction. A peptide-lysozyme protein fusion demonstrated specific, covalent labeling by the Hydrazide Reactive (HyRe) peptides in crude bacterial cell lysates, sufficient for the specific detection of an over-expressed protein fusion. Chemical synthesis of a short HyRe tag variant and subsequent reaction with two structurally distinct hydrazide probes produced covalent adducts observable by MALDI-TOF MS and MS/MS. Rather than isolating reactive carbonyl-containing peptides, we observed reaction with the N–terminal His of HyRe tag 114, amino acid sequence HKSNHSSKNRE, which attacks the hydrazide carbonyl at neutral pH. However, at the pH used during selection wash steps (<6.0), an alternative imine-containing product is formed that can be reduced with sodium cyanoborohydride. MSMS further reveals that this low pH product forms an adduct on Ser6. Further optimization of the novel bimolecular reaction described here could provide a useful tool for in vivo protein labeling and bioconjugate synthesis. The reported selection and screening methods could be widely applicable to the identification of peptides capable of other site-specific protein labeling reactions with bioorthogonal reagents. PMID:21905743

  11. 2-(4-bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, a new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum: determination of reaction parameters and characterization of an active site peptide

    SciTech Connect

    Herndon, C.S.; Hartman, F.C.

    1984-03-10

    Reductive amination of ribulose-P/sub 2/ with p-phenylenediamine in the presence of sodium cyanoborohydride yielded an epimeric mixture which was resolved by chromatography. Subsequent bromoacetylation of the isolated amino bisphosphates gave reagents A and B (ribo and arabino epimers of 2-(4-bromoacetamido)anilino-2-deoxypentitol 1.5-bisphosphate) which were competitive inhibitors of the carboxylase with K/sub i/ values of 705 and 104 ..mu..M, respectively. Reagent A exhibited no time-dependent effects on the carboxylase in either the deactivated or activated state. Incubation of the enzyme with reagent B in the presence of the essential activators CO/sub 2/ and Mg/sup 2 +/, however, resulted in an irreversible, time-dependent loss of activity, with a K/sub inact/ of 125 ..mu..M and a minimal half-time of 7.3 min. Covalent incorporation of (/sup 14/C)reagent B was directly proportional to the loss of activity, with total inactivation correlating with an incorporation of 1.1 mol of reagent/mol of subunit. Inclusion of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate protected against inactivation with a concomitant reduction in incorporation. Neither reagent affected the activity of spinach carboxylase. Fractionation of (/sup 14/C)reagent B-modified enzyme on DEAE-cellulose, subsequent to carboxymethylation and tryptic digestion, revealed two major radioactive peaks of approximately equal area. Digestion of each peak with alkaline phosphatase and rechromatography on DEAE-cellulose resulted in pure peptides I and II. The peptides were identical except in the site of labeling: peptide I contained a modified cysteinyl residue while peptide II contained a modified histidyl residue. 60 references, 7 figures, tables.

  12. A biologically active peptide mimetic of N-acetylgalactosamine/galactose

    PubMed Central

    Eggink, Laura L; Hoober, J Kenneth

    2009-01-01

    Background Glycosylated proteins and lipids are important regulatory factors whose functions can be altered by addition or removal of sugars to the glycan structure. The glycans are recognized by sugar-binding lectins that serve as receptors on the surface of many cells and facilitate initiation of an intracellular signal that changes the properties of the cells. We identified a peptide that mimics the ligand of an N-acetylgalactosamine (GalNAc)-specific lectin and asked whether the peptide would express specific biological activity. Findings A 12-mer phage display library was screened with a GalNAc-specific lectin to identify an amino acid sequence that binds to the lectin. Phage particles that were eluted from the lectin with free GalNAc were considered to have been bound to a GalNAc-binding site. Peptides were synthesized with the selected sequence as a quadravalent structure to facilitate receptor crosslinking. Treatment of human peripheral blood mononuclear cells for 24 h with the peptide stimulated secretion of interleukin-8 (IL-8) but not of IL-1β, IL-6, IL-10, or tumor necrosis factor-α (TNF-α). The secretion of IL-21 was stimulated as strongly with the peptide as with interferon-γ. Conclusion The data indicate that the quadravalent peptide has biological activity with a degree of specificity. These effects occurred at concentrations in the nanomolar range, in contrast to free sugars that generally bind to proteins in the micro- to millimolar range. PMID:19284521

  13. Biologically Active and Antimicrobial Peptides from Plants

    PubMed Central

    Salas, Carlos E.; Badillo-Corona, Jesus A.; Ramírez-Sotelo, Guadalupe; Oliver-Salvador, Carmen

    2015-01-01

    Bioactive peptides are part of an innate response elicited by most living forms. In plants, they are produced ubiquitously in roots, seeds, flowers, stems, and leaves, highlighting their physiological importance. While most of the bioactive peptides produced in plants possess microbicide properties, there is evidence that they are also involved in cellular signaling. Structurally, there is an overall similarity when comparing them with those derived from animal or insect sources. The biological action of bioactive peptides initiates with the binding to the target membrane followed in most cases by membrane permeabilization and rupture. Here we present an overview of what is currently known about bioactive peptides from plants, focusing on their antimicrobial activity and their role in the plant signaling network and offering perspectives on their potential application. PMID:25815307

  14. Biologically active and antimicrobial peptides from plants.

    PubMed

    Salas, Carlos E; Badillo-Corona, Jesus A; Ramírez-Sotelo, Guadalupe; Oliver-Salvador, Carmen

    2015-01-01

    Bioactive peptides are part of an innate response elicited by most living forms. In plants, they are produced ubiquitously in roots, seeds, flowers, stems, and leaves, highlighting their physiological importance. While most of the bioactive peptides produced in plants possess microbicide properties, there is evidence that they are also involved in cellular signaling. Structurally, there is an overall similarity when comparing them with those derived from animal or insect sources. The biological action of bioactive peptides initiates with the binding to the target membrane followed in most cases by membrane permeabilization and rupture. Here we present an overview of what is currently known about bioactive peptides from plants, focusing on their antimicrobial activity and their role in the plant signaling network and offering perspectives on their potential application.

  15. Site-selective chemical cleavage of peptide bonds.

    PubMed

    Elashal, Hader E; Raj, Monika

    2016-05-01

    Site-selective cleavage of extremely unreactive peptide bonds is a very important chemical modification that provides invaluable information regarding protein sequence, and it acts as a modulator of protein structure and function for therapeutic applications. For controlled and selective cleavage, a daunting task, chemical reagents must selectively recognize or bind to one or more amino acid residues in the peptide chain and selectively cleave a peptide bond. Building on this principle, we have developed an approach that utilizes a chemical reagent to selectively modify the serine residue in a peptide chain and leads to the cleavage of a peptide backbone at the N-terminus of the serine residue. After cleavage, modified residues can be converted back to the original fragments. This method exhibits broad substrate scope and selectively cleaves various bioactive peptides with post-translational modifications (e.g. N-acetylation and -methylation) and mutations (d- and β-amino acids), which are a known cause of age related diseases.

  16. The peptide agonist-binding site of the glucagon-like peptide-1 (GLP-1) receptor based on site-directed mutagenesis and knowledge-based modelling

    PubMed Central

    Dods, Rachel L.; Donnelly, Dan

    2015-01-01

    Glucagon-like peptide-1 (7–36)amide (GLP-1) plays a central role in regulating blood sugar levels and its receptor, GLP-1R, is a target for anti-diabetic agents such as the peptide agonist drugs exenatide and liraglutide. In order to understand the molecular nature of the peptide–receptor interaction, we used site-directed mutagenesis and pharmacological profiling to highlight nine sites as being important for peptide agonist binding and/or activation. Using a knowledge-based approach, we constructed a 3D model of agonist-bound GLP-1R, basing the conformation of the N-terminal region on that of the receptor-bound NMR structure of the related peptide pituitary adenylate cyclase-activating protein (PACAP21). The relative position of the extracellular to the transmembrane (TM) domain, as well as the molecular details of the agonist-binding site itself, were found to be different from the model that was published alongside the crystal structure of the TM domain of the glucagon receptor, but were nevertheless more compatible with published mutagenesis data. Furthermore, the NMR-determined structure of a high-potency cyclic conformationally-constrained 11-residue analogue of GLP-1 was also docked into the receptor-binding site. Despite having a different main chain conformation to that seen in the PACAP21 structure, four conserved residues (equivalent to His-7, Glu-9, Ser-14 and Asp-15 in GLP-1) could be structurally aligned and made similar interactions with the receptor as their equivalents in the GLP-1-docked model, suggesting the basis of a pharmacophore for GLP-1R peptide agonists. In this way, the model not only explains current mutagenesis and molecular pharmacological data but also provides a basis for further experimental design. PMID:26598711

  17. [Biologically Active Peptides of King Crab Hepatopancreas].

    PubMed

    Bogdanov, V V; Berezin, B B; Il'ina, A P; Yamskova, V P; Yamskov, I A

    2015-01-01

    Substances of a peptide nature isolated from the hepatopancreas of the king crab Paralithodes camtschaticus exhibited physicochemical properties and membranotropic and specific activities similar to those of membranotropic homeostatic tissue-specific bioregulators previously found in different mammalian and plant tissues. Their biological effect on vertebrate tissues was demonstrated on a model of roller organotypic cultivation of Pleurodeles waltl newt liver tissue. PMID:26353409

  18. Glucagon-like peptides activate hepatic gluconeogenesis.

    PubMed

    Mommsen, T P; Andrews, P C; Plisetskaya, E M

    1987-07-13

    Piscine (anglerfish, catfish, coho salmon) glucagon-like peptides (GLPs), applied at 3.5 nM, stimulate (1.1-1.9-fold) flux through gluconeogenesis above control levels in isolated trout and salmon hepatocytes. Human GLP-1 and GLP-2 also activate gluconeogenesis, but to a lesser degree than their piscine counterparts. Minor increases of substrate oxidation are noticed at times of peak gluconeogenic activation through GLPs. These hormones, which are derived from the same precursor peptide as glucagon are more potent activators of gluconeogenesis than glucagon when applied at equimolar concentrations, and do not appear to employ cAMP or cGMP as the intracellular messenger in hepatic tissue. PMID:3109952

  19. Peptides and proteins with antimicrobial activity.

    PubMed

    Coutinho, Henrique Douglas Melo; Lôbo, Katiuscia Menezes; Bezerra, Denise Aline Casimiro; Lôbo, Inalzuir

    2008-01-01

    The increase of microbial resistance to antibiotics has led to a continuing search for newer and more effective drugs. Antimicrobial peptides are generally found in animals, plants, and microorganisms and are of great interest to medicine, pharmacology, and the food industry. These peptides are capable of inhibiting pathogenic microorganisms. They can attack parasites, while causing little or no harm to the host cells. The defensins are peptides found in granules in the polymorphonuclear neutrophils (PMNs) and are responsible for the defense of the organism. Several animal defensins, like dermaseptin, antileukoprotease, protegrin, and others, have had their activities and efficacy tested and been shown to be effective against bacteria, fungi, and protists; there are also specific defensins from invertebrates, e.g., drosomycin and heliomicin; from plants, e.g., the types A and B; and the bacteriocins, e.g., acrocin, marcescin, etc. The aim of the present work was to compile a comprehensive bibliographic review of the diverse potentially antimicrobial peptides in an effort to systematize the current knowledge on these substances as a contribution for further researches. The currently available bibliography does not give a holistic approach on this subject. The present work intends to show that the mechanism of defense represented by defensins is promising from the perspective of its application in the treatment of infectious diseases in human, animals and plants.

  20. Peptide Bacteriocins--Structure Activity Relationships.

    PubMed

    Etayash, Hashem; Azmi, Sarfuddin; Dangeti, Ramana; Kaur, Kamaljit

    2015-01-01

    With the growing concerns in the scientific and health communities over increasing levels of antibiotic resistance, antimicrobial peptide bacteriocins have emerged as promising alternatives to conventional small molecule antibiotics. A substantial attention has recently focused on the utilization of bacteriocins in food preservation and health safety. Despite the fact that a large number of bacteriocins have been reported, only a few have been fully characterized and structurally elucidated. Since knowledge of the molecular structure is a key for understanding the mechanism of action and therapeutic effects of peptide, we centered our focus in this review on the structure-activity relationships of bacteriocins with a particular focus in seven bacteriocins, namely, nisin, microcin J25, microcin B17, microcin C, leucocin A, sakacin P, and pediocin PA-1. Significant structural changes responsible for the altered activity of the recent bacteriocin analogues are discussed here. PMID:26265354

  1. Catalytic Activities Of [GADV]-Peptides

    NASA Astrophysics Data System (ADS)

    Oba, Takae; Fukushima, Jun; Maruyama, Masako; Iwamoto, Ryoko; Ikehara, Kenji

    2005-10-01

    We have previously postulated a novel hypothesis for the origin of life, assuming that life on the earth originated from “[GADV]-protein world”, not from the “RNA world” (see Ikehara's review, 2002). The [GADV]-protein world is constituted from peptides and proteins with random sequences of four amino acids (glycine [G], alanine [A], aspartic acid [D] and valine [V]), which accumulated by pseudo-replication of the [GADV]-proteins. To obtain evidence for the hypothesis, we produced [GADV]-peptides by repeated heat-drying of the amino acids for 30 cycles ([GADV]-P30) and examined whether the peptides have some catalytic activities or not. From the results, it was found that the [GADV]-P30 can hydrolyze several kinds of chemical bonds in molecules, such as umbelliferyl-β-D-galactoside, glycine-p-nitroanilide and bovine serum albumin. This suggests that [GADV]-P30 could play an important role in the accumulation of [GADV]-proteins through pseudo-replication, leading to the emergence of life. We further show that [GADV]-octapaptides with random sequences, but containing no cyclic compounds as diketepiperazines, have catalytic activity, hydrolyzing peptide bonds in a natural protein, bovine serum albumin. The catalytic activity of the octapeptides was much higher than the [GADV]-P30 produced through repeated heat-drying treatments. These results also support the [GADV]-protein-world hypothesis of the origin of life (see Ikehara's review, 2002). Possible steps for the emergence of life on the primitive earth are presented.

  2. Elastase and suppressor active peptide activity following burn injury.

    PubMed

    Ozkan, A N; Pinney, E; Hoyt, D B; Ninnemann, J; Hansbrough, J

    1988-02-01

    Proteolytic enzyme activity following trauma and inflammation plays a key role in the pathophysiology of injury. The precise mechanisms involved in the induction of protease release has not been determined. We show here that sera from burn patients with greater than 40% TBSA have significantly elevated levels of active elastase which correspond with significantly increased levels of suppressor active peptide (SAP) and suppression of neutrophil chemotaxis. The elastase inhibitory capacity of serum from burned or blunt trauma patients was within normal range, suggesting that the primary elastase inhibitor, alpha-1-proteinase inhibitor, is functionally active. Additionally, granulocytes exposed to suppressor active peptide in vitro resulted in a markedly elevated release of elastase into the culture supernatants. These data suggest that the suppressor peptide is capable of not only suppressing immune function but is also a potent mediator for the induction of proteolytic enzyme release from leukocytes.

  3. Antiendotoxin activity of cationic peptide antimicrobial agents.

    PubMed Central

    Gough, M; Hancock, R E; Kelly, N M

    1996-01-01

    The endotoxin from gram-negative bacteria consists of a molecule lipopolysaccharide (LPS) which can be shed by bacteria during antimicrobial therapy. A resulting syndrome, endotoxic shock, is a leading cause of death in the developed world. Thus, there is great interest in the development of antimicrobial agents which can reverse rather than promote sepsis, especially given the recent disappointing clinical performance of antiendotoxin therapies. We describe here two small cationic peptides, MBI-27 and MBI-28, which have both antiendotoxic and antibacterial activities in vitro and in vivo in animal models. We had previously demonstrated that these peptides bind to LPS with an affinity equivalent to that of polymyxin B. Consistent with this, the peptides blocked the ability of LPS and intact cells to induce the endotoxic shock mediator, tumor necrosis factor (TNF), upon incubation with the RAW 264.7 murine macrophage cell line. MBI-28 was equivalent to polymyxin B in its ability to block LPS induction of TNF by this cell line, even when added 60 min after the TNF stimulus. Furthermore, MBI-28 offered significant protection in a galactosamine-sensitized mouse model of lethal endotoxic shock. This protection correlated with the ability of MBI-28 to reduce LPS-induced circulating TNF by nearly 90% in this mouse model. Both MBI-27 and MBI-28 demonstrated antibacterial activity against gram-negative bacteria in vitro and in vivo against Pseudomonas aeruginosa infections in neutropenic mice. PMID:8945527

  4. Prediction of Antimicrobial Activity of Synthetic Peptides by a Decision Tree Model

    PubMed Central

    Lira, Felipe; Perez, Pedro S.; Baranauskas, José A.

    2013-01-01

    Antimicrobial resistance is a persistent problem in the public health sphere. However, recent attempts to find effective substitutes to combat infections have been directed at identifying natural antimicrobial peptides in order to circumvent resistance to commercial antibiotics. This study describes the development of synthetic peptides with antimicrobial activity, created in silico by site-directed mutation modeling using wild-type peptides as scaffolds for these mutations. Fragments of antimicrobial peptides were used for modeling with molecular modeling computational tools. To analyze these peptides, a decision tree model, which indicated the action range of peptides on the types of microorganisms on which they can exercise biological activity, was created. The decision tree model was processed using physicochemistry properties from known antimicrobial peptides available at the Antimicrobial Peptide Database (APD). The two most promising peptides were synthesized, and antimicrobial assays showed inhibitory activity against Gram-positive and Gram-negative bacteria. Colossomin C and colossomin D were the most inhibitory peptides at 5 μg/ml against Staphylococcus aureus and Escherichia coli. The methods described in this work and the results obtained are useful for the identification and development of new compounds with antimicrobial activity through the use of computational tools. PMID:23455341

  5. Interaction of the protein C activation peptide with platelets.

    PubMed

    Martínez-Cruz, Ruth; Canseco, María Del S Pina; Lopez-Martínez, Jael; Cruz, Pedro A Hernández; Pérez-Campos, Eduardo; Cruz, Margarito Martínez; Alva, Félix Córdoba; Majluf-Cruz, Abraham; Zenteno, Edgar; Ruiz-Argüelles, Alejandro

    2007-01-01

    The peptide NH(2)-DTEDQEDQVDPR-COOH is released during activation of protein C zymogen. We measured the effect of a synthetic peptide with an amino acid sequence similar to that of the natural peptide on platelets from healthy individuals using platelet aggregometry. We found that this synthetic peptide inhibits platelet aggregation induced by thrombin; furthermore, it diminishes mobilization of intraplatelet calcium. Molecular docking showed weak interaction between the synthetic peptide and thrombin. Our findings suggest that this synthetic peptide may interact with a receptor located on the platelet cell membrane.

  6. Copper(II) complexes of terminally free alloferon peptide mutants containing two different histidyl (H(1) and H(6) or H(9) or H(12)) binding sites Structure Stability and Biological Activity.

    PubMed

    Matusiak, Agnieszka; Kuczer, Mariola; Czarniewska, Elżbieta; Urbański, Arkadiusz; Rosiński, Grzegorz; Kowalik-Jankowska, Teresa

    2015-10-01

    Mono- and dinuclear copper(II) complexes of the alloferon 1 with point mutations H9A/H12A H(1)GVSGH(6)GQA(9)GVA(12)G, H6A/H12A H(1)GVSGA(6)GQH(9)GVA(12)G and H6A/H9A H(1)GVSGA(6)GQA(9)GVH(12)G have been studied by potentiometric, UV-visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at metal-to-ligand molar ratios 1:1 and 2:1 was obtained. For all systems studied in the 5 - 6.5 pH range, the CuL complex dominates with 3N{NH2,NIm-H(1),NIm-H(6 or 9 or 12)} binding site. The stability of the CuL complexes for the ligands studied varies according to the H9A/H12A>H6A/H12A>H6A/H9A series. For the dinuclear systems the amine/imidazole nitrogen donor atoms of the histidine residue H(1) and the imidazole nitrogen atoms of H(6) or H(9) or H(12) can be considered as independent metal-binding sites in the species formed. The stability of the dinuclear complexes is higher when two coordinated copper(II) ions are closer to each other. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 have been studied. The H6A/H9A, H6A/H12A peptides displayed lower hemocytotoxic activity compared to that of alloferon 1, while the H9A/H12A analogue was not active. Among the copper(II) complexes, the most active was the Cu(II)-H9A/H12A complex formed at pH7.4 with 3N{NH2,NIm-H(1),NIm-H(6)} (CuL) and 3N{NH2,N(-),NIm-H(6)} and/or 4N{NH2,NIm-H(1),N(-),NIm-H(6)} (CuH-1L) binding sites. The Cu(II)-H6A/H9A and Cu(II)-H6A/H12A complexes were not active.

  7. Copper(II) complexes of terminally free alloferon peptide mutants containing two different histidyl (H(1) and H(6) or H(9) or H(12)) binding sites Structure Stability and Biological Activity.

    PubMed

    Matusiak, Agnieszka; Kuczer, Mariola; Czarniewska, Elżbieta; Urbański, Arkadiusz; Rosiński, Grzegorz; Kowalik-Jankowska, Teresa

    2015-10-01

    Mono- and dinuclear copper(II) complexes of the alloferon 1 with point mutations H9A/H12A H(1)GVSGH(6)GQA(9)GVA(12)G, H6A/H12A H(1)GVSGA(6)GQH(9)GVA(12)G and H6A/H9A H(1)GVSGA(6)GQA(9)GVH(12)G have been studied by potentiometric, UV-visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at metal-to-ligand molar ratios 1:1 and 2:1 was obtained. For all systems studied in the 5 - 6.5 pH range, the CuL complex dominates with 3N{NH2,NIm-H(1),NIm-H(6 or 9 or 12)} binding site. The stability of the CuL complexes for the ligands studied varies according to the H9A/H12A>H6A/H12A>H6A/H9A series. For the dinuclear systems the amine/imidazole nitrogen donor atoms of the histidine residue H(1) and the imidazole nitrogen atoms of H(6) or H(9) or H(12) can be considered as independent metal-binding sites in the species formed. The stability of the dinuclear complexes is higher when two coordinated copper(II) ions are closer to each other. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 have been studied. The H6A/H9A, H6A/H12A peptides displayed lower hemocytotoxic activity compared to that of alloferon 1, while the H9A/H12A analogue was not active. Among the copper(II) complexes, the most active was the Cu(II)-H9A/H12A complex formed at pH7.4 with 3N{NH2,NIm-H(1),NIm-H(6)} (CuL) and 3N{NH2,N(-),NIm-H(6)} and/or 4N{NH2,NIm-H(1),N(-),NIm-H(6)} (CuH-1L) binding sites. The Cu(II)-H6A/H9A and Cu(II)-H6A/H12A complexes were not active. PMID:26184757

  8. Role of peptide bond in the realization of biological activity of short peptides.

    PubMed

    Khavinson, V Kh; Tarnovskaya, S I; Lin'kova, N S; Chervyakova, N A; Nichik, T E; Elashkina, E V; Chalisova, N I

    2015-02-01

    We performed a comparative analysis of biological activity of Lys-Glu peptide and its amino acid constituents. It was established that Lys-Glu stimulated proliferation of splenic cells in organotypic culture, while the mixture of glutamic acid and lysine inhibited culture growth. Using the method of molecular docking, we showed that glutamic acid, lysine, and Lys-Glu peptide can interact with different DNA sequences. The energy of interaction and the most beneficial localization of glutamic acid, lysine, and Lys-Glu peptide in DNA molecule was calculated. We demonstrated the interaction of the peptide and amino acids with DNA along the minor groove. The energy of DNA interaction with the peptide is higher than with individual amino acids. The peptide bonds increase the interaction of Lys-Glu peptide with DNA, which potentiates the biological effect on cell proliferation in organotypic culture of splenic cells.

  9. Miniaturizing VEGF: Peptides mimicking the discontinuous VEGF receptor-binding site modulate the angiogenic response

    PubMed Central

    De Rosa, Lucia; Finetti, Federica; Diana, Donatella; Di Stasi, Rossella; Auriemma, Sara; Romanelli, Alessandra; Fattorusso, Roberto; Ziche, Marina; Morbidelli, Lucia; D’Andrea, Luca Domenico

    2016-01-01

    The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor. PMID:27498819

  10. Miniaturizing VEGF: Peptides mimicking the discontinuous VEGF receptor-binding site modulate the angiogenic response

    NASA Astrophysics Data System (ADS)

    De Rosa, Lucia; Finetti, Federica; Diana, Donatella; di Stasi, Rossella; Auriemma, Sara; Romanelli, Alessandra; Fattorusso, Roberto; Ziche, Marina; Morbidelli, Lucia; D’Andrea, Luca Domenico

    2016-08-01

    The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor.

  11. Miniaturizing VEGF: Peptides mimicking the discontinuous VEGF receptor-binding site modulate the angiogenic response.

    PubMed

    De Rosa, Lucia; Finetti, Federica; Diana, Donatella; Di Stasi, Rossella; Auriemma, Sara; Romanelli, Alessandra; Fattorusso, Roberto; Ziche, Marina; Morbidelli, Lucia; D'Andrea, Luca Domenico

    2016-01-01

    The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor. PMID:27498819

  12. Miniaturizing VEGF: Peptides mimicking the discontinuous VEGF receptor-binding site modulate the angiogenic response.

    PubMed

    De Rosa, Lucia; Finetti, Federica; Diana, Donatella; Di Stasi, Rossella; Auriemma, Sara; Romanelli, Alessandra; Fattorusso, Roberto; Ziche, Marina; Morbidelli, Lucia; D'Andrea, Luca Domenico

    2016-08-08

    The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor.

  13. Site-directed isotope labeling and ATR-FTIR difference spectroscopy of bacteriorhodopsin: the peptide carbonyl group of Tyr 185 is structurally active during the bR-->N transition.

    PubMed

    Ludlam, C F; Sonar, S; Lee, C P; Coleman, M; Herzfeld, J; RajBhandary, U L; Rothschild, K J

    1995-01-10

    The largest secondary structural change occurs in the bacteriorhodopsin (bR) photocycle during the M-->N transition. In this work site-directed isotope labeling (SDIL) and attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy were used to investigate this conformational change. L-Tyrosine containing a 13C isotope at the carbonyl carbon was selectively incorporated at Tyr 57, Tyr 147, and Tyr 185 by SDIL. This involves the cell-free expression of bR in the presence of Escherichia coli suppressor tRNA(CUATyr) aminoacylated with L-[1-13C]Tyr. ATR-FTIR difference spectroscopy reveals that of the 11 tyrosines, only the peptide carbonyl group of Tyr 185 undergoes a significant structural change during the bR-->N transition. Along with other spectroscopic evidence, this result suggests that the Tyr 185-Pro 186 region of the protein is structurally active and may function as a hinge which facilitates the tilt of the cytoplasmic portion of the F-helix in bacteriorhodopsin during the M-->N transition.

  14. Activity of Novel Synthetic Peptides against Candida albicans.

    PubMed

    Lum, Kah Yean; Tay, Sun Tee; Le, Cheng Foh; Lee, Vannajan Sanghiran; Sabri, Nadia Hanim; Velayuthan, Rukumani Devi; Hassan, Hamimah; Sekaran, Shamala Devi

    2015-01-01

    Candida spp. are the most common causes of fungal infections worldwide. Among the Candida species, Candida albicans remains the predominant species that causes invasive candidiasis in most countries. In this study, we used two peptides, KABT-AMP and uperin 3.6 as templates to develop novel antifungal peptides. Their anticandidal activity was assessed using a combination of MIC, time-killing assay and biofilm reduction assay. Hybrid peptides, KU2 and KU3 containing a mixed backbone of KABT-AMP and Uperin 3.6 demonstrated the most potent anticandidal activity with MIC values ranging from 8-16 mg/L. The number of Trp residues and the amphipathic structure of peptides probably enhanced the anticandidal activity of peptides. Increasing the cationicity of the uperin 3.6 analogues resulted in reduced MIC from the range of 64-128 mg/L to 16-64 mg/L and this was also correlated with the antibiofilm activity and killing kinetics of the peptides. Peptides showed synergistic effects when used in combination with conventional antifungals. Peptides demonstrated low haemolytic activity but significant toxicity on two normal human epithelial cell lines. This study provides us with a better understanding on the structure-activity relationship and the balance between cationicity and hydrophobicity of the peptides although the therapeutic application of the peptides is limited. PMID:25965506

  15. The latest developments in synthetic peptides with immunoregulatory activities.

    PubMed

    Zhou, Chun-lei; Lu, Rong; Lin, Gang; Yao, Zhi

    2011-02-01

    In the past few years, many researches have provided us with much data demonstrating the abilities of synthetic peptides to impact immune response in vitro and in vivo. These peptides were designed according to the structure of some important protein molecules which play a key role in immune response, so they act with specific targets. The class I and II MHC-derived peptides inhibit the TCR recognition of antigen peptide-MHC complex. Rationally designed CD80 and CD154-binding peptides block the interaction between cell surface costimulatory molecules on antigen-presenting cells (APCs) and T cells. Some peptides were designed to inhibit the activities of cell signal proteins, including JNK, NF-κB and NFAT. Some peptide antagonists competitively bind to important cytokines and inhibit their activities, such as TNF-α, TGF-β and IL-1β inhibitory peptides. Adhesion molecule ICAM-1 derived peptides block the T cell adhesion and activation. These immunoregulatory peptides showed therapeutic effect in several animal models, including collagen-induced arthritis (CIA), autoimmune cystitis model, murine skin transplant model and cardiac allograft model. These results give us important implications for the development of a novel therapy for immune mediated diseases. PMID:20979984

  16. Fatty acid conjugation enhances the activities of antimicrobial peptides.

    PubMed

    Li, Zhining; Yuan, Penghui; Xing, Meng; He, Zhumei; Dong, Chuanfu; Cao, Yongchang; Liu, Qiuyun

    2013-04-01

    Antimicrobial peptides are small molecules that play a crucial role in innate immunity in multi-cellular organisms, and usually expressed and secreted constantly at basal levels to prevent infection, but local production can be augmented upon an infection. The clock is ticking as rising antibiotic abuse has led to the emergence of many drug resistance bacteria. Due to their broad spectrum antibiotic and antifungal activities as well as anti-viral and anti-tumor activities, efforts are being made to develop antimicrobial peptides into future microbial agents. This article describes some of the recent patents on antimicrobial peptides with fatty acid conjugation. Potency and selectivity of antimicrobial peptide can be modulated with fatty acid tails of variable length. Interaction between membranes and antimicrobial peptides was affected by fatty acid conjugation. At concentrations above the critical miscelle concentration (CMC), propensity of solution selfassembly hampered binding of the peptide to cell membranes. Overall, fatty acid conjugation has enhanced the activities of antimicrobial peptides, and occasionally it rendered inactive antimicrobial peptides to be bioactive. Antimicrobial peptides can not only be used as medicine but also as food additives.

  17. Folding and activity of hybrid sequence, disulfide-stabilized peptides

    SciTech Connect

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

    1990-08-01

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

  18. Membrane-active peptides from marine organisms--antimicrobials, cell-penetrating peptides and peptide toxins: applications and prospects.

    PubMed

    Ponnappan, Nisha; Budagavi, Deepthi Poornima; Yadav, Bhoopesh Kumar; Chugh, Archana

    2015-03-01

    Marine organisms are known to be a rich and unique source of bioactive compounds as they are exposed to extreme conditions in the oceans. The present study is an attempt to briefly describe some of the important membrane-active peptides (MAPs) such as antimicrobial peptides (AMPs), cell-penetrating peptides (CPPs) and peptide toxins from marine organisms. Since both AMPs and CPPs play a role in membrane perturbation and exhibit interchangeable role, they can speculatively fall under the broad umbrella of MAPs. The study focuses on the structural and functional characteristics of different classes of marine MAPs. Further, AMPs are considered as a potential remedy to antibiotic resistance acquired by several pathogens. Peptides from marine organisms show novel post-translational modifications such as cysteine knots, halogenation and histidino-alanine bridge that enable these peptides to withstand harsh marine environmental conditions. These unusual modifications of AMPs from marine organisms are expected to increase their half-life in living systems, contributing to their increased bioavailability and stability when administered as drug in in vivo systems. Apart from AMPs, marine toxins with membrane-perturbing properties could be essentially investigated for their cytotoxic effect on various pathogens and their cell-penetrating activity across various mammalian cells. The current review will help in identifying the MAPs from marine organisms with crucial post-translational modifications that can be used as template for designing novel therapeutic agents and drug-delivery vehicles for treatment of human diseases.

  19. Screening of Pre-miRNA-155 Binding Peptides for Apoptosis Inducing Activity Using Peptide Microarrays.

    PubMed

    Pai, Jaeyoung; Hyun, Soonsil; Hyun, Ji Young; Park, Seong-Hyun; Kim, Won-Je; Bae, Sung-Hun; Kim, Nak-Kyoon; Yu, Jaehoon; Shin, Injae

    2016-01-27

    MicroRNA-155, one of the most potent miRNAs that suppress apoptosis in human cancer, is overexpressed in numerous cancers, and it displays oncogenic activity. Peptide microarrays, constructed by immobilizing 185 peptides containing the C-terminal hydrazide onto epoxide-derivatized glass slides, were employed to evaluate peptide binding properties of pre-miRNA-155 and to identify its binding peptides. Two peptides, which were identified based on the results of peptide microarray and in vitro Dicer inhibition studies, were found to inhibit generation of mature miRNA-155 catalyzed by Dicer and to enhance expression of miRNA-155 target genes in cells. In addition, the results of cell experiments indicate that peptide inhibitors promote apoptotic cell death via a caspase-dependent pathway. Finally, observations made in NMR and molecular modeling studies suggest that a peptide inhibitor preferentially binds to the upper bulge and apical stem-loop region of pre-miRNA-155, thereby suppressing Dicer-mediated miRNA-155 processing. PMID:26771315

  20. Review: Production and functionality of active peptides from milk.

    PubMed

    Muro Urista, C; Álvarez Fernández, R; Riera Rodriguez, F; Arana Cuenca, A; Téllez Jurado, A

    2011-08-01

    In recent years, research on the production of active peptides obtained from milk and their potential functionality has grown, to a great extent. Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions or conditions, and they may ultimately have an influence on health. Individual proteins of casein or milk-derived products such as cheese and yogurt have been used as a protein source to study the isolation and activity of peptides with several applications. Currently, the milk whey waste obtained in the production of cheese also represents a protein source from which active peptides could be isolated with potential industrial applications. The active properties of milk peptides and the results found with regard to their physiological effects have led to the classification of peptides as belonging to the group of ingredients of protein nature, appropriate for use in functional foods or pharmaceutical formulations. In this study, the main peptides obtained from milk protein and the past research studies about its production and biological activities will be explained. Second, an analysis will be made on the methods to determinate the biological activities, the separation of bioactive peptides and its structure identification. All of these form the base required to obtain synthetic peptides. Finally, we explain the experimental animal and human trials done in the past years. Nevertheless, more research is required on the design and implementation of equipment for the industrial production and separation of peptides. In addition, different authors suggest that more emphasis should therefore be given to preclinical studies, proving that results are consistent and that effects are demonstrated repeatedly by several research human groups.

  1. Self-assembly of cationic multidomain peptide hydrogels: supramolecular nanostructure and rheological properties dictate antimicrobial activity.

    PubMed

    Jiang, Linhai; Xu, Dawei; Sellati, Timothy J; Dong, He

    2015-12-01

    Hydrogels are an important class of biomaterials that have been widely utilized for a variety of biomedical/medical applications. The biological performance of hydrogels, particularly those used as wound dressing could be greatly advanced if imbued with inherent antimicrobial activity capable of staving off colonization of the wound site by opportunistic bacterial pathogens. Possessing such antimicrobial properties would also protect the hydrogel itself from being adversely affected by microbial attachment to its surface. We have previously demonstrated the broad-spectrum antimicrobial activity of supramolecular assemblies of cationic multi-domain peptides (MDPs) in solution. Here, we extend the 1-D soluble supramolecular assembly to 3-D hydrogels to investigate the effect of the supramolecular nanostructure and its rheological properties on the antimicrobial activity of self-assembled hydrogels. Among designed MDPs, the bactericidal activity of peptide hydrogels was found to follow an opposite trend to that in solution. Improved antimicrobial activity of self-assembled peptide hydrogels is dictated by the combined effect of supramolecular surface chemistry and storage modulus of the bulk materials, rather than the ability of individual peptides/peptide assemblies to penetrate bacterial cell membrane as observed in solution. The structure-property-activity relationship developed through this study will provide important guidelines for designing biocompatible peptide hydrogels with built-in antimicrobial activity for various biomedical applications.

  2. Rapid Identification of Protein Kinase Phosphorylation Site Motifs Using Combinatorial Peptide Libraries.

    PubMed

    Miller, Chad J; Turk, Benjamin E

    2016-01-01

    Eukaryotic protein kinases phosphorylate substrates at serine, threonine, and tyrosine residues that fall within the context of short sequence motifs. Knowing the phosphorylation site motif for a protein kinase facilitates designing substrates for kinase assays and mapping phosphorylation sites in protein substrates. Here, we describe an arrayed peptide library protocol for rapidly determining kinase phosphorylation consensus sequences. This method uses a set of peptide mixtures in which each of the 20 amino acid residues is systematically substituted at nine positions surrounding a central site of phosphorylation. Peptide mixtures are arrayed in multiwell plates and analyzed by radiolabel assay with the kinase of interest. The preferred sequence is determined from the relative rate of phosphorylation of each peptide in the array. Consensus peptides based on these sequences typically serve as efficient and specific kinase substrates for high-throughput screening or incorporation into biosensors.

  3. Antimicrobial activity of cationic peptides in endodontic procedures

    PubMed Central

    Winfred, Sofi Beaula; Meiyazagan, Gowri; Panda, Jiban J.; Nagendrababu, Venkateshbabu; Deivanayagam, Kandaswamy; Chauhan, Virander S.; Venkatraman, Ganesh

    2014-01-01

    Objectives: The present study aimed to investigate the antimicrobial and biofilm inhibition activity of synthetic antimicrobial peptides (AMPs) against microbes such as Enterococcus faecalis, Staphylococcus aureus, and Candida albicans which are involved in endodontic infections. Materials and Methods: Agar diffusion test was done to determine the activity of peptides. The morphological changes in E. faecalis and reduction in biofilm formation after treatment with peptides were observed using scanning electron microscope. The efficacy of peptides using an ex vivo dentinal model was determined by polymerase chain reaction and confocal laser scanning microscopy. Platelet aggregation was done to determine the biocompatibility of peptides. Results: Among 11 peptides, two of the amphipathic cationic peptides were found to be highly active against E. faecalis, S. aureus, C. albicans. Efficacy results using dentinal tubule model showed significant reduction in microbial load at 400 μm depth. The peptides were also biocompatible. Conclusion: These results suggest that synthetic AMPs have the potential to be developed as antibacterial agents against microorganisms involved in dental infections and thus could prevent the spread and persistence of endodontic infections improving treatment outcomes and teeth preservation. PMID:24966779

  4. Peptides of the Constant Region of Antibodies Display Fungicidal Activity

    PubMed Central

    Polonelli, Luciano; Ciociola, Tecla; Magliani, Walter; Zanello, Pier Paolo; D'Adda, Tiziana; Galati, Serena; De Bernardis, Flavia; Arancia, Silvia; Gabrielli, Elena; Pericolini, Eva; Vecchiarelli, Anna; Arruda, Denise C.; Pinto, Marcia R.; Travassos, Luiz R.; Pertinhez, Thelma A.; Spisni, Alberto; Conti, Stefania

    2012-01-01

    Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents. PMID:22470523

  5. Atrial Natriuretic Peptide Inhibits Spontaneous Contractile Activity of Lymph Nodes.

    PubMed

    Lobov, G I; Pan'kova, M N

    2016-06-01

    Atrial natriuretic peptide dose-dependently inhibited spontaneous phase and tonic activity of smooth muscle strips from the capsule of isolated bovine mesenteric lymph nodes. Pretreatment with L-NAME, diclofenac, and methylene blue had practically no effect on the peptide-induced relaxation responses. In contrast, glibenclamide significantly reduced the inhibitory effect of atrial natriuretic peptide. We suppose that the NO-dependent and cyclooxygenase signaling pathways are not involved in implementation of the inhibitory effects of atrial natriuretic peptide. ATP-sensitive K(+)-channels of the smooth muscle cell membrane are the last component in the signaling pathway leading to relaxation of smooth muscles of the lymph node capsule caused by atrial natriuretic peptide; activation of these channels leads to membrane hyperpolarization and smooth muscle relaxation. PMID:27383173

  6. Antioxidant activities of a peptide derived from chicken dark meat.

    PubMed

    Fukada, Yoko; Mizutani, Saki; Nomura, Sarika; Hara, Wakana; Matsui, Riko; Nagai, Kumiko; Murakami, Yuki; Washio, Nanami; Ikemoto, Narumi; Terashima, Masaaki

    2016-05-01

    Antioxidant activities against hypochlorite ions and peroxyl radicals of a chicken dark meat hydrolysate digested with pepsin were examined with the myoglobin method based on the structure change of myoglobin due to redox reaction with reactive oxygen species (ROS). A peptide that showed strong antioxidant activity against the peroxyl radical was isolated from the hydrolysate using HPLC equipped with a hydrophobic-interacting column. The sequence of the first five amino acid residues of the peptide was determined as YASGR (Tyr-Ala-Ser-Gly-Arg), and this sequence matched with the amino acid residues 143-147 of chicken β-actin (GenBank: CAA25004.1). The synthetic peptide YASGR showed very high antioxidant activity against the peroxyl radical. Antioxidant activities of the free amino acids, confirmed that the tyrosine residue of this peptide was possibly responsible for antioxidant activity. PMID:27407214

  7. Antimicrobial peptides: a review of how peptide structure impacts antimicrobial activity

    NASA Astrophysics Data System (ADS)

    Soares, Jason W.; Mello, Charlene M.

    2004-03-01

    Antimicrobial peptides (AMPs) have been discovered in insects, mammals, reptiles, and plants to protect against microbial infection. Many of these peptides have been isolated and studied exhaustively to decipher the molecular mechanisms that impart protection against infectious bacteria, fungi, and viruses. Unfortunately, the molecular mechanisms are still being debated within the scientific community but valuable clues have been obtained through structure/function relationship studies1. Biophysical studies have revealed that cecropins, isolated from insects and pigs, exhibit random structure in solution but undergo a conformational change to an amphipathic α-helix upon interaction with a membrane surface2. The lack of secondary structure in solution results in an extremely durable peptide able to survive exposure to high temperatures, organic solvents and incorporation into fibers and films without compromising antibacterial activity. Studies to better understand the antimicrobial action of cecropins and other AMPs have provided insight into the importance of peptide sequence and structure in antimicrobial activities. Therefore, enhancing our knowledge of how peptide structure imparts function may result in customized peptide sequences tailored for specific applications such as targeted cell delivery systems, novel antibiotics and food preservation additives. This review will summarize the current state of knowledge with respect to cell binding and antimicrobial activity of AMPs focusing primarily upon cecropins.

  8. Concise site-specific synthesis of DTPA-peptide conjugates: application to imaging probes for the chemokine receptor CXCR4.

    PubMed

    Masuda, Ryo; Oishi, Shinya; Ohno, Hiroaki; Kimura, Hiroyuki; Saji, Hideo; Fujii, Nobutaka

    2011-05-15

    Diethylenetriaminepentaacetic acid (DTPA) is a useful chelating agent for radionuclides such as (68)Ga, (99m)Tc and (111)In, which are applicable to nuclear medicine imaging. In this study, we established a facile synthetic protocol for the production of mono-DTPA-conjugated peptide probes. A novel monoreactive DTPA precursor reagent was synthesized in two steps using the chemistry of the o-nitrobenzenesulfonyl (Ns) protecting group, and under mild conditions this DTPA precursor was incorporated onto an N(ε)-bromoacetylated Lys of a protected peptide resin. The site-specific DTPA conjugation was facilitated by using a highly acid-labile 4-methyltrityl (Mtt) protecting group for the target site of the bioactive peptide during the solid-phase synthesis. A combination of both techniques yielded peptides with disulfide bonds, such as octreotide and polyphemusin II-derived CXCR4 antagonists. DTPA-peptide conjugates were purified in a single step following cleavage from the resin and disulfide bond formation. This site-specific on-resin construction strategy was used for the design and synthesis of a novel In-DTPA-labeled CXCR4 antagonist, which exhibited highly potent inhibitory activity against SDF-1-CXCR4 binding.

  9. Signal peptides are allosteric activators of the protein translocase

    PubMed Central

    Gouridis, Giorgos; Karamanou, Spyridoula; Gelis, Ioannis; Kalodimos, Charalampos G.; Economou, Anastassios

    2010-01-01

    Extra-cytoplasmic polypeptides are usually synthesized as “preproteins” carrying aminoterminal, cleavable signal peptides1 and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA2,3. Most proteins destined for the periplasm and beyond are exported post-translationally by SecA2,3. Preprotein targeting to SecA is thought to involve signal peptides4 and chaperones like SecB5,6. Here we reveal that signal peptides have a novel role beyond targeting: they are essential allosteric activators of the translocase. Upon docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, “triggering” that drives the translocase to a lower activation energy state; then “trapping” that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus and, finally, “secretion” during which trapped mature domains undergo multiple turnovers of translocation in segments7. A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases. PMID:19924216

  10. PEP-SiteFinder: a tool for the blind identification of peptide binding sites on protein surfaces.

    PubMed

    Saladin, Adrien; Rey, Julien; Thévenet, Pierre; Zacharias, Martin; Moroy, Gautier; Tufféry, Pierre

    2014-07-01

    Peptide-protein interactions are important to many processes of life, particularly for signal transmission or regulatory mechanisms. When no information is known about the interaction between a protein and a peptide, it is of interest to propose candidate sites of interaction at the protein surface, to assist the design of biological experiments to probe the interaction, or to serve as a starting point for more focused in silico approaches. PEP-SiteFinder is a tool that will, given the structure of a protein and the sequence of a peptide, identify protein residues predicted to be at peptide-protein interface. PEP-SiteFinder relies on the 3D de novo generation of peptide conformations given its sequence. These conformations then undergo a fast blind rigid docking on the complete protein surface, and we have found, as the result of a benchmark over 41 complexes, that the best poses overlap to some extent the experimental patch of interaction for close to 90% complexes. In addition, PEP-SiteFinder also returns a propensity index we have found informative about the confidence of the prediction. The PEP-SiteFinder web server is available at http://bioserv.rpbs.univ-paris-diderot.fr/PEP-SiteFinder.

  11. Cys-Gly specific dipeptidase Dug1p from S. cerevisiae binds promiscuously to di-, tri-, and tetra-peptides: Peptide-protein interaction, homology modeling, and activity studies reveal a latent promiscuity in substrate recognition.

    PubMed

    Kaur, Hardeep; Datt, Manish; Ekka, Mary Krishna; Mittal, Monica; Singh, Appu Kumar; Kumaran, Sangaralingam

    2011-02-01

    Dug1p is a recently identified novel dipeptidase and plays an important role in glutathione (GSH) degradation. To understand the mechanism of its substrate recognition and specificity towards Cys-Gly dipeptides, we characterized the solution properties of Dug1p and studied the thermodynamics of Dug1p-peptide interactions. In addition, we used homology modeling and ligand docking approaches to get structural insights into Dug1p-peptide interaction. Dug1p exists as dimer and the stoichiometry of peptide-Dug1p complex is 2:1 indicating each monomer in the dimer binds to one peptide. Thermodynamic studies indicate that the free energy change for Dug1p-peptide complex formation is similar (▵G(bind) ∼ -7.0 kcal/mol) for a variety of peptides of different composition and length (22 peptides). Three-dimensional model of Dug1p is constructed and docking of peptides to the modeled structure suggests that hydrogen bonding to active site residues (E172, E171, and D137) lock the N-terminal of the peptide into the binding site. Dug1p recognizes peptides in a metal independent manner and peptide binding is not sensitive to salts (dlogK/dlog[salt] ∼ 0) over a range of [NaCl] (0.02-0.5 M), [ZnCl(2)], and [MnCl(2)] (0-0.5 mM). Our results indicate that promiscuity in peptide binding results from the locking of peptide N-terminus into the active site. These observations were supported by our competitive inhibition activity assays. Dug1p activity towards Cys-Gly peptide is significantly reduced (∼ 70%) in the presence of Glu-Cys-Gly. Therefore, Dug1p can recognize a variety of oligopeptides, but has evolved with post-binding screening potential to hydrolyze Cys-Gly peptides selectively.

  12. Marine Peptides and Their Anti-Infective Activities

    PubMed Central

    Kang, Hee Kyoung; Seo, Chang Ho; Park, Yoonkyung

    2015-01-01

    Marine bioresources are a valuable source of bioactive compounds with industrial and nutraceutical potential. Numerous clinical trials evaluating novel chemotherapeutic agents derived from marine sources have revealed novel mechanisms of action. Recently, marine-derived bioactive peptides have attracted attention owing to their numerous beneficial effects. Moreover, several studies have reported that marine peptides exhibit various anti-infective activities, such as antimicrobial, antifungal, antimalarial, antiprotozoal, anti-tuberculosis, and antiviral activities. In the last several decades, studies of marine plants, animals, and microbes have revealed tremendous number of structurally diverse and bioactive secondary metabolites. However, the treatments available for many infectious diseases caused by bacteria, fungi, and viruses are limited. Thus, the identification of novel antimicrobial peptides should be continued, and all possible strategies should be explored. In this review, we will present the structures and anti-infective activity of peptides isolated from marine sources (sponges, algae, bacteria, fungi and fish) from 2006 to the present. PMID:25603351

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

    PubMed Central

    Velmurugan, Punitha; Jonnalagadda, Raghava Rao; Unni Nair, Balachandran

    2015-01-01

    Collagenase is an important enzyme which plays an important role in degradation of collagen in wound healing, cancer metastasis and even in embryonic development. However, the mechanism of this degradation has not yet been completely understood. In the field of biomedical and protein engineering, the design and development of new peptide based materials is of main concern. In the present work an attempt has been made to study the effect of DAla in collagen like peptide (imino-poor region of type I collagen) on the structure and stability of peptide against enzyme hydrolysis. Effect of replacement of DAla in the collagen like peptide has been studied using circular dichroic spectroscopy (CD). Our findings suggest that, DAla substitution leads to conformational changes in the secondary structure and favours the formation of polyproline II conformation than its L-counterpart in the imino-poor region of collagen like peptides. Change in the chirality of alanine at the cleavage site of collagenase in the imino-poor region inhibits collagenolytic activity. This may find application in design of peptides and peptidomimics for enzyme-substrate interaction, specifically with reference to collagen and other extra cellular matrix proteins. PMID:25973613

  14. Comparative simulation studies of native and single-site mutant human beta-defensin-1 peptides.

    PubMed

    Toubar, Rabab A; Zhmurov, Artem; Barsegov, Valeri; Marx, Kenneth A

    2013-01-01

    Human defensins play important roles in a broad range of biological functions, such as microbial defense and immunity. Yet, little is known about their molecular properties, i.e. secondary structure stability, structural variability, important side chain interactions, surface charge distribution, and resistance to thermal fluctuations, and how these properties are related to their functions. To assess these factors, we studied the native human β-defensin-1 monomer and dimer as well as several single-site mutants using molecular dynamics simulations. The results showed that disulfide bonds are important determinants in maintaining the defensins' structural integrity, as no structural transitions were observed at 300 K and only minor structural unfolding was detected upon heating to 500 K. The α-helix was less thermally stable than the core β-sheet structure held together by hydrogen bonds and hydrophobic interactions. The monomer α-helix stability was directly correlated, whereas the end-to-end distance was inversely correlated to the experimentally measured β-defensin-1 chemotactic activity, in the order: mutant 2 (Gln24Glu) > mutant 3 (Lys31Ala) = wild type > mutant 1 (Asn4Ala). The structural stability of the β-defensin-1 dimer species exhibited an inverse correlation to their chemotactic activity. In dimers formed by mutants 2 and 3, we observed sliding of one monomer upon the surface of the other in the absence of unbinding. This dynamic sliding feature may enhance the molecular oligomerization of β-defensin-1 peptides contributing to their antibacterial activity. It could also help these peptides orient correctly in the CC chemokine receptor 6 binding site, thereby initiating their chemotactic activity. In agreement with this notion, the remarkable sliding behavior was observed only for the mutants with the highest chemotactic activity.

  15. A Peptide-Coated Gold Nanocluster Exhibits Unique Behavior in Protein Activity Inhibition.

    PubMed

    An, Deyi; Su, Jiguo; Weber, Jeffrey K; Gao, Xueyun; Zhou, Ruhong; Li, Jingyuan

    2015-07-01

    Gold nanoclusters (AuNCs) can be primed for biomedical applications through functionalization with peptide coatings. Often anchored by thiol groups, such peptide coronae not only serve as passivators but can also endow AuNCs with additional bioactive properties. In this work, we use molecular dynamics simulations to study the structure of a tridecapeptide-coated Au25 cluster and its subsequent interactions with the enzyme thioredoxin reductase 1, TrxR1. We find that, in isolation, both the distribution and conformation of the coating peptides fluctuate considerably. When the coated AuNC is placed around TrxR1, however, the motion of the highly charged peptide coating (+5e/peptide) is quickly biased by electrostatic attraction to the protein; the asymmetric coating acts to guide the nanocluster's diffusion toward the enzyme's negatively charged active site. After the AuNC comes into contact with TrxR1, its peptide corona spreads over the protein surface to facilitate stable binding with protein. Though individual salt bridge interactions between the tridecapeptides and TrxR1 are transient in nature, the cooperative binding of the peptide-coated AuNC is very stable, overall. Interestingly, the biased corona peptide motion, the spreading and the cooperation between peptide extensions observed in AuNC binding are reminiscent of bacterial stimulus-driven approaching and adhesion mechanisms mediated by cilia. The prevailing AuNC binding mode we characterize also satisfies a notable hydrophobic interaction seen in the association of thioredoxin to TrxR1, providing a possible explanation for the AuNC binding specificity observed in experiments. Our simulations thus suggest this peptide-coated AuNC serves as an adept thioredoxin mimic that extends an array of auxiliary structural components capable of enhancing interactions with the target protein in question.

  16. [Biologically Active Peptides Isolated from Dill Anethum graveolens L].

    PubMed

    Kulikova, O G; Maltsev, D I; Ilyina, A P; Burdina, A V; Yamskova, V P; Yamskov, I A

    2015-01-01

    Peptide mixtures with molecular weights of 1000-2000 Da and in vivo membrano-trophic activity against mouse hepatocyte culture at very low concentrations were isolated from dill Anethum graveolens L. leaves. It has been found that plant peptides in aqueous solution formed larger nanosized particles of approximately 90 nm with a secondary structure mainly composed of β-structures and random coil structures. We demonstrated that peptides isolated from A. graveolens in vitro at an ultra-low dosage affected the size of the area of pigmented cells of amphibian liver, which are analogous to Kupffer cells of the mammalian liver, using roller organotypic newt liver culture models. PMID:26204780

  17. Immunomodulatory effects of endogenous and synthetic peptides activating opioid receptors.

    PubMed

    Pomorska, Dorota K; Gach, Katarzyna; Janecka, Anna

    2014-01-01

    The main role of endogenous opioid peptides is the modulation of pain. Opioid peptides exert their analgesic activity by binding to the opioid receptors distributed widely in the central nervous system (CNS). However, opioid receptors are also found on tissues and organs outside the CNS, including the cells of the immune system, indicating that opioids are capable of exerting additional effects in periphery. Morphine, which is a gold standard in the treatment of chronic pain, is well-known for its immunosuppressive effects. Much less is known about the immunomodulatory effects exerted by endogenous (enkephalins, endorphins, dynorphins and endomorphins) and synthetic peptides activating opioid receptors. In this review we tried to summarize opioid peptide-mediated modulation of immune cell functions which can be stimulatory as well as inhibitory. PMID:25553430

  18. How Membrane-Active Peptides Get into Lipid Membranes.

    PubMed

    Sani, Marc-Antoine; Separovic, Frances

    2016-06-21

    The structure-function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely diverse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular

  19. How Membrane-Active Peptides Get into Lipid Membranes.

    PubMed

    Sani, Marc-Antoine; Separovic, Frances

    2016-06-21

    The structure-function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely diverse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular

  20. Acetylcholinesterase accelerates assembly of amyloid-beta-peptides into Alzheimer's fibrils: possible role of the peripheral site of the enzyme.

    PubMed

    Inestrosa, N C; Alvarez, A; Pérez, C A; Moreno, R D; Vicente, M; Linker, C; Casanueva, O I; Soto, C; Garrido, J

    1996-04-01

    Acetylcholinesterase (AChE), an important component of cholinergic synapses, colocalizes with amyloid-beta peptide (A beta) deposits of Alzheimer's brain. We report here that bovine brain AChE, as well as the human and mouse recombinant enzyme, accelerates amyloid formation from wild-type A beta and a mutant A beta peptide, which alone produces few amyloid-like fibrils. The action of AChE was independent of the subunit array of the enzyme, was not affected by edrophonium, an active site inhibitor, but it was affected by propidium, a peripheral anionic binding site ligand. Butyrylcholinesterase, an enzyme that lacks the peripheral site, did not affect amyloid formation. Furthermore, AChE is a potent amyloid-promoting factor when compared with other A beta-associated proteins. Thus, in addition to its role in cholinergic synapses, AChE may function by accelerating A beta formation and could play a role during amyloid deposition in Alzheimer's brain.

  1. Peptide reporters of kinase activity in whole cell lysates

    PubMed Central

    Wu, Ding; Sylvester, Juliesta E.; Parker, Laurie L.; Zhou, Guangchang; Kron, Stephen J.

    2010-01-01

    Kinase assays are used to screen for small-molecule inhibitors that may show promise as targeted pharmaceutical therapies. Using cell lysates instead of purified kinases provides a more accurate estimate of inhibitor sensitivity and selectivity in a biological setting. This review summarizes the range of homogeneous (solution-phase) and heterogeneous (solid-supported) formats available for using peptide substrates to monitor kinase activities in cell lysates. With a focus on heterogeneous kinase assays, the peptide substrate Abltide is used as a model to optimize presentation geometries and the modular arrangement of short sequences for kinase recognition. We present results from peptides immobilized on two- and three-dimensional surfaces such as hydrogels on 96-well plates and glass slides, and fluorescent Luminex beads. We discuss methods to increase assay sensitivity using chemifluorescent ELISAs, antibody-based recognition, and label-free mass spectrometry. Monitoring the activity of specific kinases in cell lysates presents challenges that can be overcome by manipulating peptide substrates to optimize assay conditions. In particular, signal-to-background ratios were improved by 1) adding long branched hydrophilic linkers between the substrate and the surface, 2) changing the orientation of peptides relative to the surface, and 3) including peptide ligands in cis or in trans to recruit kinases to the surface. By improving the accessibility of immobilized peptide substrates to kinases in solution, the apparent rate of phosphorylation increased and assays were more sensitive to changes in endogenous kinase activities. These strategies can be generalized to improve the reactivity of most peptide substrates used in heterogeneous kinase assays with cell lysates. PMID:20593469

  2. Utilization of synthetic peptides to evaluate the importance of substrate interaction at the proteolytic site of Escherichia coli Lon protease.

    PubMed

    Patterson-Ward, Jessica; Tedesco, Johnathan; Hudak, Jason; Fishovitz, Jennifer; Becker, James; Frase, Hilary; McNamara, Kirsten; Lee, Irene

    2009-09-01

    Lon, also known as protease La, is an ATP-dependent protease functioning to degrade many unstructured proteins. Currently, very little is known about the substrate determinants of Lon at the proteolytic site. Using synthetic peptides constituting different regions of the endogenous protein substrate lambdaN, we demonstrated that the proteolytic site of Escherichia coli Lon exhibits a certain level of localized sequence specificity. Using an alanine positional scanning approach, we discovered a set of discontinuous substrate determinants surrounding the scissile Lon cleavage site in a model peptide substrate, which function to influence the k(cat) of the peptidase activity of Lon. We further investigated the mode of peptide interaction with the proteolytically inactive Lon mutant S679A in the absence and presence of ADP or AMPPNP by 2-dimensional nuclear magnetic resonance spectroscopy, and discovered that the binding interaction between protein and peptide varies with the nucleotide bound to the enzyme. This observation is suggestive of a substrate translocation step, which likely limits the turnover of the proteolytic reaction. The contribution of the identified substrate determinants towards the kinetics of ATP-dependent degradation of lambdaN and truncated lambdaN mutants by Lon was also examined. Our results indicated that Lon likely recognizes numerous discontinuous substrate determinants throughout lambdaN to achieve substrate promiscuity.

  3. Metastable atom-activated dissociation mass spectrometry of phosphorylated and sulfonated peptides in negative ion mode.

    PubMed

    Cook, Shannon L; Jackson, Glen P

    2011-06-01

    The dissociation behavior of phosphorylated and sulfonated peptide anions was explored using metastable atom-activated dissociation mass spectrometry (MAD-MS) and collision-induced dissociation (CID). A beam of high kinetic energy helium (He) metastable atoms was exposed to isolated phosphorylated and sulfonated peptides in the 3- and 2- charge states. Unlike CID, where phosphate losses are dominant, the major dissociation channels observed using MAD were C(α) - C peptide backbone cleavages and neutral losses of CO(2), H(2)O, and [CO(2) + H(2)O] from the charge reduced (oxidized) product ion, consistent with an electron detachment dissociation (EDD) mechanism such as Penning ionization. Regardless of charge state or modification, MAD provides ample backbone cleavages with little modification loss, which allows for unambiguous PTM site determination. The relative abundance of certain fragment ions in MAD is also demonstrated to be somewhat sensitive to the number and location of deprotonation sites, with backbone cleavage somewhat favored adjacent to deprotonated sites like aspartic acid residues. MAD provides a complementary dissociation technique to CID, ECD, ETD, and EDD for peptide sequencing and modification identification. MAD offers the unique ability to analyze highly acidic peptides that contain few to no basic amino acids in either negative or positive ion mode.

  4. Computer simulation of the active site of human serum cholinesterase

    SciTech Connect

    Kefang Jiao; Song Li; Zhengzheng Lu

    1996-12-31

    The first 3D-structure of acetylchelinesterase from Torpedo California electric organ (T.AChE) was published by JL. Sussman in 1991. We have simulated 3D-structure of human serum cholinesterase (H.BuChE) and the active site of H.BuChE. It is discovered by experiment that the residue of H.BuChE is still active site after a part of H.BuChE is cut. For example, the part of 21KD + 20KD is active site of H.BuChE. The 20KD as it is. Studies on these peptides by Hemelogy indicate that two active peptides have same negative electrostatic potential maps diagram. These negative electrostatic areas attached by acetyl choline with positive electrostatic potency. We predict that 147...236 peptide of AChE could be active site because it was as 20KD as with negative electrostatic potential maps. We look forward to proving from other ones.

  5. Localization of the cross-linking sites of RGD and KQAGDV peptides to the isolated fibrinogen receptor, the human platelet integrin glycoprotein IIb/IIIa. Influence of peptide length.

    PubMed

    Calvete, J J; Schäfer, W; Mann, K; Henschen, A; González-Rodríguez, J

    1992-06-15

    The non-covalent and Ca(2+)-dependent heterodimer GPIIb/IIIa, formed by platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa), also known as the integrin alpha IIb beta 3, is the inducible receptor for fibrinogen and other adhesive proteins on the surface of activated platelets. A fraction of the isolated GPIIb/IIIa in solution binds RGD or KQAGDV inhibitory peptides and, upon peptide removal, apparently acquires the capacity to bind fibrinogen ('activated' GPIIb/IIIa) [Du, X., Plow, E. F., Frelinger, A. L., III, O'Toole, T. E., Loftus, J. C. & Ginsberg, M. H. (1991) Cell 65, 409-416]. Photoaffinity labelling was used here to study the ligand binding site(s) of GPIIb/IIIa in solution, for which the peptides CKRKRKRKRRGDV (alpha 1), CGRGDF (alpha 2), CYHHLGGAKQAGDV (gamma 1) and CGAKQAGDV (gamma 2) were synthesized with a photoactivable cross-linker group and a fluorescent reporter group attached to the N-terminal cysteine residue. Contrary to the situation in activated platelets, both GPIIb and GPIIIa were equally labelled by the four peptides and the cross-linking sites were localized by protein chemical analyses of the fluorescently labelled tryptic peptides of both subunits. Thus, the localization of the cross-linking sites in GPIIb varies considerably with the peptide length and is very different from that localization observed in activated platelets: alpha 2 and gamma 2 were found cross-linked to the N-terminal of both the heavy (GPIIbH 42-73) and the light (GPIIbL2 30-75) chains of GPIIb; while the longer peptides alpha 1 and gamma 1 were cross-linked to the C-terminal of GPIIbH within the 696-724 and 752-768 peptide stretches, respectively. On the other hand, the cross-linking sites of the four inhibitory peptides in GPIIIa were found mainly within the proteolysis susceptible region, between the N-terminal (GPIIIa 1-52) and the core (GPIIb 423-622) highly disulphide-bonded domains, observing that the longer the peptide the closer the cross-linking site is to

  6. Processing of peptide and hormone precursors at the dibasic cleavage sites.

    PubMed

    Rholam, Mohamed; Fahy, Christine

    2009-07-01

    Many functionally important cellular peptides and proteins, including hormones, neuropeptides, and growth factors, are synthesized as inactive precursor polypeptides, which require post-translational proteolytic processing to become biologically active polypeptides. This is achieved by the action of a relatively small number of proteases that belong to a family of seven subtilisin-like proprotein convertases (PCs) including furin. In view of this, this review focuses on the importance of privileged secondary structures and of given amino acid residues around basic cleavage sites in substrate recognition by these endoproteases. In addition to their participation in normal cell functions, PCs are crucial for the initiation and progress of many important diseases. Hence, these proteases constitute potential drug targets in medicine. Accordingly, this review also discusses the approaches used to shed light on the cleavage preference and the substrate specificity of the PCs, a prerequisite to select which PCs are promising drug targets in each disease. PMID:19300906

  7. Processing of peptide and hormone precursors at the dibasic cleavage sites.

    PubMed

    Rholam, Mohamed; Fahy, Christine

    2009-07-01

    Many functionally important cellular peptides and proteins, including hormones, neuropeptides, and growth factors, are synthesized as inactive precursor polypeptides, which require post-translational proteolytic processing to become biologically active polypeptides. This is achieved by the action of a relatively small number of proteases that belong to a family of seven subtilisin-like proprotein convertases (PCs) including furin. In view of this, this review focuses on the importance of privileged secondary structures and of given amino acid residues around basic cleavage sites in substrate recognition by these endoproteases. In addition to their participation in normal cell functions, PCs are crucial for the initiation and progress of many important diseases. Hence, these proteases constitute potential drug targets in medicine. Accordingly, this review also discusses the approaches used to shed light on the cleavage preference and the substrate specificity of the PCs, a prerequisite to select which PCs are promising drug targets in each disease.

  8. Mucin-like peptides from Echinococcus granulosus induce antitumor activity.

    PubMed

    Noya, Verónica; Bay, Sylvie; Festari, María Florencia; García, Enrique P; Rodriguez, Ernesto; Chiale, Carolina; Ganneau, Christelle; Baleux, Françoise; Astrada, Soledad; Bollati-Fogolín, Mariela; Osinaga, Eduardo; Freire, Teresa

    2013-09-01

    There is substantial evidence suggesting that certain parasites can have antitumor properties. We evaluated mucin peptides derived from the helminth Echinococcus granulosus (denominated Egmuc) as potential inducers of antitumor activity. We present data showing that Egmuc peptides were capable of inducing an increase of activated NK cells in the spleen of immunized mice, a fact that was correlated with the capacity of splenocytes to mediate killing of tumor cells. We demonstrated that Egmuc peptides enhance LPS-induced maturation of dendritic cells in vitro by increasing the production of IL-12p40p70 and IL-6 and that Egmuc-treated DCs may activate NK cells, as judged by an increased expression of CD69. This evidence may contribute to the design of tumor vaccines and open new horizons in the use of parasite-derived molecules in the fight against cancer.

  9. Self-assembly of cationic multidomain peptide hydrogels: supramolecular nanostructure and rheological properties dictate antimicrobial activity

    NASA Astrophysics Data System (ADS)

    Jiang, Linhai; Xu, Dawei; Sellati, Timothy J.; Dong, He

    2015-11-01

    Hydrogels are an important class of biomaterials that have been widely utilized for a variety of biomedical/medical applications. The biological performance of hydrogels, particularly those used as wound dressing could be greatly advanced if imbued with inherent antimicrobial activity capable of staving off colonization of the wound site by opportunistic bacterial pathogens. Possessing such antimicrobial properties would also protect the hydrogel itself from being adversely affected by microbial attachment to its surface. We have previously demonstrated the broad-spectrum antimicrobial activity of supramolecular assemblies of cationic multi-domain peptides (MDPs) in solution. Here, we extend the 1-D soluble supramolecular assembly to 3-D hydrogels to investigate the effect of the supramolecular nanostructure and its rheological properties on the antimicrobial activity of self-assembled hydrogels. Among designed MDPs, the bactericidal activity of peptide hydrogels was found to follow an opposite trend to that in solution. Improved antimicrobial activity of self-assembled peptide hydrogels is dictated by the combined effect of supramolecular surface chemistry and storage modulus of the bulk materials, rather than the ability of individual peptides/peptide assemblies to penetrate bacterial cell membrane as observed in solution. The structure-property-activity relationship developed through this study will provide important guidelines for designing biocompatible peptide hydrogels with built-in antimicrobial activity for various biomedical applications.Hydrogels are an important class of biomaterials that have been widely utilized for a variety of biomedical/medical applications. The biological performance of hydrogels, particularly those used as wound dressing could be greatly advanced if imbued with inherent antimicrobial activity capable of staving off colonization of the wound site by opportunistic bacterial pathogens. Possessing such antimicrobial properties would

  10. Peptide Synthesis through Cell-Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release.

    PubMed

    Liutkus, Mantas; Fraser, Samuel A; Caron, Karine; Stigers, Dannon J; Easton, Christopher J

    2016-05-17

    Chlorinated analogues of Leu and Ile are incorporated during cell-free expression of peptides fused to protein, by exploiting the promiscuity of the natural biosynthetic machinery. They then act as sites for clean and efficient release of the peptides simply by brief heat treatment. Dehydro analogues of Leu and Ile are similarly incorporated as latent sites for peptide release through treatment with iodine under cold conditions. These protocols complement enzyme-catalyzed methods and have been used to prepare calcitonin, gastrin-releasing peptide, cholecystokinin-7, and prolactin-releasing peptide prohormones, as well as analogues substituted with unusual amino acids, thus illustrating their practical utility as alternatives to more traditional chemical peptide synthesis. PMID:26918308

  11. Definition of endotoxin binding sites in horseshoe crab factor C recombinant sushi proteins and neutralization of endotoxin by sushi peptides.

    PubMed

    Tan, N S; Ng, M L; Yau, Y H; Chong, P K; Ho, B; Ding, J L

    2000-09-01

    Three truncated fragments, harboring different sushi domains, namely, sushi123, sushi1, and sushi3 domains, of Factor C were produced as biologically active secreted recombinant proteins. Sushi1 and 3 each has a high-affinity LPS binding site with K:(d) of 10(-9) to 10(-10) M. Positive cooperativity in sushi123 resulted in a 1000-fold increase in K:(d)2. The core LPS binding region of sushi1 and 3 reside in two 34-mer peptides, S1 and S3. A rigidly held disulfide-bonded structure is not essential but is important for LPS binding, as confirmed by a 100- to 10000-fold decrease in affinity. Both S1 and S3 can inhibit LAL reaction and LPS-induced hTNF-alpha secretion with different potency. LAL assay revealed that at least two molecules of S1 bind cooperatively to one LPS molecule, with Hill's coefficient of 2.42. The LPS binding by S3 is independent and noncooperative. The modified SDelta1 and SDelta3 peptides exhibited increased LPS neutralization potential although its LPS binding affinities indicated only a 10-fold improvement. Hence, the structural difference of the four sushi peptides conferred different efficiencies in LPS neutralization without altering their binding affinity for LPS. Circular dichroism spectrometry revealed that the four peptides underwent conformational change in the presence of lipid A, transitioning from a random coil to either an alpha-helical or beta-sheet structure. Two factors are critical for the sensitivity of Factor C to LPS: 1) the presence of multiple binding sites for LPS on a single Factor C molecule; and 2) high positive cooperativity in LPS binding. The results showed that in the design of an improved LPS binding and neutralizing peptide, charge balance of the peptide is a critical parameter in addition to its structure.

  12. Salt site performance assessment activities

    SciTech Connect

    Kircher, J.F.; Gupta, S.K.

    1983-01-01

    During this year the first selection of the tools (codes) for performance assessments of potential salt sites have been tentatively selected and documented; the emphasis has shifted from code development to applications. During this period prior to detailed characterization of a salt site, the focus is on bounding calculations, sensitivity and with the data available. The development and application of improved methods for sensitivity and uncertainty analysis is a focus for the coming years activities and the subject of a following paper in these proceedings. Although the assessments to date are preliminary and based on admittedly scant data, the results indicate that suitable salt sites can be identified and repository subsystems designed which will meet the established criteria for protecting the health and safety of the public. 36 references, 5 figures, 2 tables.

  13. Binding sites of atrial natriuretic peptide in tree shrew adrenal gland

    SciTech Connect

    Fuchs, E.; Shigematsu, K.; Saavedra, J.M.

    1986-09-01

    Adrenal gland binding sites for atrial natriuretic peptide-(99-126) (ANP) were quantitated in tree shrew (Tupaia belangeri) by incubation of adrenal sections with (3-(/sup 125/I)-iodotyrosyl28) atrial natriuretic peptide-(99-126), followed by autoradiography with computerized microdensitometry. In the adrenal glands, there are three types of ANP binding sites. One is located in the zona glomerulosa (BMax 84 +/- 6 fmol/mg protein; Kd 122 +/- 9 pM); the second in the zona fasciculata and reticularis (BMax 29 +/- 2 fmol/mg protein; Kd 153 +/- 6 pM) and the third in the adrenal medulla (BMax 179 +/- 1 fmol/mg protein; Kd 70 +/- 2 pM). Besides the influence of ANP on the regulation of adrenocortical mineralcorticoid and glucocorticoid secretion our findings raise the possibility for a local site of action of atrial natriuretic peptide in the regulation of adrenomedullary catecholamines in the tree shrew, primates and man.

  14. PEP-SiteFinder: a tool for the blind identification of peptide binding sites on protein surfaces

    PubMed Central

    Saladin, Adrien; Rey, Julien; Thévenet, Pierre; Zacharias, Martin; Moroy, Gautier; Tufféry, Pierre

    2014-01-01

    Peptide–protein interactions are important to many processes of life, particularly for signal transmission or regulatory mechanisms. When no information is known about the interaction between a protein and a peptide, it is of interest to propose candidate sites of interaction at the protein surface, to assist the design of biological experiments to probe the interaction, or to serve as a starting point for more focused in silico approaches. PEP-SiteFinder is a tool that will, given the structure of a protein and the sequence of a peptide, identify protein residues predicted to be at peptide–protein interface. PEP-SiteFinder relies on the 3D de novo generation of peptide conformations given its sequence. These conformations then undergo a fast blind rigid docking on the complete protein surface, and we have found, as the result of a benchmark over 41 complexes, that the best poses overlap to some extent the experimental patch of interaction for close to 90% complexes. In addition, PEP-SiteFinder also returns a propensity index we have found informative about the confidence of the prediction. The PEP-SiteFinder web server is available at http://bioserv.rpbs.univ-paris-diderot.fr/PEP-SiteFinder. PMID:24803671

  15. Tumor-Specific Peptide, Selected from a Phage Peptide Library, Enhances Antitumor Activity of Lactaptin

    PubMed Central

    Makartsova, Anna A.; Fomin, Alexandr S.; Nushtaeva, Anna A.; Koval, Olga A.

    2016-01-01

    A recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, induces apoptosis in cultured tumor cells. The tumor suppression efficacy of RL2 was shown against mouse hepatoma-1 cells and MDA-MB-231 human breast adenocarcinoma cells. The RL2-based therapeutic drug lactaptin is distributed evenly throughout the organism, which reduces its antitumor efficacy. In the current study, we obtained a genetic construct that allows production of the recombinant fusion protein T3-RL2, consisting of RL2 and T3 peptide (YTYDPWLIFPAN), in E. coli cells. T3 peptide was selected from a phage peptide library as a result of two screenings: in vitro using MDA-MB-231 cell culture and in vivo using a mouse xenograft model of breast cancer MDA-MB-231. It was shown that the displayed peptide T3 provides binding and internalization of phage particles by MDA-MB-231 cells and their specific accumulation in MDA-MB-231 tumor tissue. In addition, based on the nucleotide sequences coding RL2 and the known tumor-targeting peptide iRGD, we obtained genetic constructs that provide synthesis of fusion proteins RL2-iRGD and RL-iRGD-His. We studied the cytotoxic activity of fusion proteins T3-RL2, RL2-iRGD and RL-iRGD-His in vitro using MDA-MB-231 and MCF-7 human adenocarcinoma cells. The in vitro results showed that the fusion proteins inhibit proliferation of both cell cultures, and their cytotoxic activity is higher than that of RL2. In vivo experiments on the study of the antitumor efficacy of the obtained fusion proteins demonstrated that T3-RL2 protein significantly inhibits MDA-MB-231 tumor growth in a xenograft model compared with RL2, while the antitumor effect of RL2-iRGD and RL-iRGD-His proteins is comparable to the effect of RL2. PMID:27513518

  16. Tumor-Specific Peptide, Selected from a Phage Peptide Library, Enhances Antitumor Activity of Lactaptin.

    PubMed

    Nemudraya, Anna A; Makartsova, Anna A; Fomin, Alexandr S; Nushtaeva, Anna A; Koval, Olga A; Richter, Vladimir A; Kuligina, Elena V

    2016-01-01

    A recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, induces apoptosis in cultured tumor cells. The tumor suppression efficacy of RL2 was shown against mouse hepatoma-1 cells and MDA-MB-231 human breast adenocarcinoma cells. The RL2-based therapeutic drug lactaptin is distributed evenly throughout the organism, which reduces its antitumor efficacy. In the current study, we obtained a genetic construct that allows production of the recombinant fusion protein T3-RL2, consisting of RL2 and T3 peptide (YTYDPWLIFPAN), in E. coli cells. T3 peptide was selected from a phage peptide library as a result of two screenings: in vitro using MDA-MB-231 cell culture and in vivo using a mouse xenograft model of breast cancer MDA-MB-231. It was shown that the displayed peptide T3 provides binding and internalization of phage particles by MDA-MB-231 cells and their specific accumulation in MDA-MB-231 tumor tissue. In addition, based on the nucleotide sequences coding RL2 and the known tumor-targeting peptide iRGD, we obtained genetic constructs that provide synthesis of fusion proteins RL2-iRGD and RL-iRGD-His. We studied the cytotoxic activity of fusion proteins T3-RL2, RL2-iRGD and RL-iRGD-His in vitro using MDA-MB-231 and MCF-7 human adenocarcinoma cells. The in vitro results showed that the fusion proteins inhibit proliferation of both cell cultures, and their cytotoxic activity is higher than that of RL2. In vivo experiments on the study of the antitumor efficacy of the obtained fusion proteins demonstrated that T3-RL2 protein significantly inhibits MDA-MB-231 tumor growth in a xenograft model compared with RL2, while the antitumor effect of RL2-iRGD and RL-iRGD-His proteins is comparable to the effect of RL2. PMID:27513518

  17. 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

    2015-12-01

    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. PMID:26583259

  18. OMP Peptides Activate the DegS Stress-Sensor Protease by a Relief of Inhibition Mechanism

    SciTech Connect

    Sohn, Jungsan; Grant, Robert A.; Sauer, Robert T.; MIT

    2010-03-19

    In the E. coli periplasm, C-terminal peptides of misfolded outer-membrane porins (OMPs) bind to the PDZ domains of the trimeric DegS protease, triggering cleavage of a transmembrane regulator and transcriptional activation of stress genes. We show that an active-site DegS mutation partially bypasses the requirement for peptide activation and acts synergistically with mutations that disrupt contacts between the protease and PDZ domains. Biochemical results support an allosteric model, in which these mutations, active-site modification, and peptide/substrate binding act in concert to stabilize proteolytically active DegS. Cocrystal structures of DegS in complex with different OMP peptides reveal activation of the protease domain with varied conformations of the PDZ domain and without specific contacts from the bound OMP peptide. Taken together, these results indicate that the binding of OMP peptides activates proteolysis principally by relieving inhibitory contacts between the PDZ domain and the protease domain of DegS.

  19. Cyclolinopeptides, cyclic peptides from flaxseed with osteoclast differentiation inhibitory activity.

    PubMed

    Kaneda, Toshio; Yoshida, Haruka; Nakajima, Yuki; Toishi, Minako; Nugroho, Alfarius Eko; Morita, Hiroshi

    2016-04-01

    Flaxseed (Linum usitatissimum seed) is widely used in food and natural health products. In our search for osteoclast differentiation inhibitors, some cyclic peptides isolated from flaxseed, known as the cyclolinopeptides, were discovered to have osteoclast differentiation inhibition activity. The osteoclast differentiation inhibition activity of cyclolinopeptides A-I (1-9) and their related derivatives (10-14) are described herein. Cyclolinopeptides F, H and I (6, 8 and 9), in particular, showed potent osteoclast differentiation inhibition activity. PMID:26923696

  20. Inhibition of NF-kappaB activity by plasmid expressed alphaMSH peptide.

    PubMed

    Etemad-Moghadam, Bijan; Chen, Hongmin; Yin, Peng; Aziz, Nazneen; Hedley, Mary Lynne

    2002-04-01

    Alpha-Melanocyte Stimulating Hormone (alphaMSH) is a neuroimmunomodulatory peptide with remarkable anti-inflammatory properties. Daily or twice daily administration of the peptide reduces the symptoms of several inflammatory animal disease models and the peptide has demonstrated safety in human trials. Unfortunately, the pharmacokinetics of peptide delivery are not favorable from the pharmaceutical perspective. For this reason, plasmid-based vectors were created that constitutively express the immunomodulatory peptide. The fusion constructs encode the 13 amino acids of alphaMSH in frame with the first domain of serum albumin, separated by a linker and furin cleavage sites. The fusion proteins were expressed and processed in human fetal kidney (293) cells. Supernatant from B16/F10 cells transfected with the constructs stimulated secretion of melanin from melanocytes. Furthermore, transfected cytoskeletal muscle (Sol8) cells secreted bioactive alphaMSH that reduced NF-kappaB-mediated transcriptional activation of a luciferase reporter gene. The activity of these vectors provides tools and the impetus for testing the constructs in several animal models of chronic inflammation. PMID:11960637

  1. Peptides from the scorpion Vaejovis punctatus with broad antimicrobial activity.

    PubMed

    Ramírez-Carreto, Santos; Jiménez-Vargas, Juana María; Rivas-Santiago, Bruno; Corzo, Gerardo; Possani, Lourival D; Becerril, Baltazar; Ortiz, Ernesto

    2015-11-01

    The antimicrobial potential of two new non-disulfide bound peptides, named VpAmp1.0 (LPFFLLSLIPSAISAIKKI, amidated) and VpAmp2.0 (FWGFLGKLAMKAVPSLIGGNKSSSK) is here reported. These are 19- and 25-aminoacid-long peptides with +2 and +4 net charges, respectively. Their sequences correspond to the predicted mature regions from longer precursors, putatively encoded by cDNAs derived from the venom glands of the Mexican scorpion Vaejovis punctatus. Both peptides were chemically synthesized and assayed against a variety of microorganisms, including pathogenic strains from clinical isolates and strains resistant to conventional antibiotics. Two shorter variants, named VpAmp1.1 (FFLLSLIPSAISAIKKI, amidated) and VpAmp2.1 (FWGFLGKLAMKAVPSLIGGNKK), were also synthesized and tested. The antimicrobial assays revealed that the four synthetic peptides effectively inhibit the growth of both Gram-positive (Staphylococcus aureus and Streptococcus agalactiaea) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria, with MICs in the range of 2.5-24.0 μM; yeasts (Candida albicans and Candida glabrata) with MICs of 3.1-50.0 μM; and two clinically isolated strains of Mycobacterium tuberculosis-including a multi-drug resistant one- with MICs in the range of 4.8-30.5 μM. A comparison between the activities of the original peptides and their derivatives gives insight into the structural/functional role of their distinctive residues.

  2. Novel Antimicrobial Peptides with High Anticancer Activity and Selectivity

    PubMed Central

    Chen, Kuan-Hao; Yu, Hui-Yuan; Chih, Ya-Han; Cheng, Hsi-Tsung; Chou, Yu-Ting; Cheng, Jya-Wei

    2015-01-01

    We describe a strategy to boost anticancer activity and reduce normal cell toxicity of short antimicrobial peptides by adding positive charge amino acids and non-nature bulky amino acid β-naphthylalanine residues to their termini. Among the designed peptides, K4R2-Nal2-S1 displayed better salt resistance and less toxicity to hRBCs and human fibroblast than Nal2-S1 and K6-Nal2-S1. Fluorescence microscopic studies indicated that the FITC-labeled K4R2-Nal2-S1 preferentially binds cancer cells and causes apoptotic cell death. Moreover, a significant inhibition in human lung tumor growth was observed in the xenograft mice treated with K4R2-Nal2-S1. Our strategy provides new opportunities in the development of highly effective and selective antimicrobial and anticancer peptide-based therapeutics. PMID:25970292

  3. Monitoring Criminal Activity through Invisible Fluorescent "Peptide Coding" Taggants.

    PubMed

    Gooch, James; Goh, Hilary; Daniel, Barbara; Abbate, Vincenzo; Frascione, Nunzianda

    2016-04-19

    Complementing the demand for effective crime reduction measures are the increasing availability of commercial forensic "taggants", which may be used to physically mark an object in order to make it uniquely identifiable. This study explores the use of a novel "peptide coding" reagents to establish evidence of contact transfer during criminal activity. The reagent, containing a fluorophore dispersed within an oil-based medium, also includes a unique synthetic peptide sequence that acts as a traceable "code" to identify the origin of the taggant. The reagent is detectable through its fluorescent properties, which then allows the peptide to be recovered by swabbing and extracted for electrospray ionization-mass spectrometry (ESI-MS) analysis via a simple liquid-liquid extraction procedure. The performance of the reagent in variable conditions that mimic the limits of a real world use are investigated. PMID:27010696

  4. Functional mimicry of a discontinuous antigenic site by a designed synthetic peptide.

    PubMed

    Villén, Judit; Borràs, Eva; Schaaper, Wim M M; Meloen, Rob H; Dávila, Mercedes; Domingo, Esteban; Giralt, Ernest; Andreu, David

    2002-03-01

    Functional reproduction of the discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate each of the three protein loops that define the antigenic site into a single molecule. The site D mimics were designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulfide-linked heterodimers that consist of a larger unit containing VP2(71-84), followed by a polyproline module and by VP3(52-62), and a smaller unit corresponding to VP1(188-194) (VP=viral protein). Guinea pig antisera to the peptides recognized the viral particle and competed with site D-specific monoclonal antibodies, while inoculation with a simple (not covalently joined to one another) admixture of the three VP1-VP3 sequences yielded no detectable virus-specific serum conversion. Similar results have been reproduced in two bovines. Antisera to the peptides also moderately neutralize FMDV in cell cultures and partially protect guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent, peptide-based vaccine against FMDV. PMID:11921395

  5. Synthetic peptides as functional mimics of a viral discontinuous antigenic site.

    PubMed

    Villén, J; Borràs, E; Schaaper, W M; Meloen, R H; Dávila, M; Domingo, E; Giralt, E; Andreu, D

    2001-01-01

    Functional reproduction of discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate into a single molecule each of the three protein loops that define the antigenic site. The site D mimics are designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulphide-linked heterodimers that consist of a larger unit containing VP2(71-84), followed by a polyproline module and by VP3(52-62), and a smaller unit corresponding to VP1(188-194). Guinea pig antisera to the peptides recognize the viral particle and compete with site D-specific monoclonal antibodies, while inoculation with a simple (non-covalently bound) admixture of the three VP1-VP3 sequences yields no detectable virus-specific serum conversion. Similar results have been reproduced in two cattle. Antisera to the peptides are also moderately neutralizing of FMDV in cell culture and partially protective of guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent peptide-based vaccine against FMDV. PMID:11851326

  6. Anionic activators for differential sensing with cell-penetrating peptides.

    PubMed

    Montenegro, Javier; Matile, Stefan

    2011-02-01

    The design, synthesis, and evaluation of small peptides with one to three negative charges and one to three hydrazides as key components of membrane-based synthetic sensing systems are reported. Their spontaneous reaction with hydrophobic aldehydes or ketones gives rapid access to small collections of amphiphilic anions. These anionic amphiphiles can activate polycations as anion transporters in lipid-bilayer membranes. Odorants are used as representative hydrophobic aldehydes and ketones, and cell-penetrating peptides (CPPs) as polycationic transporters in fluorogenic vesicles. Different activities obtained with different counterion activators are used to generate multidimensional patterns that can be recognized by principal component and hierarchical cluster analysis to extract unique "fingerprints" for individual analytes (including enantiomers, cis-trans isomers or perfumes as illustrative analyte mixtures). Comparison of the peptide activators reveals that carboxylates perform better than phosphonates. Gemini-like activators containing two carboxylates and two hydrophobic hydrazone tails are best, whereas excessive charges and tails give weaker activities. This result differs from cationic activators of polyanionic transporters such as DNA, which worked best with octopus amphiphiles with one cationic head and four hydrophobic tentacles.

  7. Structure-Activity Relationship of Chlorotoxin-Like Peptides

    PubMed Central

    Ali, Syed Abid; Alam, Mehtab; Abbasi, Atiya; Undheim, Eivind A. B.; Fry, Bryan Grieg; Kalbacher, Hubert; Voelter, Wolfgang

    2016-01-01

    Animal venom (e.g., scorpion) is a rich source of various protein and peptide toxins with diverse physio-/pharmaco-logical activities, which generally exert their action via target-specific modulation of different ion channel functions. Scorpion venoms are among the most widely-known source of peptidyl neurotoxins used for callipering different ion channels, such as; Na+, K+, Ca+, Cl−, etc. A new peptide of the chlorotoxin family (i.e., Bs-Tx7) has been isolated, sequenced and synthesized from scorpion Buthus sindicus (family Buthidae) venom. This peptide demonstrates 66% with chlorotoxin (ClTx) and 82% with CFTR channel inhibitor (GaTx1) sequence identities reported from Leiurus quinquestriatus hebraeus venom. The toxin has a molecular mass of 3821 Da and possesses four intra-chain disulphide bonds. Amino acid sequence analysis of Bs-Tx7 revealed the presence of a scissile peptide bond (i.e., Gly-Ile) for human MMP2, whose activity is increased in the case of tumour malignancy. The effect of hMMP2 on Bs-Tx7, or vice versa, observed using the FRET peptide substrate with methoxycoumarin (Mca)/dinitrophenyl (Dnp) as fluorophore/quencher, designed and synthesized to obtain the lowest Km value for this substrate, showed approximately a 60% increase in the activity of hMMP2 upon incubation of Bs-Tx7 with the enzyme at a micromolar concentration (4 µM), indicating the importance of this toxin in diseases associated with decreased MMP2 activity. PMID:26848686

  8. Structure-Activity Relationship of Chlorotoxin-Like Peptides.

    PubMed

    Ali, Syed Abid; Alam, Mehtab; Abbasi, Atiya; Undheim, Eivind A B; Fry, Bryan Grieg; Kalbacher, Hubert; Voelter, Wolfgang

    2016-02-02

    Animal venom (e.g., scorpion) is a rich source of various protein and peptide toxins with diverse physio-/pharmaco-logical activities, which generally exert their action via target-specific modulation of different ion channel functions. Scorpion venoms are among the most widely-known source of peptidyl neurotoxins used for callipering different ion channels, such as; Na⁺, K⁺, Ca⁺, Cl(-), etc. A new peptide of the chlorotoxin family (i.e., Bs-Tx7) has been isolated, sequenced and synthesized from scorpion Buthus sindicus (family Buthidae) venom. This peptide demonstrates 66% with chlorotoxin (ClTx) and 82% with CFTR channel inhibitor (GaTx1) sequence identities reported from Leiurus quinquestriatus hebraeus venom. The toxin has a molecular mass of 3821 Da and possesses four intra-chain disulphide bonds. Amino acid sequence analysis of Bs-Tx7 revealed the presence of a scissile peptide bond (i.e., Gly-Ile) for human MMP2, whose activity is increased in the case of tumour malignancy. The effect of hMMP2 on Bs-Tx7, or vice versa, observed using the FRET peptide substrate with methoxycoumarin (Mca)/dinitrophenyl (Dnp) as fluorophore/quencher, designed and synthesized to obtain the lowest Km value for this substrate, showed approximately a 60% increase in the activity of hMMP2 upon incubation of Bs-Tx7 with the enzyme at a micromolar concentration (4 µM), indicating the importance of this toxin in diseases associated with decreased MMP2 activity.

  9. Data-independent-acquisition mass spectrometry for identification of targeted-peptide site-specific modifications.

    PubMed

    Porter, Caleb J; Bereman, Michael S

    2015-09-01

    We present a novel strategy based on data-independent acquisition coupled to targeted data extraction for the detection and identification of site-specific modifications of targeted peptides in a completely unbiased manner. This method requires prior knowledge of the site of the modification along the peptide backbone from the protein of interest, but not the mass of the modification. The procedure, named multiplex adduct peptide profiling (MAPP), consists of three steps: 1) A fragment-ion tag is extracted from the data, consisting of the b-type and y-type ion series from the N and C-terminus, respectively, up to the amino-acid position that is believed to be modified; 2) MS1 features are matched to the fragment-ion tag in retention-time space, using the isolation window as a pre-filter to enable calculation of the mass of the modification; and 3) modified fragment ions are overlaid with the unmodified fragment ions to verify the mass calculated in step 2. We discuss the development, applications, and limitations of this new method for detection of unknown peptide modifications. We present an application of the method in profiling adducted peptides derived from abundant proteins in biological fluids with the ultimate objective of detecting biomarkers of exposure to reactive species.

  10. Characterization and in vitro activity of a branched peptide boronic acid that interacts with HIV-1 RRE RNA.

    PubMed

    Wynn, Jessica E; Zhang, Wenyu; Tebit, Denis M; Gray, Laurie R; Hammarskjold, Marie-Louise; Rekosh, David; Santos, Webster L

    2016-09-01

    A branched peptide containing multiple boronic acids was found to bind RRE IIB selectively and inhibit HIV-1 p24 capsid production in a dose-dependent manner. Structure-activity relationship studies revealed that branching in the peptide is crucial for the low micromolar binding towards RRE IIB, and the peptide demonstrates selectivity towards RRE IIB in the presence of tRNA. Footprinting studies suggest a binding site on the upper stem and internal loop regions of the RNA, which induces enzymatic cleavage of the internal loops of RRE IIB upon binding. PMID:27091070

  11. The Use of Chromium(III) to Supercharge Peptides by Protonation at Low Basicity Sites

    PubMed Central

    Feng, Changgeng; Commodore, Juliette J.; Cassady, Carolyn J.

    2014-01-01

    The addition of chromium(III) nitrate to solutions of peptides with seven or more residues greatly increases the formation of doubly protonated peptides, [M+2H]2+, by electrospray ionization. The test compound heptaalanine has only one highly basic site (the N-terminal amino group) and undergoes almost exclusive single protonation using standard solvents. When Cr(III) is added to the solution, abundant [M+2H]2+ forms, which involves protonation of the peptide backbone or the C-terminus. Salts of Al(III), Mn(II), Fe(III), Fe(II), Cu(II), Zn (II), Rh(III), La(III), Ce(IV), and Eu(III) were also studied. While several metal ions slightly enhance protonation, Cr(III) has by far the greatest ability to generate [M+2H]2+. Cr(III) does not supercharge peptide methyl esters, which suggests that the mechanism involves interaction of Cr(III) with a carboxylic acid group. Other factors may include the high acidity of hexaaquochromium(III) and the resistance of Cr(III) to reduction. Nitrate salts enhance protonation more than chloride salts and a molar ratio of 10:1 Cr(III):peptide produces the most intense [M+2H]2+. Cr(III) also supercharges numerous other small peptides, including highly acidic species. For basic peptides, Cr(III) increases the charge state (2+ versus 1+) and causes the number of peptide molecules being protonated to double or triple. Chromium(III) does not supercharge the proteins cytochrome c and myoglobin. The ability of Cr(III) to enhance [M+2H]2+ intensity may prove useful in tandem mass spectrometry because of the resulting overall increase in signal-to-noise ratio, the fact that [M+2H]2+ generally dissociate more readily than [M+H]+, and the ability to produce [M+2H]2+ precursors for electron-based dissociation techniques. PMID:25395012

  12. The Use of Chromium(III) to Supercharge Peptides by Protonation at Low Basicity Sites

    NASA Astrophysics Data System (ADS)

    Feng, Changgeng; Commodore, Juliette J.; Cassady, Carolyn J.

    2015-02-01

    The addition of chromium(III) nitrate to solutions of peptides with seven or more residues greatly increases the formation of doubly protonated peptides, [M + 2H]2+, by electrospray ionization. The test compound heptaalanine has only one highly basic site (the N-terminal amino group) and undergoes almost exclusive single protonation using standard solvents. When Cr(III) is added to the solution, abundant [M + 2H]2+ forms, which involves protonation of the peptide backbone or the C-terminus. Salts of Al(III), Mn(II), Fe(III), Fe(II), Cu(II), Zn (II), Rh(III), La(III), Ce(IV), and Eu(III) were also studied. Although several metal ions slightly enhance protonation, Cr(III) has by far the greatest ability to generate [M + 2H]2+. Cr(III) does not supercharge peptide methyl esters, which suggests that the mechanism involves interaction of Cr(III) with a carboxylic acid group. Other factors may include the high acidity of hexa-aquochromium(III) and the resistance of Cr(III) to reduction. Nitrate salts enhance protonation more than chloride salts and a molar ratio of 10:1 Cr(III):peptide produces the most intense [M + 2H]2+. Cr(III) also supercharges numerous other small peptides, including highly acidic species. For basic peptides, Cr(III) increases the charge state (2+ versus 1+) and causes the number of peptide molecules being protonated to double or triple. Chromium(III) does not supercharge the proteins cytochrome c and myoglobin. The ability of Cr(III) to enhance [M + 2H]2+ intensity may prove useful in tandem mass spectrometry because of the resulting overall increase in signal-to-noise ratio, the fact that [M + 2H]2+ generally dissociate more readily than [M + H]+, and the ability to produce [M + 2H]2+ precursors for electron-based dissociation techniques.

  13. Peptide-modified optical filters for detecting protease activity.

    PubMed

    Kilian, Kristopher A; Böcking, Till; Gaus, Katharina; Gal, Michael; Gooding, J Justin

    2007-11-01

    The organic derivatization of silicon-based nanoporous photonic crystals is presented as a method to immobilize peptides for the detection of protease enzymes in solution. A narrow-line-width rugate filter, a one-dimensional photonic crystal, is fabricated that exhibits a high-reflectivity optical resonance that is sensitive to small changes in the refractive index at the pore walls. To immobilize peptide in the pore of the photonic crystal, the hydrogen-terminated silicon surface was first modified with the alkene 10-succinimidyl undecenoate via hydrosilylation. The monolayer with the succinimide ester moiety at the distal end served the dual function of protecting the underlying silicon from oxidation as well as providing a surface suitable for subsequent derivatization with amines. The surface was further modified with 1-aminohexa(ethylene glycol) (EG(6)) to resist nonspecific adsorption of proteins common in complex biological samples. The distal hydroxyl of the EG(6) is activated using the solid-phase coupling reagent disuccinimidyl carbonate for selective immobilization of peptides as protease recognition elements. X-ray photoelectron spectroscopy analysis reveals high activation and coupling efficiency at each stage of the functionalization. Exposure of the peptide-modified crystals to the protease subtilisin in solution causes a change in the refractive index, resulting in a shift of the resonance to shorter wavelengths, indicating cleavage of organic material within the pores. The lowest detected concentration of enzyme was 37 nM (7.4 pmol in 200 microL).

  14. A branched peptide mimotope of the nicotinic receptor binding site is a potent synthetic antidote against the snake neurotoxin alpha-bungarotoxin.

    PubMed

    Bracci, Luisa; Lozzi, Luisa; Pini, Alessandro; Lelli, Barbara; Falciani, Chiara; Niccolai, Neri; Bernini, Andrea; Spreafico, Adriano; Soldani, Patrizia; Neri, Paolo

    2002-08-13

    We previously produced synthetic peptides mimicking the snake neurotoxin binding site of the nicotinic receptor. These peptide mimotopes bind the snake neurotoxin alpha-bungarotoxin with higher affinity than peptides reproducing native receptor sequences and inhibit toxin binding to nicotinic receptors in vitro; yet their efficiency in vivo is low. Here we synthesized one of the peptide mimotopes in a tetrabranched MAP form. The MAP peptide binds alpha-bungarotoxin in solution and inhibits its binding to the receptor with a K(A) and an IC(50) similar to the monomeric peptide. Nonetheless, it is at least 100 times more active in vivo. The MAP completely neutralizes toxin lethality when injected in mice at a dose compatible with its use as a synthetic antidote in humans. The in vivo efficacy of the tetrameric peptide cannot be ascribed to a kinetic and thermodynamic effect and is probably related to different pharmacokinetic behavior of the tetrameric molecule, with respect to the monomer. Our findings bring new perspectives to the therapeutic use of multimeric peptides.

  15. Binding Site Prediction of Proteins with Organic Compounds or Peptides Using GALAXY Web Servers.

    PubMed

    Heo, Lim; Lee, Hasup; Baek, Minkyung; Seok, Chaok

    2016-01-01

    We introduce two GALAXY web servers called GalaxySite and GalaxyPepDock that predict protein complex structures with small organic compounds and peptides, respectively. GalaxySite predicts ligands that may bind the input protein and generates complex structures of the protein with the predicted ligands from the protein structure given as input or predicted from the input sequence. GalaxyPepDock takes a protein structure and a peptide sequence as input and predicts structures for the protein-peptide complex. Both GalaxySite and GalaxyPepDock rely on available experimentally resolved structures of protein-ligand complexes evolutionarily related to the target. With the continuously increasing size of the protein structure database, the probability of finding related proteins in the database is increasing. The servers further relax the complex structures to refine the structural aspects that are missing in the available structures or that are not compatible with the given protein by optimizing physicochemical interactions. GalaxyPepDock allows conformational change of the protein receptor induced by peptide binding. The atomistic interactions with ligands predicted by the GALAXY servers may offer important clues for designing new molecules or proteins with desired binding properties. PMID:27094284

  16. Structure and Activity of Human Mitochondrial Peptide Deformylase, a Novel Cancer Target

    SciTech Connect

    Escobar-Alvarez, Sindy; Goldgur, Yehuda; Yang, Guangli; Ouerfelli, Ouathek; Li, Yueming; Scheinberg, David A.

    2009-07-21

    Peptide deformylase proteins (PDFs) participate in the N-terminal methionine excision pathway of newly synthesized peptides. We show that the human PDF (HsPDF) can deformylate its putative substrates derived from mitochondrial DNA-encoded proteins. The first structural model of a mammalian PDF (1.7 A), HsPDF, shows a dimer with conserved topology of the catalytic residues and fold as non-mammalian PDFs. The HsPDF C-terminus topology and the presence of a helical loop (H2 and H3), however, shape a characteristic active site entrance. The structure of HsPDF bound to the peptidomimetic inhibitor actinonin (1.7 A) identified the substrate-binding site. A defined S1' pocket, but no S2' or S3' substrate-binding pockets, exists. A conservation of PDF-actinonin interaction across PDFs was observed. Despite the lack of true S2' and S3' binding pockets, confirmed through peptide binding modeling, enzyme kinetics suggest a combined contribution from P2'and P3' positions of a formylated peptide substrate to turnover.

  17. Vasoactive intestinal peptide and electrical activity influence neuronal survival

    SciTech Connect

    Brenneman, D.E.; Eiden, L.E.

    1986-02-01

    Blockage of electrical activity in dissociated spinal cord cultures results in a significant loss of neurons during a critical period in development. Decreases in neuronal cell numbers and SVI-labeled tetanus toxin fixation produced by electrical blockage with tetrodotoxin (TTX) were prevented by addition of vasoactive intestinal peptide (VIP) to the nutrient medium. The most effective concentration of VIP was 0.1 nM. At higher concentrations, the survival-enhancing effect of VIP on TTX-treated cultures was attenuated. Addition of the peptide alone had no significant effect on neuronal cell counts or tetanus toxin fixation. With the same experimental conditions, two closely related peptides, PHI-27 (peptide, histidyl-isoleucine amide) and secretin, were found not to increase the number of neurons in TTX-treated cultures. Interference with VIP action by VIP antiserum resulted in neuronal losses that were not significantly different from those observed after TTX treatment. These data indicate that under conditions of electrical blockade a neurotrophic action of VIP on neuronal survival can be demonstrated.

  18. Prediction of Signal Peptide Cleavage Sites with Subsite-Coupled and Template Matching Fusion Algorithm.

    PubMed

    Zhang, Shao-Wu; Zhang, Ting-He; Zhang, Jun-Nan; Huang, Yufei

    2014-03-01

    Fast and effective prediction of signal peptides (SP) and their cleavage sites is of great importance in computational biology. The approaches developed to predict signal peptide can be roughly divided into machine learning based, and sliding windows based. In order to further increase the prediction accuracy and coverage of organism for SP cleavage sites, we propose a novel method for predicting SP cleavage sites called Signal-CTF that utilizes machine learning and sliding windows, and is designed for N-termial secretory proteins in a large variety of organisms including human, animal, plant, virus, bacteria, fungi and archaea. Signal-CTF consists of three distinct elements: (1) a subsite-coupled and regularization function with a scaled window of fixed width that selects a set of candidates of possible secretion-cleavable segment for a query secretory protein; (2) a sum fusion system that integrates the outcomes from aligning the cleavage site template sequence with each of the aforementioned candidates in a scaled window of fixed width to determine the best candidate cleavage sites for the query secretory protein; (3) a voting system that identifies the ultimate signal peptide cleavage site among all possible results derived from using scaled windows of different width. When compared with Signal-3L and SignalP 4.0 predictors, the prediction accuracy of Signal-CTF is 4-12 %, 10-25 % higher than that of Signal-3L for human, animal and eukaryote, and SignalP 4.0 for eukaryota, Gram-positive bacteria and Gram-negative bacteria, respectively. Comparing with PRED-SIGNAL and SignalP 4.0 predictors on the 32 archaea secretory proteins of used in Bagos's paper, the prediction accuracy of Signal-CTF is 12.5 %, 25 % higher than that of PRED-SIGNAL and SignalP 4.0, respectively. The predicting results of several long signal peptides show that the Signal-CTF can better predict cleavage sites for long signal peptides than SignalP, Phobius, Philius, SPOCTOPUS, Signal

  19. HIV-1 Env gp120 Structural Determinants for Peptide Triazole Dual Receptor Site Antagonism

    PubMed Central

    Tuzer, Ferit; Madani, Navid; Kamanna, Kantharaju; Zentner, Isaac; LaLonde, Judith; Holmes, Andrew; Upton, Elizabeth; Rajagopal, Srivats; McFadden, Karyn; Contarino, Mark; Sodroski, Joseph; Chaiken, Irwin

    2013-01-01

    Despite advances in HIV therapy, viral resistance and side-effects with current drug regimens require targeting new components of the virus. Dual antagonist peptide triazoles (PT) are a novel class of HIV-1 inhibitors that specifically target the gp120 component of the viral spike and inhibit its interaction with both of its cell surface protein ligands, namely the initial receptor CD4 and the co-receptor (CCR5/CXCR4), thus preventing viral entry. Following an initial survey of 19 gp120 alanine mutants by ELISA, we screened 11 mutants for their importance in binding to, and inhibition by the PT KR21 using surface plasmon resonance. Key mutants were purified and tested for their effects on the peptide’s affinity and its ability to inhibit binding of CD4 and the co-receptor surrogate mAb 17b. Effects of the mutations on KR21 viral neutralization were measured by single-round cell infection assays. Two mutations, D474A and T257A, caused large-scale loss of KR21 binding, as well as losses in both CD4/17b and viral inhibition by KR21. A set of other Ala mutants revealed more moderate losses in direct binding affinity and inhibition sensitivity to KR21. The cluster of sensitive residues defines a PT functional epitope. This site is in a conserved region of gp120 that overlaps the CD4 binding site and is distant from the co-receptor/17b binding site, suggesting an allosteric mode of inhibition for the latter. The arrangement and sequence conservation of the residues in the functional epitope explain the breadth of antiviral activity, and improve the potential for rational inhibitor development. PMID:23011758

  20. Biological activity and biotechnological aspects of peptide nucleic acid.

    PubMed

    Lundin, Karin E; Good, Liam; Strömberg, Roger; Gräslund, Astrid; Smith, C I Edvard

    2006-01-01

    During the latest decades a number of different nucleic acid analogs containing natural nucleobases on a modified backbone have been synthesized. An example of this is peptide nucleic acid (PNA), a DNA mimic with a noncyclic peptide-like backbone, which was first synthesized in 1991. Owing to its flexible and neutral backbone PNA displays very good hybridization properties also at low-ion concentrations and has subsequently attracted large interest both in biotechnology and biomedicine. Numerous modifications have been made, which could be of value for particular settings. However, the original PNA does so far perform well in many diverse applications. The high biostability makes it interesting for in vivo use, although the very limited diffusion over lipid membranes requires further modifications in order to make it suitable for treatment in eukaryotic cells. The possibility to use this nucleic acid analog for gene regulation and gene editing is discussed. Peptide nucleic acid is now also used for specific genetic detection in a number of diagnostic techniques, as well as for site-specific labeling and hybridization of functional molecules to both DNA and RNA, areas that are also discussed in this chapter.

  1. Biological activities of selected peptides: skin penetration ability of copper complexes with peptides.

    PubMed

    Mazurowska, Lena; Mojski, Miroslaw

    2008-01-01

    This study concerning the permeability through skin barriers of copper complexes with peptides is an important part of the research on their biological activity. The transport of copper complexes through the skin is essential in treatment of dermatological dysfunctions connected to the deficiency of these elements in the skin. During the last several years, a special interest in transepidermal copper delivery has been observed. This is the reason why copper compounds have been used as active compounds in care cosmetics. Yet, the transport process of copper complexes with tripeptides, glycyl-histidyl-lysine GHK, or gamma-glutamyl-cysteinyl-glycine GSH through the stratum corneum has received very little attention in the literature so far. The penetration ability of GHK-Cu and GSH-Cu through the stratum corneum and the influence of the complexes with tripeptide on the copper ion transport process is the key factor in their cosmetic and pharmaceutical activity. The in vitro penetration process was studied in the model system, a Franz diffusion cell with a liposome membrane, where liquid crystalline systems of physicochemical properties similar to the ones of the intercellular cement of stratum corneum were used as a standard model of a skin barrier. The results obtained demonstrated that copper complexes permeate through the membranes modeling the horny lipid layer and showed the influence of peptides on the dynamics of copper ion diffusion. PMID:18350235

  2. The vaccine-site microenvironment induced by injection of incomplete Freund's adjuvant, with or without melanoma peptides

    PubMed Central

    Harris, Rebecca C.; Chianese-Bullock, Kimberly A.; Petroni, Gina R.; Schaefer, Jochen T.; Brill, Louis B.; Molhoek, Kerrington R.; Deacon, Donna H.; Patterson, James W.; Slingluff, Craig L.

    2011-01-01

    Cancer vaccines have not been optimized. They depend on adjuvants to create an immunogenic microenvironment for antigen presentation. However, remarkably little is understood about cellular and molecular changes induced by these adjuvants in the vaccine microenvironment. We hypothesized that vaccination induces dendritic cell activation in the dermal vaccination microenvironment but that regulatory processes may also limit the effectiveness of repeated vaccination. We evaluated biopsies from immunization sites in two clinical trials of melanoma patients. In one study (Mel38), patients received one injection with an adjuvant mixture alone, comprised of incomplete Freund's adjuvant (IFA) plus granulocyte-macrophage colony stimulating factor (GM-CSF). In a second study, patients received multiple vaccinations with melanoma peptide antigens plus IFA. Single injections with adjuvant alone induced dermal inflammatory infiltrates consisting of B cells, T cells, mature dendritic cells (DC) and vessels resembling high endothelial venules (HEV). These cellular aggregates usually lacked organization and were transient. In contrast, multiple repeated vaccinations with peptides in adjuvant induced more organized and persistent lymphoid aggregates containing separate B and T cell areas, mature DC, HEV-like vessels, and lymphoid chemokines. Within these structures, there are proliferating CD4+ and CD8+ T lymphocytes, as well as FoxP3+CD4+ lymphocytes, suggesting a complex interplay of lymphoid expansion and regulation within the dermal immunization microenvironment. Further study of the physiology of the vaccine site microenvironment promises to identify opportunities for enhancing cancer vaccine efficacy by modulating immune activation and regulation at the site of vaccination. PMID:22130163

  3. The vaccine-site microenvironment induced by injection of incomplete Freund's adjuvant, with or without melanoma peptides.

    PubMed

    Harris, Rebecca C; Chianese-Bullock, Kimberly A; Petroni, Gina R; Schaefer, Jochen T; Brill, Louis B; Molhoek, Kerrington R; Deacon, Donna H; Patterson, James W; Slingluff, Craig L

    2012-01-01

    Cancer vaccines have not been optimized. They depend on adjuvants to create an immunogenic microenvironment for antigen presentation. However, remarkably little is understood about cellular and molecular changes induced by these adjuvants in the vaccine microenvironment. We hypothesized that vaccination induces dendritic cell (DC) activation in the dermal vaccination microenvironment but that regulatory processes may also limit the effectiveness of repeated vaccination. We evaluated biopsies from immunization sites in 2 clinical trials of melanoma patients. In 1 study (Mel38), patients received 1 injection with an adjuvant mixture alone, composed of incomplete Freund's adjuvant (IFA) plus granulocyte-macrophage colony stimulating factor (GM-CSF). In a second study, patients received multiple vaccinations with melanoma peptide antigens plus IFA. Single injections with adjuvant alone induced dermal inflammatory infiltrates consisting of B cells, T cells, mature DCs, and vessels resembling high endothelial venules (HEVs). These cellular aggregates usually lacked organization and were transient. In contrast, multiple repeated vaccinations with peptides in adjuvant induced more organized and persistent lymphoid aggregates containing separate B and T cell areas, mature DCs, HEV-like vessels, and lymphoid chemokines. Within these structures, there are proliferating CD4and CD8 T lymphocytes, as well as FoxP3CD4 lymphocytes, suggesting a complex interplay of lymphoid expansion and regulation within the dermal immunization microenvironment. Further study of the physiology of the vaccine site microenvironment promises to identify opportunities for enhancing cancer vaccine efficacy by modulating immune activation and regulation at the site of vaccination. PMID:22130163

  4. Allosterically Regulated Phosphatase Activity from Peptide-PNA Conjugates Folded Through Hybridization.

    PubMed

    Machida, Takuya; Dutt, Som; Winssinger, Nicolas

    2016-07-18

    The importance of spatial organization in short peptide catalysts is well recognized. We synthesized and screened a library of peptides flanked by peptide nucleic acids (PNAs) such that the peptide would be constrained in a hairpin loop upon hybridization. A screen for phosphatase activity led to the discovery of a catalyst with >25-fold rate acceleration over the linear peptide. We demonstrated that the hybridization-enforced folding of the peptide is necessary for activity, and designed a catalyst that is allosterically controlled using a complementary PNA sequence. PMID:27320214

  5. Identification of sites in adenovirus hexon for foreign peptide incorporation.

    PubMed

    Wu, Hongju; Han, Tie; Belousova, Natalya; Krasnykh, Victor; Kashentseva, Elena; Dmitriev, Igor; Kataram, Manjula; Mahasreshti, Parameshwar J; Curiel, David T

    2005-03-01

    Adenovirus type 5 (Ad5) is one of the most promising vectors for gene therapy applications. Genetic engineering of Ad5 capsid proteins has been employed to redirect vector tropism, to enhance infectivity, or to circumvent preexisting host immunity. As the most abundant capsid protein, hexon modification is particularly attractive. However, genetic modification of hexon often results in failure of rescuing viable viruses. Since hypervariable regions (HVRs) are nonconserved among hexons of different serotypes, we investigated whether the HVRs could be used for genetic modification of hexon by incorporating oligonucleotides encoding six histidine residues (His6) into different HVRs in the Ad5 genome. The modified viruses were successfully rescued, and the yields of viral production were similar to that of unmodified Ad5. A thermostability assay suggested the modified viruses were stable. The His6 epitopes were expressed in all modified hexon proteins as assessed by Western blotting assay, although the intensity of the reactive bands varied. In addition, we examined the binding activity of anti-His tag antibody to the intact virions with the enzyme-linked immunosorbent assay and found the His6 epitopes incorporated in HVR2 and HVR5 could bind to anti-His tag antibody. This suggested the His6 epitopes in HVR2 and HVR5 were exposed on virion surfaces. Finally, we examined the infectivities of the modified Ad vectors. The His6 epitopes did not affect the native infectivity of Ad5 vectors. In addition, the His6 epitopes did not appear to mediate His6-dependent viral infection, as assessed in two His6 artificial receptor systems. Our study provided valuable information for studies involving hexon modification. PMID:15731232

  6. Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase

    SciTech Connect

    Yip, Wing-Kin; Dong, Jian-Guo; Yang, S.F. ); Kenny, J.W.; Thompson, G.A. )

    1990-10-01

    The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase the key enzyme in ethylene biosynthesis, is inactivated by its substrate S-adenosylmethionine (AdoMet). Apple ACC synthase was purified with an immunoaffinity gel, and its active site was probed with NaB{sup 3}H{sub 4} or Ado({sup 14}C)Met. Peptide sequencing of both {sup 3}H- and {sup 14}C-labeled peptides revealed a common dodecapeptide of Ser-Leu-Ser-Xaa-Asp-Leu-Gly-Leu-Pro-Gly-Phe-Arg, where Xaa was the modified, radioactive residue in each case. Acid hydrolysis of the {sup 3}H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the {sup 14}C-labeled peptide indicated a protonated molecular ion at m/z 1390.6, from which the mass of Xaa was calculated to be 229, a number that is equivalent to the mass of a lysine residue alkylated by the 2-aminobutyrate portion of AdoMet, as we previously proposed. These results indicate that the same active-site lysine binds the PLP and convalently links to the 2-aminobutyrate portion of AdoMet during inactivation. The active site of tomato ACC synthase was probed in the same manner with Ado ({sup 14}C)Met. Sequencing of the tomato active-site peptide revealed two highly conserved dodecapeptides; the minor peptide possessed a sequence identical to that of the apple enzyme, whereas the major peptide differed from the minor peptide in that methionine replaced leucine at position 6.

  7. Characterization of antimicrobial peptide activity by electrochemical impedance spectroscopy

    PubMed Central

    Chang, William K.; Wimley, William C.; Searson, Peter C.; Hristova, Kalina; Merzlyakov, Mikhail

    2008-01-01

    Summary Electrochemical impedance spectroscopy performed on surface-supported bilayer membranes allows for the monitoring of changes in membrane properties, such as thickness, ion permeability, and homogeneity, after exposure to antimicrobial peptides (AMPs). We show that two model cationic peptides, very similar in sequence but different in activity, induce dramatically different changes in membrane properties as probed by impedance spectroscopy. Moreover, the impedance results excluded the “barrel-stave” and the “toroidal pore” models of AMP mode of action, and are more consistent with the “carpet” and the “detergent” models. The impedance data provide important new insights about the kinetics and the scale of the peptide action which currently are not addressed by the “carpet” and the “detergent” models. The method presented not only provides additional information about the mode of action of a particular AMP, but offers a means of characterizing AMP activity in reproducible, well-defined quantitative terms. PMID:18657512

  8. Influence of metallocene substitution on the antibacterial activity of multivalent peptide conjugates.

    PubMed

    Hoffknecht, Barbara C; Prochnow, Pascal; Bandow, Julia E; Metzler-Nolte, Nils

    2016-07-01

    Peptide dendrimers and derivatisation of peptides with metallocenes showed promising results in the search for new antibacterial agents. The two concepts are combined in this work leading to multivalent, metallocene-containing peptide derivates. These new peptides were synthesised utilising microwave assisted, copper(I) catalyzed alkyne-azide cycloaddition (CuAAC, "click" chemistry). Twelve new peptide conjugates, containing either a ferrocenoyl group or a ruthenocenoyl group on so-called ultrashort (i.e. < 5 amino acids) peptides, and ranging from monovalent to trivalent conjugates, were synthesised and their antibacterial activity was investigated by minimal inhibitory concentration (MIC) assays on five different bacterial strains. The antibacterial activity was compared to the same peptide conjugates without metallocenes. The resulting MIC values showed a significant enhancement of the antibacterial activity of these peptide conjugates against Gram-positive bacteria by the metallocenoyl groups. Additionally, the compounds with two metallocenoyl groups presented the best antibacterial activities overall. PMID:26988572

  9. Data for identification of GPI-anchored peptides and ω-sites in cancer cell lines

    PubMed Central

    Masuishi, Yusuke; Kimura, Yayoi; Arakawa, Noriaki; Hirano, Hisashi

    2016-01-01

    We present data obtained using a focused proteomics approach to identify the glycosylphosphatidylinositol (GPI)-anchored peptides in 19 human cancer cell lines. GPI-anchored proteins (GPI-APs), which localize to the outer leaflet of the membrane microdomains commonly referred to as lipid rafts play important roles in diverse biological processes. Due to the complex structure of the GPI-anchor moiety, it has been difficult to identify GPI-anchored peptide sequences on the proteomic scale by database searches using tools such as MASCOT. Here we provide data from 73 ω-sites derived from 49 GPI-APs in 19 human cancer cell lines. This article contains data related to the research article entitled “Identification of glycosylphosphatidylinositol-anchored proteins and ω-sites using TiO2-based affinity purification followed by hydrogen fluoride treatment” (Masuishi et al., 2016) [1]. PMID:27141528

  10. Data for identification of GPI-anchored peptides and ω-sites in cancer cell lines.

    PubMed

    Masuishi, Yusuke; Kimura, Yayoi; Arakawa, Noriaki; Hirano, Hisashi

    2016-06-01

    We present data obtained using a focused proteomics approach to identify the glycosylphosphatidylinositol (GPI)-anchored peptides in 19 human cancer cell lines. GPI-anchored proteins (GPI-APs), which localize to the outer leaflet of the membrane microdomains commonly referred to as lipid rafts play important roles in diverse biological processes. Due to the complex structure of the GPI-anchor moiety, it has been difficult to identify GPI-anchored peptide sequences on the proteomic scale by database searches using tools such as MASCOT. Here we provide data from 73 ω-sites derived from 49 GPI-APs in 19 human cancer cell lines. This article contains data related to the research article entitled "Identification of glycosylphosphatidylinositol-anchored proteins and ω-sites using TiO2-based affinity purification followed by hydrogen fluoride treatment" (Masuishi et al., 2016) [1]. PMID:27141528

  11. Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor.

    PubMed

    Parameswaran, N; Disa, J; Spielman, W S; Brooks, D P; Nambi, P; Aiyar, N

    2000-02-18

    Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8-37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11, 11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3, 2-de]indeno[4,5-h]-2

  12. Two Peptides Derived from the Nerve Growth Factor Precursor Are Biologically Active

    PubMed Central

    Dicou, Eleni; Pflug, Beth; Magazin, Marilyn; Lehy, Thérèse; Djakiew, Daniel; Ferrara, Pascual; Nerrière, Véronique; Harvie, Douglas

    1997-01-01

    This report provides evidence that the proregion of the NGF precursor protein contains two novel bioactive peptides. The presence of pairs of basic amino acid (aa) residues in the NGF proregion suggests that two or three peptides other than NGF may be generated by proteolytic cleavage. Synthetic peptides of 29 aa (LIP1) and 38aa (LIP2) corresponding to the sequences −71 to −43 and −40 to −3 of the proNGF, respectively, were used in this study. ELISA specific for these two peptides revealed their presence in the rat intestine. LIP1 was localized by immunohistochemistry in endocrine cells of the intestinal epithelium, and LIP2 was immunoprecipitated from an intestinal extract. We also provide evidence for the presence of specific receptors for LIP2 in several cell lines. Scatchard analysis indicated the presence of a low affinity binding site with a Kd of ∼10−7 M and a high affinity binding site of 10−9 M. Cross-linking studies revealed receptor forms of about 140 kD and 93 kD in a prostatic adenocarcinoma cell line. LIP1 and LIP2 induced rapid F-actin redistribution in PC12 cells within 2 min of incubation, which suggests a role of LIP1 and LIP2 in the process of neurite outgrowth. Furthermore, both propeptides induced rapid tyrosine phosphorylation of the Trk protein in both prostatic adenocarcinoma cells and PC12 cells, thus implicating trk in their mechanism of action. These results support our hypothesis that two peptides within the NGF precursor protein are biologically active. PMID:9015309

  13. Cytolytic and antibacterial activity of synthetic peptides derived from amoebapore, the pore-forming peptide of Entamoeba histolytica.

    PubMed Central

    Leippe, M; Andrä, J; Müller-Eberhard, H J

    1994-01-01

    The pore-forming peptide amoebapore is considered part of the cytolytic armament of pathogenic Entamoeba histolytica. Amoebapore is composed of 77 amino acid residues arranged in four alpha-helical domains. For structure-function analysis, synthetic peptides were constructed corresponding to these four domains: H1 (residues 1-22), H2 (25-39), H3 (40-64), and H4 (67-77). The peptides H1 and H3, representing two highly amphipathic alpha-helical regions of amoebapore, possessed pore-forming activity. Peptide H3 displayed cytolytic and antibacterial functions similar to those of natural amoebapore. The most potent antibacterial activity and the broadest activity spectrum were expressed by H1-Mel, a hybrid molecule composed of the N-terminal alpha-helix of amoebapore and the C-terminal hexapeptide of melittin from the venom of Apis mellifera. Images PMID:8146160

  14. Swedish mutant APP-based BACE1 binding site peptide reduces APP β-cleavage and cerebral Aβ levels in Alzheimer’s mice

    PubMed Central

    Li, Song; Hou, Huayan; Mori, Takashi; Sawmiller, Darrell; Smith, Adam; Tian, Jun; Wang, Yanjiang; Giunta, Brian; Sanberg, Paul R.; Zhang, Sheqing; Tan, Jun

    2015-01-01

    BACE1 initiates amyloid-β (Aβ) generation and the resultant cerebral amyloidosis, as a characteristic of Alzheimer’s disease (AD). Thus, inhibition of BACE1 has been the focus of a large body of research. The most recent clinical trials highlight the difficulty involved in this type of anti-AD therapy as evidenced by side effects likely due to the ubiquitous nature of BACE1, which cleaves multiple substrates. The human Swedish mutant form of amyloid protein precursor (APPswe) has been shown to possess a higher affinity for BACE1 compared to wild-type APP (APPwt). We pursued a new approach wherein harnessing this greater affinity to modulate BACE1 APP processing activity. We found that one peptide derived from APPswe, containing the β-cleavage site, strongly inhibits BACE1 activity and thereby reduces Aβ production. This peptide, termed APPswe BACE1 binding site peptide (APPsweBBP), was further conjugated to the fusion domain of the HIV-1 Tat protein (TAT) at the C-terminus to facilitate its biomembrane-penetrating activity. APPwt and APPswe over-expressing CHO cells treated with this TAT-conjugated peptide resulted in a marked reduction of Aβ and a significant increase of soluble APPα. Intraperitoneal administration of this peptide to 5XFAD mice markedly reduced β-amyloid deposits as well as improved hippocampal-dependent learning and memory. PMID:26091071

  15. Identification of the active-site serine in human lecithin: cholesterol acyltransferase

    SciTech Connect

    Farooqui, J.; Wohl, R.C.; Kezdy, F.J.; Scanu, A.M.

    1987-05-01

    Lecithin:cholesterol acyltransferase (LCAT) from human plasma reacts stoichiometrically with diisopropylphosphorofluoridate (DFP) resulting in the complete loss of transacylase activity. Purified LCAT was covalently labeled with (TH) DFP and the labeled protein was reduced and carboxymethylated. Cyanogen bromide cleavage followed by gel permeation chromatography yielded a peptide of 4-5 KDa (LCAT CNBr-III) containing most of the radioactive label. Preliminary studies comparing the amino acid composition of the LCAT-CNBr-III with the sequence of LCAT indicate that this peptide corresponds to fragment 168-220. Automated Edman degradation of the radioactive peptide recovered a radioactive PTC-amino acid at cycle 14. Of all predicted CNBr fragments only peptide 168-220 contained a serine at residue 14 from the amino terminus of the peptide. The authors conclude that serine 181 is the active site serine of LCAT.

  16. Antimicrobial activity of antihypertensive food-derived peptides and selected alanine analogues.

    PubMed

    McClean, Stephen; Beggs, Louise B; Welch, Robert W

    2014-03-01

    This study evaluated four food-derived peptides with known antihypertensive activities for antimicrobial activity against pathogenic microorganisms, and assessed structure-function relationships using alanine analogues. The peptides (EVSLNSGYY, barley; PGTAVFK, soybean; TTMPLW, α-casein; VHLPP, α-zein) and the six alanine substitution peptides of PGTAVFK were synthesised, characterised and evaluated for antimicrobial activity using the bacteria, Escherichia coli, Staphylococcus aureus, and Micrococcus luteus and the yeast, Candida albicans. The peptides TTMPLW and PGTAVFK inhibited growth of all four microorganisms tested, with activities of a similar order of magnitude to ampicillin and ethanol controls. EVSLNSGYY inhibited the growth of the bacteria, but VHLPP showed no antimicrobial activity. The alanine analogue, PGAAVFK showed the highest overall antimicrobial activity and PGTAVFA showed no activity; overall, the activities of the analogues were consistent with their structures. Some peptides with antihypertensive activity also show antimicrobial activity, suggesting that food-derived peptides may exert beneficial effects via a number of mechanisms.

  17. Modulation of host defense peptide-mediated human mast cell activation by LPS

    PubMed Central

    Gupta, Kshitij; Subramanian, Hariharan; Ali, Hydar

    2016-01-01

    Human β-defensin3 (hBD3) and the cathelicidin LL-37 are host defense peptides (HDPs) that directly kill microbes and display immunomodulatory/wound healing properties via the activation of chemokine, formylpeptide and epidermal growth factor receptors on monocytes and epithelial cells. A C-terminal 14 amino acid hBD3 peptide with all Cys residues replaced with Ser (CHRG01) and an LL-37 peptide consisting of residues 17-29 (FK-13) display antimicrobial activity but lack immunomodulatory property. Surprisingly, we found that CHRG01 and FK-13 caused Ca2+ mobilization and degranulation in human mast cells via a novel G protein coupled receptor (GPCR) known as Mas-related gene-X2 (MrgX2). At local sites of bacterial infection, the negatively charged LPS likely interacts with cationic HDPs to inhibit their activity and thus providing a mechanism for pathogens to escape the host defense mechanisms. We found that LPS caused almost complete inhibition of hBD3 and LL-37-induced Ca2+ mobilization and mast cell degranulation. In contrast, it had no effect on CHRG01 and FK-13-induced mast cell responses. These findings suggest that HDP derivatives that kill microbes, harness mast cell’s host defense and wound healing properties via the activation of MrgX2 but are resistant to inhibition by LPS could be utilized for the treatment of antibiotic-resistant microbial infections. PMID:26511058

  18. Activities of calcitonin gene-related peptide (CGRP) and related peptides at the CGRP receptor

    SciTech Connect

    Maton, P.N.; Pradhan, T.; Zhou, Z.C.; Gardner, J.D.; Jensen, R.T. )

    1990-05-01

    In guinea pig pancreatic acini rat calcitonin gene-related peptide (CGRP) increased amylase release 2-fold, salmon calcitonin had an efficacy of only 44% of that of CGRP and (Tyr0)CGRP(28-37) and human calcitonin had no actions. (Tyr0)CGRP(28-37), but not human calcitonin, antagonized the actions of CGRP in pancreatic acini with an IC50 of 3 microM. (Tyr0)CGRP(28-37) produced a parallel rightward shift in the dose-response curve for CGRP-stimulated amylase secretion. The inhibition was specific for CGRP and was reversible. Studies with 125I-CGRP demonstrated that CGRP, salmon calcitonin and (Tyr0)CGRP, but not human calcitonin, interacted with CGRP receptors on pancreatic acini. These results indicate that various CGRP-related peptides demonstrate different relationships between their abilities to occupy the CGRP receptor and to affect biologic activity, with CGRP itself being a full agonist, salmon calcitonin a partial agonist, (Tyr0)CGRP(28-37) a competitive antagonist, and human calcitonin having no actions.

  19. Insights into the Binding Sites of Organometallic Ruthenium Anticancer Compounds on Peptides Using Ultra-High Resolution Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Wills, Rebecca H.; Habtemariam, Abraha; Lopez-Clavijo, Andrea F.; Barrow, Mark P.; Sadler, Peter J.; O'Connor, Peter B.

    2014-04-01

    The binding sites of two ruthenium(II) organometallic complexes of the form [(η6-arene)Ru( N, N)Cl]+, where arene/ N, N = biphenyl (bip)/bipyridine (bipy) for complex AH076, and biphenyl (bip)/ o-phenylenediamine ( o-pda) for complex AH078, on the peptides angiotensin and bombesin have been investigated using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Fragmentation was performed using collisionally activated dissociation (CAD), with, in some cases, additional data being provided by electron capture dissociation (ECD). The primary binding sites were identified as methionine and histidine, with further coordination to phenylalanine, potentially through a π-stacking interaction, which has been observed here for the first time. This initial peptide study was expanded to investigate protein binding through reaction with insulin, on which the binding sites proposed are histidine, glutamic acid, and tyrosine. Further reaction of the ruthenium complexes with the oxidized B chain of insulin, in which two cysteine residues are oxidized to cysteine sulfonic acid (Cys-SO3H), and glutathione, which had been oxidized with hydrogen peroxide to convert the cysteine to cysteine sulfonic acid, provided further support for histidine and glutamic acid binding, respectively.

  20. Parallel activities and interactions between antimicrobial peptides and complement in host defense at the airway epithelial surface.

    PubMed

    Hiemstra, Pieter S

    2015-11-01

    Antimicrobial peptides and complement components contribute to host defense as well as inflammation and tissue injury in the respiratory tract. The airway epithelial surface is the main site of action of these immune effectors, and airway epithelial cells contribute markedly to their local production. Whereas both antimicrobial peptides and complement display overlapping functions, it is increasingly clear that both effector mechanisms also interact. Furthermore, excessive or uncontrolled release of antimicrobial peptides as well as complement activation may contribute to inflammatory lung diseases. Therefore, further knowledge of interactions between these systems may provide more insight into the pathogenesis of a range of lung diseases. In this review, recent findings on the functions, collaborations and other interactions between antimicrobial peptides and complement are discussed with a specific focus on the airway epithelium.

  1. Improved Identification and Relative Quantification of Sites of Peptide and Protein Oxidation for Hydroxyl Radical Footprinting

    NASA Astrophysics Data System (ADS)

    Li, Xiaoyan; Li, Zixuan; Xie, Boer; Sharp, Joshua S.

    2013-11-01

    Protein oxidation is typically associated with oxidative stress and aging and affects protein function in normal and pathological processes. Additionally, deliberate oxidative labeling is used to probe protein structure and protein-ligand interactions in hydroxyl radical protein footprinting (HRPF). Oxidation often occurs at multiple sites, leading to mixtures of oxidation isomers that differ only by the site of modification. We utilized sets of synthetic, isomeric "oxidized" peptides to test and compare the ability of electron-transfer dissociation (ETD) and collision-induced dissociation (CID), as well as nano-ultra high performance liquid chromatography (nanoUPLC) separation, to quantitate oxidation isomers with one oxidation at multiple adjacent sites in mixtures of peptides. Tandem mass spectrometry by ETD generates fragment ion ratios that accurately report on relative oxidative modification extent on specific sites, regardless of the charge state of the precursor ion. Conversely, CID was found to generate quantitative MS/MS product ions only at the higher precursor charge state. Oxidized isomers having multiple sites of oxidation in each of two peptide sequences in HRPF product of protein Robo-1 Ig1-2, a protein involved in nervous system axon guidance, were also identified and the oxidation extent at each residue was quantified by ETD without prior liquid chromatography (LC) separation. ETD has proven to be a reliable technique for simultaneous identification and relative quantification of a variety of functionally different oxidation isomers, and is a valuable tool for the study of oxidative stress, as well as for improving spatial resolution for HRPF studies.

  2. Autoradiographic localization of peptide YY and neuropeptide Y binding sites in the medulla oblongata

    SciTech Connect

    Leslie, R.A.; McDonald, T.J.; Robertson, H.A.

    1988-09-01

    Peptide YY is a highly potent emetic when given intravenously in dogs. We hypothesized that the area postrema, a small brain stem nucleus that acts as a chemoreceptive trigger zone for vomiting and lies outside the blood-brain barrier, might have receptors that PYY would bind to, in order to mediate the emetic response. We prepared (/sup 125/I)PYY and used autoradiography to show that high affinity binding sites for this ligand were highly localized in the area postrema and related nuclei of the dog medulla oblongata. Furthermore, the distribution of (/sup 125/I)PYY binding sites in the rat medulla oblongata was very similar to that in the dog; the distribution of (/sup 125/I)PYY binding sites throughout the rat brain was seen to be similar to the distribution of (/sup 125/I)NPY binding sites.

  3. Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor

    PubMed Central

    Booe, Jason M.; Walker, Christopher S.; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A.; Warner, Margaret L.; Bill, Roslyn M.; Harris, Paul W.; Brimble, Margaret A.; Poyner, David R.; Hay, Debbie L.; Pioszak, Augen A.

    2015-01-01

    Summary Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes. PMID:25982113

  4. High throughput peptide mapping method for analysis of site specific monoclonal antibody oxidation.

    PubMed

    Li, Xiaojuan; Xu, Wei; Wang, Yi; Zhao, Jia; Liu, Yan-Hui; Richardson, Daisy; Li, Huijuan; Shameem, Mohammed; Yang, Xiaoyu

    2016-08-19

    Oxidation of therapeutic monoclonal antibodies (mAbs) often occurs on surface exposed methionine and tryptophan residues during their production in cell culture, purification, and storage, and can potentially impact the binding to their targets. Characterization of site specific oxidation is critical for antibody quality control. Antibody oxidation is commonly determined by peptide mapping/LC-MS methods, which normally require a long (up to 24h) digestion step. The prolonged sample preparation procedure could result in oxidation artifacts of susceptible methionine and tryptophan residues. In this paper, we developed a rapid and simple UV based peptide mapping method that incorporates an 8-min trypsin in-solution digestion protocol for analysis of oxidation. This method is able to determine oxidation levels at specific residues of a mAb based on the peptide UV traces within <1h, from either TBHP treated or UV light stressed samples. This is the simplest and fastest method reported thus far for site specific oxidation analysis, and can be applied for routine or high throughput analysis of mAb oxidation during various stability and degradation studies. By using the UV trace, the method allows more accurate measurement than mass spectrometry and can be potentially implemented as a release assay. It has been successfully used to monitor antibody oxidation in real time stability studies.

  5. High throughput peptide mapping method for analysis of site specific monoclonal antibody oxidation.

    PubMed

    Li, Xiaojuan; Xu, Wei; Wang, Yi; Zhao, Jia; Liu, Yan-Hui; Richardson, Daisy; Li, Huijuan; Shameem, Mohammed; Yang, Xiaoyu

    2016-08-19

    Oxidation of therapeutic monoclonal antibodies (mAbs) often occurs on surface exposed methionine and tryptophan residues during their production in cell culture, purification, and storage, and can potentially impact the binding to their targets. Characterization of site specific oxidation is critical for antibody quality control. Antibody oxidation is commonly determined by peptide mapping/LC-MS methods, which normally require a long (up to 24h) digestion step. The prolonged sample preparation procedure could result in oxidation artifacts of susceptible methionine and tryptophan residues. In this paper, we developed a rapid and simple UV based peptide mapping method that incorporates an 8-min trypsin in-solution digestion protocol for analysis of oxidation. This method is able to determine oxidation levels at specific residues of a mAb based on the peptide UV traces within <1h, from either TBHP treated or UV light stressed samples. This is the simplest and fastest method reported thus far for site specific oxidation analysis, and can be applied for routine or high throughput analysis of mAb oxidation during various stability and degradation studies. By using the UV trace, the method allows more accurate measurement than mass spectrometry and can be potentially implemented as a release assay. It has been successfully used to monitor antibody oxidation in real time stability studies. PMID:27432793

  6. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    PubMed

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  7. Tuning the anticancer activity of a novel pro-apoptotic peptide using gold nanoparticle platforms

    PubMed Central

    Akrami, Mohammad; Balalaie, Saeed; Hosseinkhani, Saman; Alipour, Mohsen; Salehi, Fahimeh; Bahador, Abbas; Haririan, Ismaeil

    2016-01-01

    Pro-apoptotic peptides induce intrinsic apoptosis pathway in cancer cells. However, poor cellular penetration of the peptides is often associated with limited therapeutic efficacy. In this report, a series of peptide-gold nanoparticle platforms were developed to evaluate the anticancer activity of a novel alpha-lipoic acid-peptide conjugate, LA-WKRAKLAK, with respect to size and shape of nanoparticles. Gold nanoparticles (AuNPs) were found to enhance cell internalization as well as anticancer activity of the peptide conjugates. The smaller nanospheres showed a higher cytotoxicity, morphological change and cellular uptake compared to larger nanospheres and nanorods, whereas nanorods showed more hemolytic activity compared to nanospheres. The findings suggested that the anticancer and biological effects of the peptides induced by intrinsic apoptotic pathway were tuned by peptide-functionalized gold nanoparticles (P-AuNPs) as a function of their size and shape. PMID:27491007

  8. Antifungal Activity and Action Mechanism of Histatin 5-Halocidin Hybrid Peptides against Candida ssp.

    PubMed

    Han, Juhye; Jyoti, Md Anirban; Song, Ho-Yeon; Jang, Woong Sik

    2016-01-01

    The candidacidal activity of histatin 5 is initiated through cell wall binding, followed by translocation and intracellular targeting, while the halocidin peptide exerts its activity by attacking the Candida cell membrane. To improve antimicrobial activities and to understand the killing mechanism of two peptides, six hybrid peptides were designed by conjugating histatin 5 and halocidin. A comparative approach was established to study the activity, salt tolerance, cell wall glucan binding assay, cytotoxicity, generation of ROS and killing kinetics. CD spectrometry was conducted to evaluate secondary structures of these hybrid peptides. Furthermore the cellular localization of hybrid peptides was investigated by confocal fluorescence microscopy. Of the six hybrid congeners, di-PH2, di-WP2 and HHP1 had stronger activities than other hybrid peptides against all tested Candida strains. The MIC values of these peptides were 1-2, 2-4 and 2-4 μg/ml, respectively. Moreover, none of the hybrid peptides was cytotoxic in the hemolytic assay and cell-based cytotoxicity assay. Confocal laser microscopy showed that di-PH2 and HHP1 were translocated into cytoplasm whereas di-WP2 was accumulated on surface of C. albicans to exert their candidacidal activity. All translocated peptides (Hst 5, P113, di-PH2) were capable of generating intracellular ROS except HHP1. Additionally, the KFH residues at C-terminal end of these peptides were assumed for core sequence for active translocation.

  9. Antifungal Activity and Action Mechanism of Histatin 5-Halocidin Hybrid Peptides against Candida ssp

    PubMed Central

    Han, Juhye; Jyoti, Md. Anirban; Song, Ho-Yeon; Jang, Woong Sik

    2016-01-01

    The candidacidal activity of histatin 5 is initiated through cell wall binding, followed by translocation and intracellular targeting, while the halocidin peptide exerts its activity by attacking the Candida cell membrane. To improve antimicrobial activities and to understand the killing mechanism of two peptides, six hybrid peptides were designed by conjugating histatin 5 and halocidin. A comparative approach was established to study the activity, salt tolerance, cell wall glucan binding assay, cytotoxicity, generation of ROS and killing kinetics. CD spectrometry was conducted to evaluate secondary structures of these hybrid peptides. Furthermore the cellular localization of hybrid peptides was investigated by confocal fluorescence microscopy. Of the six hybrid congeners, di-PH2, di-WP2 and HHP1 had stronger activities than other hybrid peptides against all tested Candida strains. The MIC values of these peptides were 1–2, 2–4 and 2–4 μg/ml, respectively. Moreover, none of the hybrid peptides was cytotoxic in the hemolytic assay and cell-based cytotoxicity assay. Confocal laser microscopy showed that di-PH2 and HHP1 were translocated into cytoplasm whereas di-WP2 was accumulated on surface of C. albicans to exert their candidacidal activity. All translocated peptides (Hst 5, P113, di-PH2) were capable of generating intracellular ROS except HHP1. Additionally, the KFH residues at C-terminal end of these peptides were assumed for core sequence for active translocation. PMID:26918792

  10. Antifungal Activity and Action Mechanism of Histatin 5-Halocidin Hybrid Peptides against Candida ssp.

    PubMed

    Han, Juhye; Jyoti, Md Anirban; Song, Ho-Yeon; Jang, Woong Sik

    2016-01-01

    The candidacidal activity of histatin 5 is initiated through cell wall binding, followed by translocation and intracellular targeting, while the halocidin peptide exerts its activity by attacking the Candida cell membrane. To improve antimicrobial activities and to understand the killing mechanism of two peptides, six hybrid peptides were designed by conjugating histatin 5 and halocidin. A comparative approach was established to study the activity, salt tolerance, cell wall glucan binding assay, cytotoxicity, generation of ROS and killing kinetics. CD spectrometry was conducted to evaluate secondary structures of these hybrid peptides. Furthermore the cellular localization of hybrid peptides was investigated by confocal fluorescence microscopy. Of the six hybrid congeners, di-PH2, di-WP2 and HHP1 had stronger activities than other hybrid peptides against all tested Candida strains. The MIC values of these peptides were 1-2, 2-4 and 2-4 μg/ml, respectively. Moreover, none of the hybrid peptides was cytotoxic in the hemolytic assay and cell-based cytotoxicity assay. Confocal laser microscopy showed that di-PH2 and HHP1 were translocated into cytoplasm whereas di-WP2 was accumulated on surface of C. albicans to exert their candidacidal activity. All translocated peptides (Hst 5, P113, di-PH2) were capable of generating intracellular ROS except HHP1. Additionally, the KFH residues at C-terminal end of these peptides were assumed for core sequence for active translocation. PMID:26918792

  11. The Structure of the Amyloid-[beta] Peptide High-Affinity Copper II Binding Site in Alzheimer Disease

    SciTech Connect

    Streltsov, Victor A.; Titmuss, Stephen J.; Epa, V. Chandana; Barnham, Kevin J.; Masters, Colin L.; Varghese, Joseph N.

    2008-11-03

    Neurodegeneration observed in Alzheimer disease (AD) is believed to be related to the toxicity from reactive oxygen species (ROS) produced in the brain by the amyloid-{beta} (A{beta}) protein bound primarily to copper ions. The evidence for an oxidative stress role of A{beta}-Cu redox chemistry is still incomplete. Details of the copper binding site in A{beta} may be critical to the etiology of AD. Here we present the structure determined by combining x-ray absorption spectroscopy (XAS) and density functional theory analysis of A{beta} peptides complexed with Cu{sup 2+} in solution under a range of buffer conditions. Phosphate-buffered saline buffer salt (NaCl) concentration does not affect the high-affinity copper binding mode but alters the second coordination sphere. The XAS spectra for truncated and full-length A{beta}-Cu{sup 2+} peptides are similar. The novel distorted six-coordinated (3N3O) geometry around copper in the A{beta}-Cu{sup 2+} complexes include three histidines: glutamic, or/and aspartic acid, and axial water. The structure of the high-affinity Cu{sup 2+} binding site is consistent with the hypothesis that the redox activity of the metal ion bound to A{beta} can lead to the formation of dityrosine-linked dimers found in AD.

  12. Vasoactive intestinal peptide binding sites and fibers in the brain of the pigeon Columba livia: An autoradiographic and immunohistochemical study

    SciTech Connect

    Hof, P.R.; Dietl, M.M.; Charnay, Y.; Martin, J.L.; Bouras, C.; Palacios, J.M.; Magistretti, P.J. )

    1991-03-15

    The distribution of vasoactive intestinal peptide (VIP) binding sites in the pigeon brain was examined by in vitro autoradiography on slide-mounted sections. A fully characterized monoiodinated form of VIP, which maintains the biological activity of the native peptide, was used throughout this study. The highest densities of binding sites were observed in the hyperstriatum dorsale, archistriatum, auditory field L of neostriatum, area corticoidea dorsolateralis and temporo-parieto-occipitalis, area parahippocampalis, tectum opticum, nucleus dorsomedialis anterior thalami, and in the periventricular area of the hypothalamus. Lower densities of specific binding occurred in the neostriatum, hyperstriatum ventrale and nucleus septi lateralis, dorsolateral area of the thalamus, and lateral and posteromedial hypothalamus. Very low to background levels of VIP binding were detected in the ectostriatum, paleostriatum primitivum, paleostriatum augmentatum, lobus parolfactorius, nucleus accumbens, most of the brainstem, and the cerebellum. The distribution of VIP-containing fibers and terminals was examined by indirect immunofluorescence using a polyclonal antibody against porcine VIP. Fibers and terminals were observed in the area corticoidea dorsolateralis, area parahippocampalis, hippocampus, hyperstriatum accessorium, hyperstriatum dorsale, archistriatum, tuberculum olfactorium, nuclei dorsolateralis and dorsomedialis of the thalamus, and throughout the hypothalamus and the median eminence. Long projecting fibers were visualized in the tractus septohippocampalis. In the brainstem VIP immunoreactive fibers and terminals were observed mainly in the substantia grisea centralis, fasciculus longitudinalis medialis, lemniscus lateralis, and in the area surrounding the nuclei of the 7th, 9th, and 10th cranial nerves.

  13. Identification of the structural determinants for anticancer activity of a ruthenium arene peptide conjugate.

    PubMed

    Meier, Samuel M; Novak, Maria; Kandioller, Wolfgang; Jakupec, Michael A; Arion, Vladimir B; Metzler-Nolte, Nils; Keppler, Bernhard K; Hartinger, Christian G

    2013-07-01

    Organometallic Ru(arene)-peptide bioconjugates with potent in vitro anticancer activity are rare. We have prepared a conjugate of a Ru(arene) complex with the neuropeptide [Leu(5)]-enkephalin. [Chlorido(η(6)-p-cymene)(5-oxo-κO-2-{(4-[(N-tyrosinyl-glycinyl-glycinyl-phenylalanyl-leucinyl-NH2)propanamido]-1H-1,2,3-triazol-1-yl)methyl}-4H-pyronato-κO)ruthenium(II)] (8) shows antiproliferative activity in human ovarian carcinoma cells with an IC50 value as low as 13 μM, whereas the peptide or the Ru moiety alone are hardly cytotoxic. The conjugation strategy for linking the Ru(cym) (cym=η(6)-p-cymene) moiety to the peptide involved N-terminal modification of an alkyne-[Leu(5)]-enkephalin with a 2-(azidomethyl)-5-hydroxy-4H-pyran-4-one linker, using Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC), and subsequent metallation with the Ru(cym) moiety. The ruthenium-bioconjugate was characterized by high resolution top-down electrospray ionization mass spectrometry (ESI-MS) with regard to peptide sequence, linker modification and metallation site. Notably, complete sequence coverage was obtained and the Ru(cym) moiety was confirmed to be coordinated to the pyronato linker. The ruthenium-bioconjugate was analyzed with respect to cytotoxicity-determining constituents, and through the bioconjugate models [{2-(azidomethyl)-5-oxo-κO-4H-pyronato-κO}chloride (η(6)-p-cymene)ruthenium(II)] (5) and [chlorido(η(6)-p-cymene){5-oxo-κO-2-([(4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl]methyl)-4H-pyronato-κO}ruthenium(II)] (6) the Ru(cym) fragment with a triazole-carrying pyronato ligand was identified as the minimal unit required to achieve in vitro anticancer activity.

  14. Caffeic acid phenethyl ester (CAPE), an active component of propolis, inhibits Helicobacter pylori peptide deformylase activity.

    PubMed

    Cui, Kunqiang; Lu, Weiqiang; Zhu, Lili; Shen, Xu; Huang, Jin

    2013-05-31

    Helicobacter pylori (H. pylori) is a major causative factor for gastrointestinal illnesses, H. pylori peptide deformylase (HpPDF) catalyzes the removal of formyl group from the N-terminus of nascent polypeptide chains, which is essential for H. pylori survival and is considered as a promising drug target for anti-H. pylori therapy. Propolis, a natural antibiotic from honeybees, is reported to have an inhibitory effect on the growth of H. pylori in vitro. In addition, previous studies suggest that the main active constituents in the propolis are phenolic compounds. Therefore, we evaluated a collection of phenolic compounds derived from propolis for enzyme inhibition against HpPDF. Our study results show that Caffeic acid phenethyl ester (CAPE), one of the main medicinal components of propolis, is a competitive inhibitor against HpPDF, with an IC50 value of 4.02 μM. Furthermore, absorption spectra and crystal structural characterization revealed that different from most well known PDF inhibitors, CAPE block the substrate entrance, preventing substrate from approaching the active site, but CAPE does not have chelate interaction with HpPDF and does not disrupt the metal-dependent catalysis. Our study provides valuable information for understanding the potential anti-H. pylori mechanism of propolis, and CAPE could be served as a lead compound for further anti-H. pylori drug discovery. PMID:23611786

  15. Caffeic acid phenethyl ester (CAPE), an active component of propolis, inhibits Helicobacter pylori peptide deformylase activity.

    PubMed

    Cui, Kunqiang; Lu, Weiqiang; Zhu, Lili; Shen, Xu; Huang, Jin

    2013-05-31

    Helicobacter pylori (H. pylori) is a major causative factor for gastrointestinal illnesses, H. pylori peptide deformylase (HpPDF) catalyzes the removal of formyl group from the N-terminus of nascent polypeptide chains, which is essential for H. pylori survival and is considered as a promising drug target for anti-H. pylori therapy. Propolis, a natural antibiotic from honeybees, is reported to have an inhibitory effect on the growth of H. pylori in vitro. In addition, previous studies suggest that the main active constituents in the propolis are phenolic compounds. Therefore, we evaluated a collection of phenolic compounds derived from propolis for enzyme inhibition against HpPDF. Our study results show that Caffeic acid phenethyl ester (CAPE), one of the main medicinal components of propolis, is a competitive inhibitor against HpPDF, with an IC50 value of 4.02 μM. Furthermore, absorption spectra and crystal structural characterization revealed that different from most well known PDF inhibitors, CAPE block the substrate entrance, preventing substrate from approaching the active site, but CAPE does not have chelate interaction with HpPDF and does not disrupt the metal-dependent catalysis. Our study provides valuable information for understanding the potential anti-H. pylori mechanism of propolis, and CAPE could be served as a lead compound for further anti-H. pylori drug discovery.

  16. Patenting activity in synthesis of lipid nanotubes and peptide nanotubes.

    PubMed

    Zhou, Yong

    2007-01-01

    Lipid nanotubes (LNTs) and peptide nanotubes (PNTs) are especially intriguing and noncovalent self-assemblies of amphiphiles. They have hydrophilically internal and external membrane surfaces, and can provide the wide scope for chemical modifications, in sharp contrast to carbon nanotubes. These unique properties make themselves as ideal candidates for a variety of applications in chemistry, biochemistry, materials science and medicine. Patenting the LNTs and PNTs is quite active recently. This mini-review provides a brief outline of patenting activity in synthesis of the LNTs and PNTs since 1980s. The key point of the present review aims to create an optimistic circulation between the basic research achievement and potential application of this sub-field of nanotechnology, promoting each other in their future development.

  17. Collagen-binding VEGF mimetic peptide: Structure, matrix interaction, and endothelial cell activation

    NASA Astrophysics Data System (ADS)

    Chan, Tania R.

    Long term survival of artificial tissue constructs depends greatly on proper vascularization. In nature, differentiation of endothelial cells and formation of vasculature are directed by dynamic spatio-temporal cues in the extracellular matrix that are difficult to reproduce in vitro. In this dissertation, we present a novel bifunctional peptide that mimics matrix-bound vascular endothelial growth factor (VEGF), which can be used to encode spatially controlled angiogenic signals in collagen-based scaffolds. The peptide, QKCMP, contains a collagen mimetic domain (CMP) that binds to type I collagen by a unique triple helix hybridization mechanism and a VEGF mimetic domain (QK) with pro-angiogenic activity. We demonstrate QKCMP's ability to hybridize with native and heat denatured collagens through a series of binding studies on collagen and gelatin substrates. Circular dichroism experiments show that the peptide retains the triple helical structure vital for collagen binding, and surface plasmon resonance study confirms the molecular interaction between the peptide and collagen strands. Cell culture studies demonstrate QKCMP's ability to induce endothelial cell morphogenesis and network formation as a matrix-bound factor in 2D and 3D collagen scaffolds. We also show that the peptide can be used to spatially modify collagen-based substrates to promote localized endothelial cell activation and network formation. To probe the biological events that govern these angiogenic cellular responses, we investigated the cell signaling pathways activated by collagen-bound QKCMP and determined short and long-term endothelial cell response profiles for p38, ERK1/2, and Akt signal transduction cascades. Finally, we present our efforts to translate the peptide's in vitro bioactivity to an in vivo burn injury animal model. When implanted at the wound site, QKCMP functionalized biodegradable hydrogels induce enhanced neovascularization in the granulation tissue. The results show QKCMP

  18. Synthetic peptides.

    PubMed

    Francis, M J

    1996-01-01

    Efforts to produce more stable and defined vaccines have concentrated on studying, in detail, the immune response to many infectious diseases in order to identify the antigenic sites on the pathogens that are involved in stimulating protective immumty. Armed with this knowledge, it is possible to mimic such sites by producing short chains of amino acids (peptides) and to use these as the basis for novel vaccines. The earliest documented work on peptide immunization is actually for a plant virus, tobacco mosaic virus. In 1963, Anderer (1) demonstrated that rabbit antibodies to an isolated hexapeptide fragment from the virus-coat protein coupled to bovine serum albumm would neutralize the infectious vn-us in culture. Two years later, he used a synthetically produced copy of the same peptide to confirm this observation. This was pioneering work, and it was over 10 years before the next example of a peptide that elicited antivirus antibody appeared following work by Sela and his colleagues (2) on a virus, MS2 bacteriophage, which infects bacteria. The emergence of more accessible techniques for sequencing proteins in 1977, coupled with the ability to synthesize readily peptides already developed in 1963, heralded a decade of intensive research into experimental peptide vaccines. The first demonstration that peptides could elicit protective immunity in vivo, in addition to neutralizing activity in vitro, was obtained using a peptide from the VP1 coat protein of foot-and-mouth disease virus (FMDV) in 1982, with the guinea pig as a laboratory animal model (3, 4). PMID:21359696

  19. Synthesis, characterization and anti-cancer activity of a peptide nucleolipid bioconjugate.

    PubMed

    Rana, Niki; Huang, Suiying; Patel, Pradeepkumar; Samuni, Uri; Sabatino, David

    2016-08-01

    The synthesis, characterization and anti-cancer activity of a novel peptide nucleolipid bioconjugate is reported in this study. The prerequisite 5'-carboxy derived nucleolipid was synthesized following a five-step solution-phase approach and then coupled to the cytotoxic D-(KLAKLAK)2 sequence by solid-phase bioconjugation. The biophysical and structural properties of the peptide-nucleolipid bioconjugate were evaluated and compared to the peptide controls. These characterization studies revealed that the amphiphilic peptides favored helical-type secondary structures and well-defined nanoparticle formulations that were found to be contributive towards their biological activity. The peptide-nucleolipid bioconjugate displayed greater lethality in comparison to the native D-(KLAKLAK)2AK sequence when treated within the human A549 non-small cell lung carcinoma cell line. Thus, the amphiphilic peptide-nucleolipid forms a new class of anti-cancer peptides that may be developed into promising leads in the fight against cancer.

  20. Peptide-Coated Liposomal Fasudil Enhances Site Specific Vasodilation in Pulmonary Arterial Hypertension

    PubMed Central

    2015-01-01

    This study sought to develop a liposomal delivery system of fasudil—an investigational drug for the treatment of pulmonary arterial hypertension (PAH)—that will preferentially accumulate in the PAH lungs. Liposomal fasudil was prepared by film-hydration method, and the drug was encapsulated by active loading. The liposome surface was coated with a targeting moiety, CARSKNKDC, a cyclic peptide; the liposomes were characterized for size, polydispersity index, zeta potential, and storage and nebulization stability. The in vitro drug release profiles and uptake by TGF-β activated pulmonary arterial smooth muscle cells (PASMC) and alveolar macrophages were evaluated. The pharmacokinetics were monitored in male Sprague–Dawley rats, and the pulmonary hemodynamics were studied in acute and chronic PAH rats. The size, polydispersity index (PDI), and zeta potential of the liposomes were 206–216 nm, 0.058–0.084, and −20–42.7 mV, respectively. The formulations showed minimal changes in structural integrity when nebulized with a commercial microsprayer. The optimized formulation was stable for >4 weeks when stored at 4 °C. Fasudil was released in a continuous fashion over 120 h with a cumulative release of 76%. Peptide-linked liposomes were taken up at a higher degree by TGF-β activated PASMCs; but alveolar macrophages could not engulf peptide-coated liposomes. The formulations did not injure the lungs; the half-life of liposomal fasudil was 34-fold higher than that of plain fasudil after intravenous administration. Peptide-linked liposomal fasudil, as opposed to plain liposomes, reduced the mean pulmonary arterial pressure by 35–40%, without influencing the mean systemic arterial pressure. This study establishes that CAR-conjugated inhalable liposomal fasudil offers favorable pharmacokinetics and produces pulmonary vasculature specific dilatation. PMID:25333706

  1. High Throughput Screening Methods for Assessing Antibiofilm and Immunomodulatory Activities of Synthetic Peptides

    PubMed Central

    Haney, Evan F.; Mansour, Sarah; Hilchie, Ashley L.; de la Fuente-Núñez, César; Hancock, Robert E.W.

    2015-01-01

    The recent observation that certain cationic peptides possess potent antibiofilm activity demonstrated that small peptides could be used to treat biofilm-associated infections. Other so-called innate defense regulator peptides possess potent immunomodulatory properties such as leukocyte recruitment and suppression of harmful inflammation. A peptide that directly targets biofilm cells while favourably modulating the immune response would be particularly advantageous for treating serious skin infections caused by S. aureus. In the present work, using SPOT-synthesized peptide arrays on cellulose membranes, we outline a strategy for systematically assessing the antibiofilm activity of hundreds of IDR-1002 (VQRWLIVWRIRK-NH2) and IDR-HH2 (VQLRIRVAVIRA-NH2) peptide variants against MRSA biofilms. In addition, the ability of these peptides to stimulate production of a monocyte chemoattractant protein (MCP-1) and suppress LPS-induced interleukin (IL)-1β production in human peripheral blood mononuclear cells (PBMCs) was evaluated. These results informed the synthesis of second-generation peptides resulting in a new peptide, IDR-2009 (KWRLLIRWRIQK-NH2), with enhanced MCP-1 stimulatory activity, favourable IL-1β suppression characteristics and strong antibiofilm activity against MRSA and Pseudomonas aeruginosa biofilms. This work provides a proof-of-concept that multiple peptide activities can be optimized simultaneously to generate novel sequences that possess a variety of biological properties. PMID:25836992

  2. Design of host defence peptides for antimicrobial and immunity enhancing activities.

    PubMed

    McPhee, Joseph B; Scott, Monisha G; Hancock, Robert E W

    2005-05-01

    Host defense peptides are a vital component of the innate immune systems of humans, other mammals, amphibians, and arthropods. The related cationic antimicrobial peptides are also produced by many species of bacteria and function as part of the antimicrobial arsenal to help the producing organism reduce competition for resources from sensitive species. The antimicrobial activities of many of these peptides have been extensively characterized and the structural requirements for these activities are also becoming increasingly clear. In addition to their known antimicrobial role, many host defense peptides are also involved in a plethora of immune functions in the host. In this review, we examine the role of structure in determining antimicrobial activity of certain prototypical cationic peptides and ways that bacteria have evolved to usurp these activities. We also review recent literature on what structural components are related to these immunomodulatory effects. It must be stressed however that these studies, and the area of peptide research, are still in their infancy.

  3. Biological activity of Tat (47-58) peptide on human pathogenic fungi

    SciTech Connect

    Jung, Hyun Jun; Park, Yoonkyung; Hahm, Kyung-Soo; Lee, Dong Gun . E-mail: dglee222@knu.ac.kr

    2006-06-23

    Tat (47-58) peptide, a positively charged Arginine-rich peptide derived from HIV-1 regulatory protein Tat, is known for a peptidic delivery factor as a cell-penetrating peptide on mammalian cells. In this study, antifungal effect and its mode of action of Tat peptide were investigated on fungal cells. The results indicate that Tat peptide exhibits antifungal activity against pathogenic fungal cells without hemolytic effect on human erythrocytes. To understand the mechanism(s) of Tat peptide, the cellular distribution of the peptide was investigated. Tat peptide internalized in the fungal cells without any damage to cell membrane when examined using an artificial liposome (PC/cholesterol; 10:1, w/w). Moreover, flow cytometry analysis exhibited the uptake of Tat peptide by energy- and salt-independent pathway, and confocal scanning microscopy displayed that this peptide accumulated in the nucleus of fungal cells rapidly without any impediment by time or temperature, which generally influence on the viral infections. After penetration into the nuclear, the peptide affected the process of cell cycle of Candida albicans through the arrest at G1 phase.

  4. Eel calcitonin binding site distribution and antinociceptive activity in rats

    SciTech Connect

    Guidobono, F.; Netti, C.; Sibilia, V.; Villa, I.; Zamboni, A.; Pecile, A.

    1986-03-01

    The distribution of binding site for (/sup 125/I)-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing (/sup 125/I)-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain.

  5. Sandmeyer reaction repurposed for the site-selective, non-oxidizing radioiodination of fully-deprotected peptides: studies on the endogenous opioid peptide α-neoendorphin.

    PubMed

    Pickett, Julie E; Nagakura, Kunihiko; Pasternak, Anna R; Grinnell, Steven G; Majumdar, Susruta; Lewis, Jason S; Pasternak, Gavril W

    2013-08-01

    Standard radioiodination methods lack site-selectivity and either mask charges (Bolton-Hunter) or involve oxidative reaction conditions (chloramine-T). Opioid peptides are very sensitive to certain structural modifications, making these labeling methods untenable. In our model opioid peptide, α-neoendorphin, we replaced a tyrosyl hydroxyl with an iodine, and in cell lines stably expressing mu, delta, or kappa opioid receptors, we saw no negative effects on binding. We then optimized a repurposed Sandmeyer reaction using copper(I) catalysts with non-redoxing/non-nucleophilic ligands, bringing the radiochemical yield up to around 30%, and site-selectively incorporated radioactive iodine into this position under non-oxidizing reaction conditions, which should be broadly compatible with most peptides. The (125)I- and (131)I-labeled versions of the compound bound with high affinity to opioid receptors in mouse brain homogenates, thus demonstrating the general utility of the labeling strategy and of the peptide for exploring opioid binding sites. PMID:23796454

  6. Diagnosing the Protonation Site of b 2 Peptide Fragment Ions using IRMPD in the X-H (X = O, N, and C) Stretching Region

    NASA Astrophysics Data System (ADS)

    Sinha, Rajeev K.; Erlekam, Undine; Bythell, Benjamin J.; Paizs, Béla; Maître, Philippe

    2011-09-01

    Charge-directed fragmentation has been shown to be the prevalent dissociation step for protonated peptides under the low-energy activation (eV) regime. Thus, the determination of the ion structure and, in particular, the characterization of the protonation site(s) of peptides and their fragments is a key approach to substantiate and refine peptide fragmentation mechanisms. Here we report on the characterization of the protonation site of oxazolone b 2 ions formed in collision-induced dissociation (CID) of the doubly protonated tryptic model-peptide YIGSR. In support of earlier work, here we provide complementary IR spectra in the 2800-3800 cm-1 range acquired on a table-top laser system. Combining this tunable laser with a high power CO2 laser to improve spectroscopic sensitivity, well resolved bands are observed, with an excellent correspondence to the IR absorption bands of the ring-protonated oxazolone isomer as predicted by quantum chemical calculations. In particular, it is shown that a band at 3445 cm-1, corresponding to the asymmetric N-H stretch of the (nonprotonated) N-terminal NH2 group, is a distinct vibrational signature of the ring-protonated oxazolone structure.

  7. Epitope-based peptide vaccine design and target site depiction against Ebola viruses: an immunoinformatics study.

    PubMed

    Khan, M A; Hossain, M U; Rakib-Uz-Zaman, S M; Morshed, M N

    2015-07-01

    Ebola viruses (EBOVs) have been identified as an emerging threat in recent year as it causes severe haemorrhagic fever in human. Epitope-based vaccine design for EBOVs remains a top priority because a mere progress has been made in this regard. Another reason is the lack of antiviral drug and licensed vaccine although there is a severe outbreak in Central Africa. In this study, we aimed to design an epitope-based vaccine that can trigger a significant immune response as well as to prognosticate inhibitor that can bind with potential drug target sites using various immunoinformatics and docking simulation tools. The capacity to induce both humoral and cell-mediated immunity by T cell and B cell was checked for the selected protein. The peptide region spanning 9 amino acids from 42 to 50 and the sequence TLASIGTAF were found as the most potential B and T cell epitopes, respectively. This peptide could interact with 12 HLAs and showed high population coverage up to 80.99%. Using molecular docking, the epitope was further appraised for binding against HLA molecules to verify the binding cleft interaction. In addition with this, the allergenicity of the epitopes was also evaluated. In the post-therapeutic strategy, docking study of predicted 3D structure identified suitable therapeutic inhibitor against targeted protein. However, this computational epitope-based peptide vaccine designing and target site prediction against EBOVs open up a new horizon which may be the prospective way in Ebola viruses research; the results require validation by in vitro and in vivo experiments.

  8. Mapping of a cholinergic binding site by means of synthetic peptides, monoclonal antibodies, and. alpha. -bungarotoxin

    SciTech Connect

    Conti-Tronconi, B.M.; Tang, Fen; Diethelm, B.M.; Spencer, S.R. ); Reinhardt-Maelicke, S.; Maelicke, A. )

    1990-07-03

    Previous studies by several laboratories have identified a narrow sequence region of the nicotinic acetylcholine receptor (AChR) {alpha} subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, the authors used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to bind {alpha}-bungarotoxin ({alpha}-BTX) and several {alpha}-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the {alpha}-subunit, and bind to AChR in a mutually exclusive fashion with {alpha}-BTX. The binding of WF5 and W2 to Torpedo AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. Peptides {alpha}181-200 and {alpha}55-74 both inhibited binding of {sup 125}I-{alpha}-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunits bound {alpha}-BTX or WF6, or interfered with their binding. Therefore, the cholinergic binding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR {alpha} subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region.

  9. Characterization of a transport activity for long-chain peptides in barley mesophyll vacuoles.

    PubMed

    Ramos, Magali Schnell; Abele, Rupert; Nagy, Réka; Grotemeyer, Marianne Suter; Tampé, Robert; Rentsch, Doris; Martinoia, Enrico

    2011-04-01

    The plant vacuole is the largest compartment in a fully expanded plant cell. While only very limited metabolic activity can be observed within the vacuole, the majority of the hydrolytic activities, including proteolytic activities reside in this organelle. Since it is assumed that protein degradation by the proteasome results in the production of peptides with a size of 3-30 amino acids, we were interested to show whether the tonoplast exhibits a transport activity, which could deliver these peptides into the vacuole for final degradation. It is shown here that isolated barley mesophyll vacuoles take up peptides of 9-27 amino acids in a strictly ATP-dependent manner. Uptake is inhibited by vanadate, but not by NH(+)(4), while GTP could partially substitute for ATP. The apparent affinity for the 9 amino acid peptide was 15 μM, suggesting that peptides are efficiently transferred to the vacuole in vivo. Inhibition experiments showed that peptides with a chain length below 10 amino acids did not compete as efficiently as longer peptides for the uptake of the 9 amino acid peptide. Our results suggest that vacuoles contain at least one peptide transporter that belongs to the ABC-type transporters, which efficiently exports long-chain peptides from the cytosol into the vacuole for final degradation.

  10. Human 20S proteasome activity towards fluorogenic peptides of various chain lengths.

    PubMed

    Rut, Wioletta; Drag, Marcin

    2016-09-01

    The proteasome is a multicatalytic protease responsible for the degradation of misfolded proteins. We have synthesized fluorogenic substrates in which the peptide chain was systematically elongated from two to six amino acids and evaluated the effect of peptide length on all three catalytic activities of human 20S proteasome. In the cases of five- and six-membered peptides, we have also synthesized libraries of fluorogenic substrates. Kinetic analysis revealed that six-amino-acid substrates are significantly better for chymotrypsin-like and caspase-like activity than shorter peptidic substrates. In the case of trypsin-like activity, a five-amino-acid substrate was optimal. PMID:27176742

  11. Human 20S proteasome activity towards fluorogenic peptides of various chain lengths.

    PubMed

    Rut, Wioletta; Drag, Marcin

    2016-09-01

    The proteasome is a multicatalytic protease responsible for the degradation of misfolded proteins. We have synthesized fluorogenic substrates in which the peptide chain was systematically elongated from two to six amino acids and evaluated the effect of peptide length on all three catalytic activities of human 20S proteasome. In the cases of five- and six-membered peptides, we have also synthesized libraries of fluorogenic substrates. Kinetic analysis revealed that six-amino-acid substrates are significantly better for chymotrypsin-like and caspase-like activity than shorter peptidic substrates. In the case of trypsin-like activity, a five-amino-acid substrate was optimal.

  12. Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites.

    PubMed Central

    Burck, P J; Berg, D H; Luk, T P; Sassmannshausen, L M; Wakulchik, M; Smith, D P; Hsiung, H M; Becker, G W; Gibson, W; Villarreal, E C

    1994-01-01

    The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation of its C terminus, is an essential step in virion maturation. The proteinase responsible for this cleavage is the amino-terminal half of the protein encoded by the UL80a open reading fame. We have obtained high expression levels of this 256-amino-acid HCMV proteinase, assemblin, in Escherichia coli. In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 amino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present. Both the 28-kDa proteinase and the 15-kDa protein were purified by a two-step chromatographic procedure utilizing anion exchange in urea and dithiothreitol and size exclusion in NaSCN and dithiothreitol. Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule. Removal of the urea by dialysis with retention of the reducing agent yielded an active proteinase. Addition of glycerol to 50% enhanced the activity. The HCMV proteinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mimics of the maturational (M)- and release (R)-site sequences, respectively, in the UL80a-encoded protein. The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein. The Km value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (kcat) of the M-site peptide mimic substrate by the proteinase was six to eight times faster. The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded protein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates. The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor concentrations. The purified 15-kDa protein

  13. Directed Evolution of an LBP/CD14 Inhibitory Peptide and Its Anti-Endotoxin Activity

    PubMed Central

    Fang, Li; Xu, Zhi; Wang, Guan-song; Ji, Fu-yun; Mei, Chun-xia; Liu, Juan; Wu, Guo-ming

    2014-01-01

    Background LPS-binding protein (LBP) and its ligand CD14 are located upstream of the signaling pathway for LPS-induced inflammation. Blocking LBP and CD14 binding might prevent LPS-induced inflammation. In previous studies, we obtained a peptide analog (MP12) for the LBP/CD14 binding site and showed that this peptide analog had anti-endotoxin activity. In this study, we used in vitro directed evolution for this peptide analog to improve its in vivo and in vitro anti-endotoxin activity. Methods We used error-prone PCR (ep-PCR) and induced mutations in the C-terminus of LBP and attached the PCR products to T7 phages to establish a mutant phage display library. The positive clones that competed with LBP for CD14 binding was obtained by screening. We used both in vivo and in vitro experiments to compare the anti-endotoxin activities of a polypeptide designated P1 contained in a positive clone and MP12. Results 11 positive clones were obtained from among target phages. Sequencing showed that 9 positive clones had a threonine (T) to methionine (M) mutation in amino acid 287 of LBP. Compared to polypeptide MP12, polypeptide P1 significantly inhibited LPS-induced TNF-α expression and NF-κB activity in U937 cells (P<0.05). Compared to MP12, P1 significantly improved arterial oxygen pressure, an oxygenation index, and lung pathology scores in LPS-induced ARDS rats (P<0.05). Conclusion By in vitro directed evolution of peptide analogs for the LBP/CD14 binding site, we established a new polypeptide (P1) with a threonine (T)-to-methionine (M) mutation in amino acid 287 of LBP. This polypeptide had high anti-endotoxin activity in vitro and in vivo, which suggested that amino acid 287 in the C-terminus of LBP may play an important role in LBP binding with CD14. PMID:25025695

  14. CABS-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site

    PubMed Central

    Kurcinski, Mateusz; Jamroz, Michal; Blaszczyk, Maciej; Kolinski, Andrzej; Kmiecik, Sebastian

    2015-01-01

    Protein–peptide interactions play a key role in cell functions. Their structural characterization, though challenging, is important for the discovery of new drugs. The CABS-dock web server provides an interface for modeling protein–peptide interactions using a highly efficient protocol for the flexible docking of peptides to proteins. While other docking algorithms require pre-defined localization of the binding site, CABS-dock does not require such knowledge. Given a protein receptor structure and a peptide sequence (and starting from random conformations and positions of the peptide), CABS-dock performs simulation search for the binding site allowing for full flexibility of the peptide and small fluctuations of the receptor backbone. This protocol was extensively tested over the largest dataset of non-redundant protein–peptide interactions available to date (including bound and unbound docking cases). For over 80% of bound and unbound dataset cases, we obtained models with high or medium accuracy (sufficient for practical applications). Additionally, as optional features, CABS-dock can exclude user-selected binding modes from docking search or to increase the level of flexibility for chosen receptor fragments. CABS-dock is freely available as a web server at http://biocomp.chem.uw.edu.pl/CABSdock. PMID:25943545

  15. Chemical synthesis of idiotopes. Evidence that antisera to the same JH1 peptide detect multiple binding site-associated idiotopes

    PubMed Central

    1984-01-01

    In an attempt to better understand the molecular basis of idiotypy, we have generated several site-specific antisera through immunization of animals with synthetic peptides corresponding to the (JH1) heavy chain joining segment 1 of the mouse heavy chain variable (VH) region. These anti-peptide sera identify several idiotypic determinants present on intact hybridoma and myeloma immunoglobulins. Expression of at least three of these idiotopes is correlated with the antigen specificity of the family of immunoglobulins bearing the determinant. Use of synthetic peptides may prove a powerful technique in the generation of molecularly defined antiidiotypic reagents. PMID:6201582

  16. Dissociation behavior of a bifunctional tempo-active ester reagent for peptide structure analysis by free radical initiated peptide sequencing (FRIPS) mass spectrometry.

    PubMed

    Ihling, Christian; Falvo, Francesco; Kratochvil, Isabel; Sinz, Andrea; Schäfer, Mathias

    2015-02-01

    We have synthesized a homobifunctional active ester cross-linking reagent containing a TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy) moiety connected to a benzyl group (Bz), termed TEMPO-Bz-linker. The aim for designing this novel cross-linker was to facilitate MS analysis of cross-linked products by free radical initiated peptide sequencing (FRIPS). The TEMPO-Bz-linker was reacted with all 20 proteinogenic amino acids as well as with model peptides to gain detailed insights into its fragmentation mechanism upon collision activation. The final goal of this proof-of-principle study was to evaluate the potential of the TEMPO-Bz-linker for chemical cross-linking studies to derive 3D-structure information of proteins. Our studies were motivated by the well documented instability of the central NO-C bond of TEMPO-Bz reagents upon collision activation. The fragmentation of this specific bond was investigated in respect to charge states and amino acid composition of a large set of precursor ions resulting in the identification of two distinct fragmentation pathways. Molecular ions with highly basic residues are able to keep the charge carriers located, i.e. protons or sodium cations, and consequently decompose via a homolytic cleavage of the NO-C bond of the TEMPO-Bz-linker. This leads to the formation of complementary open-shell peptide radical cations, while precursor ions that are protonated at the TEMPO-Bz-linker itself exhibit a charge-driven formation of even-electron product ions upon collision activation. MS(3) product ion experiments provided amino acid sequence information and allowed determining the cross-linking site. Our study fully characterizes the CID behavior of the TEMPO-Bz-linker and demonstrates its potential, but also its limitations for chemical cross-linking applications utilizing the special features of open-shell peptide ions on the basis of selective tandem MS analysis.

  17. The Structure-Activity Relationship of the Antioxidant Peptides from Natural Proteins.

    PubMed

    Zou, Tang-Bin; He, Tai-Ping; Li, Hua-Bin; Tang, Huan-Wen; Xia, En-Qin

    2016-01-12

    Peptides derived from dietary proteins, have been reported to display significant antioxidant activity, which may exert notably beneficial effects in promoting human health and in food processing. Recently, much research has focused on the generation, separation, purification and identification of novel peptides from various protein sources. Some researchers have tried to discover the structural characteristics of antioxidant peptides in order to lessen or avoid the tedious and aimless work involving the ongoing generated peptide preparation schemes. This review aims to summarize the current knowledge on the relationship between the structural features of peptides and their antioxidant activities. The relationship between the structure of the precursor proteins and their abilities to release antioxidant fragments will also be summarized and inferred. The preparation methods and antioxidant capacity evaluation assays of peptides and a prediction scheme of quantitative structure-activity relationship (QSAR) will also be pointed out and discussed.

  18. Structure-function analysis of the glioma targeting NFL-TBS.40-63 peptide corresponding to the tubulin-binding site on the light neurofilament subunit.

    PubMed

    Berges, Raphael; Balzeau, Julien; Takahashi, Masayuki; Prevost, Chantal; Eyer, Joel

    2012-01-01

    We previously reported that a 24 amino acid peptide (NFL-TBS.40-63) corresponding to the tubulin-binding site located on the light neurofilament subunit, selectively enters in glioblastoma cells where it disrupts their microtubule network and inhibits their proliferation. Here, we analyzed the structure-function relationships using an alanine-scanning strategy, in order to identify residues essential for these biological activities. We showed that the majority of modified peptides present a decreased or total loss to penetrate in these cells, or to alter microtubules. Correspondingly, circular dichroism measurements showed that this peptide forms either β-sheet or α-helix structures according to the solvent and that alanine substitution modified or destabilized the structure, in relation with changes in the biological activities. Moreover, substitution of serine residues by phosphoserine or aspartic acid concomitantly decreased the cell penetrating activity and the structure stability. These results indicate the importance of structure for the activities, including selectivity to glioblastoma cells of this peptide, and its regulation by phosphorylation.

  19. Design of Embedded-Hybrid Antimicrobial Peptides with Enhanced Cell Selectivity and Anti-Biofilm Activity

    PubMed Central

    Xu, Wei; Zhu, Xin; Tan, Tingting; Li, Weizhong; Shan, Anshan

    2014-01-01

    Antimicrobial peptides have attracted considerable attention because of their broad-spectrum antimicrobial activity and their low prognostic to induce antibiotic resistance which is the most common source of failure in bacterial infection treatment along with biofilms. The method to design hybrid peptide integrating different functional domains of peptides has many advantages. In this study, we designed an embedded-hybrid peptide R-FV-I16 by replacing a functional defective sequence RR7 with the anti-biofilm sequence FV7 embedded in the middle position of peptide RI16. The results demonstrated that the synthetic hybrid the peptide R-FV-I16 had potent antimicrobial activity over a wide range of Gram-negative and Gram-positive bacteria, as well as anti-biofilm activity. More importantly, R-FV-I16 showed lower hemolytic activity and cytotoxicity. Fluorescent assays demonstrated that R-FV-I16 depolarized the outer and the inner bacterial membranes, while scanning electron microscopy and transmission electron microscopy further indicated that this peptide killed bacterial cells by disrupting the cell membrane, thereby damaging membrane integrity. Results from SEM also provided evidence that R-FV-I16 inherited anti-biofilm activity from the functional peptide sequence FV7. Embedded-hybrid peptides could provide a new pattern for combining different functional domains and showing an effective avenue to screen for novel antimicrobial agents. PMID:24945359

  20. Protocols to test the activity of antimicrobial peptides against the honey bee pathogen Paenibacillus larvae.

    PubMed

    Khilnani, Jasmin C; Wing, Helen J

    2015-10-01

    Paenibacillus larvae is the causal agent of the honey bee disease American Foulbrood. Two enhanced protocols that allow the activity of antimicrobial peptides to be tested against P. larvae are presented. Proof of principle experiments demonstrate that the honey bee antimicrobial peptide defensin 1 is active in both assays. PMID:26210039

  1. Catalysis: Elusive active site in focus

    NASA Astrophysics Data System (ADS)

    Labinger, Jay A.

    2016-08-01

    The identification of the active site of an iron-containing catalyst raises hopes of designing practically useful catalysts for the room-temperature conversion of methane to methanol, a potential fuel for vehicles. See Letter p.317

  2. An integrated, peptide-based approach to site-specific protein immobilization for detection of biomolecular interactions.

    PubMed

    Kruis, Ilmar C; Löwik, Dennis W P M; Boelens, Wilbert C; van Hest, Jan C M; Pruijn, Ger J M

    2016-09-21

    We have developed an integrated solution for the site-specific immobilization of proteins on a biosensor surface, which may be widely applicable for high throughput analytical purposes. The gold surface of a biosensor was coated with an anti-fouling layer of zwitterionic peptide molecules from which leucine zipper peptides protrude. Proteins of interest, the autoantigenic proteins La and U1A, were immobilized via a simple incubation procedure by using the complementary leucine zipper sequence as a genetically fused binding tag. This tag forms a strong coiled-coil interaction that is stable during multiple consecutive measurements and under common regeneration conditions. Visualization of the immobilized proteins of interest via antibody binding with multiplex surface plasmon resonance imaging demonstrated 2.5 times higher binding responses than when these proteins were randomly attached to the surface via the commonly applied activated ester-mediated coupling. The proteins could also be immobilized in a leucine zipper-dependent manner directly from complex mixtures like bacterial lysates, eliminating the need for laborious purification steps. This method allows the production of uniform functional protein arrays by control over immobilized protein orientation and geometry and is compatible with high-throughput procedures.

  3. Computer-based design of novel HIV-1 entry inhibitors: neomycin conjugated to arginine peptides at two specific sites.

    PubMed

    Berchanski, Alexander; Lapidot, Aviva

    2009-03-01

    Aminoglycoside-arginine conjugates (AAC and APAC) are multi-target inhibitors of human immunodeficiency virus type-1 (HIV-1). Here, we predict new conjugates of neomycin with two arginine peptide chains binding at specific sites on neomycin [poly-arginine-neomycin-poly-arginine (PA-Neo-PA)]. The rationale for the design of such compounds is to separate two short arginine peptides with neomycin, which may extend the binding region of the CXC chemokine receptor type 4 (CXCR4). We used homology models of CXCR4 and unliganded envelope glycoprotein 120 (HIV-1(IIIB) gp120) and docked PA-Neo-PAs and APACs to these using a multistep docking procedure. The results indicate that PA-Neo-PAs spread over two negatively charged patches of CXCR4. PA-Neo-PA-CXCR4 complexes are energetically more favorable than AACs/APAC-CXCR4 complexes. Notably, our CXCR4 model and docking procedure can be applied to predict new compounds that are either inhibitors of gp120-CXCR4 binding without affecting stromal cell-derived factor 1 alpha (SDF-1 alpha) chemotaxis activity, or inhibitors of SDF-1 alpha-CXCR4 binding resulting in an anti-metastasis effect. We also predict that PA-Neo-PAs and APACs can interfere with CD4-gp120 binding in unliganded conformation.

  4. Multifunctional antimicrobial proteins and peptides: natural activators of immune systems.

    PubMed

    Niyonsaba, François; Nagaoka, Isao; Ogawa, Hideoki; Okumura, Ko

    2009-01-01

    In addition to the physical barrier of the stratum corneum, cutaneous innate immunity also includes the release of various humoral mediators, such as cytokines and chemokines, recruitment and activation of phagocytes, and the production of antimicrobial proteins/peptides (AMPs). AMPs form an innate epithelial chemical shield, which provides a front-line component in innate immunity to inhibit microbial invasion; however, this might be an oversimplification of the diverse functions of these molecules. In fact, apart from exhibiting a broad spectrum of microbicidal properties, it is increasingly evident that AMPs display additional activities that are related to the stimulation and modulation of the cutaneous immune system. These diverse functions include chemoattraction and activation of immune and/or inflammatory cells, the production and release of cytokines and chemokines, acceleration of angiogenesis, promotion of wound healing, neutralization of harmful microbial products, and bridging of both innate and adaptive immunity. Thus, better understanding of the functions of AMPs in skin and identification of their signaling mechanisms may offer new strategies for the development of potential therapeutics for the treatment of infection- and/or inflammation-related skin diseases. Here, we briefly outline the structure, regulation of expression, and multifunctional roles of principal skin-derived AMPs.

  5. Characterization of a new peptide agonist of the protease-activated receptor-1.

    PubMed

    Mao, Yingying; Jin, Jianguo; Kunapuli, Satya P

    2008-01-15

    A new peptide (TFRRRLSRATR), derived from the c-terminal of human platelet P2Y(1) receptor, was synthesized and its biological function was evaluated. This peptide activated platelets in a concentration-dependent manner, causing shape change, aggregation, secretion and calcium mobilization. Of the several receptor antagonists tested, only BMS200261, a protease activated receptor 1 (PAR-1) specific antagonist, totally abolished the peptide-induced platelet aggregation, secretion and calcium mobilization. The TFRRR-peptide-pretreated washed platelets failed to aggregate in response to SFLLRN (10 microM) but not to AYPGKF (500 microM). In addition, in mouse platelets, peptide concentrations up to 600 microM failed to cause platelet activation, indicating that the TFRRR-peptide activated platelets through the PAR-1 receptor, rather than through the PAR-4 receptor. The shape change induced by 10 microM peptide was totally abolished by Y-27632, an inhibitor of p160(ROCK) which is a downstream mediator of G12/13 pathways. The TFRRR-peptide, YFLLRNP, and the physiological agonist thrombin selectively activated G12/13 pathways at low concentrations and began to activate both Gq and G12/13 pathways with increasing concentrations. Similar to SFLLRN, the TFRRR-peptide caused phosphorylation of Akt and Erk in a P2Y(12) receptor-dependent manner, and p-38 MAP kinase activation in a P2Y(12)-independent manner. The effects of this peptide are elicited by the first six amino acids (TFRRRL) whereas the remaining peptide (LSRATR), TFERRN, or TFEERN had no effects on platelets. We conclude that TFRRRL activates human platelets through PAR-1 receptors. PMID:17950254

  6. Antimicrobial Activity of Novel Synthetic Peptides Derived from Indolicidin and Ranalexin against Streptococcus pneumoniae

    PubMed Central

    Jindal, Hassan Mahmood; Le, Cheng Foh; Mohd Yusof, Mohd Yasim; Velayuthan, Rukumani Devi; Lee, Vannajan Sanghiran; Zain, Sharifuddin Md; Isa, Diyana Mohd; Sekaran, Shamala Devi

    2015-01-01

    Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics in order to defeat multidrug-resistant bacteria such as Streptococcus pneumoniae. In this study, thirteen antimicrobial peptides were designed based on two natural peptides indolicidin and ranalexin. Our results revealed that four hybrid peptides RN7-IN10, RN7-IN9, RN7-IN8, and RN7-IN6 possess potent antibacterial activity against 30 pneumococcal clinical isolates (MIC 7.81-15.62µg/ml). These four hybrid peptides also showed broad spectrum antibacterial activity (7.81µg/ml) against S. aureus, methicillin resistant S. aureus (MRSA), and E. coli. Furthermore, the time killing assay results showed that the hybrid peptides were able to eliminate S. pneumoniae within less than one hour which is faster than the standard drugs erythromycin and ceftriaxone. The cytotoxic effects of peptides were tested against human erythrocytes, WRL-68 normal liver cell line, and NL-20 normal lung cell line. The results revealed that none of the thirteen peptides have cytotoxic or hemolytic effects at their MIC values. The in silico molecular docking study was carried out to investigate the binding properties of peptides with three pneumococcal virulent targets by Autodock Vina. RN7IN6 showed a strong affinity to target proteins; autolysin, pneumolysin, and pneumococcal surface protein A (PspA) based on rigid docking studies. Our results suggest that the hybrid peptides could be suitable candidates for antibacterial drug development. PMID:26046345

  7. Plasmin substrate binding site cooperativity guides the design of potent peptide aldehyde inhibitors.

    PubMed

    Swedberg, Joakim E; Harris, Jonathan M

    2011-10-01

    Perioperative bleeding is a cause of major blood loss and is associated with increased rates of postoperative morbidity and mortality. To combat this, antifibrinolytic inhibitors of the serine protease plasmin are commonly used to reduce bleeding during surgery. The most effective and previously widely used of these is the broad range serine protease inhibitor aprotinin. However, adverse clinical outcomes have led to use of alternative serine lysine analogues to inhibit plasmin. These compounds suffer from low selectivity and binding affinity. Consequently, a concerted effort to discover potent and selective plasmin inhibitors has developed. This study used a noncombinatorial peptide library to define plasmin's extended substrate specificity and guide the design of potent transition state analogue inhibitors. The various substrate binding sites of plasmin were found to exhibit a higher degree of cooperativity than had previously been appreciated. Peptide sequences capitalizing on these features produced high-affinity inhibitors of plasmin. The most potent of these, Lys-Met(sulfone)-Tyr-Arg-H [KM(O(2))YR-H], inhibited plasmin with a K(i) of 3.1 nM while maintaining 25-fold selectivity over plasma kallikrein. Furthermore, 125 nM (0.16 μg/mL) KM(O(2))YR-H attenuated fibrinolysis in vitro with an efficacy similar to that of 15 nM (0.20 μg/mL) aprotinin. To date, this is the most potent peptide inhibitor of plasmin that exhibits selectivity against plasma kallikrein, making this compound an attractive candidate for further therapeutic development. PMID:21877690

  8. Antimicrobial peptides: natural templates for synthetic membrane-active compounds.

    PubMed

    Giuliani, A; Pirri, G; Bozzi, A; Di Giulio, A; Aschi, M; Rinaldi, A C

    2008-08-01

    The innate immunity of multicellular organisms relies in large part on the action of antimicrobial peptides (AMPs) to resist microbial invasion. Crafted by evolution into an extremely diversified array of sequences and folds, AMPs do share a common amphiphilic 3-D arrangement. This feature is directly linked with a common mechanism of action that predominantly (although not exclusively) develops upon interaction of peptides with cell membranes of target cells. This minireview reports on current understanding of the modes of interaction of AMPs with biological and model membranes, especially focusing on recent insights into the folding and oligomerization requirements of peptides to bind and insert into lipid membranes and exert their antibiotic effects. Given the potential of AMPs to be developed into a new class of anti-infective agents, emphasis is placed on how the information on peptide-membrane interactions could direct the design and selection of improved biomimetic synthetic peptides with antibiotic properties.

  9. Antimicrobial activity of mosquito cecropin peptides against Francisella.

    PubMed

    Kaushal, Akanksha; Gupta, Kajal; Shah, Ruhee; van Hoek, Monique L

    2016-10-01

    Francisella tularensis is the cause of the zoonotic disease tularemia. In Sweden and Scandinavia, epidemiological studies have implicated mosquitoes as a vector. Prior research has demonstrated the presence of Francisella DNA in infected mosquitoes but has not shown definitive transmission of tularemia from a mosquito to a mammalian host. We hypothesized that antimicrobial peptides, an important component of the innate immune system of higher organisms, may play a role in mosquito host-defense to Francisella. We established that Francisella sp. are susceptible to two cecropin antimicrobial peptides derived from the mosquito Aedes albopictus as well as Culex pipiens. We also demonstrated induced expression of Aedes albopictus antimicrobial peptide genes by Francisella infection C6/36 mosquito cell line. We demonstrate that mosquito antimicrobial peptides act against Francisella by disrupting the cellular membrane of the bacteria. Thus, it is possible that antimicrobial peptides may play a role in the inability of mosquitoes to establish an effective natural transmission of tularemia. PMID:27235883

  10. Amyloidogenic amyloid-β-peptide variants induce microbial agglutination and exert antimicrobial activity.

    PubMed

    Spitzer, Philipp; Condic, Mateja; Herrmann, Martin; Oberstein, Timo Jan; Scharin-Mehlmann, Marina; Gilbert, Daniel F; Friedrich, Oliver; Grömer, Teja; Kornhuber, Johannes; Lang, Roland; Maler, Juan Manuel

    2016-01-01

    Amyloid-β (Aβ) peptides are the main components of the plaques found in the brains of patients with Alzheimer's disease. However, Aβ peptides are also detectable in secretory compartments and peripheral blood contains a complex mixture of more than 40 different modified and/or N- and C-terminally truncated Aβ peptides. Recently, anti-infective properties of Aβ peptides have been reported. Here, we investigated the interaction of Aβ peptides of different lengths with various bacterial strains and the yeast Candida albicans. The amyloidogenic peptides Aβ1-42, Aβ2-42, and Aβ3p-42 but not the non-amyloidogenic peptides Aβ1-40 and Aβ2-40 bound to microbial surfaces. As observed by immunocytochemistry, scanning electron microscopy and Gram staining, treatment of several bacterial strains and Candida albicans with Aβ peptide variants ending at position 42 (Aβx-42) caused the formation of large agglutinates. These aggregates were not detected after incubation with Aβx-40. Furthermore, Aβx-42 exerted an antimicrobial activity on all tested pathogens, killing up to 80% of microorganisms within 6 h. Aβ1-40 only had a moderate antimicrobial activity against C. albicans. Agglutination of Aβ1-42 was accelerated in the presence of microorganisms. These data demonstrate that the amyloidogenic Aβx-42 variants have antimicrobial activity and may therefore act as antimicrobial peptides in the immune system. PMID:27624303

  11. Amyloidogenic amyloid-β-peptide variants induce microbial agglutination and exert antimicrobial activity

    PubMed Central

    Spitzer, Philipp; Condic, Mateja; Herrmann, Martin; Oberstein, Timo Jan; Scharin-Mehlmann, Marina; Gilbert, Daniel F.; Friedrich, Oliver; Grömer, Teja; Kornhuber, Johannes; Lang, Roland; Maler, Juan Manuel

    2016-01-01

    Amyloid-β (Aβ) peptides are the main components of the plaques found in the brains of patients with Alzheimer’s disease. However, Aβ peptides are also detectable in secretory compartments and peripheral blood contains a complex mixture of more than 40 different modified and/or N- and C-terminally truncated Aβ peptides. Recently, anti-infective properties of Aβ peptides have been reported. Here, we investigated the interaction of Aβ peptides of different lengths with various bacterial strains and the yeast Candida albicans. The amyloidogenic peptides Aβ1-42, Aβ2-42, and Aβ3p-42 but not the non-amyloidogenic peptides Aβ1-40 and Aβ2-40 bound to microbial surfaces. As observed by immunocytochemistry, scanning electron microscopy and Gram staining, treatment of several bacterial strains and Candida albicans with Aβ peptide variants ending at position 42 (Aβx-42) caused the formation of large agglutinates. These aggregates were not detected after incubation with Aβx-40. Furthermore, Aβx-42 exerted an antimicrobial activity on all tested pathogens, killing up to 80% of microorganisms within 6 h. Aβ1-40 only had a moderate antimicrobial activity against C. albicans. Agglutination of Aβ1-42 was accelerated in the presence of microorganisms. These data demonstrate that the amyloidogenic Aβx-42 variants have antimicrobial activity and may therefore act as antimicrobial peptides in the immune system. PMID:27624303

  12. Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

    PubMed Central

    Hammond, S. E.; Hanna, P. C.

    1998-01-01

    The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase. PMID:9573135

  13. Fidelity of targeting to chloroplasts is not affected by removal of the phosphorylation site from the transit peptide.

    PubMed

    Nakrieko, Kerry-Ann; Mould, Ruth M; Smith, Alison G

    2004-02-01

    Phosphorylation of the transit peptide of several chloroplast-targeted proteins enables the binding of 14-3-3 proteins. The complex that forms, together with Hsp70, has been demonstrated to be an intermediate in the chloroplast protein import pathway in vitro[May, T. & Soll, J. (2000) Plant Cell 12, 53-63]. In this paper we report that mutagenesis (in order to remove the phosphorylation site) of the transit peptide of the small subunit of ribulose bisphosphate carboxylase/oxygenase did not affect its ability to target green fluorescent protein to chloroplasts in vivo. We also found no mistargeting to other organelles such as mitochondria. Similar alterations to the transit peptides of histidyl- or cysteinyl-tRNA synthetase, which are dual-targeted to chloroplasts and mitochondria, had no effect on their ability to target green fluorescent protein in vivo. Thus, phosphorylation of the transit peptide is not responsible for the specificity of chloroplast import.

  14. Synthesis and antiviral activity of PB1 component of the influenza A RNA polymerase peptide fragments.

    PubMed

    Matusevich, O V; Egorov, V V; Gluzdikov, I A; Titov, M I; Zarubaev, V V; Shtro, A A; Slita, A V; Dukov, M I; Shurygina, A-P S; Smirnova, T D; Kudryavtsev, I V; Vasin, A V; Kiselev, O I

    2015-01-01

    This study is devoted to the antiviral activity of peptide fragments from the PB1 protein - a component of the influenza A RNA polymerase. The antiviral activity of the peptides synthesized was studied in MDCK cell cultures against the pandemic influenza strain A/California/07/2009 (H1N1) pdm09. We found that peptide fragments 6-13, 6-14, 26-30, 395-400, and 531-540 of the PB1 protein were capable of suppressing viral replication in cell culture. Terminal modifications i.e. N-acetylation and C-amidation increased the antiviral properties of the peptides significantly. Peptide PB1 (6-14) with both termini modified showed maximum antiviral activity, its inhibitory activity manifesting itself during the early stages of viral replication. It was also shown that the fluorescent-labeled analog of this peptide was able to penetrate into the cell. The broad range of virus-inhibiting activity of PB1 (6-14) peptide was confirmed using a panel of influenza A viruses of H1, H3 and H5 subtypes including those resistant to oseltamivir, the leading drug in anti-influenza therapy. Thus, short peptide fragments of the PB1 protein could serve as leads for future development of influenza prevention and/or treatment agents.

  15. Practical 4′-Phosphopantetheine Active Site Discovery from Proteomic Samples

    PubMed Central

    Meier, Jordan L.; Patel, Anand D.; Niessen, Sherry; Meehan, Michael; Kersten, Roland; Yang, Jane Y.; Rothmann, Michael; Cravatt, Benjamin F.; Dorrestein, Pieter

    2011-01-01

    Polyketide and nonribosomal peptides constitute important classes of small molecule natural products. Due to the proven biological activities of these compounds, novel methods for discovery and study of the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymes responsible for their production remains an area of intense interest, and proteomic approaches represent a relatively unexplored avenue. While these enzymes may be distinguished from the proteomic milieu by their use of the 4′-phosphopantetheine (PPant) posttranslational modification, proteomic detection of PPant peptides is hindered by their low abundance and labile nature which leaves them unassigned using traditional database searching. Here we address key experimental and computational challenges to facilitate practical discovery of this important posttranslational modification during shotgun proteomics analysis using low-resolution ion-trap mass spectrometers. Activity-based enrichment maximizes MS input of PKS/NRPS peptides, while targeted fragmentation detects putative PPant active sites. An improved data analysis pipeline allows experimental identification and validation of these PPant peptides directly from MS2 data. Finally, a machine learning approach is developed to directly detect PPant peptides from only MS2 fragmentation data. By providing new methods for analysis of an often cryptic posttranslational modification, these methods represent a first step towards the study of natural product biosynthesis in proteomic settings. PMID:21067235

  16. Peptide array on cellulose support--a screening tool to identify peptides with dipeptidyl-peptidase IV inhibitory activity within the sequence of α-lactalbumin.

    PubMed

    Lacroix, Isabelle M E; Li-Chan, Eunice C Y

    2014-11-13

    The inhibition of the enzyme dipeptidyl-peptidase IV (DPP-IV) is an effective pharmacotherapeutic approach for the management of type 2 diabetes. Recent findings have suggested that dietary proteins, including bovine α-lactalbumin, could be precursors of peptides able to inhibit DPP-IV. However, information on the location of active peptide sequences within the proteins is far from being comprehensive. Moreover, the traditional approach to identify bioactive peptides from foods can be tedious and long. Therefore, the objective of this study was to use peptide arrays to screen α-lactalbumin-derived peptides for their interaction with DPP-IV. Deca-peptides spanning the entire α-lactalbumin sequence, with a frame shift of 1 amino acid between successive sequences, were synthesized on cellulose membranes using "SPOT" technology, and their binding to and inhibition of DPP-IV was studied. Among the 114 α-lactalbumin-derived decamers investigated, the peptides 60WCKDDQNPHS69 (αK(i) = 76 µM), 105LAHKALCSEK114 (K(i) = 217 µM) and 110LCSEKLDQWL119 (K(i) = 217 µM) were among the strongest DPP-IV inhibitors. While the SPOT- and traditionally-synthesized peptides showed consistent trends in DPP-IV inhibitory activity, the cellulose-bound peptides' binding behavior was not correlated to their ability to inhibit the enzyme. This research showed, for the first time, that peptide arrays are useful screening tools to identify DPP-IV inhibitory peptides from dietary proteins.

  17. Activation of p53-dependent responses in tumor cells treated with a PARC-interacting peptide

    SciTech Connect

    Vitali, Roberta; Cesi, Vincenzo; Tanno, Barbara; Ferrari-Amorotti, Giovanna; Dominici, Carlo; Calabretta, Bruno; Raschella, Giuseppe

    2008-04-04

    We tested the activity of a p53 carboxy-terminal peptide containing the PARC-interacting region in cancer cells with wild type cytoplasmic p53. Peptide delivery was achieved by fusing it to the TAT transduction domain (TAT-p53-C-ter peptide). In a two-hybrid assay, the tetramerization domain (TD) of p53 was necessary and sufficient to bind PARC. The TAT-p53-C-ter peptide disrupted the PARC-p53 complex. Peptide treatment caused p53 nuclear relocation, p53-dependent changes in gene expression and enhancement of etoposide-induced apoptosis. These studies suggest that PARC-interacting peptides are promising candidates for the enhancement of p53-dependent apoptosis in tumors with wt cytoplasmic p53.

  18. Identification of Synthetic and Natural Host Defense Peptides with Leishmanicidal Activity

    PubMed Central

    Marr, A. K.; Cen, S.; Hancock, R. E. W.

    2016-01-01

    Leishmania parasites are a major public health problem worldwide. Effective treatment of leishmaniasis is hampered by the high incidence of adverse effects to traditional drug therapy and the emergence of resistance to current therapeutics. A vaccine is currently not available. Host defense peptides have been investigated as novel therapeutic agents against a wide range of pathogens. Here we demonstrate that the antimicrobial peptide LL-37 and the three synthetic peptides E6, L-1018, and RI-1018 exhibit leishmanicidal activity against promastigotes and intramacrophage amastigotes of Leishmania donovani and Leishmania major. We also report that the Leishmania protease/virulence factor GP63 confers protection to Leishmania from the cytolytic properties of all l-form peptides (E6, L-1018, and LL-37) but not the d-form peptide RI-1018. The results suggest that RI-1018, E6, and LL-37 are promising peptides to develop further into components for antileishmanial therapy. PMID:26883699

  19. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  20. Immunohistochemical localization of the calcitonin gene-related peptide binding site in the primate trigeminovascular system using functional antagonist antibodies.

    PubMed

    Miller, Silke; Liu, Hantao; Warfvinge, Karin; Shi, Licheng; Dovlatyan, Mary; Xu, Cen; Edvinsson, Lars

    2016-07-22

    Calcitonin gene-related peptide (CGRP) is a potent vasodilator and a neuromodulator implicated in the pathophysiology of migraine. It binds to the extracellular domains of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein (RAMP) 1 that together form the CGRP receptor. Antagonist antibodies against CGRP and its binding site at the receptor are clinically effective in preventing migraine attacks. The blood-brain barrier penetration of these antagonist antibodies is limited, suggesting that a potential peripheral site of action is sufficient to prevent migraine attacks. To further understand the sites of CGRP-mediated signaling in migraine, we used immunohistochemical staining with recently developed antagonist antibodies specifically recognizing a fusion protein of the extracellular domains of RAMP1 and CLR that comprise the CGRP binding pocket at the CGRP receptor in monkey and man. We confirmed binding of the antagonist antibodies to human vascular smooth muscle cells (VSMCs) of dural meningeal arteries and neurons in the trigeminal ganglion, both of which are likely sites of action for therapeutic antibodies in migraine patients. We further used one of these antibodies for detailed mapping on cynomolgus monkey tissue and found antagonist antibody binding sites at multiple levels in the trigeminovascular system: in the dura mater VSMCs, in neurons and satellite glial cells in the trigeminal ganglion, and in neurons in the spinal trigeminal nucleus caudalis. These data reinforce and clarify our understanding of CGRP receptor localization in a pattern consistent with a role for CGRP receptors in trigeminal sensitization and migraine pathology. PMID:27155150

  1. Gram-positive bacterial cell envelopes: The impact on the activity of antimicrobial peptides.

    PubMed

    Malanovic, Nermina; Lohner, Karl

    2016-05-01

    A number of cationic antimicrobial peptides, effectors of innate immunity, are supposed to act at the cytoplasmic membrane leading to permeabilization and eventually membrane disruption. Thereby, interaction of antimicrobial peptides with anionic membrane phospholipids is considered to be a key factor in killing of bacteria. Recently, evidence was provided that killing takes place only when bacterial cell membranes are completely saturated with peptides. This adds to an ongoing debate, which role cell wall components such as peptidoglycan, lipoteichoic acid and lipopolysaccharide may play in the killing event, i.e. if they rather entrap or facilitate antimicrobial peptides access to the cytoplasmic membrane. Therefore, in this review we focused on the impact of Gram-positive cell wall components for the mode of action and activity of antimicrobial peptides as well as in innate immunity. This led us to conclude that interaction of antimicrobial peptides with peptidoglycan may not contribute to a reduction of their antimicrobial activity, whereas interaction with anionic lipoteichoic acids may reduce the local concentration of antimicrobial peptides on the cytoplasmic membrane necessary for sufficient destabilization of the membranes and bacterial killing. Further affinity studies of antimicrobial peptides toward the different cell wall as well as membrane components will be needed to address this problem on a quantitative level. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

  2. Antiaging activity of low molecular weight peptide from Paphia undulate

    NASA Astrophysics Data System (ADS)

    Chen, Xin; Cai, Bingna; Chen, Hua; Pan, Jianyu; Chen, Deke; Sun, Huili

    2013-05-01

    Low molecular weight peptide (LMWP) was prepared from clam Paphia undulate and its antiaging effect on D-galactose-induced acute aging in rats, aged Kunming mice, ultraviolet-exposed rats, and thermally injured rats was investigated. P. undulate flesh was homogenized and digested using papain under optimal conditions, then subjected to Sephadex G-25 chromatography to isolate the LMWP. Administration of LMWP significantly reversed D-galactose-induced oxidative stress by increasing the activities of glutathione peroxidase (GPx) and catalase (CAT), and by decreasing the level of malondialdehyde (MDA). This process was accompanied by increased collagen synthesis. The LMWP prevented photoaging and promoted dermis recovery and remission of elastic fiber hyperplasia. Furthermore, treatment with the LMWP helped to regenerate elastic fibers and the collagen network, increased superoxide dismutase (SOD) in the serum and significantly decreased MDA. Thermal scald-induced inflammation and edema were also relieved by the LWMP, while wound healing in skin was promoted. These results suggest that the LMWP from P. undulate could serve as a new antiaging substance in cosmetics.

  3. Antifungal Activity of 14-Helical β-Peptides against Planktonic Cells and Biofilms of Candida Species.

    PubMed

    Raman, Namrata; Lee, Myung-Ryul; Lynn, David M; Palecek, Sean P

    2015-01-01

    Candida albicans is the most prevalent cause of fungal infections and treatment is further complicated by the formation of drug resistant biofilms, often on the surfaces of implanted medical devices. In recent years, the incidence of fungal infections by other pathogenic Candida species such as C. glabrata, C. parapsilosis and C. tropicalis has increased. Amphiphilic, helical β-peptide structural mimetics of natural antimicrobial α-peptides have been shown to exhibit specific planktonic antifungal and anti-biofilm formation activity against C. albicans in vitro. Here, we demonstrate that β-peptides are also active against clinically isolated and drug resistant strains of C. albicans and against other opportunistic Candida spp. Different Candida species were susceptible to β-peptides to varying degrees, with C. tropicalis being the most and C. glabrata being the least susceptible. β-peptide hydrophobicity directly correlated with antifungal activity against all the Candida clinical strains and species tested. While β-peptides were largely ineffective at disrupting existing Candida biofilms, hydrophobic β-peptides were able to prevent the formation of C. albicans, C. glabrata, C. parapsilosis and C. tropicalis biofilms. The broad-spectrum antifungal activity of β-peptides against planktonic cells and in preventing biofilm formation suggests the promise of this class of molecules as therapeutics. PMID:26287212

  4. Activities of Venom Proteins and Peptides with Possible Therapeutic Applications from Bees and WASPS.

    PubMed

    Ye, Xiujuan; Guan, Suzhen; Liu, Jiwen; Ng, Charlene C W; Chan, Gabriel H H; Sze, Stephen C W; Zhang, Kalin Y; Naude, Ryno; Rolka, Krzysztof; Wong, Jack Ho; Ng, Tzi Bun

    2016-01-01

    The variety of proteins and peptides isolated from honey bee venom and wasp venom includes melittin, adiapin, apamine, bradykinin, cardiopep, mast cell degranulating peptide, mastoparan, phospholipase A2 and secapin. Some of the activities they demonstrate may find therapeutic applications. PMID:27323949

  5. How water molecules affect the catalytic activity of hydrolases--a XANES study of the local structures of peptide deformylase.

    PubMed

    Cui, Peixin; Wang, Yu; Chu, Wangsheng; Guo, Xiaoyun; Yang, Feifei; Yu, Meijuan; Zhao, Haifeng; Dong, Yuhui; Xie, Yaning; Gong, Weimin; Wu, Ziyu

    2014-12-12

    Peptide deformylase (PDF) is a prokaryotic enzyme that catalyzes the deformylation of nascent peptides generated during protein synthesis and water molecules play a key role in these hydrolases. Using X-ray absorption near edge spectroscopy (XANES) and ab initio calculations we accurately probe the local atomic environment of the metal ion binding in the active site of PDF at different pH values and with different metal ions. This new approach is an effective way to monitor existing correlations among functions and structural changes. We show for the first time that the enzymatic activity depends on pH values and metal ions via the bond length of the nearest coordinating water (Wat1) to the metal ion. Combining experimental and theoretical data we may claim that PDF exhibits an enhanced enzymatic activity only when the distance of the Wat1 molecule with the metal ion falls in the limited range from 2.15 to 2.55 Å.

  6. Peptide-cleaving agents for human islet amyloid polypeptide containing substrate recognition site based on quinoxaline: cleavage efficiency enhanced by lowering substrate concentration.

    PubMed

    Chei, Woosuk; Ju, Heeyeon; Suh, Junghun

    2012-02-15

    Oligomers of human islet amyloid polypeptide (h-IAPP) are believed to be the pathogenic species for type 2 diabetes mellitus. Peptide-cleaving agents selective for oligomers of h-IAPP were synthesized by using quinoxaline derivatives as recognition sites attached to the Co(III) complex of cyclen in this study. When the initial concentration of h-IAPP was lowered from 4.0 to 0.20 μM, cleavage yield of the new agents was enhanced by 3 times reaching 16-22 mol%. This shows that the agents would have significant activities at subnano molar concentrations if the concentration of h-IAPP is lowered to the in vivo values. This further indicates that the peptide-cleaving agents prepared previously in this laboratory possess sufficiently high activity for application as a new therapeutic option for Alzheimer's disease, type 2 diabetes mellitus, and Parkinson's disease.

  7. Activity of antimicrobial peptide mimetics in the oral cavity: I. Activity against biofilms of Candida albicans.

    PubMed

    Hua, J; Yamarthy, R; Felsenstein, S; Scott, R W; Markowitz, K; Diamond, G

    2010-12-01

    Naturally occurring antimicrobial peptides hold promise as therapeutic agents against oral pathogens such as Candida albicans but numerous difficulties have slowed their development. Synthetic, non-peptidic analogs that mimic the properties of these peptides have many advantages and exhibit potent, selective antimicrobial activity. Several series of mimetics (with molecular weight < 1000) were developed and screened against oral Candida strains as a proof-of-principle for their antifungal properties. One phenylalkyne and several arylamide compounds with reduced mammalian cytotoxicities were found to be active against C. albicans. These compounds demonstrated rapid fungicidal activity in liquid culture even in the presence of saliva, and demonstrated synergy with standard antifungal agents. When assayed against biofilms grown on denture acrylic, the compounds exhibited potent fungicidal activity as measured by metabolic and fluorescent viability assays. Repeated passages in sub-minimum inhibitory concentration levels did not lead to resistant Candida, in contrast to fluconazole. Our results demonstrate the proof-of principle for the use of these compounds as anti-Candida agents, and their further testing is warranted as novel anti-Candida therapies.

  8. Activity of Antimicrobial Peptide Mimetics in the Oral Cavity: I. Activity Against Biofilms of Candida albicans

    PubMed Central

    Hua, Jianyuan; Yamarthy, Radha; Felsenstein, Shaina; Scott, Richard W.; Markowitz, Kenneth; Diamond, Gill

    2010-01-01

    Summary Naturally occurring antimicrobial peptides hold promise as therapeutic agents against oral pathogens such as Candida albicans, however numerous difficulties have slowed their development. Synthetic, non-peptidic analogs that mimic the properties of these peptides have many advantages and exhibit potent, selective antimicrobial activity. Several series of mimetics (MW <1,000) were developed and screened against oral Candida strains as a proof-of-principle for their antifungal properties. One phenylalkyne and several arylamide compounds with reduced mammalian cytotoxicities were found to be active against C. albicans. These compounds demonstrated rapid fungicidal activity in liquid culture even in the presence of saliva, and demonstrated synergy with standard antifungal agents. When assayed against biofilms grown on denture acrylic, the compounds exhibited potent fungicidal activity as measured by metabolic and fluorescent viability assays. Repeated passages in sub-MIC levels did not lead to resistant Candida in contrast to fluconazole. Our results demonstrate the proof-of principle for the use of these compounds as anti-Candida agents, and their further testing is warranted as novel anti-Candida therapies. PMID:21040515

  9. Antibody Constant Region Peptides Can Display Immunomodulatory Activity through Activation of the Dectin-1 Signalling Pathway

    PubMed Central

    Cenci, Elio; Monari, Claudia; Magliani, Walter; Ciociola, Tecla; Conti, Stefania; Gatti, Rita; Bistoni, Francesco; Polonelli, Luciano; Vecchiarelli, Anna

    2012-01-01

    We previously reported that a synthetic peptide with sequence identical to a CDR of a mouse monoclonal antibody specific for difucosyl human blood group A exerted an immunomodulatory activity on murine macrophages. It was therapeutic against systemic candidiasis without possessing direct candidacidal properties. Here we demonstrate that a selected peptide, N10K, putatively deriving from the enzymatic cleavage of the constant region (Fc) of human IgG1, is able to induce IL-6 secretion and pIkB-α activation. More importantly, it causes an up-regulation of Dectin-1 expression. This leads to an increased activation of β-glucan-induced pSyk, CARD9 and pIkB-α, and an increase in the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-1β and TNF-α. The increased activation of this pathway coincides with an augmented phagocytosis of non opsonized Candida albicans cells by monocytes. The findings suggest that some Fc-peptides, potentially deriving from the proteolysis of immunoglobulins, may cause an unexpected immunoregulation in a way reminiscent of innate immunity molecules. PMID:22952831

  10. Antimicrobial and antibiofilm activity of cathelicidins and short, synthetic peptides against Francisella.

    PubMed

    Amer, Lilian S; Bishop, Barney M; van Hoek, Monique L

    2010-05-28

    Francisella infects the lungs causing pneumonic tularemia. Focusing on the lung's host defense, we have examined antimicrobial peptides as part of the innate immune response to Francisella infection. Interest in antimicrobial peptides, such as the cathelicidins, has grown due their potential therapeutic applications and the increasing problem of bacterial resistance to commonly used antibiotics. Only one human cathelicidin, LL-37, has been characterized. Helical cathelicidins have also been discovered in snakes including the Chinese King Cobra, Naja atra (NA-CATH). Four synthetic 11-residue peptides (ATRA-1, -2, -1A and -1P) containing variations of a repeated motif within NA-CATH were designed. We hypothesized that these smaller synthetic peptides could have excellent antimicrobial effectiveness with shorter length (and less cost), making them strong potential candidates for development into broad-spectrum antimicrobial compounds. We tested the susceptibility of F. novicida to four ATRA peptides, LL-37, and NA-CATH. Two of the ATRA peptides had high antimicrobial activity (microM), while the two proline-containing ATRA peptides had low activity. The ATRA peptides did not show significant hemolytic activity even at high peptide concentration, indicating low cytotoxicity against host cells. NA-CATH killed Francisella bacteria more quickly than LL-37. However, LL-37 was the most effective peptide against F. novicida (EC50=50 nM). LL-37 mRNA was induced in A549 cells by Francisella infection. We recently demonstrated that F. novicida forms in vitro biofilms. LL-37 inhibited F. novicida biofilm formation at sub-antimicrobial concentrations. Understanding the properties of these peptides, and their endogenous expression in the lung could lead to potential future therapeutic interventions for this lung infection. PMID:20399752

  11. Zinc(II) Binding Site to the Amyloid-β Peptide: Insights from Spectroscopic Studies with a Wide Series of Modified Peptides

    PubMed Central

    2016-01-01

    The Zn(II) ion has been linked to Alzheimer’s disease (AD) due to its ability to modulate the aggregating properties of the amyloid-β (Aβ) peptide, where Aβ aggregation is a central event in the etiology of the disease. Delineating Zn(II) binding properties to Aβ is thus a prerequisite to better grasp its potential role in AD. Because of (i) the flexibility of the Aβ peptide, (ii) the multiplicity of anchoring sites, and (iii) the silent nature of the Zn(II) ion in most classical spectroscopies, this is a difficult task. To overcome these difficulties, we have investigated the impact of peptide alterations (mutations, N-terminal acetylation) on the Zn(Aβ) X-ray absorption spectroscopy fingerprint and on the Zn(II)-induced modifications of the Aβ peptides’ NMR signatures. We propose a tetrahedrally bound Zn(II) ion, in which the coordination sphere is made by two His residues and two carboxylate side chains. Equilibria between equivalent ligands for one Zn(II) binding position have also been observed, the predominant site being made by the side chains of His6, His13 or His14, Glu11, and Asp1 or Glu3 or Asp7, with a slight preference for Asp1. PMID:27665863

  12. New Milk Protein-Derived Peptides with Potential Antimicrobial Activity: An Approach Based on Bioinformatic Studies

    PubMed Central

    Dziuba, Bartłomiej; Dziuba, Marta

    2014-01-01

    New peptides with potential antimicrobial activity, encrypted in milk protein sequences, were searched for with the use of bioinformatic tools. The major milk proteins were hydrolyzed in silico by 28 enzymes. The obtained peptides were characterized by the following parameters: molecular weight, isoelectric point, composition and number of amino acid residues, net charge at pH 7.0, aliphatic index, instability index, Boman index, and GRAVY index, and compared with those calculated for known 416 antimicrobial peptides including 59 antimicrobial peptides (AMPs) from milk proteins listed in the BIOPEP database. A simple analysis of physico-chemical properties and the values of biological activity indicators were insufficient to select potentially antimicrobial peptides released in silico from milk proteins by proteolytic enzymes. The final selection was made based on the results of multidimensional statistical analysis such as support vector machines (SVM), random forest (RF), artificial neural networks (ANN) and discriminant analysis (DA) available in the Collection of Anti-Microbial Peptides (CAMP database). Eleven new peptides with potential antimicrobial activity were selected from all peptides released during in silico proteolysis of milk proteins. PMID:25141106

  13. Quantitative structure-activity relationships and docking studies of calcitonin gene-related peptide antagonists.

    PubMed

    Kyani, Anahita; Mehrabian, Mohadeseh; Jenssen, Håvard

    2012-02-01

    Defining the role of calcitonin gene-related peptide in migraine pathogenesis could lead to the application of calcitonin gene-related peptide antagonists as novel migraine therapeutics. In this work, quantitative structure-activity relationship modeling of biological activities of a large range of calcitonin gene-related peptide antagonists was performed using a panel of physicochemical descriptors. The computational studies evaluated different variable selection techniques and demonstrated shuffling stepwise multiple linear regression to be superior over genetic algorithm-multiple linear regression. The linear quantitative structure-activity relationship model revealed better statistical parameters of cross-validation in comparison with the non-linear support vector regression technique. Implementing only five peptide descriptors into this linear quantitative structure-activity relationship model resulted in an extremely robust and highly predictive model with calibration, leave-one-out and leave-20-out validation R(2) of 0.9194, 0.9103, and 0.9214, respectively. We performed docking of the most potent calcitonin gene-related peptide antagonists with the calcitonin gene-related peptide receptor and demonstrated that peptide antagonists act by blocking access to the peptide-binding cleft. We also demonstrated the direct contact of residues 28-37 of the calcitonin gene-related peptide antagonists with the receptor. These results are in agreement with the conclusions drawn from the quantitative structure-activity relationship model, indicating that both electrostatic and steric factors should be taken into account when designing novel calcitonin gene-related peptide antagonists. PMID:21974743

  14. Site-directed mutation of arginine 282 to glutamate uncouples the movement of peptides and protons by the rabbit proton-peptide cotransporter PepT1.

    PubMed

    Meredith, David

    2004-04-16

    A conserved positive residue in the seventh transmembrane domain of the mammalian proton-coupled di- and tripeptide transporter PepT1 has been shown by site-directed mutagenesis to be a key residue for protein function. Substitution of arginine 282 with a glutamate residue (R282E-PepT1) gave a protein at the plasma membrane of Xenopus laevis oocytes that was able to transport the non-hydrolyzable dipeptide [3H]d-Phe-l-Gln, although unlike the wild type, the rate of transport by R282E-PepT1 was independent of the extracellular pH level, and the substrate could not be accumulated above equilibrium. The binding affinity of the mutant transport protein was unchanged from the wild type. Thus, R282E-Pept1 appears to have been changed from a proton-driven to a facilitated transporter for peptides. In addition, peptide transport by R282E-PepT1 still induced depolarization as measured by microelectrode recordings of membrane potential. A more detailed study by two-electrode voltage clamping revealed that R282E-PepT1 behaved as a peptide-gated non-selective cation channel with the ion selectivity series lithium > sodium > N-methyl-d-glucamine at pH 7.4. There was also a proton conductance (comparing pH 7.4 and 8.4), and at pH 5.5 the predominant conductance was for potassium ions. Therefore, it can be concluded that changing arginine 282 to a glutamate not only uncouples the cotransport of protons and peptides of the wild-type PepT1 but also creates a peptide-gated cation channel in the protein.

  15. New peptide architectures through C–H activation stapling between tryptophan–phenylalanine/tyrosine residues

    PubMed Central

    Mendive-Tapia, Lorena; Preciado, Sara; García, Jesús; Ramón, Rosario; Kielland, Nicola; Albericio, Fernando; Lavilla, Rodolfo

    2015-01-01

    Natural peptides show high degrees of specificity in their biological action. However, their therapeutical profile is severely limited by their conformational freedom and metabolic instability. Stapled peptides constitute a solution to these problems and access to these structures lies on a limited number of reactions involving the use of non-natural amino acids. Here, we describe a synthetic strategy for the preparation of unique constrained peptides featuring a covalent bond between tryptophan and phenylalanine or tyrosine residues. The preparation of such peptides is achieved in solution and on solid phase directly from the corresponding sequences having an iodo-aryl amino acid through an intramolecular palladium-catalysed C–H activation process. Moreover, complex topologies arise from the internal stapling of cyclopeptides and double intramolecular arylations within a linear peptide. Finally, as a proof of principle, we report the application to this new stapling method to relevant biologically active compounds. PMID:25994485

  16. Elucidation of the Molecular Basis of Cholecystokinin Peptide Docking to Its Receptor Using Site-Specific Intrinsic Photoaffinity Labeling and Molecular Modeling†

    PubMed Central

    Dong, Maoqing; Lam, Polo C.-H.; Pino, Delia I.; Abagyan, Ruben; Miller, Laurence J.

    2012-01-01

    G protein-coupled receptors represent the largest family of receptors and the major target of current drug development efforts. Understanding of the mechanisms of ligand binding and activation of these receptors remains limited, despite recent advances in structural determination of family members. This work focuses on the use of photoaffinity labeling and molecular modeling to elucidate the structural basis of binding a natural peptide ligand to a family A G protein-coupled receptor, the type 1 cholecystokinin receptor. Two photolabile cholecystokinin analogues were developed and characterized as representing high-affinity, fully biologically active probes with sites of covalent attachment at positions 28 and 31. The sites of receptor labeling were identified by purification, proteolytic peptide mapping, and radiochemical sequencing of labeled wild-type and mutant cholecystokinin receptors. The position 28 probe labeled second extracellular loop residue Leu199, while the position 31 probe labeled first extracellular loop residue Phe107. Along with five additional spatial approximation constraints coming from previous photoaffinity labeling studies and 12 distance restraints from fluorescence resonance energy transfer studies, these were built into two homology models of the cholecystokinin receptor, based on the recent crystal structures of the β2-adrenergic receptor and A2a-adenosine receptor. The resultant agonist ligand-occupied receptor models fully accommodate all existing experimental data and represent the best refined models of a peptide hormone receptor in this important family. PMID:19441839

  17. Amyloid beta-peptide possesses a transforming growth factor-beta activity.

    PubMed

    Huang, S S; Huang, F W; Xu, J; Chen, S; Hsu, C Y; Huang, J S

    1998-10-16

    Amyloid beta-peptide (Abeta) of 39-42 amino acid residues is a major constituent of Alzheimer's disease neurite plaques. Abeta aggregates (fibrils) are believed to be responsible for neuronal damage and dysfunction, as well as microglia and astrocyte activation in disease lesions by multiple mechanisms. Since Abeta aggregates possess the multiple valencies of an FAED motif (20th to 23rd amino acid residues), which resembles the putative transforming growth factor-beta (TGF-beta) active site motif, we hypothesize that Abeta monomers and Abeta aggregates may function as TGF-beta antagonists and partial agonists, analogous to previously described monovalent and multivalent TGF-beta peptide antagonists and agonists (Huang, S. S., Liu, Q., Johnson, F. E., Konish, Y., and Huang, J. S. (1997) J. Biol. Chem. 272, 27155-27159). Here, we report that the Abeta monomer, Abeta-(1-40) and its fragment, containing the motif inhibit radiolabeled TGF-beta binding to cell-surface TGF-beta receptors in mink lung epithelial cells (Mv1Lu cells). Abeta-(1-40)-bovine serum albumin conjugate (Abeta-(1-40)-BSA), a multivalent synthetic analogue of Abeta aggregates, exhibited cytotoxicity toward bovine cerebral endothelial cells and rat post-mitotic differentiated hippocampal neuronal cells (H19-7 cells) and inhibitory activities of radiolabeled TGF-beta binding to TGF-beta receptors and TGF-beta-induced plasminogen activator inhibitor-1 expression, that were approximately 100-670 times more potent than those of Abeta-(1-40) monomers. At less than micromolar concentrations, Abeta-(1-40)-BSA but not Abeta-(1-40) monomers inhibited proliferation of Mv1Lu cells. Since TGF-beta is an organizer of responses to neurodegeneration and is also found in neurite plaques, the TGF-beta antagonist and partial agonist activities of Abeta monomers and aggregates may play an important role in the pathogenesis of the disease.

  18. Variance Component Analysis of a Multi-Site Study for the Reproducibility of Multiple Reaction Monitoring Measurements of Peptides in Human Plasma

    PubMed Central

    Xia, Jessie Q.; Sedransk, Nell; Feng, Xingdong

    2011-01-01

    Background In the Addona et al. paper (Nature Biotechnology 2009), a large-scale multi-site study was performed to quantify Multiple Reaction Monitoring (MRM) measurements of proteins spiked in human plasma. The unlabeled signature peptides derived from the seven target proteins were measured at nine different concentration levels, and their isotopic counterparts were served as the internal standards. Methodology/Principal Findings In this paper, the sources of variation are analyzed by decomposing the variance into parts attributable to specific experimental factors: technical replicates, sites, peptides, transitions within each peptide, and higher-order interaction terms based on carefully built mixed effects models. The factors of peptides and transitions are shown to be major contributors to the variance of the measurements considering heavy (isotopic) peptides alone. For the light (12C) peptides alone, in addition to these factors, the factor of study*peptide also contributes significantly to the variance of the measurements. Heterogeneous peptide component models as well as influence analysis identify the outlier peptides in the study, which are then excluded from the analysis. Using a log-log scale transformation and subtracting the heavy/isotopic peptide [internal standard] measurement from the peptide measurements (i.e., taking the logarithm of the peak area ratio in the original scale establishes that), the MRM measurements are overall consistent across laboratories following the same standard operating procedures, and the variance components related to sites, transitions and higher-order interaction terms involving sites have greatly reduced impact. Thus the heavy peptides have been effective in reducing apparent inter-site variability. In addition, the estimates of intercepts and slopes of the calibration curves are calculated for the sub-studies. Conclusions/Significance The MRM measurements are overall consistent across laboratories following the same

  19. Site-specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution

    PubMed Central

    Silva, R. A. G. D.; Kubelka, Jan; Bour, Petr; Decatur, Sean M.; Keiderling, Timothy A.

    2000-01-01

    Understanding the detailed mechanism of protein folding requires dynamic, site-specific stereochemical information. The short time response of vibrational spectroscopies allows evaluation of the distribution of populations in rapid equilibrium as the peptide unfolds. Spectral shifts associated with isotopic labels along with local stereochemical sensitivity of vibrational circular dichroism (VCD) allow determination of the segment sequence of unfolding. For a series of alanine-rich peptides that form α-helices in aqueous solution, we used isotopic labeling and VCD to demonstrate that the α-helix noncooperatively unwinds from the ends with increasing temperature. For these blocked peptides, the C-terminal is frayed at 5°C. Ab initio level theoretical simulations of the IR and VCD band shapes are used to analyze the spectra and to confirm the conformation of the labeled components. The VCD signals associated with the labeled residues are amplified by coupling to the nonlabeled parts of the molecule. Thus small labeled segments are detectable and stereochemically defined in moderately large peptides in this report of site-specific peptide VCD conformational analysis. PMID:10880566

  20. Histidine at the active site of Neurospora tyrosinase.

    PubMed

    Pfiffner, E; Lerch, K

    1981-10-13

    The involvement of histidyl residues as potential ligands to the binuclear active-site copper of Neurospora tyrosinase was explored by dye-sensitized photooxidation. The enzymatic activity of the holoenzyme was shown to be unaffected by exposure to light in the presence of methylene blue; however, irradiation of the apoenzyme under the same conditions led to a progressive loss of its ability to be reactivated with Cu2+. This photoinactivation was paralleled by a decrease in the histidine content whereas the number of histidyl residues in the holoenzyme remained constant. Copper measurements of photooxidized, reconstituted apoenzyme demonstrated the loss of binding of one copper atom per mole of enzyme as a consequence of photosensitized oxidation of three out of nine histidine residues. Their sequence positions were determined by a comparison of the relative yields of the histidine containing peptides of photooxidized holo- and apotyrosinases. The data obtained show the preferential modification of histidyl residues 188, 193, and 289 and suggest that they constitute metal ligands to one of the two active-site copper atoms. Substitution of copper by cobalt was found to afford complete protection of the histidyl residues from being modified by dye-sensitized photooxidation. PMID:6458322

  1. Anti-tumoral activity of human salivary peptides.

    PubMed

    da Costa, João Pinto; Carvalhais, Virginia; Amado, Francisco; Silva, Artur; Nogueira-Ferreira, Rita; Ferreira, Rita; Helguero, Luísa; Vitorino, Rui

    2015-09-01

    Chemotherapy continues to be the standard treatment for advanced or metastasized cancer. However, commonly used chemotherapeutic agents may induce damage in healthy cells and tissues. Thus, in recent years, there has been an increased focus on the development of new, efficient anticancer drugs exhibiting low toxicity and that are not affected by mechanisms of chemoresistance. In the present work, we tested synthetic and naturally obtained human salivary peptides against breast, prostate, colon, osteosarcoma and bladder cancer cell lines (T47-D, PC-3, HT-29, MG63, T-24, respectively). Results have showed that there is a reduced cell population increase that is peptide-, cell- and possibly pathway-specific, with the most potent effect observed in observed in T-47D breast cancer cells. Protein expression and microscopy results further indicate that, in this cell line, the peptide with the sequence GPPPQGGRPQG (GG peptide) interferes with the ability of cell adhesion proteins to stabilize adherens junctions, such as E-cadherin, leading to apoptosis. These promising results encourage future works aimed at disclosing the vast potential of salivary peptides as new therapeutic agents.

  2. Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin

    SciTech Connect

    Kim, Dae Hong; Lee, Ik Hwan; Nam, Seung Taek; Hong, Ji; Zhang, Peng; Hwang, Jae Sam; Seok, Heon; Choi, Hyemin; Lee, Dong Gun; Kim, Jae Il; Kim, Ho

    2014-06-06

    Highlights: • 11-mer peptide Lumbricusin, a defensin like peptide, is isolated from earthworm. • We here demonstrated that Lumbricusin has neurotropic and neuroprotective effects. • p27 degradation by Lumbricusin mediates effects of Lumbricusin on neuronal cells. - Abstract: We recently isolated a polypeptide from the earthworm Lumbricus terrestris that is structurally similar to defensin, a well-known antibacterial peptide. An 11-mer antibacterial peptide (NH{sub 2}-RNRRWCIDQQA), designated Lumbricusin, was synthesized based on the amino acid sequence of the isolated polypeptide. Since we previously reported that CopA3, a dung beetle peptide, enhanced neuronal cell proliferation, we here examined whether Lumbricusin exerted neurotropic and/or neuroprotective effects. Lumbricusin treatment induced a time-dependent increase (∼51%) in the proliferation of human neuroblastoma SH-SY5Y cells. Lumbricusin also significantly inhibited the apoptosis and decreased viability induced by treatment with 6-hydroxy dopamine, a Parkinson’s disease-mimicking agent. Immunoblot analyses revealed that Lumbricusin treatment increased ubiquitination of p27{sup Kip1} protein, a negative regulator of cell-cycle progression, in SH-SY5Y cells, and markedly promoted its degradation. Notably, adenoviral-mediated over-expression of p27{sup Kip1} significantly blocked the antiapoptotic effect of Lumbricusin in 6-hydroxy dopamine-treated SH-SY5Y cells. These results suggest that promotion of p27{sup Kip1} degradation may be the main mechanism underlying the neuroprotective and neurotropic effects of Lumbricusin.

  3. Neutrophil attractant/activating peptide-1/interleukin-8 in term and preterm parturition.

    PubMed

    Romero, R; Ceska, M; Avila, C; Mazor, M; Behnke, E; Lindley, I

    1991-10-01

    The neutrophil is the leukocyte most frequently recruited into the amniotic fluid in cases of microbial invasion of the amniotic cavity. Neutrophil attractant/activating peptide-1/interleukin-8 is a newly identified cytokine that is capable of inducing selective neutrophil chemotaxis and activation. The purpose of this study was to examine the relationship between amniotic fluid concentrations of neutrophil attractant/activating peptide-1/interleukin-8, microbial invasion of the amniotic cavity, and parturition (term and preterm). Amniotic fluid neutrophil attractant/activating peptide-1/interleukin-8 was measured with an immunoassay validated for human amniotic fluid (sensitivity 0.3 ng/ml). Fluid was obtained from women in the following groups: midtrimester (n = 38), term not in labor (n = 38), term in active labor (n = 67), and preterm labor with intact membranes (n = 62). Fluid was cultured for aerobic and anaerobic bacterial and Mycoplasma. Sterile amniotic fluid from most women in the midtrimester of pregnancy and women at term not in labor did not contain immunoreactive neutrophil attractant/activating peptide-1/interleukin-8. Microbial invasion of the amniotic cavity was associated with increased concentrations of neutrophil attractant/activating peptide-1/interleukin-8. The amniotic fluid of women with preterm labor and sterile amniotic fluid who had preterm delivery contained higher neutrophil attractant/activating peptide-1/interleukin-8 levels than did the amniotic fluid of women who responded to tocolysis and had delivery at term. Term parturition is associated with increased concentrations of neutrophil attractant/activating peptide-1/interleukin-8 in the amniotic fluid. We conclude that neutrophil attractant/activating peptide-1/interleukin-8 is part of the host response to microbial invasion of the amniotic cavity and that increased amniotic fluid availability of this cytokine occurs in term and preterm parturition. PMID:1951537

  4. Determination of antioxidant activity of bioactive peptide fractions obtained from yogurt.

    PubMed

    Aloğlu, H Sanlıdere; Oner, Z

    2011-11-01

    In this study, physicochemical and microbiological properties of traditional and commercial yogurt samples were determined during 4 wk of storage. Proteolytic activity, which occurs during the storage period of yogurt samples, was also determined. Peptide fractions obtained from yogurts were investigated and the effect of proteolysis on peptide release during storage was determined. The antioxidant activities of peptides released from yogurt water-soluble extracts (WSE) and from HPLC fractions were determined by 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. The antioxidant activity of WSE from traditional yogurt was greater than that of WSE from commercial yogurts. In analysis by the ABTS method, mean values increased from 7.697 to 8.739 mM Trolox/g in commercial yogurts, and from 10.115 to 13.182 mM Trolox/g in traditional yogurts during storage. Antioxidant activities of peptides released from HPLC fractions of selected yogurt samples increased 10 to 200 times. In all yogurt samples, the greatest antioxidant activity was shown in the F2 fraction. After further fractionation of yogurt samples, the fractions coded as F2.2, F2.3, F4.3, and F4.4 had the highest antioxidant activity values. Total antioxidant activity of yogurts was low but after purification of peptides by fractionation in HPLC, peptide fractions with high antioxidant activity were obtained.

  5. Major Peptides from Amaranth (Amaranthus cruentus) Protein Inhibit HMG-CoA Reductase Activity

    PubMed Central

    Soares, Rosana Aparecida Manólio; Mendonça, Simone; de Castro, Luíla Ívini Andrade; Menezes, Amanda Caroline Cardoso Corrêa Carlos; Arêas, José Alfredo Gomes

    2015-01-01

    The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol biosynthesis. A protein isolate was prepared, and an enzymatic hydrolysis that simulated the in vivo digestion of the protein was performed. After hydrolysis, the peptide mixture was filtered through a 3 kDa membrane. The peptide profile of this mixture was determined by reversed phase high performance chromatography (RP-HPLC), and the peptide identification was performed by LC-ESI MS/MS. Three major peptides under 3 kDa were detected, corresponding to more than 90% of the peptides of similar size produced by enzymatic hydrolysis. The sequences identified were GGV, IVG or LVG and VGVI or VGVL. These peptides had not yet been described for amaranth protein nor are they present in known sequences of amaranth grain protein, except LVG, which can be found in amaranth α‑amylase. Their ability to inhibit the activity of HMG-CoA reductase was determined, and we found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect. PMID:25690031

  6. Synthesis and Antibacterial Activities of Antibacterial Peptides with a Spiropyran Fluorescence Probe

    PubMed Central

    Chen, Lei; Zhu, Yu; Yang, Danling; Zou, Rongfeng; Wu, Junchen; Tian, He

    2014-01-01

    In this report, antibacterial peptides1-3 were prepared with a spiropyran fluorescence probe. The probe exhibits a change in fluorescence when isomerized from a colorless spiro-form (spiropyran, Sp) to a colored open-form (merocyanine, Mc) under different chemical environments, which can be used to study the mechanism of antimicrobial activity. Peptides 1-3 exhibit a marked decrease in antimicrobial activity with increasing alkyl chain length. This is likely due to the Sp-Mc isomers in different polar environments forming different aggregate sizes in TBS, as demonstrated by time-dependent dynamic light scattering (DLS). Moreover, peptides 1-3 exhibited low cytotoxicity and hemolytic activity. These probe-modified peptides may provide a novel approach to study the effect of structural changes on antibacterial activity, thus facilitating the design of new antimicrobial agents to combat bacterial infection. PMID:25358905

  7. Antihypertensive activity of peptides identified in the in vitro gastrointestinal digest of pork meat.

    PubMed

    Escudero, Elizabeth; Toldrá, Fidel; Sentandreu, Miguel Angel; Nishimura, Hitoshi; Arihara, Keizo

    2012-07-01

    This study investigated the in vivo antihypertensive activity of three novel peptides identified in the in vitro digest of pork meat. These peptides were RPR, KAPVA and PTPVP and all of them showed significant antihypertensive activity after oral administration to spontaneously hypertensive rats, RPR being the peptide with the greatest in vivo activity. To our knowledge, this is the first report showing the in vivo antihypertensive action of the three peptides from nebulin (RPR) and titin (KAPVA and PTPVP), thus confirming their reported in vitro angiotensin I-converting enzyme (ACE) inhibitory activity. These findings suggest that pork meat could constitute a source of bioactive constituents that could be utilized in functional foods or nutraceuticals.

  8. Acyl silicates and acyl aluminates as activated intermediates in peptide formation on clays

    NASA Technical Reports Server (NTRS)

    White, D. H.; Kennedy, R. M.; Macklin, J.

    1984-01-01

    Glycine reacts with heating on dried clays and other minerals to give peptides in much better yield than in the absence of mineral. This reaction was proposed to occur by way of an activated intermediate such as an acyl silicate or acyl aluminate analogous to acyl phosphates involved in several biochemical reactions including peptide bond synthesis. The proposed mechanism has been confirmed by trapping the intermediate, as well as by direct spectroscopic observation of a related intermediate. The reaction of amino acids on periodically dried mineral surfaces represents a widespead, geologically realistic setting for prebiotic peptide formation via in situ activation.

  9. Preparation of an active recombinant peptide of crustacean androgenic gland hormone.

    PubMed

    Okuno, Atsuro; Hasegawa, Yuriko; Nishiyama, Makoto; Ohira, Tsuyoshi; Ko, Rinkei; Kurihara, Masaaki; Matsumoto, Shogo; Nagasawa, Hiromichi

    2002-03-01

    In crustaceans, male sexual characteristics are induced by a hormone referred to as androgenic gland hormone. We have recently cloned a candidate cDNA in the terrestrial isopod Armadillidium vulgare. In order to prove that this cDNA encodes the hormone, recombinant single-chain precursor molecules consisting of B chain, C peptide and A chain were produced using both baculovirus and bacterial expression systems. Neither recombinant precursors showed activity. Digestion of only the precursor carrying a glycan moiety with lysyl endopeptidase gave a heterodimeric peptide with hormonal activity by removing a part of C peptide. These results indicate that the cDNA encodes the hormone. PMID:11836008

  10. New cyclic peptides with osteoblastic proliferative activity from Dianthus superbus.

    PubMed

    Tong, Yun; Luo, Jian-Guang; Wang, Rui; Wang, Xiao-Bing; Kong, Ling-Yi

    2012-03-01

    Two new cyclic peptides, dianthins G-H (1 and 2), together with the known dianthin E (3), were isolated from the traditional Chinese medicinal plant Dianthus superbus. The sequences of cyclic peptides 1 and 2 were elucidated as cyclo (-Gly(1)-Pro(2)-Leu(3)-Thr(4)-Leu(5)-Phe(6)-) and cyclo (-Gly(1)-Pro(2)-Val(3)-Thr(4)-Ile(5)-Phe(6)-), on the basis of ESI tandem mass fragmentation analysis, extensive 2D NMR methods and X-ray diffraction. The isolated three compounds all increase proliferation of MC3T3-E1 cells in vitro using MTT method.

  11. Investigating the antimicrobial peptide 'window of activity' using cationic lipopeptides with hydrocarbon and fluorinated tails.

    PubMed

    Findlay, Brandon; Zhanel, George G; Schweizer, Frank

    2012-07-01

    To probe the effect of carbon-fluorine bonds on antimicrobial peptide-membrane interactions, 24 cationic lipopeptides were created. The collection of lipopeptides was built from two different peptide sequences, KGK and KKK, with a variety of different lipids selected to probe the effectiveness of both hydrocarbon and fluorinated tails. The antimicrobial activity of each peptide was tested against a mixture of pathogenic and reference bacterial strains, with the cationic disinfectant benzalkonium chloride as a positive control. Non-specific interactions with hydrophobic proteins were assessed by repeating antimicrobial testing in the presence of bovine serum albumin (BSA), and the toxicity of the lipopeptides was assessed by measuring lysis of ovine erythrocytes. Peptide sequence had a moderate effect on activity, with the most active peptide (C16-KGK) inhibiting the growth of two Staphylococcus epidermidis strains at ≤ 0.25 μg/mL. Tail composition was less important than the overall hydrophobicity, with the most active fluorinated tails equivalent to moderately active hydrocarbon tails. The activity of all peptides was significantly reduced by the presence of BSA, and haemolysis was closely correlated with antimicrobial activity.

  12. Modeling of protein-peptide interactions using the CABS-dock web server for binding site search and flexible docking.

    PubMed

    Blaszczyk, Maciej; Kurcinski, Mateusz; Kouza, Maksim; Wieteska, Lukasz; Debinski, Aleksander; Kolinski, Andrzej; Kmiecik, Sebastian

    2016-01-15

    Protein-peptide interactions play essential functional roles in living organisms and their structural characterization is a hot subject of current experimental and theoretical research. Computational modeling of the structure of protein-peptide interactions is usually divided into two stages: prediction of the binding site at a protein receptor surface, and then docking (and modeling) the peptide structure into the known binding site. This paper presents a comprehensive CABS-dock method for the simultaneous search of binding sites and flexible protein-peptide docking, available as a user's friendly web server. We present example CABS-dock results obtained in the default CABS-dock mode and using its advanced options that enable the user to increase the range of flexibility for chosen receptor fragments or to exclude user-selected binding modes from docking search. Furthermore, we demonstrate a strategy to improve CABS-dock performance by assessing the quality of models with classical molecular dynamics. Finally, we discuss the promising extensions and applications of the CABS-dock method and provide a tutorial appendix for the convenient analysis and visualization of CABS-dock results. The CABS-dock web server is freely available at http://biocomp.chem.uw.edu.pl/CABSdock/.

  13. Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody.

    PubMed

    Kong, Rui; Xu, Kai; Zhou, Tongqing; Acharya, Priyamvada; Lemmin, Thomas; Liu, Kevin; Ozorowski, Gabriel; Soto, Cinque; Taft, Justin D; Bailer, Robert T; Cale, Evan M; Chen, Lei; Choi, Chang W; Chuang, Gwo-Yu; Doria-Rose, Nicole A; Druz, Aliaksandr; Georgiev, Ivelin S; Gorman, Jason; Huang, Jinghe; Joyce, M Gordon; Louder, Mark K; Ma, Xiaochu; McKee, Krisha; O'Dell, Sijy; Pancera, Marie; Yang, Yongping; Blanchard, Scott C; Mothes, Walther; Burton, Dennis R; Koff, Wayne C; Connors, Mark; Ward, Andrew B; Kwong, Peter D; Mascola, John R

    2016-05-13

    The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. Here, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showed that the N-terminal portion of the fusion peptide can be solvent-exposed. These results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design. PMID:27174988

  14. Multiple biological activities for two peptides derived from the nerve growth factor precursor

    SciTech Connect

    Dicou, Eleni . E-mail: dicou@ipmc.cnrs.fr

    2006-09-01

    ProNGF can be cleaved proteolytically at dibasic residues and liberates two other peptides beside NGF, LIP1 a 29 amino acid (aa) peptide and LIP2 a 38 aa peptide. These peptides were found present in the rat intestine and shown to induce rapid phosphorylation of the Trk receptor in cell lines. The present study describes several novel biological properties for these peptides. They exert an anti-proliferative effect on the mitogenic activity of estrogen and IGF in MCF-7 cells. They protect against in vivo induction of excitotoxic lesions by the glutamatergic analogue ibotenate injected into the developing mouse brain and against in vitro NMDA-induced cell death in primary neuronal cultures. They bind to murine microglial cells and induce phosphorylation of Akt. These results suggest a role for LIP1 and LIP2 in cell survival.

  15. [Biologically active peptides derived from food proteins as the food components with cardioprotective properties].

    PubMed

    Iwaniak, Anna; Darewicz, Małgorzata; Minkiewicz, Piotr; Protasiewicz, Monika; Borawska, Justyna

    2014-06-01

    Food proteins are the source of peptides with many biological activities. One of them is their impact on blood circulatory system. This group of peptides includes the ones with the ability to reduce the blood pressure (inhibitors of angiotensin converting enzyme--ACE), antithrombotic, and to lower the cholesterol level. Among the above-mentioned peptides' bioactivities, the most of them act as the ACE inhibitors. Some of them are the functional food components and nutraceuticals and possess the status of food with special use. The main known source of antithrombotic and cholesterol lowering peptides are milk and soy proteins, respectively. However, the scientists make the efforts to find new alternative sources of peptides with the above-mentioned activities. It should be noted, that although the bioactive peptides are considered as the safe food components and thus be supportive in the cardiovascular diseases therapy, they cannot substitute the drugs. This review shows the characteristics of selected peptides with: blood pressure reducing, antithrombotic, and cholesterol level reducing activities. We focused on the sequences that were identified in food proteins as well as were tested on humans or animals.

  16. The self-assembly of redox active peptides: Synthesis and electrochemical capacitive behavior.

    PubMed

    Piccoli, Julia P; Santos, Adriano; Santos-Filho, Norival A; Lorenzón, Esteban N; Cilli, Eduardo M; Bueno, Paulo R

    2016-05-01

    The present work reports on the synthesis of a redox-tagged peptide with self-assembling capability aiming applications in electrochemically active capacitive surfaces (associated with the presence of the redox centers) generally useful in electroanalytical applications. Peptide containing ferrocene (fc) molecular (redox) group (Ac-Cys-Ile-Ile-Lys(fc)-Ile-Ile-COOH) was thus synthesized by solid phase peptide synthesis (SPPS). To obtain the electrochemically active capacitive interface, the side chain of the cysteine was covalently bound to the gold electrode (sulfur group) and the side chain of Lys was used to attach the ferrocene in the peptide chain. After obtaining the purified redox-tagged peptide, the self-assembly and redox capability was characterized by cyclic voltammetry (CV) and electrochemical impedance-based capacitance spectroscopy techniques. The obtained results confirmed that the redox-tagged peptide was successfully attached by forming an electroactive self-assembled monolayer onto gold electrode. The design of redox active self-assembly ferrocene-tagged peptide is predictably useful in the development of biosensor devices precisely to detect, in a label-free platform, those biomarkers of clinical relevance. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 357-367, 2016.

  17. Local atomic structure and oxidation processes of Cu(I) binding site in amyloid beta peptide: XAS Study

    NASA Astrophysics Data System (ADS)

    Kremennaya, M. A.; Soldatov, M. A.; Stretsov, V. A.; Soldatov, A. V.

    2016-05-01

    There are two different motifs of X-ray absorption spectra for Cu(I) K-edge in amyloid-β peptide which could be due to two different configurations of local Cu(I) environment. Two or three histidine ligands can coordinate copper ion in varying conformations. On the other hand, oxidation of amyloid-β peptide could play an additional role in local copper environment. In order to explore the peculiarities of local atomic and electronic structure of Cu(I) binding sites in amyloid-β peptide the x-ray absorption spectra were simulated for various Cu(I) environments including oxidized amyloid-β and compared with experimental data.

  18. Selective Deletion of the Internal Lysine Residue from the Peptide Sequence by Collisional Activation

    NASA Astrophysics Data System (ADS)

    Banerjee, Shibdas; Mazumdar, Shyamalava

    2012-11-01

    The gas-phase peptide ion fragmentation chemistry is always the center of attraction in proteomics to analyze the amino acid sequence of peptides and proteins. In this work, we describe the formation of an anomalous fragment ion, which corresponds to the selective deletion of the internal lysine residue from a series of lysine containing peptides upon collisional activation in the ion trap. We detected several water-loss fragment ions and the maximum number of water molecules lost from a particular fragment ion was equal to the number of lysine residues in that fragment. As a consequence of this water-loss phenomenon, internal lysine residues were found to be deleted from the peptide ion. The N,N-dimethylation of all the amine functional groups of the peptide stopped the internal lysine deletion reaction, but selective N-terminal α-amino acetylation had no effect on this process indicating involvement of the side chains of the lysine residues. The detailed mechanism of the lysine deletion was investigated by multistage CID of the modified and unmodified peptides, by isotope labeling and by energy resolved CID studies. The results suggest that the lysine deletion might occur through a unimolecular multistep mechanism involving a seven-membered cyclic imine intermediate formed by the loss of water from a lysine residue in the protonated peptide. This intermediate subsequently undergoes degradation reaction to deplete the interior imine ring from the peptide backbone leading to the deletion of an internal lysine residue.

  19. Enhanced stability and activity of an antimicrobial peptide in conjugation with silver nanoparticle.

    PubMed

    Pal, Indrani; Brahmkhatri, Varsha P; Bera, Swapna; Bhattacharyya, Dipita; Quirishi, Yasrib; Bhunia, Anirban; Atreya, Hanudatta S

    2016-12-01

    The conjugation of nanoparticles with antimicrobial peptides (AMP) is emerging as a promising route to achieve superior antimicrobial activity. However, the nature of peptide-nanoparticle interactions in these systems remains unclear. This study describes a system consisting of a cysteine containing antimicrobial peptide conjugated with silver nanoparticles, in which the two components exhibit a dynamic interaction resulting in a significantly enhanced stability and biological activity compared to that of the individual components. This was investigated using NMR spectroscopy in conjunction with other biophysical techniques. Using fluorescence assisted cell sorting and membrane mimics we carried out a quantitative comparison of the activity of the AMP-nanoparticle system and the free peptide. Taken together, the study provides new insights into nanoparticle-AMP interactions at a molecular level and brings out the factors that will be useful for consideration while designing new conjugates with enhanced functionality. PMID:27585423

  20. Unexpected Opioid Activity Profiles of Analogs of the Novel Peptide Kappa Opioid Receptor Ligand CJ-15,208

    PubMed Central

    Aldrich, Jane V.; Kulkarni, Santosh S.; Senadheera, Sanjeewa N.; Ross, Nicolette C.; Reilley, Kate J.; Eans, Shainnel O.; Ganno, Michelle L.; Murray, Thomas F.; McLaughlin, Jay P.

    2013-01-01

    An alanine scan was performed on the novel kappa opioid receptor (KOR) peptide ligand CJ-15,208 to determine which residues contribute to the potent in vivo agonist activity observed for the parent peptide. These cyclic tetrapeptides were synthesized by a combination of solid phase peptide synthesis of the linear precursors, followed by cyclization in solution. Like the parent peptide, each of the analogs exhibited agonist activity and KOR antagonist activity in an antinociceptive assay in vivo. Unlike the parent peptide, the agonist activity of the potent analogs was mediated predominantly if not exclusively by mu opioid receptors (MOR). Thus analogs 2 and 4, in which one of the phenylalanine residues was replaced by alanine, exhibited both potent MOR agonist activity and KOR antagonist activity in vivo. These peptides represent novel lead compounds for the development of peptide-based opioid analgesics. PMID:21761566

  1. CXC chemokines connective tissue activating peptide-III and neutrophil activating peptide-2 are heparin/heparan sulfate-degrading enzymes.

    PubMed

    Hoogewerf, A J; Leone, J W; Reardon, I M; Howe, W J; Asa, D; Heinrikson, R L; Ledbetter, S R

    1995-02-17

    Heparan sulfate proteoglycans at cell surfaces or in extracellular matrices bind diverse molecules, including growth factors and cytokines, and it is believed that the activities of these molecules may be regulated by the metabolism of heparan sulfate. In this study, purification of a heparan sulfate-degrading enzyme from human platelets led to the discovery that the enzymatic activity residues in at least two members of the platelet basic protein (PBP) family known as connective tissue activating peptide-III (CTAP-III) and neutrophil activating peptide-2. PBP and its N-truncated derivatives, CTAP-III and neutrophil activating peptide-2, are CXC chemokines, a group of molecules involved in inflammation and wound healing. SDS-polyacrylamide gel electrophoresis analysis of the purified heparanase resulted in a single broad band at 8-10 kDa, the known molecular weight of PBP and its truncated derivatives. Gel filtration chromatography of heparanase resulted in peaks of activity corresponding to monomers, dimers, and tetramers; these higher order aggregates are known to form among the chemokines. N-terminal sequence analysis of the same preparation indicated that only PBP and truncated derivatives were present, and commercial CTAP-III from three suppliers had heparanase activity. Antisera produced in animals immunized with a C-terminal synthetic peptide of PBP inhibited heparanase activity by 95%, compared with activity of the purified enzyme in the presence of the preimmune sera. The synthetic peptide also inhibited heparanase by 95% at 250 microM, compared to the 33% inhibition of heparanase activity by two other peptides. The enzyme was determined to be an endoglucosaminidase, and it degraded both heparin and heparan sulfate with optimal activity at pH 5.8. Chromatofocusing of the purified heparanase resulted in two protein peaks: an inactive peak at pI7.3, and an active peak at pI 4.8-5.1. Sequence analysis showed that the two peaks contained identical protein

  2. In vivo characterization of activatable cell penetrating peptides for targeting protease activity in cancer

    PubMed Central

    Olson, Emilia S.; Aguilera, Todd A.; Jiang, Tao; Ellies, Lesley G.; Nguyen, Quyen T.; Wong, Edmund; Gross, Larry; Tsien, Roger Y.

    2009-01-01

    Activatable cell penetrating peptides (ACPPs) are novel in vivo targeting agents comprised of a polycationic cell penetrating peptide (CPP) connected via a cleavable linker to a neutralizing polyanion (Fig. 1A). Adsorption and uptake into cells are inhibited until the linker is proteolyzed1–3. An ACPP cleavable by matrix metalloproteinase-2 (MMP-2) in vitro was the first one demonstrated to work in a tumor model in vivo, but only HT-1080 xenografts and resected human squamous cell carcinomas were tested. Generality to other cancer types, in vivo selectivity of ACPPs for MMPs, and spatial resolution require further characterization. We now show that ACPPs can target many xenograft tumor models from different cancer sites, as well as a well thoroughly studied transgenic model of spontaneous breast cancer (mouse mammary tumor virus promoter driving polyoma middle T antigen, MMTV-PyMT). Pharmacological inhibitors and genetic knockouts indicate that current ACPPs are selective for MMP-2 and MMP-9 in the above in vivo models. In accord with the known local distribution of MMP activity, accumulation is strongest at the tumor-stromal interface in primary tumors and associated metastases, indicating better spatial resolution (<50 µm) than other currently available MMP-cleavable probes4. We also find that background uptake of ACPPs into normal tissues such as liver and kidney can be decreased by appending inert macromolecules of 30–50 KDa to the polyanionic inhibitory domain. Our results validate an approach that should generally deliver imaging agents and chemotherapeutics to sites of invasion, tumor-promoting inflammation, and metastasis. PMID:20023745

  3. Structure activity relationship modelling of milk protein-derived peptides with dipeptidyl peptidase IV (DPP-IV) inhibitory activity.

    PubMed

    Nongonierma, Alice B; FitzGerald, Richard J

    2016-05-01

    Quantitative structure activity type models were developed in an attempt to predict the key features of peptide sequences having dipeptidyl peptidase IV (DPP-IV) inhibitory activity. The models were then employed to help predict the potential of peptides, which are currently reported in the literature to be present in the intestinal tract of humans following milk/dairy product ingestion, to act as inhibitors of DPP-IV. Two models (z- and v-scale) for short (2-5 amino acid residues) bovine milk peptides, behaving as competitive inhibitors of DPP-IV, were developed. The z- and the v-scale models (p<0.05, R(2) of 0.829 and 0.815, respectively) were then applied to 56 milk protein-derived peptides previously reported in the literature to be found in the intestinal tract of humans which possessed a structural feature of DPP-IV inhibitory peptides (P at the N2 position). Ten of these peptides were synthetized and tested for their in vitro DPP-IV inhibitory properties. There was no agreement between the predicted and experimentally determined DPP-IV half maximal inhibitory concentrations (IC50) for the competitive peptide inhibitors. However, the ranking for DPP-IV inhibitory potency of the competitive peptide inhibitors was conserved. Furthermore, potent in vitro DPP-IV inhibitory activity was observed with two peptides, LPVPQ (IC50=43.8±8.8μM) and IPM (IC50=69.5±8.7μM). Peptides present within the gastrointestinal tract of human may have promise for the development of natural DPP-IV inhibitors for the management of serum glucose.

  4. Imaging site-specific peptide-targeting in tumor tissues using spectral-domain optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Ma, Lixin; Zhang, Miao; Yu, Ping

    2011-03-01

    We report imaging studies on site-specific peptide-targeting in tumor tissues using newly developed optical peptide probes and spectral-domain optical coherence tomography (SD-OCT). The system used two broadband superluminescent light emission diodes with different central wavelengths. An electro-optic modulation in the reference beam was used to get full-range deep imaging inside tumor tissues. The optical probes were based on Bombesin (BBN) that is a fourteen amino acid peptide. BBN has high binding affinity to gastrin-releasing peptide (GRP) receptors overexpressed on several human cancer cell lines. Fluorescence BBN probes were developed by conjugating the last eight residues of BBN, -Q-W-A-V-G-H-L-M-(NH2), with Alexa Flour 680 or Alexa Fluor 750 dye molecules via amino acid linker -G-G-G. The SD-OCT imaging can identify normal tissue and tumor tissue through the difference in scattering coefficient, and trace the BBN conjugate probes through the absorption of the dye molecules using the twowavelength algorithm. We performed the specific uptake and receptor-blocking experiments of the optical BBN probes in severely compromised immunodeficient mouse model bearing human PC-3 prostate tumor xenografts. Tumor and muscle tissues were collected and used for SD-OCT imaging. The SD-OCT images showed fluorescence traces of the BBN probes in the peptide-targeted tumor tissues. Our results demonstrated that SD-OCT is a potential tool for preclinical and clinical early cancer detection.

  5. Amphiphilic cationic β(3R3)-peptides: membrane active peptidomimetics and their potential as antimicrobial agents.

    PubMed

    Mosca, Simone; Keller, Janos; Azzouz, Nahid; Wagner, Stefanie; Titz, Alexander; Seeberger, Peter H; Brezesinski, Gerald; Hartmann, Laura

    2014-05-12

    We introduce a novel class of membrane active peptidomimetics, the amphiphilic cationic β(3R3)-peptides, and evaluate their potential as antimicrobial agents. The design criteria, the building block and oligomer synthesis as well as a detailed structure-activity relationship (SAR) study are reported. Specifically, infrared reflection absorption spectroscopy (IRRAS) was employed to investigate structural features of amphiphilic cationic β(3R3)-peptide sequences at the hydrophobic/hydrophilic air/liquid interface. Furthermore, Langmuir monolayers of anionic and zwitterionic phospholipids have been used to model the interactions of amphiphilic cationic β(3R3)-peptides with prokaryotic and eukaryotic cellular membranes in order to predict their membrane selectivity and elucidate their mechanism of action. Lastly, antimicrobial activity was tested against Gram-positive M. luteus and S. aureus as well as against Gram-negative E. coli and P. aeruginosa bacteria along with testing hemolytic activity and cytotoxicity. We found that amphiphilic cationic β(3R3)-peptide sequences combine high and selective antimicrobial activity with exceptionally low cytotoxicity in comparison to values reported in the literature. Overall, this study provides further insights into the SAR of antimicrobial peptides and peptidomimetics and indicates that amphiphilic cationic β(3R3)-peptides are strong candidates for further development as antimicrobial agents with high therapeutic index.

  6. Conserved sequences of sperm-activating peptide and its receptor throughout evolution, despite speciation in the sea star Asterias amurensis and closely related species.

    PubMed

    Nakachi, Mia; Hoshi, Motonori; Matsumoto, Midori; Moriyama, Hideaki

    2008-08-01

    The asteroidal sperm-activating peptides (asterosaps) from the egg jelly bind to their sperm receptor, a membrane-bound guanylate cyclase, on the tail to activate sperm in sea stars. Asterosaps are produced as single peptides and then cleaved into shorter peptides. Sperm activation is followed by the acrosome reaction, which is subfamily specific. In order to investigate the molecular details of the asterosap-receptor interaction, corresponding cDNAs have been cloned, sequenced and analysed from the Asteriinae subfamily including Asterias amurensis, A. rubens, A. forbesi and Aphelasterias japonica, as well as Distolasterias nipon from the Coscinasteriinae subfamily. Averages of 29% and 86% identity were found from the deduced amino acid sequences in asterosap and its receptor extracellular domains, respectively, across all species examined. The phylogenic tree topology for asterosap and its receptor was similar to that of the mitochondrial cytochrome c oxidase subunit I. In spite of a certain homology, the amino acid sequences exhibited speciation. Conservation was found in the asterosap residues involved in disulphide bonding and proteinase-cleaving sites. Conversely, similarities were detected between potential asterosap-binding sites and the structure of the atrial natriuretic peptide receptor. Although the sperm-activating peptide and its receptor share certain common sequences, they may serve as barriers that ensure speciation in the sea star A. amurensis and closely related species.

  7. Antimicrobial activities of chicken β-defensin (4 and 10) peptides against pathogenic bacteria and fungi

    PubMed Central

    Yacoub, Haitham A.; Elazzazy, Ahmed M.; Abuzinadah, Osama A. H.; Al-Hejin, Ahmed M.; Mahmoud, Maged M.; Harakeh, Steve M.

    2015-01-01

    Host Defense Peptides (HDPs) are small cationic peptides found in several organisms. They play a vital role in innate immunity response and immunomodulatory stimulation. This investigation was designed to study the antimicrobial activities of β-defensin peptide-4 (sAvBD-4) and 10 (sAvBD-4) derived from chickens against pathogenic organisms including bacteria and fungi. Ten bacterial strains and three fungal species were used in investigation. The results showed that the sAvBD-10 displayed a higher bactericidal potency against all the tested bacterial strains than that of sAvBD-4. The exhibited bactericidal activity was significant against almost the different bacterial strains at different peptide concentrations except for that of Pseudomonas aeruginosa (P. aeruginosa) and Streptococcus bovis (Str. bovis) strains where a moderate effect was noted. Both peptides were effective in the inactivation of fungal species tested yielding a killing rate of up to 95%. The results revealed that the synthetic peptides were resistant to salt at a concentration of 50 mM NaCl. However, they lost antimicrobial potency when applied in the presence of high salt concentrations. Based on blood hemolysis studies, a little hemolytic effect was showed in the case of both peptides even when applied at high concentrations. The data obtained from this study indicated that synthetic avian peptides exhibit strong antibacterial and antifungal activity. In conclusion, future work and research should be tailored to a better understanding of the mechanisms of action of those peptides and their potential use in the pharmaceutical industry to help reduce the incidence and impact of infectious agent and be marketed as a naturally occurring antibiotic. PMID:25941665

  8. Antimicrobial activities of chicken β-defensin (4 and 10) peptides against pathogenic bacteria and fungi.

    PubMed

    Yacoub, Haitham A; Elazzazy, Ahmed M; Abuzinadah, Osama A H; Al-Hejin, Ahmed M; Mahmoud, Maged M; Harakeh, Steve M

    2015-01-01

    Host Defense Peptides (HDPs) are small cationic peptides found in several organisms. They play a vital role in innate immunity response and immunomodulatory stimulation. This investigation was designed to study the antimicrobial activities of β-defensin peptide-4 (sAvBD-4) and 10 (sAvBD-4) derived from chickens against pathogenic organisms including bacteria and fungi. Ten bacterial strains and three fungal species were used in investigation. The results showed that the sAvBD-10 displayed a higher bactericidal potency against all the tested bacterial strains than that of sAvBD-4. The exhibited bactericidal activity was significant against almost the different bacterial strains at different peptide concentrations except for that of Pseudomonas aeruginosa (P. aeruginosa) and Streptococcus bovis (Str. bovis) strains where a moderate effect was noted. Both peptides were effective in the inactivation of fungal species tested yielding a killing rate of up to 95%. The results revealed that the synthetic peptides were resistant to salt at a concentration of 50 mM NaCl. However, they lost antimicrobial potency when applied in the presence of high salt concentrations. Based on blood hemolysis studies, a little hemolytic effect was showed in the case of both peptides even when applied at high concentrations. The data obtained from this study indicated that synthetic avian peptides exhibit strong antibacterial and antifungal activity. In conclusion, future work and research should be tailored to a better understanding of the mechanisms of action of those peptides and their potential use in the pharmaceutical industry to help reduce the incidence and impact of infectious agent and be marketed as a naturally occurring antibiotic.

  9. Stable activity of a deubiquitylating enzyme (Usp2-cc) in the presence of high concentrations of urea and its application to purify aggregation-prone peptides

    SciTech Connect

    Shahnawaz, Mohammad; Thapa, Arjun; Park, Il-Seon . E-mail: parkis@chosun.ac.kr

    2007-08-03

    Chemical synthesis of long or aggregation-prone peptide has been problematic. Its biological production has an advantage in that point, but it often forms inclusion body which creates difficulties in recovery of targets. As a deubiquitylating enzyme (Usp2-cc) was shown in this study to maintain its activity even in the presence of up to 4 M urea, target peptide was purified by a single step of chromatography after overexpression as inclusion body, solubilization in urea and cleavage by the enzyme from the fusion protein consisting of GroES (used for high expression and easy to handle), ubiquitin (as a cleavage site) and target peptide. This system is a convenient tool for production of peptides that are difficult to be chemically synthesized and biologically purified.

  10. Stable activity of a deubiquitylating enzyme (Usp2-cc) in the presence of high concentrations of urea and its application to purify aggregation-prone peptides.

    PubMed

    Shahnawaz, Mohammad; Thapa, Arjun; Park, Il-Seon

    2007-08-01

    Chemical synthesis of long or aggregation-prone peptide has been problematic. Its biological production has an advantage in that point, but it often forms inclusion body which creates difficulties in recovery of targets. As a deubiquitylating enzyme (Usp2-cc) was shown in this study to maintain its activity even in the presence of up to 4M urea, target peptide was purified by a single step of chromatography after overexpression as inclusion body, solubilization in urea and cleavage by the enzyme from the fusion protein consisting of GroES (used for high expression and easy to handle), ubiquitin (as a cleavage site) and target peptide. This system is a convenient tool for production of peptides that are difficult to be chemically synthesized and biologically purified. PMID:17560941

  11. Coordination of two high-affinity hexamer peptides to copper(II) and palladium(II) models of the peptide-metal chelation site on IMAC resins

    SciTech Connect

    Chen, Y.; Pasquinelli, R.; Ataai, M.; Koepsel, R.R.; Kortes, R.A.; Shepherd, R.E.

    2000-03-20

    The coordination of peptides Ser-Pro-His-His-Gly-Gly (SPHHGG) and (His){sub 6} (HHHHHH) to [Pd{sup II}(mida)(D{sub 2}O)] (mida{sup 2{minus}} = N-methyliminodiacetate) was studied by {sup 1}H NMR as model reactions for Cu{sup II}(iminodiacetate)-immobilized metal affinity chromatography (IMAC) sites. This is the first direct physical description of peptide coordination for IMAC. A three-site coordination is observed which involves the first, third, and fourth residues along the peptide chain. The presence of proline in position 2 of SPHHGG achieves the best molecular mechanics and bonding angles in the coordinated peptide and enhances the interaction of the serine amino nitrogen. Histidine coordination of H{sub 1}, H{sub 3}, and H{sub 4} of (His){sub 6} and H{sub 3} and H{sub 4} of SPHHGG was detected by {sup 1}H NMR contact shifts and H/D exchange of histidyl protons. The EPR spectra of SPHHGG and HHHHHH attached to the [Cu{sup II}(mida)] unit were obtained for additional modeling of IMAC sites. EPR parameters of the parent [Cu(mida)(H{sub 2}O){sub 2}] complex are representative: g{sub zz} = 2.31; g{sub yy} = 2.086; g{sub xx} = 2.053; A{sub {vert_bar}{vert_bar}} = 161 G; A{sub N} = 19G (three line, one N coupling). Increased rhombic distortion is detected relative to the starting aqua complex in the order of [Cu(mida)L] for distortion of HHHHHH > SPHHGG > (H{sub 2}O){sub 2}. The lowering of symmetry is also seen in the decrease in the N-shf coupling, presumably to the imino nitrogen of mida{sup 2{minus}} in the order 19 G (H{sub 2}O), 16 G (SPHHGG) and 11 G (HHHHHH). Visible spectra of the [Cu(mida)(SPHHGG)] and [Cu(mida)(HHHHHH)] as a function of pH indicate coordination of one histidyl donor at ca. 4.5, two in the range of pH 5--7, and two chelate ring attachments involving the terminal amino donor for SPHHGG or another histidyl donor of HHHHHH in the pH domain of 7--8 in agreement with the [Pd{sup II}(mida)L] derivatives which form the two

  12. Active Sites Environmental Monitoring Program: Action levels

    SciTech Connect

    Ashwood, J.S.; Ashwood, T.L.

    1991-10-01

    The Active Sites Environmental Monitoring Program (ASEMP) was established at Oak Ridge National Laboratory to provide for early leak detection and to monitor performance of the active low-level waste disposal facilities in Solid Waste Storage Area (SWSA) 6 and the transuranic waste storage areas in SWSA 5 North. Early leak detection is accomplished by sampling runoff, groundwater, and perched water in burial trenches. Sample results are compared to action levels that represent background contamination by naturally occurring and fallout-derived radionuclides. 15 refs., 3 figs., 12 tabs.

  13. Hierarchy of Specific Lipid-Peptide Interactions Produces the Activity of Cell-penetrating and Cell-permeating Peptides

    NASA Astrophysics Data System (ADS)

    Davis, Matthew; Parente, Daniel; Gordon, Vernita; Mishra, Abhijit; Schmidt, Nathan; Yang, Lihua; Coridan, Robert; Som, Abhigyan; Tew, Gregory; Wong, Gerard

    2008-03-01

    Protein transduction domains can cross cell membranes with high efficiency, even when carrying a variety of cargos, and thus has strong biotechnological potential. The molecular mechanism of entry, however, is not well understood. We use small-angle x-ray scattering (SAXS) and confocal microscopy to systematically study the interaction of the TAT and ANTP PTD with model membranes of variable composition. Their membrane transduction activity requires the presence of both PE and PS lipids in the membrane. Antimicrobial peptides (AMP's) are cationic amphiphiles that comprise a key component of innate immunity. Synthetic analogs of AMP's, such as the family of phenylene ethynylene antimicrobial oligomers (AMO's), recently demonstrated broad-spectrum antimicrobial activity, but the underlying molecular mechanism is unknown. PE lipid greatly enhances permeating activity of AMO in these membranes, showing the importance of specific lipid composition for the activity of cell-permeating peptides. Since bacterial cell membranes are richer in PE lipids than are eukaryotic cell membranes, this may indicate a mechanism for antimicrobial specificity.

  14. Anticancer activity of a synthetic peptide derived from harmoniasin, an antibacterial peptide from the ladybug Harmonia axyridis.

    PubMed

    Kim, In-Woo; Lee, Joon Ha; Kwon, Young-Nam; Yun, Eun-Young; Nam, Sung-Hee; Ahn, Mi-Young; Kang, Dong-Chul; Hwang, Jae Sam

    2013-08-01

    Harmoniasin is a defensin-like antimicrobial peptide identified from the ladybug Harmonia axyridis. Among the synthetic homodimer peptide analogues derived from harmoniasin, HaA4 has been found to have antibacterial activity without hemolytic activity. In this study, we investigated whether HaA4 has anticancer activity against human leukemia cell lines such as U937 and Jurkat cells. HaA4 manifested cytotoxicity and decreased the cell viability of U937 and Jurkat cells in MTS assay and LDH release assay. We found that HaA4 induced apoptotic and necrotic cell death of the leukemia cells using flow cytometric analysis, acridine orange/ethidium bromide staining and nucleosomal fragmentation of genomic DNA. Activation of caspase-7 and -9 and fragmentation of poly (ADP-ribose) polymerase was detected in the HaA4-treated leukemia cells, suggesting induction of a caspase-dependent apoptosis pathway by HaA4. Caspase-dependent apoptosis was further confirmed by reversal of the HaA4-induced viability reduction by treatment of Z-VAD-FMK, a pan-caspase inhibitor. In conclusion, HaA4 caused necrosis and caspase-dependent apoptosis in both U937 and Jurkat leukemia cells, which suggests potential utility of HaA4 as a cancer therapeutic agent. PMID:23732481

  15. Human neutrophils are activated by a peptide fragment of Clostridium difficile toxin B presumably via formyl peptide receptor.

    PubMed

    Goy, Sebastian D; Olling, Alexandra; Neumann, Detlef; Pich, Andreas; Gerhard, Ralf

    2015-06-01

    Clostridium difficile may induce antibiotic-associated diarrhoea and, in severe cases, pseudomembranous colitis characterized by tremendous neutrophil infiltration. All symptoms are caused by two exotoxins: TcdA and TcdB. We describe here the activation of isolated human blood neutrophils by TcdB and, moreover, by toxin fragments generated by limited proteolytical digestion. Kinetics and profiles of TcdB-induced rise in intracellular-free Ca(2+) and reactive oxygen species production were similar to that induced by fMLF, which activates the formyl peptide receptor (FPR) recognizing formylated bacterial peptide sequences. Transfection assays with the FPR-1 isoform hFPR26 in HEK293 cells, heterologous desensitization experiments and FPR inhibition via cyclosporine H strongly suggest activation of cells via FPR-1. Domain analyses revealed that the N-terminal glucosyltransferase domain of TcdB is a potent activator of FPR pointing towards an additional mechanism that might contribute to pathogenesis. This pro-inflammatory ligand effect can be triggered even by cleaved and, thus, non-cytotoxic toxin. In summary, we report (i) a ligand effect on neutrophils as completely new molecular mode of action, (ii) pathogenic potential of truncated or proteolytically cleaved 'non-cytotoxic' fragments and (iii) an interaction of the N-terminal glucosyltransferase domain instead of the C-terminal receptor binding domain of TcdB with target cells. PMID:25529763

  16. Proteinase-activated receptors induce nonoxidative, antimicrobial peptides and increased antimicrobial activity in human mononuclear phagocytes.

    PubMed

    Lippuner, Nadine; Morell, Bernhard; Schaffner, Andreas; Schaer, Dominik J

    2007-02-01

    As thrombin and SFLLRNPNDKYEPF (SFLLRN-14), a synthetic ligand, mainly of the proteinase-activated receptor-1 (PAR-1), induce in monocytes the synthesis and secretion of chemokines, the PAR pathway can be viewed as a mononuclear phagocyte-activating principle. Classically, antimicrobial activity of mononuclear phagocytes is the measure for activation. Here, we investigated whether thrombin or SFLLRN-14 increases the antimicrobial activity of human monocytes and compared these effects to those of IFN-gamma. Furthermore, we measured the effects of these agents on the secretion of reactive oxygen intermediates and the antimicrobial activity of acid peptide extracts from monocytes. Human monocytes were exposed to maximally active concentrations of thrombin, SFLLRN-14, and IFN-gamma. Human monocytes treated with thrombin or SFLLRN-14 and then challenged with Salmonella enterica serovar typhimurium, including its attenuated mutant phoP, or Listeria monocytogenes killed, within 3 h, significantly more bacteria than control cells, an effect comparable with or surpassing the effect of IFN-gamma. This finding establishes the proteinase-PAR pathway as a potent, alternate activation pathway of mononuclear phagocytes. Thrombin and SFLLRN-14 had no significant effects on the amount of H(2)O(2) secreted by monocytes. This was in contrast to IFN-gamma, which as expected, increased the secretion of H(2)O(2) by approximately fourfold. Thrombin and SFLLRN-14, but not IFN-gamma, however, significantly increased the antimicrobial activity of acid peptide extracts of monocytes in a radial diffusion assay. Taken together, these findings suggest that IFN-gamma and thrombin differentially regulate oxidative and nonoxidative killing systems of human monocytes. PMID:17095611

  17. Antimicrobial activity of de novo designed cationic peptides against multi-resistant clinical isolates.

    PubMed

    Faccone, Diego; Veliz, Omar; Corso, Alejandra; Noguera, Martin; Martínez, Melina; Payes, Cristian; Semorile, Liliana; Maffía, Paulo Cesar

    2014-01-01

    Antibiotic resistance is one of the main problems concerning public health or clinical practice. Antimicrobial peptides appear as good candidates for the development of new therapeutic drugs. In this study we de novo designed a group of cationic antimicrobial peptides, analyzed its physicochemical properties, including its structure by circular dichroism and studied its antimicrobial properties against a panel of clinical isolates expressing different mechanisms of resistance. Three cationic alpha helical peptides exhibited antimicrobial activity comparable to, or even better than the comparator omiganan (MBI-226).

  18. Activity Prediction and Molecular Mechanism of Bovine Blood Derived Angiotensin I-Converting Enzyme Inhibitory Peptides

    PubMed Central

    Zhang, Ting; Nie, Shaoping; Liu, Boqun; Yu, Yiding; Zhang, Yan; Liu, Jingbo

    2015-01-01

    Development of angiotensin I-converting enzyme (ACE, EC 3.4.15.1) inhibitory peptides from food protein is under extensive research as alternative for the prevention of hypertension. However, it is difficult to identify peptides released from food sources. To accelerate the progress of peptide identification, a three layer back propagation neural network model was established to predict the ACE-inhibitory activity of pentapeptides derived from bovine hemoglobin by simulated enzyme digestion. The pentapeptide WTQRF has the best predicted value with experimental IC50 23.93 μM. The potential molecular mechanism of the WTQRF / ACE interaction was investigated by flexible docking. PMID:25768442

  19. Activity prediction and molecular mechanism of bovine blood derived angiotensin I-converting enzyme inhibitory peptides.

    PubMed

    Zhang, Ting; Nie, Shaoping; Liu, Boqun; Yu, Yiding; Zhang, Yan; Liu, Jingbo

    2015-01-01

    Development of angiotensin I-converting enzyme (ACE, EC 3.4.15.1) inhibitory peptides from food protein is under extensive research as alternative for the prevention of hypertension. However, it is difficult to identify peptides released from food sources. To accelerate the progress of peptide identification, a three layer back propagation neural network model was established to predict the ACE-inhibitory activity of pentapeptides derived from bovine hemoglobin by simulated enzyme digestion. The pentapeptide WTQRF has the best predicted value with experimental IC50 23.93 μM. The potential molecular mechanism of the WTQRF / ACE interaction was investigated by flexible docking. PMID:25768442

  20. Cytotoxic activity to acute myeloid leukemia cells by Antp-TPR hybrid peptide targeting Hsp90.

    PubMed

    Horibe, Tomohisa; Kawamoto, Megumi; Kohno, Masayuki; Kawakami, Koji

    2012-07-01

    We previously reported that Antp-TPR hybrid peptide inhibited the interaction of Hsp90 with TPR2A and had selective cytotoxic activity discriminating between normal and cancer cells to induce cancer cell death. In this study, we investigated the cytotoxic activity of Antp-TPR peptide toward acute myeloid leukemia (AML) cells. It was demonstrated that Antp-TPR peptide induced AML cell death in cell lines such as U937, K562, THP-1, and HL-60 via activation of caspases 3 and 7, and disruption of mitochondrial membrane potential. Conversely, Antp-TPR peptide did not reduce the viability of normal cells including peripheral blood mononuclear cells (PBMCs), although both geldanamycin and 17-AAG, small-molecule inhibitors of Hsp90, mediated cytotoxicity to these normal cells at low concentrations. In addition, mutation analysis of TPR peptide demonstrated that the highly conserved amino acids Lys and Arg were critical to the cytotoxic activity. These results indicated that Antp-TPR hybrid peptide would provide potent and selective therapeutic options in the treatment of AML.

  1. Cytochrome c peroxidase activity of heme bound amyloid β peptides.

    PubMed

    Seal, Manas; Ghosh, Chandradeep; Basu, Olivia; Dey, Somdatta Ghosh

    2016-09-01

    Heme bound amyloid β (Aβ) peptides, which have been associated with Alzheimer's disease (AD), can catalytically oxidize ferrocytochrome c (Cyt c(II)) in the presence of hydrogen peroxide (H2O2). The rate of catalytic oxidation of Cyt(II) c has been found to be dependent on several factors, such as concentration of heme(III)-Aβ, Cyt(II) c, H2O2, pH, ionic strength of the solution, and peptide chain length of Aβ. The above features resemble the naturally occurring enzyme cytochrome c peroxidase (CCP) which is known to catalytically oxidize Cyt(II) c in the presence of H2O2. In the absence of heme(III)-Aβ, the oxidation of Cyt(II) c is not catalytic. Thus, heme-Aβ complex behaves as CCP.

  2. Cytochrome c peroxidase activity of heme bound amyloid β peptides.

    PubMed

    Seal, Manas; Ghosh, Chandradeep; Basu, Olivia; Dey, Somdatta Ghosh

    2016-09-01

    Heme bound amyloid β (Aβ) peptides, which have been associated with Alzheimer's disease (AD), can catalytically oxidize ferrocytochrome c (Cyt c(II)) in the presence of hydrogen peroxide (H2O2). The rate of catalytic oxidation of Cyt(II) c has been found to be dependent on several factors, such as concentration of heme(III)-Aβ, Cyt(II) c, H2O2, pH, ionic strength of the solution, and peptide chain length of Aβ. The above features resemble the naturally occurring enzyme cytochrome c peroxidase (CCP) which is known to catalytically oxidize Cyt(II) c in the presence of H2O2. In the absence of heme(III)-Aβ, the oxidation of Cyt(II) c is not catalytic. Thus, heme-Aβ complex behaves as CCP. PMID:27270708

  3. Structural difference at the active site of dibucaine resistant variant of human plasma cholinesterase.

    PubMed Central

    Muensch, H; Yoshida, A; Altland, K; Jensen, W; Goedde, H W

    1978-01-01

    Human plasma cholinesterase from five different genotypes -- E1U E1U, E1U E1A, E1A E1A, E1U E1S, E1A E1S, and E1U E1U C5+ -- was purified 8,000 fold from serum by a two-step procedure involving chromatography on DEAE-cellulose and preparative disc electrophoresis. The esterases were labeled with diisopropyl-1, 3-C14-fluorophosphate (DFP) aminoethylated, and digested by trypsin. The trytic digests were subjected to high voltage electrophoresis, and the radioactive peptides were detected by radioautography. Comparison of the peptides revealed different electrophoretic mobilities of the usual and atypical (dibucaine resistant) plasma cholinesterase peptides. The results are consistent with a structural abnormality of the active center in the variant enzyme. No difference was observed an the esteratic site of the enzyme with C5 component. Images Fig. 1 PMID:677127

  4. Heavy metal-activated synthesis of peptides in Chlamydomonas reinhardtii

    SciTech Connect

    Howe, G.; Merchant, S. )

    1992-01-01

    In this study, the authors have addressed the capacity of the green alga Chlamydomonas reinhardtii to produce metal-binding peptides in response to stress induced by the heavy metals Cd{sup 2+}, Hg{sup 2+}, and Ag{sup +}. Cells cultured in the presence of sublethal concentrations of Cd{sup 2+} synthesized and accumulated oligopeptides consisting solely of glutamic acid, cysteine, and glycine in an average ratio of 3:3:1. Cadmium-induced peptides were isolated in their native form as higher molecular weight peptide-metal complexes with an apparent molecular weight of approximately 6.5 {times} 10{sup 3}. The isolated complex bound cadmium (as evidenced by absorption spectroscopy) and sequestered (with a stoichiometry of 0.7 moles of cadmium per mole of cysteine) up to 70% of the total cadmium found in extracts of cadmium-treated cells. In Hg{sup 2+}-treated cells, the principal thiol-containing compound induced by Hg{sup 2+} ion was glutathione. It is possible that glutathione functions in plant cells (as it does in animal cells) to detoxify heavy metals. Cells treated with Ag{sup +} ions also synthesized a sulfur-containing component with a charge to mass ratio similar to Cd{sup 2+}-induced peptides. But, in contrast to the results obtained using Cd{sup 2+} as an inducer, these molecules did not accumulate to significant levels in Ag{sup +}-treated cells. The presence of physiological concentrations of Cu{sup 2+} in the growth medium blocked the synthesis of the Ag{sup +}-inducible component(s) and rendered cells resistant to the toxic effects of Ag{sup +}, suggesting competition between Cu{sup 2+} and Ag{sup +} ions, possibly at the level of metal uptake.

  5. Preparation and biological activity of quaternized carboxymethyl chitosan conjugated with collagen peptide.

    PubMed

    Zhu, Xiaoming; Zhou, Xiaoyu; Yi, Jiayan; Tong, Jun; Wu, Huan; Fan, Lihong

    2014-09-01

    Tissue repair is a spontaneous process which initiated on wounding. If this complex mechanism is disturbed or impaired, the use of biomaterials might increase the chance of successful healing. In this view, a water-soluble chitosan derivative, quaternized carboxymethyl chitosan (QCMC) was prepared and collagen peptides (COPs) were grafted to the backbone by carbodiimide method. The reaction conditions affecting the degree of substitution (DS) were studied including the mass ratio of collagen peptide to QCMC, reaction temperature and reaction time. The hydrogen peroxide-scavenging activity could be different by changing the DS, concentration and molecular weight. MTT assay was used to investigate the cell viability of the derivative. The results indicated that the introduction of collagen peptide into the QCMC improved its hydrogen peroxide-scavenging activity and cell viability with the DS and concentration increased. Therefore, QCMC conjugated with collagen peptides may prove beneficial to the process of the wound-healing. PMID:24995634

  6. 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

  7. A cactus-derived toxin-like cystine knot Peptide with selective antimicrobial activity.

    PubMed

    Aboye, Teshome L; Strömstedt, Adam A; Gunasekera, Sunithi; Bruhn, Jan G; El-Seedi, Hesham; Rosengren, K Johan; Göransson, Ulf

    2015-05-01

    Naturally occurring cystine knot peptides show a wide range of biological activity, and as they have inherent stability they represent potential scaffolds for peptide-based drug design and biomolecular engineering. Here we report the discovery, sequencing, chemical synthesis, three-dimensional solution structure determination and bioactivity of the first cystine knot peptide from Cactaceae (cactus) family: Ep-AMP1 from Echinopsis pachanoi. The structure of Ep-AMP1 (35 amino acids) conforms to that of the inhibitor cystine knot (or knottin) family but represents a novel diverse sequence; its activity was more than 500 times higher against bacterial than against eukaryotic cells. Rapid bactericidal action and liposome leakage implicate membrane permeabilisation as the mechanism of action. Sequence homology places Ec-AMP1 in the plant C6-type of antimicrobial peptides, but the three dimensional structure is highly similar to that of a spider neurotoxin.

  8. Site-specific modification of anti-angiogenesis peptide HM-3 by polyethylene glycol molecular weight of 20 kDa.

    PubMed

    Zhu, Beili; Xu, Han-Mei; Zhao, Liming; Huang, Xiaofeng; Zhang, Fengguo

    2010-09-01

    HM-3, an RGD modified endostatin-derived polypeptide, is a potent angiogenesis inhibitor synthesized in our laboratory. Its robust inhibitory effects on endothelial cell migration and tumour growth have been demonstrated by in vivo and in vitro activity assays. However, the drug has relatively short half-life in vivo. For the purpose of prolonging HM-3 half-life and retaining the safety and efficacy of the peptide, the study chose methoxy-polyethylene glycol-Succinimidyl Carbonate (SC-mPEG, molecular weight 20 kDa, named SC-mPEG(20k)) to specifically modify its N terminus. Compared with HM-3, the site-specific mono-PEGylated peptide PEG(20k)-HM-3 was shown the same activity in the inhibition of B16F10 tumour in vivo (the inhibitory effect of PEG(20k)-HM-3, HM-3 and Taxol were 44.35, 39.68%, respectively), while the frequency of drug-administering reduced from twice a day to once every 3 days. Its rate of in vitro degradation in serum was markedly reduced (72.78% could still be detected after 132 h). Histochemistry and immunohistochemistry analysis showed that both HM-3 and PEG(20k)-HM-3 induced large areas of continuous necrosis within tumours and significantly reduced the vessel density compared to control. It might be a breakthrough in PEG modification field to modify a small peptide with a large PEG and reach a good result.

  9. Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin.

    PubMed

    Kim, Dae Hong; Lee, Ik Hwan; Nam, Seung Taek; Hong, Ji; Zhang, Peng; Hwang, Jae Sam; Seok, Heon; Choi, Hyemin; Lee, Dong Gun; Kim, Jae Il; Kim, Ho

    2014-06-01

    We recently isolated a polypeptide from the earthworm Lumbricus terrestris that is structurally similar to defensin, a well-known antibacterial peptide. An 11-mer antibacterial peptide (NH2-RNRRWCIDQQA), designated Lumbricusin, was synthesized based on the amino acid sequence of the isolated polypeptide. Since we previously reported that CopA3, a dung beetle peptide, enhanced neuronal cell proliferation, we here examined whether Lumbricusin exerted neurotropic and/or neuroprotective effects. Lumbricusin treatment induced a time-dependent increase (∼51%) in the proliferation of human neuroblastoma SH-SY5Y cells. Lumbricusin also significantly inhibited the apoptosis and decreased viability induced by treatment with 6-hydroxy dopamine, a Parkinson's disease-mimicking agent. Immunoblot analyses revealed that Lumbricusin treatment increased ubiquitination of p27(Kip1) protein, a negative regulator of cell-cycle progression, in SH-SY5Y cells, and markedly promoted its degradation. Notably, adenoviral-mediated over-expression of p27(Kip1) significantly blocked the antiapoptotic effect of Lumbricusin in 6-hydroxy dopamine-treated SH-SY5Y cells. These results suggest that promotion of p27(Kip1) degradation may be the main mechanism underlying the neuroprotective and neurotropic effects of Lumbricusin. PMID:24796676

  10. Trichodiene synthase. Identification of active site residues by site-directed mutagenesis.

    PubMed

    Cane, D E; Shim, J H; Xue, Q; Fitzsimons, B C; Hohn, T M

    1995-02-28

    Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with [methyl-14C]methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site. The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis. The C190A mutant displayed partial but significantly reduced activity, with a reduction in kcat/Km of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive. A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F. sporotrichioides enzyme. From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified. Three of these mutants were overexpressed and purified to homogeneity. The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in Km, as well as a 200-fold reduction in kcat. In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204. Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on kcat, while increasing the Km by a factor of ca. 7-8.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7873527

  11. Oxidation Protection in Metal-Binding Peptide Motif and Its Application to Antibody for Site-Selective Conjugation

    PubMed Central

    Chung, Hye-Shin; Lee, Sunbae; Park, Soon Jae

    2016-01-01

    Here, we demonstrate that a metal ion binding motif could serve as an efficient and robust tool for site-specific conjugation strategy. Cysteine-containing metal binding motifs were constructed as single repeat or tandem repeat peptides and their metal binding characteristics were investigated. The tandem repeats of the Cysteine-Glycine-Histidine (CGH) metal ion binding motif exhibited concerted binding to Co(II) ions, suggesting that conformational transition of peptide was triggered by the sequential metal ion binding. Evaluation of the free thiol content after reduction by reducing reagent showed that metal-ion binding elicited strong retardation of cysteine oxidation in the order of Zn(II)>Ni(II)>Co(II). The CGH metal ion binding motif was then introduced to the C-terminus of antibody heavy chain and the metal ion-dependent characteristics of oxidation kinetics were investigated. As in the case of peptides, CGH-motif-introduced antibody exhibited strong dependence on metal ion binding to protect against oxidation. Zn(II)-saturated antibody with tandem repeat of CGH motif retains the cysteine reactivity as long as 22 hour even with saturating O2 condition. Metal-ion dependent fluorophore labeling clearly indicated that metal binding motifs could be employed as an efficient tool for site-specific conjugation. Whereas Trastuzumab without a metal ion binding site exhibited site-nonspecific dye conjugation, Zn(II) ion binding to antibody with a tandem repeat of CGH motif showed that fluorophores were site-specifically conjugated to the heavy chain of antibody. We believe that this strong metal ion dependence on oxidation protection and the resulting site-selective conjugation could be exploited further to develop a highly site-specific conjugation strategy for proteins that contain multiple intrinsic cysteine residues, including monoclonal antibodies. PMID:27420328

  12. Characterization of active sites in zeolite catalysts

    SciTech Connect

    Eckert, J.; Bug, A.; Nicol, J.M.

    1997-11-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). Atomic-level details of the interaction of adsorbed molecules with active sites in catalysts are urgently needed to facilitate development of more effective and/or environmentally benign catalysts. To this end the authors have carried out neutron scattering studies combined with theoretical calculations of the dynamics of small molecules inside the cavities of zeolite catalysts. The authors have developed the use of H{sub 2} as a probe of adsorption sites by observing the hindered rotations of the adsorbed H{sub 2} molecule, and they were able to show that an area near the four-rings is the most likely adsorption site for H{sub 2} in zeolite A while adsorption of H{sub 2} near cations located on six-ring sites decreases in strength as Ni {approximately} Co > Ca > Zn {approximately} Na. Vibrational and rotational motions of ethylene and cyclopropane adsorption complexes were used as a measure for zeolite-adsorbate interactions. Preliminary studies of the binding of water, ammonia, and methylamines were carried out in a number of related guest-host materials.

  13. Activation of epitope-specific memory cytotoxic T lymphocyte responses by synthetic peptides.

    PubMed

    Reali, E; Guerrini, R; Giori, B; Borghi, M; Marastoni, M; Tomatis, R; Traniello, S; Masucci, M G; Gavioli, R

    1996-08-01

    Cytotoxic T lymphocytes (CTL) recognize antigens as short peptides selected for presentation by their ability to bind to MHC class I molecules. Polyclonal Epstein-Barr virus (EBV)-specific memory CTL responses, reactivated from blood lymphocytes of HLA-A11-positive individuals by stimulation with the autologous EBV-transformed lymphoblastoid cell line (LCL), are often dominated by reactivites directed to the peptide epitope IVTDFSVIK (IVT), corresponding to amino acids 416-424 of EBV nuclear antigen-4 (EBNA4). We now report the selective activation of IVT-specific CTL by stimulation of lymphocytes with the corresponding synthetic peptide. A more than 10-fold increase in frequency of CTL clones with this specificity (from 8% to 96%) was obtained when the peptide was presented by HLA-A11-transfected T2 cells (T2/A11). Titration of synthetic peptide in cytotoxic assay demonstrated that clones activated under these conditions are as efficient as clones activated by conventional LCL stimulations. Induction of memory CTL responses required low surface density of MHC: peptide complexes, since reactivation was achieved by stimulation with T2/A11 cells pulsed with concentrations of peptide that are suboptimal for induction of target cell lysis. This protocol of activation revealed the presence of IVT-specific CTL precursors in a donor that failed to mount an IVT-specific response upon stimulation with the autologous B95.8 virus-transformed LCL. The results suggest that stimulation with synthetic peptide epitopes can be efficiently used for induction of memory CTL responses, and may be particularly helpful for the selective expansion of subdominant CTL specificities.

  14. Evidence for long-distance xylem transport of signal peptide activity from tomato roots.

    PubMed

    Neumann, Peter M

    2007-01-01

    Several types of small, endogenous signal peptides are now known to induce a wide range of local and systemic responses in plants, but how such signal peptide activity is transported over long distances remains unclear. In particular, the possible occurrence and root-to-shoot transport of signal peptide activity in the xylem does not appear to have been previously investigated. Suspension-cultured cells of wild tomato Lycopersicon peruvanium L. were used in an established bioassay for detecting nanomolar concentrations of signal peptides via the induction of alkalinizing activity. Xylem sap naturally exuded from the cut and washed stem-surfaces of de-topped tomato plants (Lycopersicon esculentum L. cv. Castlemart) was collected, partially purified, concentrated, and shown by the bioassay consistently to contain significant alkalinizing activity. Plant salinity treatment induced further small increases in activity. Subsidiary experiments indicated that the alkalinizing activity found in the xylem-sap had properties similar to those of known plant signal peptides and was root derived. Thus, it was (i) detectable within minutes, (ii) eluted similarly during HPLC chromatography, (iii) destroyed by incubation with proteases and stable in the presence of protease inhibitor cocktail, and (iv) not found in bioassays of simulated xylem sap placed on the cut stem-surfaces of non-exuding roots in order to detect any significant release of wound peptides from the stem. Further investigations of the signal peptide activity in root xylem sap could provide new insights into its identity, genes, receptors, origins, and possible hormonal roles in regulating shoot growth and development.

  15. Antigen mobility in the combining site of an anti-peptide antibody.

    PubMed

    Cheetham, J C; Raleigh, D P; Griest, R E; Redfield, C; Dobson, C M; Rees, A R

    1991-09-15

    The interaction between a high-affinity antibody, raised against a peptide incorporating the loop region of hen egg lysozyme (residues 57-84), and a peptide antigen corresponding to this sequence, has been probed by proton NMR. The two-dimensional correlated spectroscopy spectrum of the antibody-antigen complex shows sharp, well-resolved resonances from at least half of the bound peptide residues, indicating that the peptide retains considerable mobility when bound to the antibody. The strongly immobilized residues (which include Arg-61, Trp-62, Trp-63, and Ile-78) do not correspond to a contiguous region in the sequence of the peptide. Examination of the crystal structure of the protein shows that these residues, although remote in sequence, are grouped together in the protein structure, forming a hydrophobic projection on the surface of the molecule. The antibody binds hen egg lysozyme with only a 10-fold lower affinity than the peptide antigen. We propose that the peptide could bind to the antibody in a conformation that brings these groups together in a manner related to that found in the native protein, accounting for the high crossreactivity. PMID:1716767

  16. Antigen mobility in the combining site of an anti-peptide antibody.

    PubMed Central

    Cheetham, J C; Raleigh, D P; Griest, R E; Redfield, C; Dobson, C M; Rees, A R

    1991-01-01

    The interaction between a high-affinity antibody, raised against a peptide incorporating the loop region of hen egg lysozyme (residues 57-84), and a peptide antigen corresponding to this sequence, has been probed by proton NMR. The two-dimensional correlated spectroscopy spectrum of the antibody-antigen complex shows sharp, well-resolved resonances from at least half of the bound peptide residues, indicating that the peptide retains considerable mobility when bound to the antibody. The strongly immobilized residues (which include Arg-61, Trp-62, Trp-63, and Ile-78) do not correspond to a contiguous region in the sequence of the peptide. Examination of the crystal structure of the protein shows that these residues, although remote in sequence, are grouped together in the protein structure, forming a hydrophobic projection on the surface of the molecule. The antibody binds hen egg lysozyme with only a 10-fold lower affinity than the peptide antigen. We propose that the peptide could bind to the antibody in a conformation that brings these groups together in a manner related to that found in the native protein, accounting for the high crossreactivity. PMID:1716767

  17. B7H6-derived peptides trigger TNF-α-dependent immunostimulatory activity of lymphocytic NK92-MI cells.

    PubMed

    Phillips, Mariana; Romeo, Francesca; Bitsaktsis, Constantine; Sabatino, David

    2016-09-01

    The rise of biologics that can stimulate immune responses towards the eradication of tumors has led to the evolution of cancer-based immunotherapy. Representatively, B7H6 has been recently identified as a protein ligand on tumor cells that binds specifically to the NKp30 receptor and triggers NK cell-derived cytokine production, which ultimately leads to tumor cell lysis and death. In an effort to develop effective immunotherapy approaches, the rational design of a novel class of immunostimulatory peptides (IPs) derived from the binding interface of B7H6:NKp30 is described in this study. The IPs comprised the B7H6 active site sequence for NKp30 binding and immunostimulatory activity. An aminohexanoic acid linker was also introduced at the N-terminus of the peptides for FITC-labeling by Fmoc-solid phase peptide synthesis. The peptides were characterized by LCMS to confirm identities and purities >95%. The secondary structures of the peptides were examined by CD spectroscopy in H2 O, PBS and a H2 O:TFE mixture which demonstrated versatile peptide structures which transitioned from random coil (H2 O) to α-helical (PBS) and turn-type (H2 O:TFE) conformations. Their biological properties were then evaluated by flow cytometry, enzyme-linked immunosorbent assays (ELISAs), and cell death assays. The occupancy of the synthetic peptides to a human NK cell line demonstrated comparable binding relative to the natural NKp30 ligand, B7H6, and the human anti-NKp30 monoclonal antibody (mAb), in a concentration dependent manner. A competitive binding assay between the human anti-NKp30 mAb or B7H6, and the synthetic peptides, demonstrated partial displacement of the ligands upon anti-NKp30 mAb treatment, suggesting NKp30 receptor specificities by the synthetic peptides. Moreover, the immunostimulatory activity of B7H6 was demonstrated by the secretion of the pro-inflammatory cytokines tumor necrosis factor-alfa (TNF-α) and interferon gamma (IFN-γ) by the human NK cell line. The

  18. Structure diversification of vancomycin through peptide-catalyzed, site-selective lipidation: A catalysis-based approach to combat glycopeptide-resistant pathogens

    PubMed Central

    Yoganathan, Sabesan; Miller, Scott J.

    2015-01-01

    Emergence of antibiotic-resistant infections highlights the need for novel antibiotic leads, perhaps with a broader spectrum of activity. Herein, we disclose a semisynthetic, catalytic approach for structure diversification of vancomycin. We have identified three unique peptide catalysts that exhibit site-selectivity for the lipidation of the aliphatic hydroxyls on vancomycin, generating three new derivatives 9a, 9b and 9c. Incorporation of lipid chains into vancomycin scaffold provides promising improvement of its bioactivity against vancomycin-resistant enterococci (Van A and Van B phenotypes of VRE). The MICs for 9a, 9b, and 9c against MRSA and VRE (Van B phenotype) range from 0.12 to 0.25 μg/mL. We have also performed a structure activity relationship (SAR) study to investigate the effect of lipid chain length at the newly accessible G4-OH derivatization site. PMID:25671771

  19. Function-based classification of carbohydrate-active enzymes by recognition of short, conserved peptide motifs.

    PubMed

    Busk, Peter Kamp; Lange, Lene

    2013-06-01

    Functional prediction of carbohydrate-active enzymes is difficult due to low sequence identity. However, similar enzymes often share a few short motifs, e.g., around the active site, even when the overall sequences are very different. To exploit this notion for functional prediction of carbohydrate-active enzymes, we developed a simple algorithm, peptide pattern recognition (PPR), that can divide proteins into groups of sequences that share a set of short conserved sequences. When this method was used on 118 glycoside hydrolase 5 proteins with 9% average pairwise identity and representing four characterized enzymatic functions, 97% of the proteins were sorted into groups correlating with their enzymatic activity. Furthermore, we analyzed 8,138 glycoside hydrolase 13 proteins including 204 experimentally characterized enzymes with 28 different functions. There was a 91% correlation between group and enzyme activity. These results indicate that the function of carbohydrate-active enzymes can be predicted with high precision by finding short, conserved motifs in their sequences. The glycoside hydrolase 61 family is important for fungal biomass conversion, but only a few proteins of this family have been functionally characterized. Interestingly, PPR divided 743 glycoside hydrolase 61 proteins into 16 subfamilies useful for targeted investigation of the function of these proteins and pinpointed three conserved motifs with putative importance for enzyme activity. Furthermore, the conserved sequences were useful for cloning of new, subfamily-specific glycoside hydrolase 61 proteins from 14 fungi. In conclusion, identification of conserved sequence motifs is a new approach to sequence analysis that can predict carbohydrate-active enzyme functions with high precision. PMID:23524681

  20. Function-based classification of carbohydrate-active enzymes by recognition of short, conserved peptide motifs.

    PubMed

    Busk, Peter Kamp; Lange, Lene

    2013-06-01

    Functional prediction of carbohydrate-active enzymes is difficult due to low sequence identity. However, similar enzymes often share a few short motifs, e.g., around the active site, even when the overall sequences are very different. To exploit this notion for functional prediction of carbohydrate-active enzymes, we developed a simple algorithm, peptide pattern recognition (PPR), that can divide proteins into groups of sequences that share a set of short conserved sequences. When this method was used on 118 glycoside hydrolase 5 proteins with 9% average pairwise identity and representing four characterized enzymatic functions, 97% of the proteins were sorted into groups correlating with their enzymatic activity. Furthermore, we analyzed 8,138 glycoside hydrolase 13 proteins including 204 experimentally characterized enzymes with 28 different functions. There was a 91% correlation between group and enzyme activity. These results indicate that the function of carbohydrate-active enzymes can be predicted with high precision by finding short, conserved motifs in their sequences. The glycoside hydrolase 61 family is important for fungal biomass conversion, but only a few proteins of this family have been functionally characterized. Interestingly, PPR divided 743 glycoside hydrolase 61 proteins into 16 subfamilies useful for targeted investigation of the function of these proteins and pinpointed three conserved motifs with putative importance for enzyme activity. Furthermore, the conserved sequences were useful for cloning of new, subfamily-specific glycoside hydrolase 61 proteins from 14 fungi. In conclusion, identification of conserved sequence motifs is a new approach to sequence analysis that can predict carbohydrate-active enzyme functions with high precision.

  1. Chimerization of lactoferricin and lactoferrampin peptides strongly potentiates the killing activity against Candida albicans.

    PubMed

    Bolscher, Jan; Nazmi, Kamran; van Marle, Jan; van 't Hof, Wim; Veerman, Enno

    2012-06-01

    Bovine lactoferrin harbors 2 antimicrobial sequences (LFcin and LFampin), situated in close proximity in the N1-domain. To mimic their semi parallel configuration we have synthesized a chimeric peptide (LFchimera) in which these sequences are linked in a head-to-head fashion to the α- and ε-amino group, respectively, of a single lysine. In line with previously described bactericidal effects, this peptide was also a stronger candidacidal agent than the antimicrobial peptides LFcin17-30 and LFampin265-284, or a combination of these 2. Conditions that strongly reduced the candidacidal activities of LFcin17-30 and LFampin265-284, such as high ionic strength and energy depletion, had little influence on the activity of LFchimera. Freeze-fracture electron microscopy showed that LFchimera severely affected the membrane morphology, resulting in disintegration of the membrane bilayer and in an efflux of small and high molecular weight molecules such as ATP and proteins. The differential effects displayed by the chimeric peptide and a mixture of its constituent peptides clearly demonstrate the synergistic effect of linking these peptides in a fashion that allows a similar spatial arrangement as in the parent protein, suggesting that in bovine lactoferrrin the corresponding fragments act in concert in its candidacidal activity.

  2. Effect of probiotics on antioxidant and antimutagenic activities of crude peptide extract from yogurt.

    PubMed

    Sah, B N P; Vasiljevic, T; McKechnie, S; Donkor, O N

    2014-08-01

    Search for bioactive peptides is intensifying because of the risks associated with the use of synthetic therapeutics, thus peptide liberation by lactic acid bacteria and probiotics has received a great focus. However, proteolytic capacity of these bacteria is strain specific. The study was conducted to establish proteolytic activity of Lactobacillus acidophilus (ATCC® 4356™), Lactobacillus casei (ATCC® 393™) and Lactobacillus paracasei subsp. paracasei (ATCC® BAA52™) in yogurt. Crude peptides were separated by high-speed centrifugation and tested for antioxidant and antimutagenic activities. The degree of proteolysis highly correlated with these bioactivities, and its value (11.91%) for samples containing all the cultures was double that of the control. Liberated peptides showed high radical scavenging activities with 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), IC50 1.51 and 1.63mg/ml, respectively and strong antimutagenicity (26.35%). These probiotics enhanced the generation of bioactive peptides and could possibly be commercially applied in new products, or production of novel anticancer peptides.

  3. Immunomodulatory and Anti-Inflammatory Activities of Chicken Cathelicidin-2 Derived Peptides

    PubMed Central

    van Dijk, Albert; van Eldik, Mandy; Veldhuizen, Edwin J. A.; Tjeerdsma-van Bokhoven, Hanne L. M.; de Zoete, Marcel R.; Bikker, Floris J.; Haagsman, Henk P.

    2016-01-01

    Host Defence Peptides and derived peptides are promising classes of antimicrobial and immunomodulatory lead compounds. For this purpose we examined whether chicken cathelicidin-2 (CATH-2)-derived peptides modulate the function and inflammatory response of avian immune cells. Using a chicken macrophage cell line (HD11) we found that full-length CATH-2 dose-dependently induced transcription of chemokines CXCLi2/IL-8, MCP-3 and CCLi4/RANTES, but not of pro-inflammatory cytokine IL-1β. In addition, CATH-2 efficiently inhibited IL-1β and nitric oxide production by HD11 cells induced by different sources of lipopolysaccharides (LPS). N-terminal truncated CATH-2 derived peptides maintained the capacity to selectively induce chemokine transcription, but despite their high LPS affinity several analogs lacked LPS-neutralizing capacity. Substitution of phenylalanine residues by tryptophan introduced endotoxin neutralization capacity in inactive truncated CATH-2 derived peptides. In contrast, amino acid substitution of phenylalanine by tyrosine abrogated endotoxin neutralization activity of CATH-2 analogs. These findings support a pivotal role for aromatic residues in peptide-mediated endotoxin neutralization by CATH-2 analogs and were shown to be independent of LPS affinity. The capacity to modulate chemokine production and dampen endotoxin-induced pro-inflammatory responses in chicken immune cells implicates that small CATH-2 based peptides could serve as leads for the design of CATH-2 based immunomodulatory anti-infectives. PMID:26848845

  4. Antibacterial activity of casein-derived peptides isolated from rabbit (Oryctolagus cuniculus) milk.

    PubMed

    Baranyi, Maria; Thomas, Ursula; Pellegrini, Antonio

    2003-05-01

    Acid-precipitated rabbit 'whole casein' was digested by trypsin, chymotrypsin, pepsin, and clostripain to screen for possible peptides with antibacterial properties. The peptide fragments were separated by reversed-phase chromatography. The collected fractions were pooled and their antibacterial properties tested against Escherichia coli, Bacillus subtilis and Staphylococcus lentus. Three antibacterial peptide fragments derived from tryptic digestion of rabbit casein were isolated and identified. Their sequences were found as follows: HVEQLLR (residues 50-56 of beta-casein), ILPFIQSLFPFAER (residues 64-77 of beta-casein), and FHLGHLK (residues 19-25 of alpha(s1)-casein). The three peptides were synthesized and found to exert antibacterial effect against gram positive bacteria only. Proteolytic digestion of rabbit casein by chymotrypsin, pepsin and clostripain yielded several peptide fragments with antibacterial activity. Since antibiotic peptides can be released from casein during the digestion of milk proteins, our results suggest a possible antibacterial function of rabbit caseins. It is conceivable that antibacterial peptides can be generated by endopeptidases of the mammalian gastrointestinal tract possibly providing protection for new-born rabbits against aggression of micro-organisms.

  5. Synthetic peptide models for the redox-active disulfide loop of glutaredoxin. Conformational studies

    SciTech Connect

    Kishore, R.; Raghothama, S.; Balaram, P.

    1988-04-05

    Two cyclic peptide disulfides have been synthesized as models for the 14-membered redox-active disulfide loop glutaredoxin. /sup 1/H NMR studies at 270 MHz in chloroform solutions establish a type I ..beta..-turn conformation for the Pro-X segment in both peptides, stabilized by a 4 ..-->.. 1 hydrogen bond between the Cys(1) CO and Cys(4) NH groups. Nuclear Overhauser effects establish that the aromatic ring in the X = Phe peptide is oriented over the central peptide unit. In dimethyl sulfoxide solutions two conformational species are observed in slow exchange on the NMR time scale, for both peptides. These are assigned to type I and type II ..beta..-turn structures with -Pro-Tyr(Phe)-as the corner resides. The structural assignments are based on correlation of NMR parameters with model 14-membered cyclic cystine peptides with Pro-X spacers. Circular dichroism studies based on the -S-S-n-sigma* transition suggest a structural change in the disulfide bridge with changing solvent polarity, establishing conformational coupling between the peptide backbone and the disulfide linkage in these systems.

  6. Activation of p70s6k is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs.

    PubMed

    Ferrari, S; Bannwarth, W; Morley, S J; Totty, N F; Thomas, G

    1992-08-01

    Partial amino acid sequences were obtained from 22 internal tryptic peptides of rat liver p70s6k (M(r) 70,000 ribosomal protein S6 kinase), 3 of which were found to contain phosphorylated residues. To determine whether these sites were associated with p70s6k activation, the kinase was labeled to high specific activity with 32P(i) in Swiss mouse 3T3 cells. By sequential cleavage with CNBr and endoproteinase Lys-C followed by two-dimensional tryptic peptide analysis, it could be shown that all of the sites were located in a small endoproteinase Lys-C peptide of M(r) 2400. Analysis of the p70s6k protein sequence revealed a single candidate that could represent this peptide. Three tryptic peptides derived from the endoproteinase Lys-C fragment were chosen by a newly described computer program as the most likely candidates to contain the in vivo sites of phosphorylation. Synthetic peptides based on these sequences were phosphorylated either chemically or enzymatically and found to comigrate by two-dimensional thin-layer electrophoresis/chromatography with the four major in vivo labeled tryptic phosphopeptides. Three of the phosphorylation sites in these peptides were equivalent to those sequenced in the rat liver p70s6k. In addition, all four sites display the motif Ser/Thr-Pro, typical of cell cycle-regulated sites, and are clustered in a putative autoinhibitory domain of the enzyme.

  7. Establishment of Epithelial Attachment on Titanium Surface Coated with Platelet Activating Peptide

    PubMed Central

    Sugawara, Shiho; Maeno, Masahiko; Lee, Cliff; Nagai, Shigemi; Kim, David M.; Da Silva, John; Kondo, Hisatomo

    2016-01-01

    The aim of this study was to produce epithelial attachment on a typical implant abutment surface of smooth titanium. A challenging complication that hinders the success of dental implants is peri-implantitis. A common cause of peri-implantitis may results from the lack of epithelial sealing at the peri-implant collar. Histologically, epithelial sealing is recognized as the attachment of the basement membrane (BM). BM-attachment is promoted by activated platelet aggregates at surgical wound sites. On the other hand, platelets did not aggregate on smooth titanium, the surface typical of the implant abutment. We then hypothesized that epithelial BM-attachment was produced when titanium surface was modified to allow platelet aggregation. Titanium surfaces were coated with a protease activated receptor 4-activating peptide (PAR4-AP). PAR4-AP coating yielded rapid aggregation of platelets on the titanium surface. Platelet aggregates released robust amount of epithelial chemoattractants (IGF-I, TGF-β) and growth factors (EGF, VEGF) on the titanium surface. Human gingival epithelial cells, when they were co-cultured on the platelet aggregates, successfully attached to the PAR4-AP coated titanium surface with spread laminin5 positive BM and consecutive staining of the epithelial tight junction component ZO1, indicating the formation of complete epithelial sheet. These in-vitro results indicate the establishment of epithelial BM-attachment to the titanium surface. PMID:27741287

  8. Disulfide cross-linking influences symbiotic activities of nodule peptide NCR247.

    PubMed

    Shabab, Mohammed; Arnold, Markus F F; Penterman, Jon; Wommack, Andrew J; Bocker, Hartmut T; Price, Paul A; Griffitts, Joel S; Nolan, Elizabeth M; Walker, Graham C

    2016-09-01

    Interactions of rhizobia with legumes establish the chronic intracellular infection that underlies symbiosis. Within nodules of inverted repeat-lacking clade (IRLC) legumes, rhizobia differentiate into nitrogen-fixing bacteroids. This terminal differentiation is driven by host nodule-specific cysteine-rich (NCR) peptides that orchestrate the adaptation of free-living bacteria into intracellular residents. Medicago truncatula encodes a family of >700 NCR peptides that have conserved cysteine motifs. NCR247 is a cationic peptide with four cysteines that can form two intramolecular disulfide bonds in the oxidized forms. This peptide affects Sinorhizobium meliloti transcription, translation, and cell division at low concentrations and is antimicrobial at higher concentrations. By preparing the three possible disulfide-cross-linked NCR247 regioisomers, the reduced peptide, and a variant lacking cysteines, we performed a systematic study of the effects of intramolecular disulfide cross-linking and cysteines on the activities of an NCR peptide. The relative activities of the five NCR247 variants differed strikingly among the various bioassays, suggesting that the NCR peptide-based language used by plants to control the development of their bacterial partners during symbiosis is even greater than previously recognized. These patterns indicate that certain NCR bioactivities require cysteines whereas others do not. The results also suggest that NCR247 may exert some of its effects within the cell envelope whereas other activities occur in the cytoplasm. BacA, a membrane protein that is critical for symbiosis, provides protection against all bactericidal forms of NCR247. Oxidative folding protects NCR247 from degradation by the symbiotically relevant metalloprotease HrrP (host range restriction peptidase), suggesting that disulfide bond formation may additionally stabilize NCR peptides during symbiosis. PMID:27551097

  9. Testing the limits of rational design by engineering pH sensitivity into membrane active peptides

    PubMed Central

    Wiedman, Gregory

    2015-01-01

    In this work, we sought to rationally design membrane active peptides that are triggered by low pH to form macromolecular-sized pores in lipid bilayers. Such peptides could have broad utility in biotechnology and in nanomedicine as cancer therapeutics or drug delivery vehicles that promote release of macromolecules from endosomes. Our approach to rational design was to combine the properties of a pH-independent peptide, MelP5, which forms large pores allowing passage of macromolecules, with the properties of two pH-dependent membrane active peptides, pHlip and GALA. We created two hybrid sequences, MelP5_Δ4 and MelP5_Δ6 by using the distribution of acidic residues on pHlip and GALA as a guide to insert acidic amino acids into the amphipathic helix of MelP5. We show that the new peptides bind to lipid bilayers and acquire secondary structure in a pH-dependent manner. The peptides also destabilize bilayers in a pH-dependent manner, such that lipid vesicles release the small molecules ANTS/DPX at low pH only. Thus, we were successful in designing pH-triggered pore-forming peptides. However, no macro-molecular release was observed under any conditions. Therefore, we abolished the unique macromolecular poration properties of MelP5 by introducing pH-sensitivity into its sequence. We conclude that the properties of pHlip, GALA and MelP5 are additive, but only partially so. We propose that this lack of additivity is a limitation in the rational design of novel membrane active peptides, and that high-throughput approaches to discovery will be critical for continued progress in the field. PMID:25572997

  10. Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site

    SciTech Connect

    Grossman, Moran; Born, Benjamin; Heyden, Matthias; Tworowski, Dmitry; Fields, Gregg B.; Sagi, Irit; Havenith, Martina

    2011-09-18

    Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.

  11. Vv-AMP1, a ripening induced peptide from Vitis vinifera shows strong antifungal activity

    PubMed Central

    de Beer, Abré; Vivier, Melané A

    2008-01-01

    Background Latest research shows that small antimicrobial peptides play a role in the innate defense system of plants. These peptides typically contribute to preformed defense by developing protective barriers around germinating seeds or between different tissue layers within plant organs. The encoding genes could also be upregulated by abiotic and biotic stimuli during active defense processes. The peptides display a broad spectrum of antimicrobial activities. Their potent anti-pathogenic characteristics have ensured that they are promising targets in the medical and agricultural biotechnology sectors. Results A berry specific cDNA sequence designated Vv-AMP1, Vitis vinifera antimicrobial peptide 1, was isolated from Vitis vinifera. Vv-AMP1 encodes for a 77 amino acid peptide that shows sequence homology to the family of plant defensins. Vv-AMP1 is expressed in a tissue specific, developmentally regulated manner, being only expressed in berry tissue at the onset of berry ripening and onwards. Treatment of leaf and berry tissue with biotic or abiotic factors did not lead to increased expression of Vv-AMP1 under the conditions tested. The predicted signal peptide of Vv-AMP1, fused to the green fluorescent protein (GFP), showed that the signal peptide allowed accumulation of its product in the apoplast. Vv-AMP1 peptide, produced in Escherichia coli, had a molecular mass of 5.495 kDa as determined by mass spectrometry. Recombinant Vv-AMP1 was extremely heat-stable and showed strong antifungal activity against a broad spectrum of plant pathogenic fungi, with very high levels of activity against the wilting disease causing pathogens Fusarium oxysporum and Verticillium dahliae. The Vv-AMP1 peptide did not induce morphological changes on the treated fungal hyphae, but instead strongly inhibited hyphal elongation. A propidium iodide uptake assay suggested that the inhibitory activity of Vv-AMP1 might be associated with altering the membrane permeability of the fungal

  12. Site-selective recognition of peptide phosphorylation by a terbium(III) complex in aqueous solution.

    PubMed

    Wang, Xiaohui; Yang, Tao; Luo, Jian; Yang, Liu; Yao, Cheng

    2015-05-11

    A terbium(III) complex exhibits efficient selectivity for proximal diphosphorylation of peptides, accompanied with remarkable luminescence enhancement in the presence of Zn(II) ions in both buffer and protein extraction solutions from brain homogenates of mice.

  13. Activity Determinants of Helical Antimicrobial Peptides: A Large-Scale Computational Study

    PubMed Central

    He, Yi; Lazaridis, Themis

    2013-01-01

    Antimicrobial peptides (AMPs), produced by a wide range of organisms, have attracted attention due to their potential use as novel antibiotics. The majority of these peptides are cationic and are thought to function by permeabilizing the bacterial membrane, either by making pores or by dissolving it (‘carpet’ model). A key hypothesis in the literature is that antimicrobial and hemolytic activity correlate with binding affinity to anionic and zwitterionic membranes, respectively. Here we test this hypothesis by using binding free energy data collected from the literature and theoretical binding energies calculated from implicit membrane models for 53 helical AMPs. We indeed find a correlation between binding energy and biological activity, depending on membrane anionic content: antibacterial activity correlates best with transfer energy to membranes with anionic lipid fraction higher than 30% and hemolytic activity correlates best with transfer energy to a 10% anionic membrane. However, the correlations are weak, with correlation coefficient up to 0.4. Weak correlations of the biological activities have also been found with other physical descriptors of the peptides, such as surface area occupation, which correlates significantly with antibacterial activity; insertion depth, which correlates significantly with hemolytic activity; and structural fluctuation, which correlates significantly with both activities. The membrane surface coverage by many peptides at the MIC is estimated to be much lower than would be required for the ‘carpet’ mechanism. Those peptides that are active at low surface coverage tend to be those identified in the literature as pore-forming. The transfer energy from planar membrane to cylindrical and toroidal pores was also calculated for these peptides. The transfer energy to toroidal pores is negative in almost all cases while that to cylindrical pores is more favorable in neutral than in anionic membranes. The transfer energy to pores

  14. Electrochemical assay of active prostate-specific antigen (PSA) using ferrocene-functionalized peptide probes

    SciTech Connect

    Zhao, Ning; He, Yuqing; Mao, Xun; Sun, Yuhan; Zhang, Xibao; Li, Chen-Zhong; Lin, Yuehe; Liu, Guodong

    2010-03-24

    This paper presents a novel approach to electrochemically determine enzymatically active PSA using ferrocene-functionalized helix peptide (CHSSLKQK). The principle of electrochemical measurement is based on the specific proteolytic cleavage events of the FC-peptide on the gold electrode surface in the presence of PSA, resulting the change of the current signal of the electrode. The percentage of the decreased current is linear with the concentration of active PSA at the range of 0.5-40 ng/mL with a detection limit of 0.2 ng/mL. The direct transduction of peptide cleavage events into an electrical signal provides a simple, sensitive method for detecting the enzymatic activity of PSA and determining the active PSA concentration.

  15. Solution structure and antiparasitic activity of scorpine-like peptides from Hoffmannihadrurus gertschi.

    PubMed

    Flores-Solis, David; Toledano, Yanis; Rodríguez-Lima, Oscar; Cano-Sánchez, Patricia; Ramírez-Cordero, Belen Ernestina; Landa, Abraham; Rodríguez de la Vega, Ricardo C; Del Rio-Portilla, Federico

    2016-07-01

    Scorpine-like peptides are two domain peptides found in different scorpion venoms displaying various antimicrobial, cytolytic, and potassium channel-blocking activities. The relative contribution of each domain to their different activities remains to be elucidated. Here, we report the recombinant production, solution structure, and antiparasitic activity of Hge36, first identified as a naturally occurring truncated form of a Scorpine-like peptide from the venom of Hoffmannihadrurus gertschi. We also show that removing the first four residues from Hge36 renders a molecule with enhanced potassium channel-blocking and antiparasitic activities. Our results are important to rationalize the structure-function relationships of a pharmacologically versatile molecular scaffold. PMID:27314815

  16. The Peptide Microarray-Based Resonance Light Scattering Assay for Sensitively Detecting Intracellular Kinase Activity.

    PubMed

    Li, Tao; Liu, Xia; Liu, Dianjun; Wang, Zhenxin

    2016-01-01

    The peptide microarray technology is a robust, reliable, and efficient technique for large-scale determination of enzyme activities, and high-throughput profiling of substrate/inhibitor specificities of enzymes. Here, the activities of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) in different cell lysates have been detected by a peptide microarray-based resonance light scattering (RLS) assay with gold nanoparticle (GNP) probes. Highly sensitive detection of PKA activity in 0.1 μg total cell proteins of SHG-44 (human glioma cell) cell lysate (corresponding to 200 cells) is achieved by a selected peptide substrate. The experimental results also demonstrate that the RLS assay can be employed to evaluate the chemical regulation of intracellular kinase activity. PMID:26490469

  17. Analysis of the antimicrobial activities of a chemokine-derived peptide (CDAP-4) on Pseudomonas aeruginosa

    SciTech Connect

    Martinez-Becerra, Francisco; Dominguez-Ramirez, Lenin; Mendoza-Hernandez, Guillermo; Lopez-Vidal, Yolanda; Soldevila, Gloria . E-mail: garciaze@servidor.unam.mx

    2007-04-06

    Chemokines are key molecules involved in the control of leukocyte trafficking. Recently, a novel function as antimicrobial proteins has been described. CCL13 is the only member of the MCP chemokine subfamily displaying antimicrobial activity. To determine Key residues involved in its antimicrobial activity, CCL13 derived peptides were synthesized and tested against several bacterial strains, including Pseudomonas aeruginosa. One of these peptides, corresponding to the C-terminal region of CCL13 (CDAP-4) displayed good antimicrobial activity. Electron microscopy studies revealed remarkable morphological changes after CDAP-4 treatment. By computer modeling, CDAP-4 in {alpha} helical configuration generated a positive electrostatic potential that extended beyond the surface of the molecule. This feature is similar to other antimicrobial peptides. Altogether, these findings indicate that the antimicrobial activity was displayed by CCL13 resides to some extent at the C-terminal region. Furthermore, CDAP-4 could be considered a good antimicrobial candidate with a potential use against pathogens including P. aeruginosa.

  18. Diversity of Monomers in Nonribosomal Peptides: towards the Prediction of Origin and Biological Activity ▿ †

    PubMed Central

    Caboche, Ségolène; Leclère, Valérie; Pupin, Maude; Kucherov, Gregory; Jacques, Philippe

    2010-01-01

    Nonribosomal peptides (NRPs) are molecules produced by microorganisms that have a broad spectrum of biological activities and pharmaceutical applications (e.g., antibiotic, immunomodulating, and antitumor activities). One particularity of the NRPs is the biodiversity of their monomers, extending far beyond the 20 proteogenic amino acid residues. Norine, a comprehensive database of NRPs, allowed us to review for the first time the main characteristics of the NRPs and especially their monomer biodiversity. Our analysis highlighted a significant similarity relationship between NRPs synthesized by bacteria and those isolated from metazoa, especially from sponges, supporting the hypothesis that some NRPs isolated from sponges are actually synthesized by symbiotic bacteria rather than by the sponges themselves. A comparison of peptide monomeric compositions as a function of biological activity showed that some monomers are specific to a class of activities. An analysis of the monomer compositions of peptide products predicted from genomic information (metagenomics and high-throughput genome sequencing) or of new peptides detected by mass spectrometry analysis applied to a culture supernatant can provide indications of the origin of a peptide and/or its biological activity. PMID:20693331

  19. Frozen translational and rotational motion of human immunodeficiency virus transacting activator of transcription peptide-modified nanocargo on neutral lipid bilayer.

    PubMed

    Wei, Lin; Zhao, Xin; Chen, Bo; Li, Hongchang; Xiao, Lehui; Yeung, Edward S

    2013-05-21

    With time-resolved high-precision single-particle tracking methodologies, we explored the adsorption and thermal motion of transacting activator of transcription (TAT) peptide-modified nanocargo on a model lipid bilayer in the nonelectrostatic domain. We found that the lateral and rotational motion of TAT peptide-modified nanocargo could be effectively suppressed on the surface of neutral lipid membrane, a feature that cannot be explained by existing hypotheses. A semiquantitative association activation energy analysis revealed that multiple weak bonds were required for the initial adsorption process. As a result, the localized multiple TAT peptides on the surface of the nanocargo can provide a pathway for the creation of a net of peptide-lipid complexes (e.g., lipid domain). The dragging forces caused by these complexes effectively confined the thermal motion of the nanocargo on the fluid membrane that cannot be achieved by individual peptides with random spatial and conformational distributions. These interesting findings could provide insightful information for the better understanding of the intracellular internalization mechanism of TAT peptide-modified nanocargo and might shed new light on the development of highly efficient intracellular carriers for site-specific delivery of drugs and genes.

  20. 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.

  1. 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. PMID:25907065

  2. Cholesterol-Enriched Domain Formation Induced by Viral-Encoded, Membrane-Active Amphipathic Peptide.

    PubMed

    Hanson, Joshua M; Gettel, Douglas L; Tabaei, Seyed R; Jackman, Joshua; Kim, Min Chul; Sasaki, Darryl Y; Groves, Jay T; Liedberg, Bo; Cho, Nam-Joon; Parikh, Atul N

    2016-01-01

    The α-helical (AH) domain of the hepatitis C virus nonstructural protein NS5A, anchored at the cytoplasmic leaflet of the endoplasmic reticulum, plays a role in viral replication. However, the peptides derived from this domain also exhibit remarkably broad-spectrum virocidal activity, raising questions about their modes of membrane association. Here, using giant lipid vesicles, we show that the AH peptide discriminates between membrane compositions. In cholesterol-containing membranes, peptide binding induces microdomain formation. By contrast, cholesterol-depleted membranes undergo global softening at elevated peptide concentrations. Furthermore, in mixed populations, the presence of ∼100 nm vesicles of viral dimensions suppresses these peptide-induced perturbations in giant unilamellar vesicles, suggesting size-dependent membrane association. These synergistic composition- and size-dependent interactions explain, in part, how the AH domain might on the one hand segregate molecules needed for viral assembly and on the other hand furnish peptides that exhibit broad-spectrum virocidal activity.

  3. Peptide-Mediated PEGylation of Polysulfone Reduces Protein Adsorption and Leukocyte Activation.

    PubMed

    Davis, Elisabeth M; Platnich, Jaye M; Irvin, Randall T; Muruve, Daniel A

    2015-01-01

    The exposure of blood to bioincompatible materials used for dialysis triggers leukocyte activation and protein adsorption. We describe a single-step, postmanufacturing method for surface modification to create biomaterials used in medical devices and dialysis with altered surface characteristics. Peptides derived from the receptor-binding domain of the type IV pilin of Pseudomonas aeruginosa were synthesized using L and D-amino acids to generate L-K122-4, enantiomer D-K122-4, and D-retroinverso RI-K122-4 peptides. L-K122-4, D-K122-4, and RI-K122-4 peptides, but not control peptides, bound durably to the surfaces of materials used in medical devices and dialysis including silicone and polysulfone. D-K122-4 enantiomeric peptides were protease resistant on polysulfone and could remain bound to the surface for up to 28 days. To demonstrate that K122-4 peptides could be used to modify material surfaces, D-K122-4 peptide was conjugated to polyethylene glycol (D-K122-4-PEG) and applied to polysulfone. When compared with untreated material, D-K122-4-PEG reduced the surface adsorption of albumin or immunoglobulin G to polysulfone. In coincubation experiments, although uncoated polysulfone induced pro-interleukin-1β cytokine expression in leukocytes, cellular activation was prevented when leukocytes were incubated with D-K122-4-PEG-modified polysulfone. These data demonstrate the proof of principle that K122-4 peptides can be applied to modify the surface characteristics of materials used for dialysis. PMID:26181712

  4. Anti-biofilm and sporicidal activity of peptides based on wheat puroindoline and barley hordoindoline proteins.

    PubMed

    Shagaghi, Nadin; Alfred, Rebecca L; Clayton, Andrew H A; Palombo, Enzo A; Bhave, Mrinal

    2016-07-01

    The broad-spectrum activity of antimicrobial peptides (AMPs) and low probability of development of host resistance make them excellent candidates as novel bio-control agents. A number of AMPs are found to be cationic, and a small proportion of these are tryptophan-rich. The puroindolines (PIN) are small, basic proteins found in wheat grains with proposed roles in biotic defence of seeds and seedlings. Synthetic peptides based on their unique tryptophan-rich domain (TRD) display antimicrobial properties. Bacterial endospores and biofilms are highly resistant cells, with significant implications in both medical and food industries. In this study, the cationic PIN TRD-based peptides PuroA (FPVTWRWWKWWKG-NH2 ) and Pina-M (FSVTWRWWKWWKG-NH2 ) and the related barley hordoindoline (HIN) based Hina (FPVTWRWWTWWKG-NH2 ) were tested for effects on planktonic cells and biofilms of the common human pathogens including Pseudomonas aeruginosa, Listeria monocytogenes and the non-pathogenic Listeria innocua. All peptides showed significant bactericidal activity. Further, PuroA and Pina-M at 2 × MIC prevented initial biomass attachment by 85-90% and inhibited >90% of 6-h preformed biofilms of all three organisms. However Hina, with a substitution of Lys-9 with uncharged Thr, particularly inhibited Listeria biofilms. The PIN based peptides were also tested against vegetative cells and endospores of Bacillus subtilis. The results provided evidence that these tryptophan-rich peptides could kill B. subtilis even in sporulated state, reducing the number of viable spores by 4 log units. The treated spores appeared withered under scanning electron microscopy. The results establish the potential of these tryptophan-rich peptides in controlling persistent pathogens of relevance to food industries and human health. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27238815

  5. Development of a Peptide that Selectively Activates Protein Phosphatase-1 in Living Cells**

    PubMed Central

    Chatterjee, Jayanta; Beullens, Monique; Sukackaite, Rasa; Qian, Junbin; Lesage, Bart; Hart, Darren J; Bollen, Mathieu; Köhn, Maja

    2012-01-01

    The first cell-penetrating peptide that activates protein phosphatase-1 (PP1) by disrupting a subset of PP1 complexes in living cells has been developed. Activated PP1 rapidly dephosphorylates its substrates, counteracting kinase activity inside cells. Activation of PP1 can thus be a novel approach to study PP1 function and to counteract Ser/Thr kinase activity under pathologically increased kinase signaling. PMID:22962028

  6. Identification of peptides in human Hsp20 and Hsp27 that possess molecular chaperone and anti-apoptotic activities

    PubMed Central

    Nahomi, Rooban B.; DiMauro, Michael A.; Wang, Benlian; Nagaraj, Ram H.

    2015-01-01

    Previous studies have identified peptides in the ‘crystallin-domain’ of the small heat-shock protein (sHSP) α-crystallin with chaperone and anti-apoptotic activities. We found that peptides in heat-shock protein Hsp20 (G71HFSVLLDVKHFSPEEIAVK91) and Hsp27 (D93RWRVSLDVNHFAPDELTVK113) with sequence homology to α-crystallin also have robust chaperone and anti-apoptotic activities. Both peptides inhibited hyperthermic and chemically induced aggregation of client proteins. The scrambled peptides of Hsp20 and Hsp27 showed no such effects. The chaperone activities of the peptides were better than those from αA- and αB-crystallin. HeLa cells took up the FITC-conjugated Hsp20 peptide and, when the cells were thermally stressed, the peptide was translocated from the cytoplasm to the nucleus. The two peptides inhibited apoptosis in HeLa cells by blocking cytochrome c release from the mitochondria and caspase-3 activation. We found that scrambling the last four amino acids in the two peptides (KAIV in Hsp20 and KTLV in Hsp27) made them unable to enter cells and ineffective against stress-induced apoptosis. Intraperitoneal injection of the peptides prevented sodium-selenite-induced cataract formation in rats by inhibiting protein aggregation and oxidative stress. Our study has identified peptides from Hsp20 and Hsp27 that may have therapeutic benefit in diseases where protein aggregation and apoptosis are contributing factors. PMID:25332102

  7. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.; Stringer, C.D.; Milanez, S.; Lee, E.H.

    1985-01-01

    Previous affinity labeling studies and comparative sequence analyses have identified two different lysines at the active site of ribulosebisphosphate carboxylase/oxygenase and have suggested their essentiality to function. The essential lysines occupy positions 166 and 329 in the Rhodospirillum rubrum enzyme and positions 175 and 334 in the spinach enzyme. Based on the pH-dependencies of inactivations of the two enzymes by trinitrobenzene sulfonate, Lys-166 (R. rubrum enzyme) exhibits a pK/sub a/ of 7.9 and Lys-334 (spinach enzyme) exhibits a pK/sub a/ of 9.0. These low pK/sub a/ values as well as the enhanced nucleophilicities of the lysyl residues argue that both are important to catalysis rather than to substrate binding. Lys-166 may correspond to the essential base that initiates catalysis and that displays a pK/sub a/ of 7.5 in the pH-curve for V/sub max//K/sub m/. Cross-linking experiments with 4,4'-diisothiocyano-2,2'-disulfonate stilbene demonstrate that the two active-site lysines are within 12 A. 50 refs., 7 figs., 1 tab.

  8. Bactericidal activity of tracheal antimicrobial peptide against respiratory pathogens of cattle.

    PubMed

    Taha-Abdelaziz, Khaled; Perez-Casal, José; Schott, Courtney; Hsiao, Jason; Attah-Poku, Samuel; Slavić, Durđa; Caswell, Jeff L

    2013-04-15

    Tracheal antimicrobial peptide (TAP) is a β-defensin produced by mucosal epithelial cells of cattle. Although effective against several human pathogens, the activity of this bovine peptide against the bacterial pathogens that cause bovine respiratory disease have not been reported. This study compared the antibacterial effects of synthetic TAP against Mannheimia haemolytica, Histophilus somni, Pasteurella multocida, and Mycoplasma bovis. Bactericidal activity against M. bovis was not detected. In contrast, the Pasteurellaceae bacteria showed similar levels of susceptibility to that of Escherichia coli, with 0.125μg TAP inhibiting growth in a radial diffusion assay and minimum inhibitory concentrations of 1.56-6.25μg/ml in a bactericidal assay. Significant differences among isolates were not observed. Sequencing of exon 2 of the TAP gene from 23 cattle revealed a prevalent non-synonymous single nucleotide polymorphism (SNP) A137G, encoding either serine or asparagine at residue 20 of the mature peptide. The functional effect of this SNP was tested against M. haemolytica using synthetic peptides. The bactericidal effect of the asparagine-containing peptide was consistently higher than the serine-containing peptide. Bactericidal activities were similar for an acapsular mutant of M. haemolytica compared to the wild type. These findings indicate that the Pasteurellaceae bacteria that cause bovine respiratory disease are susceptible to killing by bovine TAP and appear not to have evolved resistance, whereas M. bovis appears to be resistant. A non-synonymous SNP was identified in the coding region of the TAP gene, and the corresponding peptides vary in their bactericidal activity against M. haemolytica.

  9. Physical Activity, Blood Glucose and C-Peptide in Healthy School-Children, a Longitudinal Study

    PubMed Central

    Ludvigsson, Johnny

    2016-01-01

    Aim To further elucidate the relationship between physical activity and several risk factors for development of diabetes (glucose, C-peptide and obesity) over time. Methods A prospective longitudinal study where physical activity was measured on 199 children from Kalmar and Linköping at age 8, and the same 107 children from Linköping again at age 12. Anthropometric data was collected and blood was analyzed for C-peptide and f-glucose. The children in the study were representative for the general Swedish child population, and on an average lean. Results High physical activity was related to lower C-peptide at age 8 and 12. This correlation was especially pronounced in boys, who also were more physically active than girls at both time points. The association seen at 8 years of age was similar at age 12 in most children. Children with higher BMI Z-Score had a higher fasting C-peptide (age 12) but linear regression showed that children with more steps per day were less likely to have a higher fasting C-peptide irrespective of BMI. Longitudinal follow-up showed that a decrease in physical activity increased insulin resistance and β-cell load. Conclusions Already in young children, physical activity improves insulin sensitivity and decreases the need of C-peptide over time. This seems to become even more pronounced with increasing age when children are followed longitudinally. Low physical activity increases the load on insulin producing β-cells, might increase the risk for both type 1- and 2 diabetes. PMID:27270732

  10. Molecular Design, Structural Analysis and Antifungal Activity of Derivatives of Peptide CGA-N46.

    PubMed

    Li, Rui-Fang; Lu, Zhi-Fang; Sun, Ya-Nan; Chen, Shi-Hua; Yi, Yan-Jie; Zhang, Hui-Ru; Yang, Shuo-Ye; Yu, Guang-Hai; Huang, Liang; Li, Chao-Nan

    2016-09-01

    Chromogranin A (CGA)-N46, a derived peptide of human chromogranin A, has antifungal activity. To further research the active domain of CGA-N46, a series of derivatives were designed by successively deleting amino acid from both terminus of CGA-N46, and the amino acid sequence of each derivative was analyzed by bioinformatic software. Based on the predicted physicochemical properties of the peptides, including half-life time in mammalian reticulocytes (in vitro), yeast (in vivo) and E. coli (in vivo), instability index, aliphatic index and grand average of hydropathicity (GRAVY), the secondary structure, net charge, the distribution of hydrophobic residues and hydrophilic residues, the final derivatives CGA-N15, CGA-N16, CGA-N12 and CGA-N8 were synthesized by solid-phase peptide synthesis. The results of bioinformatic analysis showed that CGA-N46 and its derivatives were α-helix, neutral or weak positive charge, hydrophilic, and CGA-N12 and CGA-N8 were more stable than the other derivatives. The results of circular dichroism confirmed that CGA-N46 and its derived peptides displayed α-helical structure in an aqueous solution and 30 mM sodium dodecylsulfate, but α-helical contents decreased in hydrophobic lipid vesicles. CGA-N15, CGA-N16, CGA-N12 and CGA-N8 had higher antifungal activities than their mother peptide CGA-N46. Among of the derived peptides, CGA-N12 showed the least hemolytic activity. In conclusion, we have successfully identified the active domain of CGA-N46 with strong antifungal activity and weak hemolytic activity, which provides the possibility to develop a new class of antibiotics.

  11. Tuning Liposome Membrane Permeability by Competitive Peptide Dimerization and Partitioning-Folding Interactions Regulated by Proteolytic Activity.

    PubMed

    Lim, Seng Koon; Sandén, Camilla; Selegård, Robert; Liedberg, Bo; Aili, Daniel

    2016-01-01

    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.

  12. 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.

  13. Tuning Liposome Membrane Permeability by Competitive Peptide Dimerization and Partitioning-Folding Interactions Regulated by Proteolytic Activity

    PubMed Central

    Lim, Seng Koon; Sandén, Camilla; Selegård, Robert; Liedberg, Bo; Aili, Daniel

    2016-01-01

    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. PMID:26892926

  14. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  15. Modulation of Backbone Flexibility for Effective Dissociation of Antibacterial and Hemolytic Activity in Cyclic Peptides.

    PubMed

    Oddo, Alberto; Thomsen, Thomas T; Britt, Hannah M; Løbner-Olesen, Anders; Thulstrup, Peter W; Sanderson, John M; Hansen, Paul R

    2016-08-11

    Bacterial resistance to antibiotic therapy is on the rise and threatens to evolve into a worldwide emergency: alternative solutions to current therapies are urgently needed. Cationic amphipathic peptides are potent membrane-active agents that hold promise as the next-generation therapy for multidrug-resistant infections. The peptides' behavior upon encountering the bacterial cell wall is crucial, and much effort has been dedicated to the investigation and optimization of this amphipathicity-driven interaction. In this study we examined the interaction of a novel series of nine-membered flexible cyclic AMPs with liposomes mimicking the characteristics of bacterial membranes. Employed techniques included circular dichroism and marker release assays, as well as microbiological experiments. Our analysis was aimed at correlating ring flexibility with their antimicrobial, hemolytic, and membrane activity. By doing so, we obtained useful insights to guide the optimization of cyclic antimicrobial peptides via modulation of their backbone flexibility without loss of activity. PMID:27563396

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

    SciTech Connect

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

    1980-10-10

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

  17. Identification of protein O-GlcNAcylation sites using electron transfer dissociation mass spectrometry on native peptides.

    PubMed

    Chalkley, Robert J; Thalhammer, Agnes; Schoepfer, Ralf; Burlingame, A L

    2009-06-01

    Protein O-GlcNAcylation occurs in all animals and plants and is implicated in modulation of a wide range of cytosolic and nuclear protein functions, including gene silencing, nutrient and stress sensing, phosphorylation signaling, and diseases such as diabetes and Alzheimer's. The limiting factor impeding rapid progress in deciphering the biological functions of protein O-GlcNAcylation has been the inability to easily identify exact residues of modification. We describe a robust, high-sensitivity strategy able to assign O-GlcNAcylation sites of native modified peptides using electron transfer dissociation mass spectrometry. We have studied the murine postsynaptic density pseudoorganelle and report the assignment of 58 modification sites from a single experiment--significantly increasing the number of sites known in the literature. Components of several repressor complexes, such as NCoR1, polyhomeotic-like protein3, and EMSY, are modified. In addition, 28 O-GlcNAc sites were found on the protein Bassoon, effectively matching the number of phosphorylation sites reported previously on this protein. This finding suggests that on certain proteins, O-GlcNAcylation may be as extensive and important as phosphorylation in regulating protein function. Three of the newly discovered O-GlcNAc sites on Bassoon have previously been reported as phosphorylation sites, highlighting the interplay of the modifications. Surprisingly, several peptides with GlcNAc modifications on asparagines within the N-X-S/T consensus sequence were also observed from membrane protein extracellular domains. This powerful strategy fulfills a long-standing need in the biological community by facilitating modification site identifications that will accelerate understanding of the biological significance of this elusive regulatory posttranslational modification.

  18. Antibacterial activities of peptides from the water-soluble extracts of Italian cheese varieties.

    PubMed

    Rizzello, C G; Losito, I; Gobbetti, M; Carbonara, T; De Bari, M D; Zambonin, P G

    2005-07-01

    Water-soluble extracts of 9 Italian cheese varieties that differed mainly for type of cheese milk, starter, technology, and time of ripening were fractionated by reversed-phase fast protein liquid chromatography, and the antimicrobial activity of each fraction was first assayed toward Lactobacillus sakei A15 by well-diffusion assay. Active fractions were further analyzed by HPLC coupled to electrospray ionization-ion trap mass spectrometry, and peptide sequences were identified by comparison with a proteomic database. Parmigiano Reggiano, Fossa, and Gorgonzola water-soluble extracts did not show antibacterial peptides. Fractions of Pecorino Romano, Canestrato Pugliese, Crescenza, and Caprino del Piemonte contained a mixture of peptides with a high degree of homology. Pasta filata cheeses (Caciocavallo and Mozzarella) also had antibacterial peptides. Peptides showed high levels of homology with N-terminal, C-terminal, or whole fragments of well known antimicrobial or multifunctional peptides reported in the literature: alphaS1-casokinin (e.g., sheep alphaS1-casein (CN) f22-30 of Pecorino Romano and cow alphaS1-CN f24-33 of Canestrato Pugliese); isracidin (e.g., sheep alphaS1-CN f10-21 of Pecorino Romano); kappacin and casoplatelin (e.g., cow kappa-CN f106-115 of Canestrato Pugliese and Crescenza); and beta-casomorphin-11 (e.g., goat beta-CN f60-68 of Caprino del Piemonte). As shown by the broth microdilution technique, most of the water-soluble fractions had a large spectrum of inhibition (minimal inhibitory concentration of 20 to 200 microg/mL) toward gram-positive and gram-negative bacterial species, including potentially pathogenic bacteria of clinical interest. Cheeses manufactured from different types of cheese milk (cow, sheep, and goat) have the potential to generate similar peptides with antimicrobial activity.

  19. Scolopendin 2, a cationic antimicrobial peptide from centipede, and its membrane-active mechanism.

    PubMed

    Lee, Heejeong; Hwang, Jae-Sam; Lee, Jaeho; Kim, Jae Il; Lee, Dong Gun

    2015-02-01

    Scolopendin 2 is a 16-mer peptide (AGLQFPVGRIGRLLRK) derived from the centipede Scolopendra subspinipes mutilans. We observed that this peptide exhibited antimicrobial activity in a salt-dependent manner against various fungal and bacterial pathogens and showed no hemolytic effect in the range of 1.6 μM to 100 μM. Circular dichroism analysis showed that the peptide has an α-helical properties. Furthermore, we determined the mechanism(s) of action using flow cytometry and by investigating the release of intracellular potassium. The results showed that the peptide permeabilized the membranes of Escherichia coli O157 and Candida albicans, resulting in loss of intracellular potassium ions. Additionally, bis-(1,3-dibutylbarbituric acid) trimethine oxonol and 3,3'-dipropylthiacarbocyanine iodide assays showed that the peptide caused membrane depolarization. Using giant unilamellar vesicles encapsulating calcein and large unilamellar vesicles containing fluorescein isothiocyanate-dextran, which were similar in composition to typical E. coli O157 and C. albicans membranes, we demonstrated that scolopendin 2 disrupts membranes, resulting in a pore size between 4.8 nm and 5.0 nm. Thus, we have demonstrated that a cationic antimicrobial peptide, scolopendin 2, exerts its broad-spectrum antimicrobial effects by forming pores in the cell membrane.

  20. Differential roles of the two-component peptides of lactocin 705 in antimicrobial activity.

    PubMed

    Cuozzo, Sergio A; Castellano, Patricia; Sesma, Fernando J M; Vignolo, Graciela M; Raya, Raul R

    2003-03-01

    Lactobacillus casei CRL705 produces a class IIb bacteriocin, lactocin 705, which relies on the complementary action of two components, Lac705alpha and Lac705beta. These peptides exert a bactericidal effect on the indicator strain Lactobacillus plantarum CRL691, with an optimal Lac705alpha/Lac705beta peptide ratio of 1 to 4. Electron microscopy studies showed that treated CRL691 cells have their cell wall severely damaged, with mesosome-like membranous formations protruding into their cytoplasm. Although less pronounced, a similar effect was also observed with the Lac705beta peptide alone. Furthermore, Lac705beta increased the inhibitory action of a diluted supernatant of L. casei CRL705, while Lac705alpha protected CRL691 cells from inhibition. Both peptides were required to dissipate the proton motive force (Deltapsi and DeltapH) of CRL691 cells. These data suggested that of the two components of lactocin 705, the Lac705alpha peptide is responsible for receptor recognition, and the Lac705beta peptide is the active component on the cell membrane of CRL691 cells. PMID:12567240

  1. Scolopendin 2, a cationic antimicrobial peptide from centipede, and its membrane-active mechanism.

    PubMed

    Lee, Heejeong; Hwang, Jae-Sam; Lee, Jaeho; Kim, Jae Il; Lee, Dong Gun

    2015-02-01

    Scolopendin 2 is a 16-mer peptide (AGLQFPVGRIGRLLRK) derived from the centipede Scolopendra subspinipes mutilans. We observed that this peptide exhibited antimicrobial activity in a salt-dependent manner against various fungal and bacterial pathogens and showed no hemolytic effect in the range of 1.6 μM to 100 μM. Circular dichroism analysis showed that the peptide has an α-helical properties. Furthermore, we determined the mechanism(s) of action using flow cytometry and by investigating the release of intracellular potassium. The results showed that the peptide permeabilized the membranes of Escherichia coli O157 and Candida albicans, resulting in loss of intracellular potassium ions. Additionally, bis-(1,3-dibutylbarbituric acid) trimethine oxonol and 3,3'-dipropylthiacarbocyanine iodide assays showed that the peptide caused membrane depolarization. Using giant unilamellar vesicles encapsulating calcein and large unilamellar vesicles containing fluorescein isothiocyanate-dextran, which were similar in composition to typical E. coli O157 and C. albicans membranes, we demonstrated that scolopendin 2 disrupts membranes, resulting in a pore size between 4.8 nm and 5.0 nm. Thus, we have demonstrated that a cationic antimicrobial peptide, scolopendin 2, exerts its broad-spectrum antimicrobial effects by forming pores in the cell membrane. PMID:25462167

  2. Exploration of structure-activity relationships at the two C-terminal residues of potent 11mer Glucagon-Like Peptide-1 receptor agonist peptides via parallel synthesis.

    PubMed

    Haque, Tasir S; Martinez, Rogelio L; Lee, Ving G; Riexinger, Douglas G; Lei, Ming; Feng, Ming; Koplowitz, Barry; Mapelli, Claudio; Cooper, Christopher B; Zhang, Ge; Huang, Christine; Ewing, William R; Krupinski, John

    2010-07-01

    We report the identification of potent agonists of the Glucagon-Like Peptide-1 receptor (GLP-1R) via evaluation of two positional scanning libraries and a two-dimensional focused library, synthesized in part on SynPhase Lanterns. These compounds are 11 amino acid peptides containing several unnatural amino acids, including (in particular) analogs of biphenylalanine (Bip) at the two C-terminal positions. Typical activities of the most potent peptides in this class are in the picomolar range in an in vitro functional assay using human GLP-1 receptor.

  3. Machine Learning Assisted Design of Highly Active Peptides for Drug Discovery

    PubMed Central

    Giguère, Sébastien; Laviolette, François; Marchand, Mario; Tremblay, Denise; Moineau, Sylvain; Liang, Xinxia; Biron, Éric; Corbeil, Jacques

    2015-01-01

    The discovery of peptides possessing high biological activity is very challenging due to the enormous diversity for which only a minority have the desired properties. To lower cost and reduce the time to obtain promising peptides, machine learning approaches can greatly assist in the process and even partly replace expensive laboratory experiments by learning a predictor with existing data or with a smaller amount of data generation. Unfortunately, once the model is learned, selecting peptides having the greatest predicted bioactivity often requires a prohibitive amount of computational time. For this combinatorial problem, heuristics and stochastic optimization methods are not guaranteed to find adequate solutions. We focused on recent advances in kernel methods and machine learning to learn a predictive model with proven success. For this type of model, we propose an efficient algorithm based on graph theory, that is guaranteed to find the peptides for which the model predicts maximal bioactivity. We also present a second algorithm capable of sorting the peptides of maximal bioactivity. Extensive analyses demonstrate how these algorithms can be part of an iterative combinatorial chemistry procedure to speed up the discovery and the validation of peptide leads. Moreover, the proposed approach does not require the use of known ligands for the target protein since it can leverage recent multi-target machine learning predictors where ligands for similar targets can serve as initial training data. Finally, we validated the proposed approach in vitro with the discovery of new cationic antimicrobial peptides. Source code freely available at http://graal.ift.ulaval.ca/peptide-design/. PMID:25849257

  4. Machine learning assisted design of highly active peptides for drug discovery.

    PubMed

    Giguère, Sébastien; Laviolette, François; Marchand, Mario; Tremblay, Denise; Moineau, Sylvain; Liang, Xinxia; Biron, Éric; Corbeil, Jacques

    2015-04-01

    The discovery of peptides possessing high biological activity is very challenging due to the enormous diversity for which only a minority have the desired properties. To lower cost and reduce the time to obtain promising peptides, machine learning approaches can greatly assist in the process and even partly replace expensive laboratory experiments by learning a predictor with existing data or with a smaller amount of data generation. Unfortunately, once the model is learned, selecting peptides having the greatest predicted bioactivity often requires a prohibitive amount of computational time. For this combinatorial problem, heuristics and stochastic optimization methods are not guaranteed to find adequate solutions. We focused on recent advances in kernel methods and machine learning to learn a predictive model with proven success. For this type of model, we propose an efficient algorithm based on graph theory, that is guaranteed to find the peptides for which the model predicts maximal bioactivity. We also present a second algorithm capable of sorting the peptides of maximal bioactivity. Extensive analyses demonstrate how these algorithms can be part of an iterative combinatorial chemistry procedure to speed up the discovery and the validation of peptide leads. Moreover, the proposed approach does not require the use of known ligands for the target protein since it can leverage recent multi-target machine learning predictors where ligands for similar targets can serve as initial training data. Finally, we validated the proposed approach in vitro with the discovery of new cationic antimicrobial peptides. Source code freely available at http://graal.ift.ulaval.ca/peptide-design/.

  5. Magnetic nanoparticles enhance the anticancer activity of cathelicidin LL-37 peptide against colon cancer cells

    PubMed Central

    Niemirowicz, Katarzyna; Prokop, Izabela; Wilczewska, Agnieszka Z; Wnorowska, Urszula; Piktel, Ewelina; Wątek, Marzena; Savage, Paul B; Bucki, Robert

    2015-01-01

    The pleiotropic activity of human cathelicidin LL-37 peptide includes an ability to suppress development of colon cancer cells. We hypothesized that the anticancer activity of LL-37 would improve when attached to the surface of magnetic nanoparticles (MNPs). Using colon cancer culture (DLD-1 cells and HT-29 cells), we evaluated the effects of MNPs, LL-37 peptide, its synthetic analog ceragenin CSA-13, and two novel nanosystems, ie, MNP@LL-37 and MNP@CSA-13, on cancer cell viability and apoptosis. Treatment of cancer cells with the LL-37 peptide linked to MNPs (MNP@LL-37) caused a greater decrease in cell viability and a higher rate of apoptosis compared with treatment using free LL-37 peptide. Additionally, we observed a strong ability of ceragenin CSA-13 and MNP@CSA-13 to induce apoptosis of DLD-1 cells. We found that both nanosystems were successfully internalized by HT-29 cells, and cathelicidin LL-37 and ceragenin CSA-13 might play a key role as novel homing molecules. These results indicate that the previously described anticancer activity of LL-37 peptide against colon cancer cells might be significantly improved using a theranostic approach. PMID:26082634

  6. Airway Epithelial Cells are the Site of Expression of a Mammalian Antimicrobial Peptide Gene

    NASA Astrophysics Data System (ADS)

    Diamond, Gill; Jones, Douglas E.; Bevins, Charles L.

    1993-05-01

    We previously reported the isolation and characterization of a broad-spectrum antimicrobial peptide from the bovine tracheal mucosa, which we called tracheal antimicrobial peptide (TAP). We now show the TAP gene is expressed throughout the adult conducting airway, from nasal to bronchiolar tissue, but not in tissues other than airway mucosa, as determined by Northern blot analysis. In situ hybridization of airway sections localizes TAP mRNA to columnar cells of the pseudostratified epithelium. We report the structural organization of the TAP gene and show that TAP is a member of a large family of related sequences with high nucleotide identity in the 5'exon. The data support the hypothesis that antimicrobial peptides contribute to host defense of the respiratory tract.

  7. Trichoplaxin - a new membrane-active antimicrobial peptide from placozoan cDNA.

    PubMed

    Simunić, Juraj; Petrov, Dražen; Bouceba, Tahar; Kamech, Nédia; Benincasa, Monica; Juretić, Davor

    2014-05-01

    A method based on the use of signal peptide sequences from antimicrobial peptide (AMP) precursors was used to mine a placozoa expressed sequence tag database and identified a potential antimicrobial peptide from Trichoplax adhaerens. This peptide, with predicted sequence FFGRLKSVWSAVKHGWKAAKSR is the first AMP from a placozoan species, and was named trichoplaxin. It was chemically synthesized and its structural properties, biological activities and membrane selectivity were investigated. It adopts an α-helical structure in contact with membrane-like environments and is active against both Gram-negative and Gram-positive bacterial species (including MRSA), as well as yeasts from the Candida genus. The cytotoxic activity, as assessed by the haemolytic activity against rat erythrocytes, U937 cell permeabilization to propidium iodide and MCF7 cell mitochondrial activity, is significantly lower than the antimicrobial activity. In tests with membrane models, trichoplaxin shows high affinity for anionic prokaryote-like membranes with good fit in kinetic studies. Conversely, there is a low affinity for neutral eukaryote-like membranes and absence of a dose dependent response. With high selectivity for bacterial cells and no homologous sequence in the UniProt, trichoplaxin is a new potential lead compound for development of broad-spectrum antibacterial drugs.

  8. Activatable iRGD-based peptide monolith: Targeting, internalization, and fluorescence activation for precise tumor imaging.

    PubMed

    Cho, Hong-Jun; Lee, Sung-Jin; Park, Sung-Jun; Paik, Chang H; Lee, Sang-Myung; Kim, Sehoon; Lee, Yoon-Sik

    2016-09-10

    A disulfide-bridged cyclic RGD peptide, named iRGD (internalizing RGD, c(CRGDK/RGPD/EC)), is known to facilitate tumor targeting as well as tissue penetration. After the RGD motif-induced targeting on αv integrins expressed near tumor tissue, iRGD encounters proteolytic cleavage to expose the CendR motif that promotes penetration into cancer cells via the interaction with neuropilin-1. Based on these proteolytic cleavage and internalization mechanism, we designed an iRGD-based monolithic imaging probe that integrates multiple functions (cancer-specific targeting, internalization and fluorescence activation) within a small peptide framework. To provide the capability of activatable fluorescence signaling, we conjugated a fluorescent dye to the N-terminal of iRGD, which was linked to the internalizing sequence (CendR motif), and a quencher to the opposite C-terminal. It turned out that fluorescence activation of the dye/quencher-conjugated monolithic peptide probe requires dual (reductive and proteolytic) cleavages on both disulfide and amide bond of iRGD peptide. Furthermore, the cleavage of the iRGD peptide leading to fluorescence recovery was indeed operative depending on the tumor-related angiogenic receptors (αvβ3 integrin and neuropilin-1) in vitro as well as in vivo. Compared to an 'always fluorescent' iRGD control probe without quencher conjugation, the dye/quencher-conjugated activatable monolithic peptide probe visualized tumor regions more precisely with lower background noise after intravenous injection, owing to the multifunctional responses specific to tumor microenvironment. All these results, along with minimal in vitro and in vivo toxicity profiles, suggest potential of the iRGD-based activatable monolithic peptide probe as a promising imaging agent for precise tumor diagnosis. PMID:27349354

  9. Activatable iRGD-based peptide monolith: Targeting, internalization, and fluorescence activation for precise tumor imaging.

    PubMed

    Cho, Hong-Jun; Lee, Sung-Jin; Park, Sung-Jun; Paik, Chang H; Lee, Sang-Myung; Kim, Sehoon; Lee, Yoon-Sik

    2016-09-10

    A disulfide-bridged cyclic RGD peptide, named iRGD (internalizing RGD, c(CRGDK/RGPD/EC)), is known to facilitate tumor targeting as well as tissue penetration. After the RGD motif-induced targeting on αv integrins expressed near tumor tissue, iRGD encounters proteolytic cleavage to expose the CendR motif that promotes penetration into cancer cells via the interaction with neuropilin-1. Based on these proteolytic cleavage and internalization mechanism, we designed an iRGD-based monolithic imaging probe that integrates multiple functions (cancer-specific targeting, internalization and fluorescence activation) within a small peptide framework. To provide the capability of activatable fluorescence signaling, we conjugated a fluorescent dye to the N-terminal of iRGD, which was linked to the internalizing sequence (CendR motif), and a quencher to the opposite C-terminal. It turned out that fluorescence activation of the dye/quencher-conjugated monolithic peptide probe requires dual (reductive and proteolytic) cleavages on both disulfide and amide bond of iRGD peptide. Furthermore, the cleavage of the iRGD peptide leading to fluorescence recovery was indeed operative depending on the tumor-related angiogenic receptors (αvβ3 integrin and neuropilin-1) in vitro as well as in vivo. Compared to an 'always fluorescent' iRGD control probe without quencher conjugation, the dye/quencher-conjugated activatable monolithic peptide probe visualized tumor regions more precisely with lower background noise after intravenous injection, owing to the multifunctional responses specific to tumor microenvironment. All these results, along with minimal in vitro and in vivo toxicity profiles, suggest potential of the iRGD-based activatable monolithic peptide probe as a promising imaging agent for precise tumor diagnosis.

  10. Purification of labeled cyanogen bromide peptides of the alpha polypeptide from sodium ion and potassium ion activated adenosinetriphosphatase modified with N-(/sup 3/H)ethylmaleimide

    SciTech Connect

    Le, D.T.

    1986-05-06

    Sodium ion and potassium ion activated adenosinetriphosphatase, isolated from canine kidney, was reacted with N-(/sup 3/H)ethylmaleimide while it was poised in three different conformations, ostensibly E2-P, E2, and E1, respectively. These assignments were made from a consideration of the particular concentrations of ligands in the respective alkylation mixtures. After a 30-min reaction, the remaining enzymatic activity was found to vary among these three different samples from 90 to 30% of that of unalkylated controls. In all cases, the alpha polypeptide was purified and subjected to digestion with cyanogen bromide, and in each digest the same two distinct radioactive peptides were identified and purified by gel filtration on a column of Sephadex LH-60. The incorporation of N-(/sup 3/H)ethylmaleimide into one of these two peptides correlated closely with enzymatic inactivation, while the incorporation into the other was most extensive when the portion of the active site to which ATP binds was unoccupied. Alkylation of the residue within the latter peptide, however, does not result in inactivation of the enzyme. Both peptides were further purified by high-pressure liquid chromatography, and their amino-terminal sequences were determined by manual dansyl Edman or solid-phase techniques. The peptide containing the sulfhydryl protected by ATP has, as its amino terminus, the lysine that reacts exclusively with fluoresceinyl 5'-isothiocyanate.

  11. Active immunizations with peptide-DC vaccines and passive transfer with antibodies protect neutropenic mice against disseminated candidiasis.

    PubMed

    Xin, Hong

    2016-01-01

    We previously report that peptide-pulsed dendritic cell (DC) vaccination, which targeting two peptides (Fba and Met6) expressed on the cell surface of Candida albicans, can induce high degree of protection against disseminated candidiasis in immunocompetent mice. Passive transfer of immune sera from the peptide immunized mice or peptide-related monoclonal antibodies demonstrated that protection was medicated by peptide-specific antibodies. In this study the efficacy of active and passive immunization against disseminated candidiasis was tested in mice with cyclophosphamide-induced neutropenia. Peptide-DC vaccines were given to mice prior to induction of neutropenia. We show active immunization with either Fba or Met6 peptide-DC vaccine significantly improved the survival and reduced the fungal burden of disseminated candidiasis in those immunocompromised mice. Importantly, we show that administration of two protective monoclonal antibodies also protect neutropenic mice against the disease, implying possibility of developing a successful passive immunotherapy strategy to treat the disease and protect against disseminated candidiasis. The results of this study are crucial as they address the fundamental questions as to whether the synthetic peptide vaccine induced immunity protects the host during a neutropenic episode. We anticipate that this peptide-vaccine study will serve as the foundation of future investigations into new peptide vaccines comprised of cell surface peptides from other medically important Candida species, as well as other fungi.

  12. Design, recombinant expression, and antibacterial activity of the cecropins-melittin hybrid antimicrobial peptides.

    PubMed

    Cao, Yu; Yu, Rong Qing; Liu, Yi; Zhou, Huo Xiang; Song, Ling Ling; Cao, Yi; Qiao, Dai Rong

    2010-09-01

    In order to evaluate their antibacterial activities and toxicities, the cecropins-melittin hybrid antimicrobial peptide, CA(1-7)-M(4-11) (CAM) and CB(1-7)-M(4-11) (CBM), were designed by APD2 database. The recombinant hybrid antimicrobial peptides were successfully expressed and purified in Pichia pastoris. Antimicrobial activity assay showed that both of the two hybrid antimicrobial peptides had strong antibacterial abilities against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis, Bacillus thuringiensis, and Salmonella derby. The potency of CAM and CBM to E. coli 25922 were 0.862 and 0.849, respectively, slightly lower than Amp's 0.957. The hemolytic assays indicated CAM and CBM had no hemolytic in vivo and in vitro, and so they had a good application prospect. PMID:20111863

  13. Peptides from cowpea present antioxidant activity, inhibit cholesterol synthesis and its solubilisation into micelles.

    PubMed

    Marques, Marcelo Rodrigues; Soares Freitas, Rosana Aparecida Manólio; Corrêa Carlos, Amanda Caroline; Siguemoto, Érica Sayuri; Fontanari, Gustavo Guadagnucci; Arêas, José Alfredo Gomes

    2015-02-01

    In previous studies, it was reported that the protein isolated from the cowpea interferes favourably in lipid metabolism, and reduces cholesterol synthesis. The present study investigated the role of cowpea peptide fractions in the micellar solubilisation of cholesterol, in the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) activity, and in the in vitro antioxidant capacity, considering the effects of thermal processing. The protein was isolated from the raw and cooked beans and digested to simulate human digestion. The peptides from the protein isolate of raw bean with molecular mass lower than 3kDa reduced 89% of the HMGCR enzymatic reaction velocity. The cooked cowpeas were more effective in inhibiting the micellar solubility of cholesterol than the raw ones but not the antioxidant activity. This is the first report that cowpea peptides inhibit cholesterol homeostasis in vitro in two distinct routes, and act as an antioxidant.

  14. Glycine-extended anglerfish peptide YG (aPY) a neuropeptide Y (NPY) homologue may be a precursor of a biologically active peptide.

    PubMed

    Balasubramaniam, A; Andrews, P C; Renugopalakrishnan, V; Rigel, D F

    1989-01-01

    The 37 residue peptide YG (aPY), isolated from anglerfish endocrine pancreas, bears distinct sequence homology to the pancreatic polypeptide family of hormones. However, instead of a carboxyl-terminal tyrosine-amide, aPY has a free carboxyl-terminus ending with glycine. Towards studying the structure-activity relationship of this hormone, we have synthesized aPY by solid phase methodology using Boc-amino acid derivatives and phenylacetamidomethyl resin. The crude peptide was purified to homogeneity in 20% yield by reversed phase chromatography. The purified peptide had the expected amino acid composition and sequence, and was found to be identical with the natural aPY by analytical HPLC and peptide mapping of proteolytic digests. Neither the snythetic nor the natural aPY exhibited the characteristic vasoconstrictor activity of the related pancreatic polypeptide family of hormones. However, [Des37-Gly]-aPY, isolated from the anglerfish pancreas, caused vasoconstriction in rats. Based on these results and by analogy to the glycine-extended gastrin peptides, it may be suggested that aPY is a precursor of a biologically active peptide, namely [Des37-Gly]-aPY-amide. PMID:2780417

  15. Glycine-extended anglerfish peptide YG (aPY) a neuropeptide Y (NPY) homologue may be a precursor of a biologically active peptide.

    PubMed

    Balasubramaniam, A; Andrews, P C; Renugopalakrishnan, V; Rigel, D F

    1989-01-01

    The 37 residue peptide YG (aPY), isolated from anglerfish endocrine pancreas, bears distinct sequence homology to the pancreatic polypeptide family of hormones. However, instead of a carboxyl-terminal tyrosine-amide, aPY has a free carboxyl-terminus ending with glycine. Towards studying the structure-activity relationship of this hormone, we have synthesized aPY by solid phase methodology using Boc-amino acid derivatives and phenylacetamidomethyl resin. The crude peptide was purified to homogeneity in 20% yield by reversed phase chromatography. The purified peptide had the expected amino acid composition and sequence, and was found to be identical with the natural aPY by analytical HPLC and peptide mapping of proteolytic digests. Neither the snythetic nor the natural aPY exhibited the characteristic vasoconstrictor activity of the related pancreatic polypeptide family of hormones. However, [Des37-Gly]-aPY, isolated from the anglerfish pancreas, caused vasoconstriction in rats. Based on these results and by analogy to the glycine-extended gastrin peptides, it may be suggested that aPY is a precursor of a biologically active peptide, namely [Des37-Gly]-aPY-amide.

  16. Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors.

    PubMed

    Swaney, Danielle L; McAlister, Graeme C; Wirtala, Matthew; Schwartz, Jae C; Syka, John E P; Coon, Joshua J

    2007-01-15

    Electron-transfer dissociation (ETD) delivers the unique attributes of electron capture dissociation to mass spectrometers that utilize radio frequency trapping-type devices (e.g., quadrupole ion traps). The method has generated significant interest because of its compatibility with chromatography and its ability to: (1) preserve traditionally labile post-translational modifications (PTMs) and (2) randomly cleave the backbone bonds of highly charged peptide and protein precursor ions. ETD, however, has shown limited applicability to doubly protonated peptide precursors, [M + 2H]2+, the charge and type of peptide most frequently encountered in "bottom-up" proteomics. Here we describe a supplemental collisional activation (CAD) method that targets the nondissociated (intact) electron-transfer (ET) product species ([M + 2H]+*) to improve ETD efficiency for doubly protonated peptides (ETcaD). A systematic study of supplementary activation conditions revealed that low-energy CAD of the ET product population leads to the near-exclusive generation of c- and z-type fragment ions with relatively high efficiency (77 +/- 8%). Compared to those formed directly via ETD, the fragment ions were found to comprise increased relative amounts of the odd-electron c-type ions (c+*) and the even-electron z-type ions (z+). A large-scale analysis of 755 doubly charged tryptic peptides was conducted to compare the method (ETcaD) to ion trap CAD and ETD. ETcaD produced a median sequence coverage of 89%-a significant improvement over ETD (63%) and ion trap CAD (77%).

  17. Production of Biologically Active Cecropin A Peptide in Rice Seed Oil Bodies

    PubMed Central

    Izquierdo, Esther; Campo, Sonia; Badosa, Esther; Rossignol, Michel; Montesinos, Emilio; San Segundo, Blanca; Coca, María

    2016-01-01

    Cecropin A is a natural antimicrobial peptide that exhibits fast and potent activity against a broad spectrum of pathogens and neoplastic cells, and that has important biotechnological applications. However, cecropin A exploitation, as for other antimicrobial peptides, is limited by their production and purification costs. Here, we report the efficient production of this bioactive peptide in rice bran using the rice oleosin 18 as a carrier protein. High cecropin A levels were reached in rice seeds driving the expression of the chimeric gene by the strong embryo-specific oleosin 18 own promoter, and targeting the peptide to the oil body organelle as an oleosin 18-cecropin A fusion protein. The accumulation of cecropin A in oil bodies had no deleterious effects on seed viability and seedling growth, as well as on seed yield. We also show that biologically active cecropin A can be easily purified from the transgenic rice seeds by homogenization and simple flotation centrifugation methods. Our results demonstrate that the oleosin fusion technology is suitable for the production of cecropin A in rice seeds, which can potentially be extended to other antimicrobial peptides to assist their exploitation. PMID:26760761

  18. Production of Biologically Active Cecropin A Peptide in Rice Seed Oil Bodies.

    PubMed

    Montesinos, Laura; Bundó, Mireia; Izquierdo, Esther; Campo, Sonia; Badosa, Esther; Rossignol, Michel; Montesinos, Emilio; San Segundo, Blanca; Coca, María

    2016-01-01

    Cecropin A is a natural antimicrobial peptide that exhibits fast and potent activity against a broad spectrum of pathogens and neoplastic cells, and that has important biotechnological applications. However, cecropin A exploitation, as for other antimicrobial peptides, is limited by their production and purification costs. Here, we report the efficient production of this bioactive peptide in rice bran using the rice oleosin 18 as a carrier protein. High cecropin A levels were reached in rice seeds driving the expression of the chimeric gene by the strong embryo-specific oleosin 18 own promoter, and targeting the peptide to the oil body organelle as an oleosin 18-cecropin A fusion protein. The accumulation of cecropin A in oil bodies had no deleterious effects on seed viability and seedling growth, as well as on seed yield. We also show that biologically active cecropin A can be easily purified from the transgenic rice seeds by homogenization and simple flotation centrifugation methods. Our results demonstrate that the oleosin fusion technology is suitable for the production of cecropin A in rice seeds, which can potentially be extended to other antimicrobial peptides to assist their exploitation. PMID:26760761

  19. Determining the Binding Sites of β-Cyclodextrin and Peptides by Electron-Capture Dissociation High Resolution Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Qi, Yulin; Geib, Timon; Volmer, Dietrich A.

    2015-07-01

    Cyclodextrins (CDs) are a group of cyclic oligosaccharides, which readily form inclusion complexes with hydrophobic compounds to increase bioavailability, thus making CDs ideal drug excipients. Recent studies have also shown that CDs exhibit a wide range of protective effects, preventing proteins from aggregation, degradation, and folding. These effects strongly depend on the binding sites on the protein surface. CDs only exhibit weak interactions with amino acids, however; conventional analytical techniques therefore usually fail to reveal the exact location of the binding sites. Moreover, some studies even suggest that CD inclusion complexes are merely electrostatic adducts. Here, electron capture dissociation (ECD) was applied in this proof-of-concept study to examine the exact nature of the CD/peptide complexes, and CD binding sites were unambiguously located for the first time via Fourier-transform ion cyclotron resonance (FTICR) tandem mass spectrometry.

  20. Tuberoinfundibular peptide of 39 residues (TIP39): molecular structure and activity for parathyroid hormone 2 receptor.

    PubMed

    Della Penna, K; Kinose, F; Sun, H; Koblan, K S; Wang, H

    2003-01-01

    The neuropeptide TIP39 was recently purified from bovine hypothalamus based on the ability of the peptide to activate the parathyroid hormone 2 receptor (PTH2R) ( Nat. Neurosci. 2 (1999) 941). PTH2R is abundantly expressed in the nervous system, and its expression pattern suggests that it may play a role in modulation of pituitary function and in nociception. Towards understanding the physiological role of TIP39 and PTH2R, we cloned human, mouse and rat TIP39 gene. Our results revealed that: (1) the mature peptide is processed from a precursor; (2) TIP39 peptide is highly conserved among species; and (3) TIP39 from all species activates adenylyl cyclase and elevates intracellular calcium levels through PTH2R. We also defined and compared the structure-activity relationship of TIP39 on both activation of adenylyl cyclase and calcium mobilization pathways through PTH2R, finding common and differential determinants of TIP39 that are required for these pathways. Furthermore, we observed that TIP39 elevates intracellular calcium levels in primary dorsal root ganglion neurons whereas the peptide inactive on PTH2R do not, suggesting that TIP39 may activate these neurons important for nociception in vivo through PTH2R-dependent mechanisms.

  1. Chicken cathelicidin-2-derived peptides with enhanced immunomodulatory and antibacterial activities against biological warfare agents.

    PubMed

    Molhoek, E Margo; van Dijk, Albert; Veldhuizen, Edwin J A; Dijk-Knijnenburg, Helma; Mars-Groenendijk, Roos H; Boele, Linda C L; Kaman-van Zanten, Wendy E; Haagsman, Henk P; Bikker, Floris J

    2010-09-01

    Host defence peptides (HDPs) are considered to be excellent candidates for the development of novel therapeutic agents. Recently, it was demonstrated that the peptide C1-15, an N-terminal segment of chicken HDP cathelicidin-2, exhibits potent antibacterial activity while lacking cytotoxicity towards eukaryotic cells. In the present study, we report that C1-15 is active against bacteria such as Bacillus anthracis and Yersinia pestis that may potentially be used by bioterrorists. Substitution of single and multiple phenylalanine (Phe) residues to tryptophan (Trp) in C1-15 resulted in variants with improved antibacterial activity against B. anthracis and Y. pestis as well as decreased salt sensitivity. In addition, these peptides exhibited enhanced neutralisation of lipopolysaccharide (LPS)-induced release of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs). The antibacterial and LPS-neutralising activities of these C1-15-derived peptides are exerted at concentrations far below the concentrations that are toxic to human PBMCs. Taken together, we show that Phe-->Trp substitutions in C1-15 variants enhances the antibacterial and LPS-neutralising activities against pathogenic bacteria, including those that may potentially be used as biological warfare agents.

  2. Kinetically Tunable Photostability of Fluorogen-Activating Peptide-Fluorogen Complexes.

    PubMed

    Saurabh, Saumya; Zhang, Ming; Mann, Victor R; Costello, Andrea M; Bruchez, Marcel P

    2015-10-01

    Ease of genetic encoding, labeling specificity, and high photostability are the most sought after qualities in a fluorophore for biological detection. Furthermore, many applications can gain from the fluorogenic nature of fluoromodules and the ability to turn on the same fluoromodules multiple times. Fluorogen-activating peptides (FAPs) bind noncovalently to their cognate fluorogens and exhibit enhanced photostability. Herein, the photostabilities of malachite green (MG)-binding and thiazole-orange-binding FAPs are compared under limiting- and excess-fluorogen conditions to establish distinct mechanisms for photostability that correspond to the dissociation rate of the FAP-fluorogen complex. FAPs with slow dissociation show evidence of dye encapsulation and protection from photo or environmental degradation and single-step bleaching at the single molecule level, whereas those with rapid dissociation show repeated cycles of binding and enhanced photostability by exchange of bleached fluorogen with a new dye. A combination of generalizable selection pressure based on bleaching, flow cytometry, and site-specific amino acid mutagenesis is used to obtain a modified FAP with enhanced photostability, due to rapid dissociation of the MG fluorogen. These studies shed light on the basic mechanisms by which noncovalent association can effect photostable labeling, and demonstrate novel reagents for photostable and intermittent labeling of biological targets. PMID:26310607

  3. Enzyme Design From the Bottom Up: An Active Nickel Electrocatalyst with a Structured Peptide Outer Coordination Sphere

    SciTech Connect

    Reback, Matthew L.; Buchko, Garry W.; Kier, Brandon L.; Ginovska-Pangovska, Bojana; Xiong, Yijia; Lense, Sheri; Hou, Jianbo; Roberts, John A.; Sorensen, Christina M.; Raugei, Simone; Squier, Thomas C.; Shaw, Wendy J.

    2014-02-03

    Functional, peptide-containing metal complexes with a well-defined peptide structure have the potential to enhance molecular catalysts via an enzyme-like outer coordination sphere. Here, we report the synthesis and characterization of an active, peptide-based metal complex built upon the well characterized hydrogen production catalyst, Ni(PPh2NPh)2. The incorporated peptide maintains its B-hairpin structure when appended to the metal core, and the electrocatalytic activity of the peptide-based metal complex (~100,000 s-1) is fully retained. The combination of an active molecular catalyst with a structured peptide outer coordination sphere provides a scaffold that permits the incorporation of features of an enzyme-like outer-coordination sphere necessary to create molecular electrocatalysts with en-hanced functionality.

  4. Alkyl isocyanates as active site-directed inactivators of guinea pig liver transglutaminase.

    PubMed

    Gross, M; Whetzel, N K; Folk, J E

    1975-10-10

    Alkyl isocyanates are effective inactivators of guinea pig liver transglutaminase. Based on the specificity of the reaction the protection against inactivation by glutamine substrate, and the essential nature of calcium for the inactivation reaction, it is concluded that these reagents act as amide substrate analogs and, thus function in an active site-specific manner. Support for the contention that inactivation results from alkyl thiocarbamate ester formation through the single active site sulfhydryl group of the enzyme is (a) the loss of one free--SH group and the incorporation of 1 mol of reagent/mol of enzyme in the reaction, (b) similarity in chemical properties of the inactive enzyme derivative formed to those previously reported for another alkyl thiocarbamoylenzyme and an alkyl thiocarbamoylcysteine derivative, and (c) the finding that labeled peptides from digests of [methyl-14C]thiocarbamoyltransglutaminase and those from digests of iodoacetamide-inactivated enzyme occupy similar positions on peptide maps. Transglutaminase was found to be inactivated neither by urethan anlogs of its active ester substrates nor by urea analogs of its amide substrates. It is concluded on the basis of these findings that inactive carbamoylenzyme derivatives are formed only by direct addition of the transglutaminase active--SH group to the isocyanate C--N double bond, and not, like several serine active site enzymes, by nucleophilic displacement with urethan analogs of substrate, or by nucleophilic displacement with urea analogs of substrate. PMID:240837

  5. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase*

    PubMed Central

    Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W.

    2016-01-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. PMID:26893379

  6. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase.

    PubMed

    Kalamajski, Sebastian; Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W

    2016-04-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.

  7. Minimal Determinants for Binding Activated G alpha from the Structure of a G alpha i1-Peptide Dimer

    SciTech Connect

    Johnston,C.; Lobanova, E.; Shavkunov, A.; Low, J.; Ramer, J.; Blasesius, R.; Fredericks, Z.; willard, F.; Kuhlman, B.; et al.

    2006-01-01

    G-Proteins cycle between an inactive GDP-bound state and an active GTP-bound state, serving as molecular switches that coordinate cellular signaling. We recently used phage display to identify a series of peptides that bind G{alpha}subunits in a nucleotide-dependent manner [Johnston, C. A., Willard, F. S., Jezyk, M. R., Fredericks, Z., Bodor, E. T., Jones, M. B., Blaesius, R., Watts, V. J., Harden, T. K., Sondek, J., Ramer, J. K., and Siderovski, D. P. (2005) Structure 13, 1069-1080]. Here we describe the structural features and functions of KB-1753, a peptide that binds selectively to GDP{center_dot}AlF{sub 4{sup -}}- and GTP{gamma}S-bound states of G{alpha}{sup i} subunits. KB-1753 blocks interaction of G{alpha}{sub transducin} with its effector, cGMP phosphodiesterase, and inhibits transducin-mediated activation of cGMP degradation. Additionally, KB-1753 interferes with RGS protein binding and resultant GAP activity. A fluorescent KB-1753 variant was found to act as a sensor for activated G{alpha} in vitro. The crystal structure of KB-1753 bound to G{alpha}{sub i1}-GDP{center_dot}AlF{sub 4{sup -}} reveals binding to a conserved hydrophobic groove between switch II and 3 helices and, along with supporting biochemical data and previous structural analyses, supports the notion that this is the site of effector interactions for G{alpha}i subunits.

  8. Histone peptide AKRHRK enhances H{sub 2}O{sub 2}-induced DNA damage and alters its site specificity

    SciTech Connect

    Midorikawa, Kaoru; Murata, Mariko; Kawanishi, Shosuke . E-mail: kawanisi@doc.medic.mie-u.ac.jp

    2005-08-12

    Histone proteins are involved in compaction of DNA and the protection of cells from oxygen toxicity. However, several studies have demonstrated that the metal-binding histone reacts with H{sub 2}O{sub 2}, leading to oxidative damage to a nucleobase. We investigated whether histone can accelerate oxidative DNA damage, using a minimal model for the N-terminal tail of histone H4, CH{sub 3}CO-AKRHRK-CONH{sub 2}, which has a metal-binding site. This histone peptide enhanced DNA damage induced by H{sub 2}O{sub 2} and Cu(II), especially at cytosine residues, and induced additional DNA cleavage at the 5'-guanine of GGG sequences. The peptide also enhanced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and ESR spin-trapping signal from H{sub 2}O{sub 2} and Cu(II). Cyclic redox reactions involving histone-bound Cu(II) and H{sub 2}O{sub 2}, may give rise to multiple production of radicals leading to multiple hits in DNA. It is noteworthy that the histone H4 peptide with specific sequence AKRHRK can cause DNA damage rather than protection under metal-overloaded condition.

  9. Acyl lipidation of a peptide: effects on activity and epidermal permeability in vitro

    PubMed Central

    Rocco, Daniel; Ross, James; Murray, Paul E; Caccetta, Rima

    2016-01-01

    Short-chain lipid conjugates can increase permeability of a small peptide across human epidermis; however, the emerging lipoaminoacid (LAA) conjugation technique is costly and can deliver mixed synthetic products of varied biological potential. LAA conjugation using a racemic mixture produces a mixture of D- and L-stereoisomers. Individual enantiomers can be produced at an extra cost. We investigated an affordable technique that produces only one synthetic product: short-chain (C7–C8) acyl lipidation. Acyl lipidation of Ala-Ala-Pro-Val, an inhibitor of human neutrophil elastase (HNE; believed to lead to abnormal tissue destruction and disease development), was investigated as an alternative to LAA conjugation. The current study aimed to assess the effects of acyl lipidation (either at the N-terminal or at the C-terminal) on neutrophil elastase activity in vitro and on transdermal delivery ex vivo. The inhibitory capacity of the acyl conjugates was compared to LAA conjugates (conjugated at the N-terminal) of the same peptide. The L-stereoisomer appears to rapidly degrade, but it represents a significantly (P<0.05) better inhibitor of HNE than the parent peptide (Ala-Ala-Pro-Val). Although the D-stereoisomer appears to permeate human epidermal skin sections in a better fashion than the L-stereoisomer, it is not a significantly better inhibitor of HNE than the parent peptide. Acyl lipidation (with a C7 lipid chain) at either end of the peptide substantially enhances the permeability of the peptide across human skin epidermis as well as significantly (P<0.005) increases its elastase inhibitory potential. Therefore, our current study indicates that acyl lipidation of a peptide is a more economical and effective alternative to LAA conjugation. PMID:27468224

  10. Acyl lipidation of a peptide: effects on activity and epidermal permeability in vitro.

    PubMed

    Rocco, Daniel; Ross, James; Murray, Paul E; Caccetta, Rima

    2016-01-01

    Short-chain lipid conjugates can increase permeability of a small peptide across human epidermis; however, the emerging lipoaminoacid (LAA) conjugation technique is costly and can deliver mixed synthetic products of varied biological potential. LAA conjugation using a racemic mixture produces a mixture of D- and L-stereoisomers. Individual enantiomers can be produced at an extra cost. We investigated an affordable technique that produces only one synthetic product: short-chain (C7-C8) acyl lipidation. Acyl lipidation of Ala-Ala-Pro-Val, an inhibitor of human neutrophil elastase (HNE; believed to lead to abnormal tissue destruction and disease development), was investigated as an alternative to LAA conjugation. The current study aimed to assess the effects of acyl lipidation (either at the N-terminal or at the C-terminal) on neutrophil elastase activity in vitro and on transdermal delivery ex vivo. The inhibitory capacity of the acyl conjugates was compared to LAA conjugates (conjugated at the N-terminal) of the same peptide. The L-stereoisomer appears to rapidly degrade, but it represents a significantly (P<0.05) better inhibitor of HNE than the parent peptide (Ala-Ala-Pro-Val). Although the D-stereoisomer appears to permeate human epidermal skin sections in a better fashion than the L-stereoisomer, it is not a significantly better inhibitor of HNE than the parent peptide. Acyl lipidation (with a C7 lipid chain) at either end of the peptide substantially enhances the permeability of the peptide across human skin epidermis as well as significantly (P<0.005) increases its elastase inhibitory potential. Therefore, our current study indicates that acyl lipidation of a peptide is a more economical and effective alternative to LAA conjugation. PMID:27468224

  11. Antiviral activity of a Bacillus sp. P34 peptide against pathogenic viruses of domestic animals

    PubMed Central

    Silva, Débora Scopel e; de Castro, Clarissa Caetano; Silva, Fábio da Silva e; Sant’anna, Voltaire; Vargas, Gilberto D’Avila; de Lima, Marcelo; Fischer, Geferson; Brandelli, Adriano; da Motta, Amanda de Souza; Hübner, Silvia de Oliveira

    2014-01-01

    P34 is an antimicrobial peptide produced by a Bacillus sp. strain isolated from the intestinal contents of a fish in the Brazilian Amazon basin with reported antibacterial activity. The aim of this work was to evaluate the peptide P34 for its in vitro antiviral properties against canine adenovirus type 2 (CAV-2), canine coronavirus (CCoV), canine distemper virus (CDV), canine parvovirus type 2 (CPV-2), equine arteritis virus (EAV), equine influenza virus (EIV), feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1). The results showed that the peptide P34 exhibited antiviral activity against EAV and FHV-1. The peptide P34 inhibited the replication of EAV by 99.9% and FHV-1 by 94.4%. Virucidal activity was detected only against EAV. When P34 and EAV were incubated for 6 h at 37 °C the viral titer reduced from 104.5 TCID50 to 102.75 TCID50, showing a percent of inhibition of 98.6%. In conclusion, our results demonstrated that P34 inhibited EAV and FHV-1 replication in infected cell cultures and it showed virucidal activity against EAV. Since there is documented resistance to the current drugs used against herpesviruses and there is no treatment for equine viral arteritis, it is advisable to search for new antiviral compounds to overcome these infections. PMID:25477947

  12. Preparation, characterization, and activity of peptide-cellulosic aerogel protease sensor from cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nanocellulosic aerogels (NA) provide a lightweight biocompatible material with structural properties of both high porosity and specific surface area for biosensor design. We report here the preparation, characterization, and activity of a peptide-nanocellulose aerogel (PA) made from unprocessed cot...

  13. The promotion of antimicrobial activity on silicon substrates using a "click" immobilized short peptide.

    PubMed

    Wang, Lin; Chen, Junjian; Shi, Lin; Shi, Zhifeng; Ren, Li; Wang, Yingjun

    2014-01-28

    We demonstrated, for the first time, that the short antimicrobial peptide Tet213 could be conjugated onto the silicon surface by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The modified surface exhibited excellent antimicrobial activity against S. aureus and E. coli, and low cytotoxicity to rat bone mesenchymal stem cells (rBMSCs).

  14. Membrane Thinning and Thickening Induced by Membrane-Active Amphipathic Peptides.

    PubMed

    Grage, Stephan L; Afonin, Sergii; Kara, Sezgin; Buth, Gernot; Ulrich, Anne S

    2016-01-01

    Membrane thinning has been discussed as a fundamental mechanism by which antimicrobial peptides can perturb cellular membranes. To understand which factors play a role in this process, we compared several amphipathic peptides with different structures, sizes and functions in their influence on the lipid bilayer thickness. PGLa and magainin 2 from X. laevis were studied as typical representatives of antimicrobial cationic amphipathic α-helices. A 1:1 mixture of these peptides, which is known to possess synergistically enhanced activity, allowed us to evaluate whether and how this synergistic interaction correlates with changes in membrane thickness. Other systems investigated here include the α-helical stress-response peptide TisB from E. coli (which forms membrane-spanning dimers), as well as gramicidin S from A. migulanus (a natural antibiotic), and BP100 (designer-made antimicrobial and cell penetrating peptide). The latter two are very short, with a circular β-pleated and a compact α-helical structure, respectively. Solid-state (2)H-NMR and grazing incidence small angle X-ray scattering (GISAXS) on oriented phospholipid bilayers were used as complementary techniques to access the hydrophobic thickness as well as the bilayer-bilayer repeat distance including the water layer in between. This way, we found that magainin 2, gramicidin S, and BP100 induced membrane thinning, as expected for amphiphilic peptides residing in the polar/apolar interface of the bilayer. PGLa, on the other hand, decreased the hydrophobic thickness only at very high peptide:lipid ratios, and did not change the bilayer-bilayer repeat distance. TisB even caused an increase in the hydrophobic thickness and repeat distance. When reconstituted as a mixture, PGLa and magainin 2 showed a moderate thinning effect which was less than that of magainin 2 alone, hence their synergistically enhanced activity does not seem to correlate with a modulation of membrane thickness. Overall, the absence of

  15. Peptide inhibition of constitutively activated epithelial Na(+) channels expressed in Xenopus oocytes.

    PubMed

    Ji, H L; Fuller, C M; Benos, D J

    1999-12-31

    The hypothesis that 30-amino acid peptides corresponding to the C-terminal portion of the beta- and/or gamma-rat epithelial sodium channel (rENaC) subunits block constitutively activated ENaC was tested by examining the effects of these peptides on wild-type (wt) rENaC (alphabetagamma-rENaC), truncated Liddle's mutants (alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC), and point mutants (alphabeta(Y)gamma-, alphabetagamma(Y)-rENaC) expressed in Xenopus oocytes. The chord conductances of alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC were 2- or 3-fold greater than for wt alphabetagamma-rENaC. Introduction of peptides into oocytes expressing alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC produced a concentration-dependent inhibition of the amiloride-sensitive Na(+) conductances, with apparent dissociation constants (K(d)) ranging from 1700 to 160 microM, depending upon whether individual peptides or their combination was used. Injection of peptides alone or in combination into oocytes expressing wt alphabetagamma-rENaC or single-point mutants did not affect the amiloride-sensitive whole-cell currents. The single channel conductances of all the mutant ENaCs were the same as that of wild type (alphabetagamma-). The single channel activities (N.P(o)) of the mutants were approximately 2.2-2.6-fold greater than wt alphabetagamma-rENaC (1.08 +/- 0.24, n = 7) and were reduced to 1.09 +/- 0.17 by 100 microM peptide mixture (n = 9). The peptides were without effect on the single channel properties of either wt or single-point mutants of rENaC. Our data demonstrate that the C-terminal peptides blocked the Liddle's truncation mutant (alphabeta(T)gamma(T)) expressed in Xenopus oocytes but not the single-point mutants (alphabeta(Y)gamma or alphabetagamma(Y)). Moreover, the blocking effect of both peptides in combination on alphabeta(T)gamma(T)-rENaC was synergistic. PMID:10608827

  16. Membrane Thinning and Thickening Induced by Membrane-Active Amphipathic Peptides

    PubMed Central

    Grage, Stephan L.; Afonin, Sergii; Kara, Sezgin; Buth, Gernot; Ulrich, Anne S.

    2016-01-01

    Membrane thinning has been discussed as a fundamental mechanism by which antimicrobial peptides can perturb cellular membranes. To understand which factors play a role in this process, we compared several amphipathic peptides with different structures, sizes and functions in their influence on the lipid bilayer thickness. PGLa and magainin 2 from X. laevis were studied as typical representatives of antimicrobial cationic amphipathic α-helices. A 1:1 mixture of these peptides, which is known to possess synergistically enhanced activity, allowed us to evaluate whether and how this synergistic interaction correlates with changes in membrane thickness. Other systems investigated here include the α-helical stress-response peptide TisB from E. coli (which forms membrane-spanning dimers), as well as gramicidin S from A. migulanus (a natural antibiotic), and BP100 (designer-made antimicrobial and cell penetrating peptide). The latter two are very short, with a circular β-pleated and a compact α-helical structure, respectively. Solid-state 2H-NMR and grazing incidence small angle X-ray scattering (GISAXS) on oriented phospholipid bilayers were used as complementary techniques to access the hydrophobic thickness as well as the bilayer-bilayer repeat distance including the water layer in between. This way, we found that magainin 2, gramicidin S, and BP100 induced membrane thinning, as expected for amphiphilic peptides residing in the polar/apolar interface of the bilayer. PGLa, on the other hand, decreased the hydrophobic thickness only at very high peptide:lipid ratios, and did not change the bilayer-bilayer repeat distance. TisB even caused an increase in the hydrophobic thickness and repeat distance. When reconstituted as a mixture, PGLa and magainin 2 showed a moderate thinning effect which was less than that of magainin 2 alone, hence their synergistically enhanced activity does not seem to correlate with a modulation of membrane thickness. Overall, the absence of a

  17. β-Boomerang Antimicrobial and Antiendotoxic Peptides: Lipidation and Disulfide Bond Effects on Activity and Structure

    PubMed Central

    Mohanram, Harini; Bhattacharjya, Surajit

    2014-01-01

    Drug-resistant Gram-negative bacterial pathogens and endotoxin- or lipopolysaccharide (LPS)-mediated inflammations are among some of the most prominent health issues globally. Antimicrobial peptides (AMPs) are eminent molecules that can kill drug-resistant strains and neutralize LPS toxicity. LPS, the outer layer of the outer membrane of Gram-negative bacteria safeguards cell integrity against hydrophobic compounds, including antibiotics and AMPs. Apart from maintaining structural integrity, LPS, when released into the blood stream, also induces inflammatory pathways leading to septic shock. In previous works, we have reported the de novo design of a set of 12-amino acid long cationic/hydrophobic peptides for LPS binding and activity. These peptides adopt β-boomerang like conformations in complex with LPS. Structure-activity studies demonstrated some critical features of the β-boomerang scaffold that may be utilized for the further development of potent analogs. In this work, β-boomerang lipopeptides were designed and structure-activity correlation studies were carried out. These lipopeptides were homo-dimerized through a disulfide bridge to stabilize conformations and for improved activity. The designed peptides exhibited potent antibacterial activity and efficiently neutralized LPS toxicity under in vitro assays. NMR structure of C4YI13C in aqueous solution demonstrated the conserved folding of the lipopeptide with a boomerang aromatic lock stabilized with disulfide bond at the C-terminus and acylation at the N-terminus. These lipo-peptides displaying bacterial sterilization and low hemolytic activity may be useful for future applications as antimicrobial and antiendotoxin molecules. PMID:24756162

  18. Enthalpy-driven nuclease-like activity and mechanism of peptide-chlorambucil conjugates.

    PubMed

    Yang, Robin C K; Huang, Jonathan T B; Chen, Yu-Ling; Hung, Chia-Chun; Liao, Mokai; Yao, Wen-Chen; Chen, Chiu-Heng; Liou, Chien-Chung; Waring, Michael J; Sheh, Leung

    2014-07-21

    We report the results of attaching the anticancer drug chlorambucil (CLB) to two high-affinity DNA binding peptides: Met-Hyp-Arg-Lys-(Py)4-Lys-Arg-NH2 (HyM-10) and Gln-Hyp-Arg-Lys-(Py)4-Lys-Arg-NH2 (HyQ-10). These CLB-peptide conjugates cleave DNA very effectively and sequence-selectively without the use of chemicals, heat, or UV irradiation. Polyacrylamide gel electrophoresis identifies the sites where CLB-HyM-10 and CLB-HyQ-10 attack a complementary pair of 5'-(32)P-labeled duplexes derived from pBR322 in the absence of piperidine or other chemical additives. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has confirmed the preferential cleavage sites as well as a novel stepwise cleavage mechanism of sequence-selective DNA cleavage. Resembling restriction endonucleases, the CLB-peptide conjugates appear to be capable of producing double strand DNA breaks. Circular dichroism studies show that CLB-HyM-10 and CLB-HyQ-10 induce significant local conformational changes in DNA via the minor groove, possibly with dimeric binding stoichiometry. The energetic basis of DNA binding by these conjugates has been investigated by isothermal titration calorimetry, revealing that the binding of both the peptides and their CLB conjugates is overwhelmingly enthalpy-driven. The maintenance of a conserved negative binding free energy in DNA-conjugate interactions is a crucial feature of the universal enthalpy-entropy compensation phenomenon. The strongly enthalpy-driven binding of CLB-peptide conjugates to preferred loci in DNA furnishes the required proximity effect to generate the observed nuclease-like sequence-selective cleavage.

  19. Proteins, peptides, polysaccharides, and nucleotides with inhibitory activity on human immunodeficiency virus and its enzymes.

    PubMed

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

    2015-12-01

    Human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome, has claimed innumerable lives in the past. Many biomolecules which suppress HIV replication and also other biomolecules that inhibit enzymes essential to HIV replication have been reported. Proteins including a variety of milk proteins, ribosome-inactivating proteins, ribonucleases, antifungal proteins, and trypsin inhibitors; peptides comprising cathelicidins, defensins, synthetic peptides, and others; polysaccharides and polysaccharopeptides; nucleosides, nucleotides, and ribozymes, demonstrated anti-HIV activity. In many cases, the mechanism of anti-HIV action has been elucidated. Strategies have been devised to augment the anti-HIV potency of these compounds.

  20. Design of Peptide and Peptidomimetic Ligands with Novel Pharmacological Activity Profiles

    PubMed Central

    Hruby, Victor J.; Cai, Minying

    2016-01-01

    Peptide hormones and neurotransmitters are of central importance in most aspects of intercellular communication and are involved in virtually all degenerative diseases. In this review, we discuss physicochemical approaches to the design of novel peptide and peptidomimetic agonists, antagonists, inverse agonists, and related compounds that have unique biological activity profiles, reduced toxic side effects, and, if desired, the ability to cross the blood-brain barrier. Designing ligands for specific biological and medical needs is emphasized, as is the close collaboration of chemists and biologists to maximize the chances for success. Special emphasis is placed on the use of conformational (φ-ψ space) and topographical (χ space) considerations in design. PMID:23294313

  1. Active Hydrogenation Catalyst with a Structured, Peptide-Based Outer-Coordination Sphere

    SciTech Connect

    Jain, Avijita; Buchko, Garry W.; Reback, Matthew L.; O'Hagan, Molly J.; Ginovska-Pangovska, Bojana; Linehan, John C.; Shaw, Wendy J.

    2012-10-05

    The synthesis, catalytic activity, and structural features of a rhodium-based hydrogenation catalyst containing a phosphine ligand coupled to a 14-residue peptide are reported. Both CD and NMR spectroscopy show that the peptide adopts a helical structure in 1:1:1 TFE/MeCN/H2O that is maintained when the peptide is attached to the ligand and when the ligand is attached to the metal complex. The metal complex hydrogenates aqueous solutions of 3-butenol to 1-butanol at 360 ± 50 turnovers/Rh/h at 294 K. This peptide- based catalyst represents a starting point for developing and characterizing a peptide-based outer-coordination sphere that can be used to introduce enzyme-like features into molecular catalysts. This work was funded by the US DOE Basic Energy Sciences, Chemical Sciences, Geoscience and Biosciences Division (AJ, JCL and WJS), the Office of Science Early Career Research Program through the Office of Basic Energy Sciences (GWB, MLR and WJS). Part of the research was conducted at the W.R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by U.S. Department of Energy’s Office of Biolog-ical and Environmental Research (BER) program located at Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battelle for the U.S. Department of Energy.

  2. Interactions of a designed peptide with lipopolysaccharide: Bound conformation and anti-endotoxic activity

    SciTech Connect

    Bhunia, Anirban; Chua, Geok Lin; Domadia, Prerna N.; Warshakoon, Hemamali; Cromer, Jens R.; David, Sunil A.; Bhattacharjya, Surajit

    2008-05-09

    Designed peptides that would selectively interact with lipopolysaccharide (LPS) or endotoxin and fold into specific conformations could serve as important scaffolds toward the development of antisepsis compounds. Here, we describe solution structure of a designed amphipathic peptide, H{sub 2}N-YVKLWRMIKFIR-CONH{sub 2} (YW12D) in complex with endotoxin as determined by transferred nuclear Overhauser effect spectroscopy. The conformation of the isolated peptide is highly flexible, but undergoes a dramatic structural stabilization in the presence of LPS. Structure calculations reveal that the peptide presents two amphipathic surfaces in its bound state to LPS whereby each surface is characterized by two positive charges and a number of aromatic and/or aliphatic residues. ITC data suggests that peptide interacts with two molecules of lipid A. In activity assays, YW12D exhibits neutralization of LPS toxicity with very little hemolysis of red blood cells. Structural and functional properties of YW12D would be applicable in designing low molecular weight non-toxic antisepsis molecules.

  3. Isolation and characterization of two antigenically active peptides from bovine β-lactoglobulin-A

    PubMed Central

    Bing, D. H.; Stavitsky, A. B.

    1968-01-01

    Bovine β-lactoglobulin-A was hydrolysed with trypsin to yield a mixture of peptides which would not precipitate with rabbit antibody against the native protein, but would still inhibit the antigen—antibody reaction. The hydrolysate was fractionated by ion exchange chromatography and found to contain seven antigenically active fractions. Two of these fractions were found to be homogeneous peptides. Each inhibited the reaction of antigen with antibody against the intact protein in haemagglutination, flocculation and passive cutaneous anaphylaxis reaction in guinea-pigs. One of the peptides had a molecular weight of 950 and the other had a molecular weight of 575. Both contained about equal numbers of polar and apolar amino acids. PMID:5673287

  4. Contribution of Amphipathicity and Hydrophobicity to the Antimicrobial Activity and Cytotoxicity of β-Hairpin Peptides

    PubMed Central

    2016-01-01

    Bacteria have acquired extensive resistance mechanisms to protect themselves against antibiotic action. Today the bacterial membrane has become one of the “final frontiers” in the search for new compounds acting on novel targets to address the threat of multi-drug resistant (MDR) and XDR bacterial pathogens. β-Hairpin antimicrobial peptides are amphipathic, membrane-binding antibiotics that exhibit a broad range of activities against Gram-positive, Gram-negative, and fungal pathogens. However, most members of the class also possess adverse cytotoxicity and hemolytic activity that preclude their development as candidate antimicrobials. We examined peptide hydrophobicity, amphipathicity, and structure to better dissect and understand the correlation between antimicrobial activity and toxicity, membrane binding, and membrane permeability. The hydrophobicity, pI, net charge at physiological pH, and amphipathic moment for the β-hairpin antimicrobial peptides tachyplesin-1, polyphemusin-1, protegrin-1, gomesin, arenicin-3, and thanatin were determined and correlated with key antimicrobial activity and toxicity data. These included antimicrobial activity against five key bacterial pathogens and two fungi, cytotoxicity against human cell lines, and hemolytic activity in human erythrocytes. Observed antimicrobial activity trends correlated with compound amphipathicity and, to a lesser extent, with overall hydrophobicity. Antimicrobial activity increased with amphipathicity, but unfortunately so did toxicity. Of note, tachyplesin-1 was found to be 8-fold more amphipathic than gomesin. These analyses identify tachyplesin-1 as a promising scaffold for rational design and synthetic optimization toward an antibiotic candidate. PMID:27331141

  5. Antiviral cationic peptides as a strategy for innovation in global health therapeutics for dengue virus: high yield production of the biologically active recombinant plectasin peptide.

    PubMed

    Rothan, Hussin A; Mohamed, Zulqarnain; Suhaeb, Abdulrazzaq M; Rahman, Noorsaadah Abd; Yusof, Rohana

    2013-11-01

    Dengue virus infects millions of people worldwide, and there is no vaccine or anti-dengue therapeutic available. Antimicrobial peptides have been shown to possess effective antiviral activity against various viruses. One of the main limitations of developing these peptides as potent antiviral drugs is the high cost of production. In this study, high yield production of biologically active plectasin peptide was inexpensively achieved by producing tandem plectasin peptides as inclusion bodies in E. coli. Antiviral activity of the recombinant peptide towards dengue serotype-2 NS2B-NS3 protease (DENV2 NS2B-NS3pro) was assessed as a target to inhibit dengue virus replication in Vero cells. Single units of recombinant plectasin were collected after applying consecutive steps of refolding, cleaving by Factor Xa, and nickel column purification to obtain recombinant proteins of high purity. The maximal nontoxic dose (MNTD) of the recombinant peptide against Vero cells was 20 μM (100 μg/mL). The reaction velocity of DENV2 NS2B-NS3pro decreased significantly after increasing concentrations of recombinant plectasin were applied to the reaction mixture. Plectasin peptide noncompetitively inhibited DENV2 NS2B-NS3pro at Ki value of 5.03 ± 0.98 μM. The percentage of viral inhibition was more than 80% at the MNTD value of plectasin. In this study, biologically active recombinant plectasin which was able to inhibit dengue protease and viral replication in Vero cells was successfully produced in E. coli in a time- and cost- effective method. These findings are potentially important in the development of potent therapeutics against dengue infection.

  6. Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids

    PubMed Central

    Kim, Ki-Hyun; Nielsen, Peter E.; Glazer, Peter M.

    2007-01-01

    Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable to form pcPNA–pcPNA duplexes but can bind to complementary DNA sequences by Watson–Crick pairing via double duplex-invasion complex formation. We show that psoralen-conjugated pcPNAs can deliver site-specific photoadducts and mediate targeted gene modification within both episomal and chromosomal DNA in mammalian cells without detectable off-target effects. Most of the induced psoralen-pcPNA mutations were single-base substitutions and deletions at the predicted pcPNA-binding sites. The pcPNA-directed mutagenesis was found to be dependent on PNA concentration and UVA dose and required matched pairs of pcPNAs. Neither of the individual pcPNAs alone had any effect nor did complementary PNA pairs of the same sequence. These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules. PMID:17977869

  7. Autoantibodies against Modified Histone Peptides in SLE Patients Are Associated with Disease Activity and Lupus Nephritis

    PubMed Central

    Dieker, Jürgen; Berden, Jo H.; Bakker, Marinka; Briand, Jean-Paul; Muller, Sylviane; Voll, Reinhard; Sjöwall, Christopher; Herrmann, Martin; Hilbrands, Luuk B.; van der Vlag, Johan

    2016-01-01

    Persistent exposure of the immune system to death cell debris leads to autoantibodies against chromatin in patients with systemic lupus erythematosus (SLE). Deposition of anti-chromatin/chromatin complexes can instigate inflammation in multiple organs including the kidney. Previously we identified specific cell death-associated histone modifications as targets of autoantibodies in SLE. In this study we addressed, in a large cohort of SLE patients and controls, the question whether plasma reactivities with specific histone peptides associated with serology and clinical features. Plasma from SLE patients with and without lupus nephritis, disease controls, and healthy controls, were tested in ELISA with histone H4 peptide acetylated at lysines 8, 12 and 16 (H4pac), H2B peptide acetylated at lysine 12 (H2Bpac), H3 peptide trimethylated at lysine 27 (H3pme), and their unmodified equivalents. SLE patients displayed a higher reactivity with the modified equivalent of each peptide. Reactivity with H4pac showed both a high sensitivity (89%) and specificity (91%) for SLE, while H2Bpac exhibited a high specificity (96%) but lower sensitivity (69%). Reactivity with H3pme appeared not specific for SLE. Anti-H4pac and anti-H2Bpac reactivity demonstrated a high correlation with disease activity. Moreover, patients reacting with multiple modified histone peptides exhibited higher SLEDAI and lower C3 levels. SLE patients with renal involvement showed higher reactivity with H2B/H2Bpac and a more pronounced reactivity with the modified equivalent of H3pme and H2Bpac. In conclusion, reactivity with H4pac and H2Bpac is specific for SLE patients and correlates with disease activity, whereas reactivity with H2Bpac is in particular associated with lupus nephritis. PMID:27780265

  8. Peptides from the inside of the antibodies are active against infectious agents and tumours.

    PubMed

    Ciociola, Tecla; Giovati, Laura; Sperindè, Martina; Magliani, Walter; Santinoli, Claudia; Conti, Giorgio; Conti, Stefania; Polonelli, Luciano

    2015-05-01

    Synthetic peptides, representative of sequences related to the complementarity determining regions and constant region of antibodies, proved to exert in vitro, ex vivo and/or in vivo antimicrobial, antiviral, anti-tumour and/or immunomodulatory activities, conceivably mediated by different mechanisms of action and regardless of the specificity and isotype of the belonging immunoglobulin. Antibody-derived peptides can show intrinsic properties of self-aggregation in β structures, able to assemble on molecular targets and dissociate spontaneously, leading to the formation of hydrogels. Whilst the self-assembled state may provide protection against proteases and the slow kinetic of dissociation assures a release of the active form over time, the receptor affinity is responsible for targeted delivery. Peptides derived from single amino acid substitution of bioactive antibody fragments, adopted as surrogates of natural point mutations, displayed further differential biological activities. Overall, these observations allow to envisage that antibodies could represent an unlimited source of new anti-infective and anti-tumour peptides.

  9. Identification of a Peptide Produced by Bifidobacterium longum CECT 7210 with Antirotaviral Activity

    PubMed Central

    Chenoll, Empar; Casinos, Beatriz; Bataller, Esther; Buesa, Javier; Ramón, Daniel; Genovés, Salvador; Fábrega, Joan; Rivero Urgell, Montserrat; Moreno Muñoz, José A.

    2016-01-01

    Rotavirus is one of the main causes of acute diarrhea and enteritis in infants. Currently, studies are underway to assess the use of probiotics to improve rotavirus vaccine protection. A previous work demonstrated that the probiotic strain Bifidobacterium longum subsp. infantis CECT 7210 is able to hinder rotavirus replication both in vitro and in vivo. The present study takes a systematic approach in order to identify the molecule directly involved in rotavirus inhibition. Supernatant protease digestions revealed both the proteinaceous nature of the active substance and the fact that the molecule responsible for inhibiting rotavirus replication is released to the supernatant. Following purification by cationic exchange chromatography, active fractions were obtained and the functional compound was identified as an 11-amino acid peptide (MHQPHQPLPPT, named 11-mer peptide) with a molecular mass of 1.282 KDa. The functionality of 11-mer was verified using the synthesized peptide in Wa, Ito, and VA70 rotavirus infections of both HT-29 and MA-104 cell lines. Finally, protease activity was detected in B. longum subsp. infantis CECT 7210 supernatant, which releases 11-mer peptide. A preliminary identification of the protease is also included in the study. PMID:27199974

  10. Why does threonine, and not serine, function as the active site nucleophile in proteasomes?

    PubMed

    Kisselev, A F; Songyang, Z; Goldberg, A L

    2000-05-19

    Proteasomes belong to the N-terminal nucleophile group of amidases and function through a novel proteolytic mechanism, in which the hydroxyl group of the N-terminal threonines is the catalytic nucleophile. However, it is unclear why threonine has been conserved in all proteasomal active sites, because its replacement by a serine in proteasomes from the archaeon Thermoplasma acidophilum (T1S mutant) does not alter the rates of hydrolysis of Suc-LLVY-amc (Seemüller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. (1995) Science 268, 579-582) and other standard peptide amide substrates. However, we found that true peptide bonds in decapeptide libraries were cleaved by the T1S mutant 10-fold slower than by wild type (wt) proteasomes. In degrading proteins, the T1S proteasome was 3.5- to 6-fold slower than the wt, and this difference increased when proteolysis was stimulated using the proteasome-activating nucleotidase (PAN) ATPase complex. With mutant proteasomes, peptide bond cleavage appeared to be rate-limiting in protein breakdown, unlike with wt. Surprisingly, a peptide ester was hydrolyzed by both particles much faster than the corresponding amide, and the T1S mutant cleaved it faster than the wt. Moreover, the T1S mutant was inactivated by the ester inhibitor clasto-lactacystin-beta-lactone severalfold faster than the wt, but reacted with nonester irreversible inhibitors at similar rates. T1A and T1C mutants were completely inactive in all these assays. Thus, proteasomes lack additional active sites, and the N-terminal threonine evolved because it allows more efficient protein breakdown than serine. PMID:10809725

  11. Control of active sites in flocculation: Concept of equivalent active sites''

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    Flocculation and dispersion of solids are strong functions of the amount and conformation of the adsorbed polymer. Regions of dispersion and flocculation of solids with particular polymer molecules may be deduced from saturation adsorption data. The concept of equivalent active sites'' is proposed to explain flocculation and dispersion behavior irrespective of the amount or conformation of the adsorbed polymer. The concept has been further extended to study the selective flocculation process.

  12. Self-assembled monolayers of Aβ peptides on Au electrodes: an artificial platform for probing the reactivity of redox active metals and cofactors relevant to Alzheimer's disease.

    PubMed

    Pramanik, Debajyoti; Sengupta, Kushal; Mukherjee, Soumya; Dey, Somdatta Ghosh; Dey, Abhishek

    2012-07-25

    The water-soluble hydrophilic part of human Aβ peptide has been extended to include a C-terminal cysteine residue. Utilizing the thiol functionality of this cysteine residue, self-assembled monolayers (SAM) of these peptides are formed on Au electrodes. Atomic force microscopy imaging confirms formation of small Aβ aggregates on the surface of the electrode. These aggregates bind redox active metals like Cu and cofactors like heme, both of which are proposed to generate toxic partially reduced oxygen species (PROS) and play a vital role in Alzheimer's disease. The spectroscopic and electrochemical properties of these Cu and heme bound Aβ SAM are similar to those reported for the soluble Cu and heme bound Aβ peptide. Experiments performed on these Aβ-SAM electrodes clearly demonstrate that (1) heme bound Aβ is kinetically more competent in reducing O(2) than Cu bound Aβ, (2) under physiological conditions the reduced Cu site produces twice as much PROS (measured in situ) than the reduced heme site, and (3) chelators like clioquinol remove Cu from these aggregates, while drugs like methylene blue inhibit O(2) reactivity of the heme cofactor. This artificial construct provides a very easy platform for investigating potential drugs affecting aggregation of human Aβ peptides and PROS generation by its complexes with redox active metals and cofactors.

  13. Medical-grade honey enriched with antimicrobial peptides has enhanced activity against antibiotic-resistant pathogens

    PubMed Central

    Kwakman, P. H. S.; de Boer, L.; Ruyter-Spira, C. P.; Creemers-Molenaar, T.; Helsper, J. P. F. G.; Vandenbroucke-Grauls, C. M. J. E.; te Velde, A. A.

    2010-01-01

    Honey has potent activity against both antibiotic-sensitive and -resistant bacteria, and is an interesting agent for topical antimicrobial application to wounds. As honey is diluted by wound exudate, rapid bactericidal activity up to high dilution is a prerequisite for its successful application. We investigated the kinetics of the killing of antibiotic-resistant bacteria by RS honey, the source for the production of Revamil® medical-grade honey, and we aimed to enhance the rapid bactericidal activity of RS honey by enrichment with its endogenous compounds or the addition of antimicrobial peptides (AMPs). RS honey killed antibiotic-resistant isolates of Pseudomonas aeruginosa, Staphylococcus epidermidis, Enterococcus faecium, and Burkholderia cepacia within 2 h, but lacked such rapid activity against methicillin-resistant S. aureus (MRSA) and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. It was not feasible to enhance the rapid activity of RS honey by enrichment with endogenous compounds, but RS honey enriched with 75 μM of the synthetic peptide Bactericidal Peptide 2 (BP2) showed rapid bactericidal activity against all species tested, including MRSA and ESBL E. coli, at up to 10–20-fold dilution. RS honey enriched with BP2 rapidly killed all bacteria tested and had a broader spectrum of bactericidal activity than either BP2 or honey alone. PMID:20927564

  14. Possible active site of the sweet-tasting protein thaumatin.

    PubMed

    Slootstra, J W; De Geus, P; Haas, H; Verrips, C T; Meloen, R H

    1995-10-01

    Epitopes on thaumatin and monellin were studied using the PEPSCAN-technology. The antibodies used were raised against thaumatin. Only antibodies that, in an ELISA, both recognized thaumatin and monellin were used in the PEPSCAN-analyses. On thaumatin two major overlapping epitopes were identified. On monellin no epitopes could be identified. The identified epitope region on thaumatin shares structural features with various peptide and protein sweeteners. It contains an aspartame-like site which is formed by Asp21 and Phe80, tips of the two extruding loops KGDAALDAGGR19-29 and CKRFGRPP77-84, which are spatially positioned next to each other. Furthermore, sub-sequences of the KGDAALDAGGR19-29 loop are similar to peptide-sweeteners such as L-Asp-D-Ala-L-Ala-methyl ester and L-Asp-D-Ala-Gly-methyl ester. Since the aspartame-like Asp21-Phe80 site and the peptide-sweetener-like sequences are also not present in non-sweet thaumatin-like proteins it is postulated that the KGDAALDAGGR19-29- and CKRFGRPP77-84 loop contain important sweet-taste determinants. This region has previously not been implicated as a sweet-taste determinant of thaumatin.

  15. Glycoprotein identification and localization of O-glycosylation sites by mass spectrometric analysis of deglycosylated/alkylaminylated peptide fragments.

    PubMed

    Hanisch, F G; Jovanovic, M; Peter-Katalinic, J

    2001-03-01

    In-gel digestion of densely O-glycosylated proteins, an essential step in proteome analysis, is often hampered by steric hindrance of the proteases. To overcome this technical problem a simple and convenient method has been developed, which combines several advantages: (1) Approximately 70% of the oligosaccharides are cleaved without significant protein hydrolysis at the optimal reaction conditions of 70% ethylamine, and quantitative cleavage is achieved with 40% methylamine, at 50 degrees C. (2) To the unsaturated derivatives of Ser and Thr the alkylamine is added as a label of previous O-glycosylation sites. (3) The alkylaminylated protein is effectively cleaved by proteolysis. (4) The modified peptides are identified by MALDI mass spectrometry under consideration of incremental mass increases. (5) The alkylamine label is stable under MALDI post-source-decay analysis as well as in collision-induced dissociation experiments allowing sequencing and peptide localization of O-glycosylation sites. Applicability of the method is evaluated with a series of synthetic glycopeptides, the densely O-glycosylated human glycophorin A, and with the mucin MUC1 from human milk fat globule membranes.

  16. Isolation and characterization of two peptides with prolactin release-inhibiting activity from porcine hypothalami.

    PubMed Central

    Schally, A V; Guoth, J G; Redding, T W; Groot, K; Rodriguez, H; Szonyi, E; Stults, J; Nikolics, K

    1991-01-01

    Two peptides with in vitro prolactin release-inhibiting activity were purified from stalk median eminence (SME) fragments of 20,000 pig hypothalami. Monolayer cultures of rat anterior pituitary cells were incubated with aliquots of chromatographic fractions and the inhibition of release of prolactin in vitro was measured by RIA in order to monitor the purification. The hypothalamic tissue extract was separated into 11 fractions by high-performance aqueous size-exclusion chromatography with one fraction showing a 4-fold increase in prolactin release-inhibiting factor (PIF) activity. This material was further purified by semipreparative reversed-phase (RP) HPLC. This process resulted in the separation of two distinct fractions that showed high PIF activity. These were further purified by semipreparative and analytical RP-HPLC to apparent homogeneity as judged by the UV absorbance profiles. Neither of the two peptides showed cross-reactivity with gonadotropin releasing hormone-associated peptide or with somatostatin-14 antibodies. Protein sequence analysis revealed that one of the PIF peptides was Trp-Cys-Leu-Glu-Ser-Ser-Gln-Cys-Gln-Asp-Leu-Ser-Thr-Glu-Ser-Asn-Leu-Leu- Ala-Cys - Ile-Arg-Ala-Cys-Lys-Pro, identical to residues 27-52 of the N-terminal region of the proopiomelanocortin (POMC) precursor (corresponding to amino acids 1-26 of the 16-kDa fragment). The sequence of the other PIF was Ala-Ser-Asp-Arg-Ser-Asn-Ala-Thr-Leu-Leu-Asp-Gly-Pro-Ser-Gly-Ala-Leu-Leu- Leu-Arg - Leu-Val-Gln-Leu-Ala-Gly-Ala-Pro-Glu-Pro-Ala-Glu-Pro-Ala-Gln-Pro-Gly-Val- Tyr, representing residues 109-147 of the vasopressin-neurophysin precursor. Synthetic peptides corresponding to the N-terminal region of POMC had significant PIF activity in vitro. PMID:2023899

  17. Therapeutic effects of cell-permeant peptides that activate G proteins downstream of growth factors

    PubMed Central

    Ma, Gary S.; Aznar, Nicolas; Kalogriopoulos, Nicholas; Midde, Krishna K.; Lopez-Sanchez, Inmaculada; Sato, Emi; Dunkel, Ying; Gallo, Richard L.; Ghosh, Pradipta

    2015-01-01

    In eukaryotes, receptor tyrosine kinases (RTKs) and trimeric G proteins are two major signaling hubs. Signal transduction via trimeric G proteins has long been believed to be triggered exclusively by G protein-coupled receptors (GPCRs). This paradigm has recently been challenged by several studies on a multimodular signal transducer, Gα-Interacting Vesicle associated protein (GIV/Girdin). We recently demonstrated that GIV’s C terminus (CT) serves as a platform for dynamic association of ligand-activated RTKs with Gαi, and for noncanonical transactivation of G proteins. However, exogenous manipulation of this platform has remained beyond reach. Here we developed cell-permeable GIV-CT peptides by fusing a TAT-peptide transduction domain (TAT-PTD) to the minimal modular elements of GIV that are necessary and sufficient for activation of Gi downstream of RTKs, and used them to engineer signaling networks and alter cell behavior. In the presence of an intact GEF motif, TAT-GIV-CT peptides enhanced diverse processes in which GIV’s GEF function has previously been implicated, e.g., 2D cell migration after scratch-wounding, invasion of cancer cells, and finally, myofibroblast activation and collagen production. Furthermore, topical application of TAT-GIV-CT peptides enhanced the complex, multireceptor-driven process of wound repair in mice in a GEF-dependent manner. Thus, TAT-GIV peptides provide a novel and versatile tool to manipulate Gαi activation downstream of growth factors in a diverse array of pathophysiologic conditions. PMID:25926659

  18. Peptides derived from central turn motifs within integrin αIIb and αV cytoplasmic tails inhibit integrin activation.

    PubMed

    Li, Xinlei; Liu, Yongqing; Haas, Thomas A

    2014-12-01

    We previously found that peptides derived from the full length of integrin αIIb and αV cytoplasmic tails inhibited their parent integrin activation, respectively. Here we showed that the cell-permeable peptides corresponding to the conserved central turn motif within αIIb and αV cytoplasmic tails, myr-KRNRPPLEED (αIIb peptide) and myr-KRVRPPQEEQ (αV peptide), similarly inhibited both αIIb and αV integrin activation. Pre-treatment with αIIb or αV peptides inhibited Mn(2+)-activated αIIbβ3 binding to soluble fibrinogen as well as the binding of αIIbβ3-expressing Chinese Hamster Ovary cells to immobilized fibrinogen. Our turn peptides also inhibited adhesion of two breast cancer cell lines (MDA-MB-435 and MCF7) to αV ligand vitronectin. These results suggest that αIIb and αV peptides share a same mechanism in regulating integrin function. Using αIIb peptide as a model, we found that replacement of RPP with AAA significantly attenuated the inhibitory activity of αIIb peptide. Furthermore, we found that αIIb peptide specifically bound to β-tubulin in cells. Our work suggests that the central motif of α tails is an anchoring point for cytoskeletons during integrin activation and integrin-mediated cell adhesion, and its function depends on the turn structure at RPP. However, post-treatment of peptides derived from the full-length tail or from the turn motif did not reverse αIIb and αV integrin activation.

  19. Peptide fragments of the dihydropyridine receptor can modulate cardiac ryanodine receptor channel activity and sarcoplasmic reticulum Ca2+ release.

    PubMed Central

    Dulhunty, Angela F; Curtis, Suzanne M; Cengia, Louise; Sakowska, Magdalena; Casarotto, Marco G

    2004-01-01

    We show that peptide fragments of the dihydropyridine receptor II-III loop alter cardiac RyR (ryanodine receptor) channel activity in a cytoplasmic Ca2+-dependent manner. The peptides were AC (Thr-793-Ala-812 of the cardiac dihydropyridine receptor), AS (Thr-671-Leu-690 of the skeletal dihydropyridine receptor), and a modified AS peptide [AS(D-R18)], with an extended helical structure. The peptides added to the cytoplasmic side of channels in lipid bilayers at > or = 10 nM activated channels when the cytoplasmic [Ca2+] was 100 nM, but either inhibited or did not affect channel activity when the cytoplasmic [Ca2+] was 10 or 100 microM. Both activation and inhibition were independent of bilayer potential. Activation by AS, but not by AC or AS(D-R18), was reduced at peptide concentrations >1 mM in a voltage-dependent manner (at +40 mV). In control experiments, channels were not activated by the scrambled AS sequence (ASS) or skeletal II-III loop peptide (NB). Resting Ca2+ release from cardiac sarcoplasmic reticulum was not altered by peptide AC, but Ca2+-induced Ca2+ release was depressed. Resting and Ca2+-induced Ca2+ release were enhanced by both the native and modified AS peptides. NMR revealed (i) that the structure of peptide AS(D-R18) is not influenced by [Ca2+] and (ii) that peptide AC adopts a helical structure, particularly in the region containing positively charged residues. This is the first report of specific functional interactions between dihydropyridine receptor A region peptides and cardiac RyR ion channels in lipid bilayers. PMID:14678014

  20. In vitro activity of CD4+ and CD8+ T lymphocytes from mice immunized with a synthetic malaria peptide.

    PubMed Central

    Rénia, L; Marussig, M S; Grillot, D; Pied, S; Corradin, G; Miltgen, F; Del Giudice, G; Mazier, D

    1991-01-01

    In previous work, a T-helper epitope was mapped within the circumsporozoite protein of the murine malaria parasite Plasmodium yoelii. A 21-mer synthetic peptide corresponding to this epitope (amino acid positions 59-79; referred to as Py1) induced a specific T-cell proliferation in BALB/c and C57BL/6 mice and provided help for the production of antibodies to peptides from the repetitive region, (Gln-Gly-Pro-Gly-Ala-Pro)n, of the P. yoelii circumsporozoite protein when mice were immunized with the Py1 peptide conjugated to the repetitive peptide. Experiments were then designed to study the in vitro antiparasite efficacy of T cells elicited in vivo by peptide immunization. T-cell activity was evaluated on cultured hepatic stages of P. yoelii. Peptide immunizations led to the preferential activation of CD8+ T cells in BALB/c mice and of both CD4+ and CD8+ T cells in C57BL/6 mice. Parasite elimination was mediated directly by these cells and did not seem to be dependent on lymphokine secretion. These data suggest that peptide-primed CD4+ T cells as well as CD8+ T cells could be cytolytic for the hepatic phase of malaria parasites. The fact that the same peptide could activate different lymphocyte populations, depending on the strain of mouse, highlights the importance of a better understanding of the fine mechanisms behind the immune responses to synthetic peptides being tested for malaria vaccine development. Images PMID:1680235

  1. Sclerotiamide: The First Non-Peptide-Based Natural Product Activator of Bacterial Caseinolytic Protease P.

    PubMed

    Lavey, Nathan P; Coker, Jesse A; Ruben, Eliza A; Duerfeldt, Adam S

    2016-04-22

    Caseinolytic protease P (ClpP) maintains essential roles in bacterial homeostasis. As such, both the inhibition and activation of this enzyme result in bactericidal activity, making ClpP a promising target for antibacterial drug development. Herein, we report the results of a fluorescence-based screen of ∼450 structurally diverse fungal and bacterial secondary metabolites. Sclerotiamide (1), a paraherquamide-related indolinone, was identified as the first non-peptide-based natural product activator of ClpP. Structure-activity relationships arising from the initial screen, preliminary biochemical evaluation of 1, and rationale for the exploitation of this chemotype to develop novel ClpP activators are presented.

  2. Autoradiographic localization of calcitonin gene-related peptide (CGRP) binding sites in human and guinea pig lung

    SciTech Connect

    Mak, J.C.; Barnes, P.J.

    1988-09-01

    /sup 125/I-Human calcitonin gene-related peptide (hCGRP) binding sites were localized in human and guinea pig lungs by an autoradiographic method. Scatchard analysis of saturation experiments from slide-mounted sections of guinea pig lung displayed specific /sup 125/I-hCGRP binding sites with a dissociation constant (Kd) of 0.72 +/- 0.05 nM (mean +/- S.E.M., n = 3) and a maximal number of binding sites (Bmax) of 133.4 +/- 5.6 fmol/mg protein. In both human and guinea pig lung, autoradiography revealed that CGRP binding sites were widely distributed, with particularly dense labeling over bronchial and pulmonary blood vessels of all sizes and alveolar walls. Airway smooth muscle and epithelium of large airways was sparsely labeled but no labeling was found over submucosal glands. This localization corresponds well to the reported pattern of CGRP-like immunoreactive innervation. The findings of localization of CGRP binding sites on bronchial and pulmonary blood vessels indicate that CGRP may be important in the regulation of airway and pulmonary blood flow.

  3. A structure-activity relationship for induction of meningeal inflammation by muramyl peptides.

    PubMed Central

    Burroughs, M; Rozdzinski, E; Geelen, S; Tuomanen, E

    1993-01-01

    Components of bacterial peptidoglycans have potent biological activities, including adjuvant effects, cytotoxicity, and induction of sleep. Mixtures of peptidoglycan components also induce inflammation in the lung, subarachnoid space, and joint, but the structural requirements for activity are unknown. Using a rabbit model for meningitis, we determined the biological activities of 14 individual muramyl peptides constituting > 90% of the peptidoglycan of the gram-negative pediatric pathogen Haemophilus influenzae. Upon intracisternal inoculation, most of the muropeptides induced leukocytosis in cerebrospinal fluid (CSF), influx of protein into CSF, or brain edema, alone or in combination. The disaccharide-tetrapeptide, the major component of all gram-negative peptidoglycans, induced CSF leukocytosis and protein influx at doses as low as 0.4 microgram (0.42 nM). Modification of the N-acetyl muramic acid or substitution of the alanine at position four in the peptide side chain decreased leukocytosis but enhanced brain edema. As the size of the muropeptide increased, the inflammatory activity decreased. Muropeptide carrying the diaminopimelyl-diaminopimelic acid cross-link specifically induced cytotoxic brain edema. These findings significantly expand the spectrum of biological activities of natural muramyl peptides and provide the basis for a structure-activity relationship for the inflammatory properties of bacterial muropeptides. PMID:8325996

  4. Biological Activity of Aminophosphonic Acids and Their Short Peptides

    NASA Astrophysics Data System (ADS)

    Lejczak, Barbara; Kafarski, Pawel

    The biological activity and natural occurrence of the aminophosphonic acids were described half a century ago. Since then the chemistry and biology of this class of compounds have developed into the separate field of phosphorus chemistry. Today it is well acknowledged that these compounds possess a wide variety of promising, and in some cases commercially useful, physiological activities. Thus, they have found applications ranging from agrochemical (with the herbicides glyphosate and bialaphos being the most prominent examples) to medicinal (with the potent antihypertensive fosinopril and antiosteoporetic bisphosphonates being examples).

  5. Enhanced Amphiphilic Profile of a Short β-Stranded Peptide Improves Its Antimicrobial Activity

    PubMed Central

    Manzo, Giorgia; Scorciapino, Mariano A.; Wadhwani, Parvesh; Bürck, Jochen; Montaldo, Nicola Pietro; Pintus, Manuela; Sanna, Roberta; Casu, Mariano; Giuliani, Andrea; Pirri, Giovanna; Luca, Vincenzo; Ulrich, Anne S.; Rinaldi, Andrea C.

    2015-01-01

    SB056 is a novel semi-synthetic antimicrobial peptide with a dimeric dendrimer scaffold. Active against both Gram-negative and -positive bacteria, its mechanism has been attributed to a disruption of bacterial membranes. The branched peptide was shown to assume a β-stranded conformation in a lipidic environment. Here, we report on a rational modification of the original, empirically derived linear peptide sequence [WKKIRVRLSA-NH2, SB056-lin]. We interchanged the first two residues [KWKIRVRLSA-NH2, β-SB056-lin] to enhance the amphipathic profile, in the hope that a more regular β-strand would lead to a better antimicrobial performance. MIC values confirmed that an enhanced amphiphilic profile indeed significantly increases activity against both Gram-positive and -negative strains. The membrane binding affinity of both peptides, measured by tryptophan fluorescence, increased with an increasing ratio of negatively charged/zwitterionic lipids. Remarkably, β-SB056-lin showed considerable binding even to purely zwitterionic membranes, unlike the original sequence, indicating that besides electrostatic attraction also the amphipathicity of the peptide structure plays a fundamental role in binding, by stabilizing the bound state. Synchrotron radiation circular dichroism and solid-state 19F-NMR were used to characterize and compare the conformation and mobility of the membrane bound peptides. Both SB056-lin and β-SB056-lin adopt a β-stranded conformation upon binding POPC vesicles, but the former maintains an intrinsic structural disorder that also affects its aggregation tendency. Upon introducing some anionic POPG into the POPC matrix, the sequence-optimized β-SB056-lin forms well-ordered β-strands once electro-neutrality is approached, and it aggregates into more extended β-sheets as the concentration of anionic lipids in the bilayer is raised. The enhanced antimicrobial activity of the analogue correlates with the formation of these extended β-sheets, which

  6. Novel biologically active peptides from the venom of Polistes rothneyi iwatai.

    PubMed

    Murata, Kazuya; Shinada, Tetsuro; Ohfune, Yasufumi; Hisada, Miki; Yasuda, Akikazu; Naoki, Hideo; Nakajima, Terumi

    2006-12-01

    Four novel peptides, polistes-mastoparan-R1, 2, 3, and polistes-protonectin, were isolated from the venom of a paper wasp, Polistes rothneyi iwatai. MALDI-TOF MS analysis of a small amount of the crude venom gave six molecular-related ion peaks. Among them, m/z 1565 was expected to be a novel peptide. Purification of the crude venom by HPLC gave two known kinins, Thr6-bradykinin and Ala-Arg-Thr6-bradykinin, and four novel peptides named polistes-mastoparan-R1, 2, and 3, and polistes-protonectin. Polistes-mastoparan-R1, 2, and 3 (Pm-R) were tetradecapeptides that possess high sequence homology with that of mastoparan. The sequence of polistes-protonectin was similar to that of protonectin isolated from a Brazilian paper wasp. Histamine-releasing activities of Pm-R1, 2, and 3 were more potent than that of mastoparan. Polistes-protonectin exhibited the most potent hemolytic activity in comparison with the four novel peptides and mastoparan.

  7. Peptide length and folding state govern the capacity of staphylococcal β-type phenol-soluble modulins to activate human formyl-peptide receptors 1 or 2.

    PubMed

    Kretschmer, Dorothee; Rautenberg, Maren; Linke, Dirk; Peschel, Andreas

    2015-04-01

    Most staphylococci produce short α-type PSMs and about twice as long β-type PSMs that are potent leukocyte attractants and toxins. PSMs are usually secreted with the N-terminal formyl group but are only weak agonists for the leukocyte FPR1. Instead, the FPR1-related FPR2 senses PSMs efficiently and is crucial for leukocyte recruitment in infection. Which structural features distinguish FPR1 from FPR2 ligands has remained elusive. To analyze which peptide properties may govern the capacities of β-type PSMs to activate FPRs, full-length and truncated variants of such peptides from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus lugdunensis were synthesized. FPR2 activation was observed even for short N- or C-terminal β-type PSM variants once they were longer than 18 aa, and this activity increased with length. In contrast, the shortest tested peptides were potent FPR1 agonists, and this property declined with increasing peptide length. Whereas full-length β-type PSMs formed α-helices and exhibited no FPR1-specific activity, the truncated peptides had less-stable secondary structures, were weak agonists for FPR1, and required N-terminal formyl-methionine residues to be FPR2 agonists. Together, these data suggest that FPR1 and FPR2 have opposed ligand preferences. Short, flexible PSM structures may favor FPR1 but not FPR2 activation, whereas longer peptides with α-helical, amphipathic properties are strong FPR2 but only weak FPR1 agonists. These findings should help to unravel the ligand specificities of 2 critical human PRRs, and they may be important for new, anti-infective and anti-inflammatory strategies.

  8. Application of Asian pumpkin (Cucurbita ficifolia) serine proteinase for production of biologically active peptides from casein.

    PubMed

    Dąbrowska, Anna; Szołtysik, Marek; Babij, Konrad; Pokora, Marta; Zambrowicz, Aleksandra; Chrzanowska, Józefa

    2013-01-01

    The main objective of this study was to determine potential application of a serine proteinase derived from Asian pumpkin for obtaining biologically active peptides from casein. The course of casein hydrolysis by three doses of the enzyme (50, 150, 300 U/mg of protein) was monitored for 24 hours by the determinations of: hydrolysis degree DH (%), free amino group content (μmole Gly/g), RP HPLC peptide profiles and by polyacrylamide gel electrophoresis. In all hydrolyzates analyzed antioxidant activities were determined using three tests: the ability to reduce iron ions in FRAP test, the ability to scavenge free radicals in DPPH test, and Fe(2+) chelating activity. The antimicrobial activity of obtained peptide fractions was determined as the ability to inhibit the growth of Escherichia coli, Bacillus cereus and Pseudomonas fluorescens in a diffusion plate test. The deepest degradation, expressed as the DH [%] and the free amino group content (67% and 7528 µmole Gly/mg, respectively), was noted in samples hydrolyzed with 300 U/ml of enzyme for 24 hours, while in other samples the determined values were about three and two times lower. The results were in agreement with the peptide profiles obtained by RP HPLC. The highest antioxidative activities determined in all tests were seen for the casein hydrolysate obtained with 300 U/mg protein of serine proteinase after 24 h of reaction (2.15 µM Trolox/mg, 96.15 µg Fe(3+)/mg, 814.97 µg Fe(2+)/mg). Antimicrobial activity was presented in three preparations. In other samples no antimicrobial activity was detected.

  9. Active site studies of Escherichia coli 2-keto-4-hydroxyglutarate aldolase

    SciTech Connect

    Vlahos, C.J.

    1987-01-01

    The data presented delineate the complete amino acid sequence of E. coli KHG aldolase and also identify Lys-133, Glu-45, and Arg-49 as aminoacyl residues required for catalytic activity. Incubation of E. coli KHG aldolase with (/sup 14/C)pyruvate in the presence of NaCNBH/sub 3/ results in the incorporation of one mol of /sup 14/C per mol of enzyme subunit. Digestion of this enzyme-adduct with trypsin, followed by purification of the peptides, allowed for the isolation of a unique radioactive peptide. Its amino acid sequence showed that the pyruvate-binding (i.e., Schiff-base forming) lysine residue is located at position 133 in the intact enzyme. E. coli KHG aldolase activity is lost when the enzyme is reacted with bromopyruvate; saturation kinetics are observed. The substrates, pyruvate and KHG, protect the enzyme from inactivation. Both facts suggest that the reagent is active-site specific. Incubation of the aldolase with (3-/sup 14/C)bromopyruvate is associated with a concomitant loss of enzymatic activity and esterification of Glu-45; if the enzyme is denatured in the presence of excess bromopyruvate, Cys-159 and Cys-180 are also alkylated. Blocking the active-site lysine residue with pyruvate prevents Glu-45 from being esterified but does not eliminate alkylation of these two cysteine residues. Woodward's Reagent K was also found to inactivate the aldolase under conditions that are usually specific for carboxyl group modification. This aldolase is also inactivated by 1,2-cyclohexanedione. Loss of enzymatic activity occurs concomitantly with modification of one arginine residue per enzyme subunit. Treatment of the aldolase with the arginine-specific reagent, 4-(oxyacetyl)phenoxyacetic acid, followed by digestion with trypsin allowed for the isolation of a unique peptide and the identification of Arg-49 as the specific residue involved.

  10. A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle.

    PubMed

    Nelson, Benjamin R; Makarewich, Catherine A; Anderson, Douglas M; Winders, Benjamin R; Troupes, Constantine D; Wu, Fenfen; Reese, Austin L; McAnally, John R; Chen, Xiongwen; Kavalali, Ege T; Cannon, Stephen C; Houser, Steven R; Bassel-Duby, Rhonda; Olson, Eric N

    2016-01-15

    Muscle contraction depends on release of Ca(2+) from the sarcoplasmic reticulum (SR) and reuptake by the Ca(2+)adenosine triphosphatase SERCA. We discovered a putative muscle-specific long noncoding RNA that encodes a peptide of 34 amino acids and that we named dwarf open reading frame (DWORF). DWORF localizes to the SR membrane, where it enhances SERCA activity by displacing the SERCA inhibitors, phospholamban, sarcolipin, and myoregulin. In mice, overexpression of DWORF in cardiomyocytes increases peak Ca(2+) transient amplitude and SR Ca(2+) load while reducing the time constant of cytosolic Ca(2+) decay during each cycle of contraction-relaxation. Conversely, slow skeletal muscle lacking DWORF exhibits delayed Ca(2+) clearance and relaxation and reduced SERCA activity. DWORF is the only endogenous peptide known to activate the SERCA pump by physical interaction and provides a means for enhancing muscle contractility. PMID:26816378

  11. Antimicrobial activity of the synthetic peptide scolopendrasin ii from the centipede Scolopendra subspinipes mutilans.

    PubMed

    Kwon, Young-Nam; Lee, Joon Ha; Kim, In-Woo; Kim, Sang-Hee; Yun, Eun-Young; Nam, Sung-Hee; Ahn, Mi-Young; Jeong, Mihye; Kang, Dong-Chul; Lee, In Hee; Hwang, Jae Sam

    2013-10-28

    The centipede Scolopendra subpinipes mutilans is a medicinally important arthropod species. However, its transcriptome is not currently available and transcriptome analysis would be useful in providing insight into a molecular level approach. Hence, we performed de novo RNA sequencing of S. subpinipes mutilans using next-generation sequencing. We generated a novel peptide (scolopendrasin II) based on a SVM algorithm, and biochemically evaluated the in vitro antimicrobial activity of scolopendrasin II against various microbes. Scolopendrasin II showed antibacterial activities against gram-positive and -negative bacterial strains, including the yeast Candida albicans and antibiotic-resistant gram-negative bacteria, as determined by a radial diffusion assay and colony count assay without hemolytic activity. In addition, we confirmed that scolopendrasin II bound to the surface of bacteria through a specific interaction with lipoteichoic acid and a lipopolysaccharide, which was one of the bacterial cell-wall components. In conclusion, our results suggest that scolopendrasin II may be useful for developing peptide antibiotics.

  12. Tumor-Penetrating Peptides

    PubMed Central

    Teesalu, Tambet; Sugahara, Kazuki N.; Ruoslahti, Erkki

    2013-01-01

    Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC), contains the integrin-binding RGD motif. RGD mediates tumor-homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR) motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular “zip code” of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies, and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is present in the

  13. Design and Characterization of a Multifunctional pH-Triggered Peptide C8 for Selective Anticancer Activity.

    PubMed

    Lu, Sheng; Bennett, W F Drew; Ding, Yong; Zhang, Lei; Fan, Helen Y; Zhao, Danyang; Zheng, Tao; Ouyang, Ping-Kai; Li, Jason; Wu, Yan; Xu, Wen; Chu, Dafeng; Yuan, Yongfang; Heerklotz, Heiko; Karttunen, Mikko; Chen, P

    2015-12-01

    Most drug delivery systems have been developed for efficient delivery to tumor sites via targeting and on-demand strategies, but the carriers rarely execute synergistic therapeutic actions. In this work, C8, a cationic, pH-triggered anticancer peptide, is developed by incorporating histidine-mediated pH-sensitivity, amphipathic helix, and amino acid pairing self-assembly design. We designed C8 to function as a pH-responsive nanostructure whose cytotoxicity can be switched on and off by its self-assembly: Noncytotoxic β-sheet fibers at high pH with neutral histidines, and positively charged monomers with membrane lytic activity at low pH. The selective activity of C8, tested for three different cancer cell lines and two noncancerous cell lines, is shown. Based on liposome leakage assays and multiscale computer simulations, its physical mechanisms of pore-forming action and selectivity are proposed, which originate from differences in the lipid composition of the cellular membrane and changes in hydrogen bonding. C8 is then investigated for its potential as a drug carrier. C8 forms a nanocomplex with ellipticine, a nonselective model anticancer drug. It selectively targets cancer cells in a pH-responsive manner, demonstrating enhanced efficacy and selectivity. This study provides a novel powerful strategy for the design and development of multifunctional self-assembling peptides for therapeutic and drug delivery applications. PMID:26474414

  14. Antagonistic Activities of Novel Peptides from Bacillus amyloliquefaciens PT14 against Fusarium solani and Fusarium oxysporum.

    PubMed

    Kim, Young Gwon; Kang, Hee Kyoung; Kwon, Kee-Deok; Seo, Chang Ho; Lee, Hyang Burm; Park, Yoonkyung

    2015-12-01

    Bacillus species have recently drawn attention due to their potential use in the biological control of fungal diseases. This paper reports on the antifungal activity of novel peptides isolated from Bacillus amyloliquefaciens PT14. Reverse-phase high-performance liquid chromatography revealed that B. amyloliquefaciens PT14 produces five peptides (PT14-1, -2, -3, -4a, and -4b) that exhibit antifungal activity but are inactive against bacterial strains. In particular, PT14-3 and PT14-4a showed broad-spectrum antifungal activity against Fusarium solani and Fusarium oxysporum. The PT14-4a N-terminal amino acid sequence was identified through Edman degradation, and a BLAST homology analysis showed it not to be identical to any other protein or peptide. PT14-4a displayed strong fungicidal activity with minimal inhibitory concentrations of 3.12 mg/L (F. solani) and 6.25 mg/L (F. oxysporum), inducing severe morphological deformation in the conidia and hyphae. On the other hand, PT14-4a had no detectable hemolytic activity. This suggests PT14-4a has the potential to serve as an antifungal agent in clinical therapeutic and crop-protection applications.

  15. Binding sites for. alpha. -bungarotoxin and the noncompetitive inhibitor phencyclidine on a synthetic peptide comprising residues 172-227 of the. alpha. -subunit of the nicotinic acetylcholine receptor

    SciTech Connect

    Donnelly-Roberts, D.L.; Lentz, T.L. )

    1991-07-30

    The binding of the competitive antagonist {alpha}-bungarotoxin ({alpha}-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the {alpha}-subunit of the Torpedo acetylcholine receptor has been characterized. {sup 125}I-{alpha}-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by {alpha}-Btx d-tubocurarine and NaCl. In the presence of 0.02% sodium dodecyl sulfate, {sup 125}I-{alpha}-Btx bound to the 56-residue peptide with a K{sub D} of 3.5 nM, as determined by equilibrium saturation binding studies. Because {alpha}Btx binds to a peptide comprising residues 173-204 with the same affinity and does not bind to a peptide comprising residues 205-227, the competitive antagonist and hence agonist binding site lies between residues 173 and 204. After photoaffinity labeling, ({sup 3}H)PCP was bound to the 172-227 peptide. ({sup 3}H)PCP binding was inhibited by chlorpromazine, tetracaine, and dibucaine. It is concluded that a high-affinity binding site for PCP is located between residues 205 and 227, which includes the first 18 residues of transmembrane segment M1, and that a low-affinity site is located in the competitive antagonist binding site between residues 173 and 204. These results show that a synthetic peptide comprising residues 172-227 of the {alpha} subunit contains three binding sites, one for {alpha}-Btx and two for PCP. Previous studies on the intact receptor indicate high-affinity PCP binding occurs in the receptor channel.

  16. Injection of Cocaine-Amphetamine Regulated Transcript (CART) peptide into the nucleus accumbens does not inhibit caffeine-induced locomotor activity: Implications for CART peptide mechanism.

    PubMed

    Job, Martin O

    2016-09-01

    Much evidence suggests that intra-nucleus accumbens (NAc) CART peptide (CART 55-102) injection inhibits locomotor activity (LMA) when there is an increase in the release and activity of dopamine (DA) in the NAc. However, this hypothesis has not been fully tested. One way to examine this is to determine if there is a lack of effect of intra-NAc CART peptide on LMA that does not involve increases in DA release in the NAc. Several studies have suggested that caffeine-induced LMA does not involve extracellular DA release in the NAc core. Therefore, in this study, we have examined the effect of injections of CART peptide (2.5μg) into the NAc core on the locomotor effects of caffeine in male Sprague-Dawley rats. Several LMA relevant doses of caffeine were used (0, 10, 20mg/kg i.p.), and an inverted U response curve was found as expected. We determined, in the same animals, that intra-NAc CART peptide had no effect on caffeine-induced LMA whereas it blunted cocaine-mediated LMA, as shown by other reports. We also extended a previous observation in mice by showing that at a LMA activating dose of caffeine there is no alteration of CART peptide levels in the NAc of rats. Our study supports the hypothesis that the inhibitory effects of CART peptide in the NAc may be exerted only under conditions of increased extracellular DA release and activity in this region. Our results also suggest that intra-NAc CART 55-102 does not generally inhibit increases in LMA due to all drugs, but has a more specific inhibitory effect on dopaminergic neurotransmission. PMID:27168116

  17. Jatrophidin I, a cyclic peptide from Brazilian Jatropha curcas L.: isolation, characterization, conformational studies and biological activity.

    PubMed

    Altei, Wanessa F; Picchi, Douglas G; Abissi, Barbara M; Giesel, Guilherme M; Flausino, Otavio; Reboud-Ravaux, Michèle; Verli, Hugo; Crusca, Edson; Silveira, Edilberto R; Cilli, Eduardo M; Bolzani, Vanderlan S

    2014-11-01

    A cyclic peptide, jatrophidin I, was isolated from the latex of Jatropha curcas L. Its structure was elucidated by extensive 2D NMR spectroscopic analysis, with additional conformational studies performed using Molecular Dynamics/Simulated Annealing (MD/SA). Jatrophidin I had moderate protease inhibition activity when compared with pepstatin A; however, the peptide was inactive in antimalarial, cytotoxic and antioxidant assays.

  18. Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide.

    PubMed

    Yung, Stephanie L; Dela Cruz, Fernando; Hamren, Sarah; Zhu, Jian; Tsutsumi, Manami; Bloom, James W; Caudle, Margaret; Roczniak, Steve; Todd, Tracey; Lemoine, Lynn; MacDougall, Margit; Shanafelt, Armen B; Pan, Clark Q

    2003-03-21

    Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.

  19. Characterization and activity of an immobilized antimicrobial peptide containing bactericidal PEG-hydrogel.

    PubMed

    Cleophas, Rik T C; Sjollema, Jelmer; Busscher, Henk J; Kruijtzer, John A W; Liskamp, Rob M J

    2014-09-01

    A single step immobilization-polymerization strategy of a highly active antimicrobial peptide into a soft hydrogel network on a poly(ethylene terephthalate) surface using thiol-ene chemistry is described. The bactericidal hydrogel was molecularly characterized via Coomassie and Lowry assay protein staining agents as well as by X-ray photoelectron spectroscopy. The bactericidal activity was established against Staphylococcus aureus and Staphylococcus epidermidis, two bacterial strains commonly associated with biomaterial infections. To gain further insight into the biological stability, the hydrogels were incubated with human serum prior to activity testing without loss of activity. These studies revealed a promising bactericidal hydrogel with good stability under physiological conditions.

  20. Design of an α-helical antimicrobial peptide with improved cell-selective and potent anti-biofilm activity

    PubMed Central

    Zhang, Shi-Kun; Song, Jin-wen; Gong, Feng; Li, Su-Bo; Chang, Hong-Yu; Xie, Hui-Min; Gao, Hong-Wei; Tan, Ying-Xia; Ji, Shou-Ping

    2016-01-01

    AR-23 is a melittin-related peptide with 23 residues. Like melittin, its high α-helical amphipathic structure results in strong bactericidal activity and cytotoxicity. In this study, a series of AR-23 analogues with low amphipathicity were designed by substitution of Ala1, Ala8 and Ile17 with positively charged residues (Arg or Lys) to study the effect of positively charged residue distribution on the biological viability of the antimicrobial peptide. Substitution of Ile17 on the nonpolar face with positively charged Lys dramatically altered the hydrophobicity, amphipathicity, helicity and the membrane-penetrating activity against human cells as well as the haemolytic activity of the peptide. However, substitution on the polar face only slightly affected the peptide biophysical properties and biological activity. The results indicate that the position rather than the number of positively charged residue affects the biophysical properties and selectivity of the peptide. Of all the analogues, A(A1R, A8R, I17K), a peptide with Ala1-Arg, Ala8-Arg and Ile17-Lys substitutions, exhibited similar bactericidal activity and anti-biofilm activity to AR-23 but had much lower haemolytic activity and cytotoxicity against mammalian cells compared with AR-23. Therefore, the findings reported here provide a rationalization for peptide design and optimization, which will be useful for the future development of antimicrobial agents. PMID:27271216

  1. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  2. Allosteric receptor activation by the plant peptide hormone phytosulfokine.

    PubMed

    Wang, Jizong; Li, Hongju; Han, Zhifu; Zhang, Heqiao; Wang, Tong; Lin, Guangzhong; Chang, Junbiao; Yang, Weicai; Chai, Jijie

    2015-09-10

    Phytosulfokine (PSK) is a disulfated pentapeptide that has a ubiquitous role in plant growth and development. PSK is perceived by its receptor PSKR, a leucine-rich repeat receptor kinase (LRR-RK). The mechanisms underlying the recognition of PSK, the activation of PSKR and the identity of the components downstream of the initial binding remain elusive. Here we report the crystal structures of the extracellular LRR domain of PSKR in free, PSK- and co-receptor-bound forms. The structures reveal that PSK interacts mainly with a β-strand from the island domain of PSKR, forming an anti-β-sheet. The two sulfate moieties of PSK interact directly with PSKR, sensitizing PSKR recognition of PSK. Supported by biochemical, structural and genetic evidence, PSK binding enhances PSKR heterodimerization with the somatic embryogenesis receptor-like kinases (SERKs). However, PSK is not directly involved in PSKR-SERK interaction but stabilizes PSKR island domain for recruitment of a SERK. Our data reveal the structural basis for PSKR recognition of PSK and allosteric activation of PSKR by PSK, opening up new avenues for the design of PSKR-specific small molecules.

  3. 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.

  4. Elucidation of peptide sequence effects that control the activity, size, and function of nanoparticles

    NASA Astrophysics Data System (ADS)

    Coppage, Ryan

    Bio-inspired nanoparticle catalysis offers the opportunity to improve on current catalytic standards with respect to turnover efficiency, organic solvent use, and thermal activation. Unfortunately, projected energy demands will soon outweigh our fuel supplies. The task of creating multifunctional catalysts that both lower thermal activation and possess a number of functions in aqueous conditions is daunting. Similar to these needs, nature has evolved to create a wide range of highly specialized catalytic processes, which incorporate inorganic materials, take place in ambient temperatures, and in an aqueous environment. These specialized biological systems provide inspiration, but are not applicable to current needs. Exploitation of these biotic-abiotic systems could allow for green, multifunctional catalysts. In the resulting works, a peptide sequence has been isolated via phage display with affinity for Pd surfaces, that forms stable, peptide-capped nanoparticles. Substitution of residues results in the tuning of both nanocatalyst activity and nanoparticle size, such that a peptide surface-controlling effect can be noted. These characteristics can be exploited to ultimately understand the binding interactions among bio-inorganic interfaces, such that a rational design of biomolecules can be realized for the synthesis of highly active, green, multifunctional nanomaterials.

  5. An antifungal peptide with antiproliferative activity toward tumor cells from red kidney beans.

    PubMed

    Li, Miao; Wang, Hexiang; Ng, Tzi Bun

    2011-06-01

    A 7.3-kDa antifungal peptide was purified from dried red kidney beans. The purification procedure entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, followed by fast protein liquid chromatography-gel filtration on Superdex 75. The peptide was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and CM-cellulose. It exhibited a molecular mass of 7.3 kDa in gel filtration and also in SDS-polyacrylamide gel electrophoresis, indicating that it is a single-chained protein. The N-terminal sequence of the peptide was DGVCFGGLANGDRT. The peptide exerted an antifungal action on Fusarium oxysporum with an IC₅₀ of 3.8±0.4 µM (mean±SD, n=3). It also inhibited mycelial growth in Mycosphaerella arachidicola. It suppressed growth of lymphoma MBL2 cells and leukemia L1210 cells with an IC₅₀ of 5.2±0.4 µM and 7.6±0.6 µM, respectively. HIV-1 reverse transcriptase was inhibited with an IC₅₀ of 40±3.2 µM. However, no activity was demonstrated toward other viral enzymes.

  6. Structure-dependent charge density as a determinant of antimicrobial activity of peptide analogues of defensin.

    PubMed

    Bai, Yang; Liu, Shouping; Jiang, Ping; Zhou, Lei; Li, Jing; Tang, Charles; Verma, Chandra; Mu, Yuguang; Beuerman, Roger W; Pervushin, Konstantin

    2009-08-01

    Defensins are small (3-5 kDa) cysteine-rich cationic proteins found in both vertebrates and invertebrates constituting the front line of host innate immunity. Despite intensive research, bactericidal and cytotoxic mechanisms of defensins are still largely unknown. Moreover, we recently demonstrated that small peptides derived from defensins are even more potent bactericidal agents with less toxicity toward host cells. In this paper, structures of three C-terminal (R36-K45) analogues of human beta-defensin-3 were studied by 1H NMR spectroscopy and extensive molecular dynamics simulations. Because of indications that these peptides might target the inner bacterial membrane, they were reconstituted in dodecylphosphocholine or dodecylphosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] mixed micelles, and lipid bicelles mimicking the phospholipid-constituted bilayer membrane of mammalian and bacterial cells. The results show that the binding affinity and partitioning into the lipid phase and the ability to dimerize and accrete well-defined structures upon interactions with lipid membranes contribute to compactization of positive charges within peptide oligomers. The peptide charge density, mediated by corresponding three-dimensional structures, was found to directly correlate with the antimicrobial activity. These novel observations may provide a new rationale for the design of improved antimicrobial agents.

  7. Redistribution of Cholesterol in Model Lipid Membranes in Response to the Membrane-Active Peptide Alamethicin

    NASA Astrophysics Data System (ADS)

    Heller, William; Qian, Shuo

    2013-03-01

    The cellular membrane is a heterogeneous, dynamic mixture of molecules and macromolecules that self-assemble into a tightly-regulated functional unit that provides a semipermeable barrier between the cell and its environment. Among the many compositional differences between mammalian and bacterial cell membranes that impact its physical properties, one key difference is cholesterol content, which is more prevalent in mammals. Cholesterol is an amphiphile that associates with membranes and serves to maintain its fluidity and permeability. Membrane-active peptides, such as the alpha-helical peptide alamethicin, interact with membranes in a concentration- and composition-dependent manner to form transmembrane pores that are responsible for the lytic action of the peptide. Through the use of small-angle neutron scattering and deuterium labeling, it was possible to observe a redistribution of the lipid and cholesterol in unilamellar vesicles in response to the presence of alamethicin at a peptide-to-lipid ratio of 1/200. The results demonstrate that the membrane remodeling powers of alamethicin reach beyond the membrane thinning effect to altering the localization of specific components in the bilayer, complementing the accepted two-state mechanism of pore formation. Research was supported by U. S. DOE-OBER (CSMB; FWP ERKP291) and the U. S. DOE-BES Scientific User Facilities Division (ORNL's SNS and HFIR).

  8. Bioprocess-centered molecular design (BMD) for the efficient production of an interfacially active peptide.

    PubMed

    Morreale, Giacomo; Lee, Eun Gyo; Jones, Daniel B; Middelberg, Anton P J

    2004-09-30

    The efficient expression and purification of an interfacially active peptide (mLac21) was achieved by using bioprocess-centered molecular design (BMD), wherein key bioprocess considerations are addressed during the initial molecular biology work. The 21 amino acid mLac21 peptide sequence is derived from the lac repressor protein and is shown to have high affinity for the oil-water interface, causing a substantial reduction in interfacial tension following adsorption. The DNA coding for the peptide sequence was cloned into a modified pET-31(b) vector to permit the expression of mLac21 as a fusion to ketosteroid isomerase (KSI). Rational iterative molecular design, taking into account the need for a scaleable bioprocess flowsheet, led to a simple and efficient bioprocess yielding mLac21 at 86% purity following ion exchange chromatography (and >98% following chromatographic polishing). This case study demonstrates that it is possible to produce acceptably pure peptide for potential commodity applications using common scaleable bioprocess unit operations. Moreover, it is shown that BMD is a powerful strategy that can be deployed to reduce bioseparation complexity.

  9. An antifungal peptide with antiproliferative activity toward tumor cells from red kidney beans.

    PubMed

    Li, Miao; Wang, Hexiang; Ng, Tzi Bun

    2011-06-01

    A 7.3-kDa antifungal peptide was purified from dried red kidney beans. The purification procedure entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, followed by fast protein liquid chromatography-gel filtration on Superdex 75. The peptide was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and CM-cellulose. It exhibited a molecular mass of 7.3 kDa in gel filtration and also in SDS-polyacrylamide gel electrophoresis, indicating that it is a single-chained protein. The N-terminal sequence of the peptide was DGVCFGGLANGDRT. The peptide exerted an antifungal action on Fusarium oxysporum with an IC₅₀ of 3.8±0.4 µM (mean±SD, n=3). It also inhibited mycelial growth in Mycosphaerella arachidicola. It suppressed growth of lymphoma MBL2 cells and leukemia L1210 cells with an IC₅₀ of 5.2±0.4 µM and 7.6±0.6 µM, respectively. HIV-1 reverse transcriptase was inhibited with an IC₅₀ of 40±3.2 µM. However, no activity was demonstrated toward other viral enzymes. PMID:21309741

  10. Probing the Mec1ATR Checkpoint Activation Mechanism with Small Peptides.

    PubMed

    Wanrooij, Paulina H; Tannous, Elias; Kumar, Sandeep; Navadgi-Patil, Vasundhara M; Burgers, Peter M

    2016-01-01

    Yeast Mec1, the ortholog of human ATR, is the apical protein kinase that initiates the cell cycle checkpoint in response to DNA damage and replication stress. The basal activity of Mec1 kinase is activated by cell cycle phase-specific activators. Three distinct activators stimulate Mec1 kinase using an intrinsically disordered domain of the protein. These are the Ddc1 subunit of the 9-1-1 checkpoint clamp (ortholog of human and Schizosaccharomyces pombe Rad9), the replication initiator Dpb11 (ortholog of human TopBP1 and S. pombe Cut5), and the multifunctional nuclease/helicase Dna2. Here, we use small peptides to determine the requirements for Mec1 activation. For Ddc1, we identify two essential aromatic amino acids in a hydrophobic environment that when fused together are proficient activators. Using this increased insight, we have been able to identify homologous motifs in S. pombe Rad9 that can activate Mec1. Furthermore, we show that a 9-amino acid Dna2-based peptide is sufficient for Mec1 activation. Studies with mutant activators suggest that binding of an activator to Mec1 is a two-step process, the first step involving the obligatory binding of essential aromatic amino acids to Mec1, followed by an enhancement in binding energy through interactions with neighboring sequences.

  11. Lysine-tagged peptide coupling onto polylactide nanoparticles coated with activated ester-based amphiphilic copolymer: a route to highly peptide-functionalized biodegradable carriers.

    PubMed

    Handké, Nadège; Ficheux, Damien; Rollet, Marion; Delair, Thierry; Mabrouk, Kamel; Bertin, Denis; Gigmes, Didier; Verrier, Bernard; Trimaille, Thomas

    2013-03-01

    Efficient biomolecule conjugation to the surface of biodegradable colloidal carriers is crucial for their targeting efficiency in drug/vaccine delivery applications. We here propose a potent strategy to drastically improve peptide immobilization on biodegradable polylactide (PLA) nanoparticles (NPs). Our approach particularly relies on the use of an amphiphilic block copolymer PLA-b-poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) (PLA-b-P(NAS-co-NVP)) as NP surface modifier, whose the N-succinimidyl (NS) ester functions of the NAS units along the polymer chain ensure N-terminal amine peptide coupling. The well-known immunostimulatory peptide sequence derived from the human interleukin 1β (IL-1β), VQGEESNDK, was coupled on the NPs of 169 nm mean diameter in phosphate buffer (pH 8, 10 mM). A maximum amount of 2 mg immobilized per gram of NPs (i.e. 0.042 peptidenm(-2)) was obtained. Introduction of a three lysine tag at the peptide N-terminus (KKKVQGEESNDK) resulted in a dramatic improvement of the immobilized peptide amounts (27.5 mg/g NP, i.e. 0.417 peptidenm(-2)). As a comparison, the density of tagged peptide achievable on surfactant free PLA NPs of similar size (140 nm), through classical EDC or EDC/NHS activation of the surface PLA carboxylic end-groups, was found to be 6 mg/g NP (i.e. 0.075 peptidenm(-2)), showing the decisive impact of the P(NAS-co-NVP)-based hairy corona for high peptide coupling. These results demonstrate that combined use of lysine tag and PLA-b-P(NAS-co-NVP) surfactant represents a valuable platform to tune and optimize surface bio-functionalization of PLA-based biodegradable carriers.

  12. [Expression, purification of recombinant cationic peptide AIK in Escherichia coli and its antitumor activity].

    PubMed

    Fan, Fangfang; Sun, Huiying; Xu, Hui; Liu, Jiawei; Zhang, Haiyuan; Li, Yilan; Ning, Xuelian; Sun, Yue; Bai, Jing; Fu, Songbin; Zhou, Chunshui

    2015-12-01

    AIK is a novel cationic peptide with potential antitumor activity. In order to construct the AIK expression vector by Gateway technology, and establish an optimal expression and purification method for recombinant AIK, a set of primers containing AttB sites were designed and used to create the AttB-TEV-FLAG-AIR fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by attB and attP mediated recombination (BP reaction), then, transferred into the destination vector pDESTl 5 by attL and attR mediated recombination (LR reaction). All the cloning was verified by both colony PCR and DNA sequencing. The BL21 F. coli transformed by the GST-AIR expression plasmid was used to express the GST-AIK fusion protein with IPTG induction and the induction conditions were optimized. GST-AIR fusion protein was purified by glutathione magnetic beads, followed by rTEV cleavage to remove GST tag and MTS assay to test the growth inhibition activity of the recombinant AIR on human leukemia HL-60 cells. We found that a high level of soluble expression of GST-AIK protein (more than 30% out of the total bacterial proteins) was achieved upon 0.1 mmol/L ITPG induction for 4 h at 37 °C in the transformed BL21 F. coli with starting OD₆₀₀ at 1.0. Through GST affinity purification and rTEV cleavage, the purity of the resulting recombinant AIK was greater than 95%. And the MTS assays on HL-60 cells confirmed that the recombinant AIK retains an antitumor activity at a level similar to the chemically synthesized AIK. Taken together, we have established a method for expression and purification of recombinant AIK with a potent activity against tumor cells, which will be beneficial for the large-scale production and application of recombinant AIK in the future. PMID:27093838

  13. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, Virginia C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program --now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history The missions will develop technology and acquire data necessary for eventual human Exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines be opportunities for the Mars community to provide input into the landing site selection process.

  14. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, V. C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program -- now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history. The missions will develop technology and acquire data necessary for eventual human exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines the opportunities for the Mars community to provide input into the landing site selection process.

  15. The antimicrobial peptide pardaxin exerts potent anti-tumor activity against canine perianal gland adenoma

    PubMed Central

    Pan, Chieh-Yu; Lin, Chao-Nan; Chiou, Ming-Tang; Yu, Chao Yuan; Chen, Jyh-Yih; Chien, Chi-Hsien

    2015-01-01

    Pardaxin is an antimicrobial peptide of 33 amino acids, originally isolated from marine fish. We previously demonstrated that pardaxin has anti-tumor activity against murine fibrosarcoma, both in vitro and in vivo. In this study, we examined the anti-tumor activity, toxicity profile, and maximally-tolerated dose of pardaxin treatment in dogs with different types of refractory tumor. Local injection of pardaxin resulted in a significant reduction of perianal gland adenoma growth between 28 and 38 days post-treatment. Surgical resection of canine histiocytomas revealed large areas of ulceration, suggesting that pardaxin acts like a lytic peptide. Pardaxin treatment was not associated with significant variations in blood biochemical parameters or secretion of immune-related proteins. Our findings indicate that pardaxin has strong therapeutic potential for treating perianal gland adenomas in dogs. These data justify the veterinary application of pardaxin, and also provide invaluable information for veterinary medicine and future human clinical trials. PMID:25544775

  16. Describing the Mechanism of Antimicrobial Peptide Action with the Interfacial Activity Model

    PubMed Central

    Wimley, William C.

    2010-01-01

    Antimicrobial peptides (AMPs) have been studied for three decades, and yet a molecular understanding of their mechanism of action is still lacking. Here we summarize current knowledge for both synthetic vesicle experiments and microbe experiments, with a focus on comparisons between the two. Microbial experiments are done at peptide:lipid ratios that are at least 4 orders of magnitude higher than vesicle-based experiments. To close the gap between the two concentration regimes, we propose an “interfacial activity model”, which is based on an experimentally testable molecular image of AMP-membrane interactions. The interfacial activity model may be useful in driving engineering and design of novel AMPs. PMID:20698568

  17. Structure and antimicrobial activity of platypus 'intermediate' defensin-like peptide.

    PubMed

    Torres, Allan M; Bansal, Paramjit; Koh, Jennifer M S; Pagès, Guilhem; Wu, Ming J; Kuchel, Philip W

    2014-05-01

    The three-dimensional structure of a chemically synthesized peptide that we have called 'intermediate' defensin-like peptide (Int-DLP), from the platypus genome, was determined by nuclear magnetic resonance (NMR) spectroscopy; and its antimicrobial activity was investigated. The overall structural fold of Int-DLP was similar to that of the DLPs and β-defensins, however the presence of a third antiparallel β-strand makes its structure more similar to the β-defensins than the DLPs. Int-DLP displayed potent antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. The four arginine residues at the N-terminus of Int-DLP did not affect the overall fold, but were important for its antimicrobial potency. PMID:24694388

  18. Measurement of lecithin-cholesterol acyltransferase activity with the use of a Peptide-proteoliposome substrate.

    PubMed

    Vaisman, Boris L; Remaley, Alan T

    2013-01-01

    Lecithin-cholesterol acyltransferase (LCAT) is the major enzyme responsible for the esterification of free cholesterol on plasma lipoproteins, which is a key step in the reverse cholesterol transport pathway. The measurement of plasma LCAT activity not only is important in the diagnosis of patients with genetic or acquired LCAT deficiency but is also valuable in calculating cardiovascular risk, as well as in research studies of lipoprotein metabolism. In this chapter, we describe a convenient LCAT assay based on the use of an apoA-I mimetic peptide. The proteoliposome substrate used in this assay for LCAT is easily made with the peptide and can be stored by deep freezing without significant loss of activity. PMID:23912995

  19. Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type.

    PubMed Central

    Tomkinson, B; Wernstedt, C; Hellman, U; Zetterqvist, O

    1987-01-01

    The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [3H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases. PMID:3313395

  20. Site-Selective Binding of Nanoparticles to Double-Stranded DNA via Peptide Nucleic Acid "Invasion"

    SciTech Connect

    Stadler, A.L.; van der Lelie, D.; Sun, D.; Maye, M. M.; Gang, O.

    2011-04-01

    We demonstrate a novel method for by-design placement of nano-objects along double-stranded (ds) DNA. A molecular intercalator, designed as a peptide nucleic acid (PNA)-DNA chimera, is able to invade dsDNA at the PNA-side due to the hybridization specificity between PNA and one of the duplex strands. At the same time, the single-stranded (ss) DNA tail of the chimera, allows for anchoring of nano-objects that have been functionalized with complementary ssDNA. The developed method is applied for interparticle attachment and for the fabrication of particle clusters using a dsDNA template. This method significantly broadens the molecular toolbox for constructing nanoscale systems by including the most conventional not yet utilized DNA motif, double helix DNA.

  1. Site-specific labeling of proteins and peptides with trans-cyclooctene containing handles capable of tetrazine ligation.

    PubMed

    Wollack, James W; Monson, Benjamin J; Dozier, Jonathan K; Dalluge, Joseph J; Poss, Kristina; Hilderbrand, Scott A; Distefano, Mark D

    2014-08-01

    There is a growing library of functionalized non-natural substrates for the enzyme protein farnesyltransferase (PFTase). PFTase covalently attaches these functionalized non-natural substrates to proteins ending in the sequence CAAX, where C is a cysteine that becomes alkylated, A represents an aliphatic amino acid, and X is Ser, Met, Ala, or Gln. Reported substrates include a variety of functionalities that allow modified proteins to undergo subsequent bioconjugation reactions. To date the most common strategy used in this approach has been copper catalyzed azide-alkyne cycloaddition (CuAAC). While being fast and bioorthogonal CuAAC has limited use in live cell experiments due to copper's toxicity.(1) Here, we report the synthesis of trans-cyclooctene geranyl diphosphate. This substrate can be synthesized from geraniol in six steps and be enzymatically transferred to peptides and proteins that end in a CAAX sequence. Proteins and peptides site-specially modified with trans-cyclooctene geranyl diphosphate were subsequently targeted for further modification via tetrazine ligation. As tetrazine ligation is bioorthogonal, fast, and is contingent on ring strain rather than the addition of a copper catalyst, this labeling strategy should prove useful for labeling proteins where the presence of copper may hinder solubility or biological reactivity.

  2. Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 2: Peptide Tags and Unnatural Amino Acids.

    PubMed

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M

    2016-04-01

    Molecular imaging using radioisotope- or fluorophore-labeled antibodies is increasingly becoming a critical component of modern precision medicine. Yet despite this promise, the vast majority of these immunoconjugates are synthesized via the random coupling of amine-reactive bifunctional probes to lysines within the antibody, a process that can result in heterogeneous and poorly defined constructs with suboptimal pharmacological properties. In an effort to circumvent these issues, the last 5 years have played witness to a great deal of research focused on the creation of effective strategies for the site-specific attachment of payloads to antibodies. These chemoselective modification methods yield immunoconjugates that are more homogenous and better defined than constructs created using traditional synthetic approaches. Moreover, site-specifically labeled immunoconjugates have also been shown to exhibit superior in vivo behavior compared to their randomly modified cousins. The over-arching goal of this two-part review is to provide a broad yet detailed account of the various site-specific bioconjugation approaches that have been used to create immunoconjugates for positron emission tomography (PET), single photon emission computed tomography (SPECT), and fluorescence imaging. In Part 1, we covered site-specific bioconjugation techniques based on the modification of cysteine residues and the chemoenzymatic manipulation of glycans. In Part 2, we will detail two families of bioconjugation approaches that leverage biochemical tools to achieve site-specificity. First, we will discuss modification methods that employ peptide tags either as sites for enzyme-catalyzed ligations or as radiometal coordination architectures. And second, we will examine bioconjugation strategies predicated on the incorporation of unnatural or non-canonical amino acids into antibodies via genetic engineering. Finally, we will compare the advantages and disadvantages of the modification

  3. Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 2: Peptide Tags and Unnatural Amino Acids

    PubMed Central

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M.

    2016-01-01

    Molecular imaging using radioisotope- or fluorophore-labeled antibodies is increasingly becoming a critical component of modern precision medicine. Yet despite this promise, the vast majority of these immunoconjugates are synthesized via the random coupling of amine-reactive bifunctional probes to lysines within the antibody, a process that can result in heterogeneous and poorly defined constructs with suboptimal pharmacological properties. In an effort to circumvent these issues, the last 5 years have played witness to a great deal of research focused on the creation of effective strategies for the site-specific attachment of payloads to antibodies. These chemoselective modification methods yield immunoconjugates that are more homogenous and better defined than constructs created using traditional synthetic approaches. Moreover, site-specifically labeled immunoconjugates have also been shown to exhibit superior in vivo behavior compared to their randomly modified cousins. The over-arching goal of this two-part review is to provide a broad yet detailed account of the various site-specific bioconjugation approaches that have been used to create immunoconjugates for positron emission tomography (PET), single photon emission computed tomography (SPECT), and fluorescence imaging. In Part 1, we covered site-specific bioconjugation techniques based on the modification of cysteine residues and the chemoenzymatic manipulation of glycans. In Part 2, we will detail two families of bioconjugation approaches that leverage biochemical tools to achieve site-specificity. First, we will discuss modification methods that employ peptide tags either as sites for enzyme-catalyzed ligations or as radiometal coordination architectures. And second, we will examine bioconjugation strategies predicated on the incorporation of unnatural or non-canonical amino acids into antibodies via genetic engineering. Finally, we will compare the advantages and disadvantages of the modification

  4. Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active Site*S

    PubMed Central

    Maresso, Anthony W.; Wu, Ruiying; Kern, Justin W.; Zhang, Rongguang; Janik, Dorota; Missiakas, Dominique M.; Duban, Mark-Eugene; Joachimiak, Andrzej; Schneewind, Olaf

    2011-01-01

    Sortases anchor surface proteins to the cell wall of Gram-positive pathogens through recognition of specific motif sequences. Loss of sortase leads to large reductions in virulence, which identifies sortase as a target for the development of antibacterials. By screening 135,625 small molecules for inhibition, we report here that aryl (β-amino)ethyl ketones inhibit sortase enzymes from staphylococci and bacilli. Inhibition of sortases occurs through an irreversible, covalent modification of their active site cysteine. Sortases specifically activate this class of molecules via β-elimination, generating a reactive olefin intermediate that covalently modifies the cysteine thiol. Analysis of the three-dimensional structure of Bacillus anthracis sortase B with and without inhibitor provides insights into the mechanism of inhibition and reveals binding pockets that can be exploited for drug discovery. PMID:17545669

  5. The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

    S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the

  6. Identification and biological activity of ovine and caprine calcitonin receptor-stimulating peptides 1 and 2.

    PubMed

    Charles, Christopher J; Katafuchi, Takeshi; Yandle, Timothy G; Minamino, Naoto

    2008-08-01

    We have recently reported the isolation of three new members of the calcitonin (CT) gene-related peptide family of peptides, the CT receptor (CT-R)-stimulating peptides (CRSPs). We now report the sequencing and characterization of ovine/caprine CRSP-1 and caprine CRSP-2. Mature ovine and caprine CRSP-1 are identical and have strong structural homology to CRSP-1s identified to date from other species. As with other CRSP-1s, ovine/caprine CRSP-1 binds to and activates the CT-R but not the CT-like receptor (CL-R) in combination with the receptor activity-modifying proteins (RAMPs). By contrast, caprine CRSP-2 does not activate any of these receptor-RAMP complexes. Intravenous infusions of ovine CRSP-1 to normal conscious sheep induced dose-dependent reduction in plasma total Ca levels (P=0.02) and corrected Ca levels (P=0.017) associated with increases in plasma cAMP (P=0.002). CRSP-1 reduced both plasma amino-terminal pro-C-type natriuretic peptide levels (P=0.006) and plasma renin activity (P=0.028). There were no significant effects observed on hemodynamic or renal indices measured. In conclusion, we have sequenced ovine/caprine CRSP-1 and caprine CRSP-2 precursors. This newly identified CRSP-1 has been shown to share the structural and biological features of CRSP-1s known to date. In vivo studies confirm that ovine CRSP-1 reduces plasma Ca levels in sheep, presumably via a cAMP-mediated mechanism. By contrast, caprine CRSP-2 did not stimulate any combination of CT-R, CL-R, and RAMPs. Accession numbers of cDNA determined in this study are caprine CRSP-1, AB364646; caprine CRSP-2, AB364647; and ovine CRSP-1, AB364648.

  7. Selective immunosuppression by administration of major histocompatibility complex (MHC) class II-binding peptides. I. Evidence for in vivo MHC blockade preventing T cell activation

    PubMed Central

    1992-01-01

    Draining lymph node cells (LNC) from mice immunized with hen egg white lysozyme (HEL) display at their surface antigen-MHC complexes able to stimulate, in the absence of any further antigen addition, HEL peptide- specific, class II-restricted T cell hybridomas. Chloroquine addition to these LNC cultures fails to inhibit antigen presentation, indicating that antigenic complexes of class II molecules and HEL peptides are formed in vivo. MHC class II restriction of antigen presentation by LNC from HEL-primed mice was verified by the use of anti-class II monoclonal antibodies. Coinjection of HEL and the I-Ak-binding peptide HEL 112-129 in mice of H-2k haplotype inhibits the ability of LNC to stimulate I-Ak-restricted, HEL 46-61-specific T cell hybridomas. Similar results are obtained in mice coinjected with the HEL peptides 46-61 and 112-129. Inhibition of T hybridoma activation can also be observed using as antigen-presenting cells irradiated, T cell-depleted LNC from mice coinjected with HEL 46-61 and HEL 112-129, ruling out the possible role of either specific or nonspecific suppressor T cells. Inhibition of T cell proliferation is associated with MHC-specific inhibition of antigen presentation and with occupancy by the competitor of class II binding sites, as measured by activation of peptide- specific T cell hybridomas. These results demonstrate that administration of MHC class II binding peptide competitors selectively inhibits antigen presentation to class II-restricted T cells, indicating competitive blockade of class II molecules in vivo. PMID:1569402

  8. Aqueous Extract of Chrysanthemum morifolium (菊花 Jú Huā) Enhances the Antimelanogenic and Antioxidative Activities of the Mixture of Soy Peptide and Collagen Peptide

    PubMed Central

    Gui, Min; Du, Jun; Guo, Jianmin; Xiao, Baiquan; Yang, Wei; Li, Minjie

    2014-01-01

    The possible synergistic effect between the aqueous extract of Chrysanthemum morifolium (菊花 Jú Huā) (AECM) and the peptide mixture (PM) containing soy peptide and collagen peptide was investigated in an ultraviolet (UV) irradiation–induced skin damage mouse model. The irradiated mice were treated with the PM or PM + AECM (containing PM and AECM), respectively. Both PM and PM + AECM groups displayed an apparent photoprotective effect on the UV-irradiated skin damage of mice. Histological evaluation demonstrated that the epidermal hyperplasia and melanocytes in the basal epidermal layer of the UV-irradiated skin in mice decreased when treated with either PM or PM + AECM. Further study showed that soy peptide, collagen peptide, and AECM also inhibited the activities of mushroom tyrosinase with IC50 values of 82.3, 28.2, and 1.6 μg/ml, respectively. Additionally, PM + AECM reduced melanogenesis by 46.2% at the concentration of 10 mg/ml in B16 mouse melanoma cells. Meanwhile, the UV-induced increase of antioxidative indicators, including glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and malondialdehyde (MDA), was reduced significantly after treatment with 1.83 g/kg/dbw of PM + AECM. This evidence supported the synergistic antioxidative effect of AECM with PM. These results demonstrated that oral intake of PM and AECM had synergistic antimelanogenic and antioxidative effects in UV-irradiated mice. PMID:25161922

  9. Aqueous Extract of Chrysanthemum morifolium ( Jú Huā) Enhances the Antimelanogenic and Antioxidative Activities of the Mixture of Soy Peptide and Collagen Peptide.

    PubMed

    Gui, Min; Du, Jun; Guo, Jianmin; Xiao, Baiquan; Yang, Wei; Li, Minjie

    2014-07-01

    The possible synergistic effect between the aqueous extract of Chrysanthemum morifolium ( Jú Huā) (AECM) and the peptide mixture (PM) containing soy peptide and collagen peptide was investigated in an ultraviolet (UV) irradiation-induced skin damage mouse model. The irradiated mice were treated with the PM or PM + AECM (containing PM and AECM), respectively. Both PM and PM + AECM groups displayed an apparent photoprotective effect on the UV-irradiated skin damage of mice. Histological evaluation demonstrated that the epidermal hyperplasia and melanocytes in the basal epidermal layer of the UV-irradiated skin in mice decreased when treated with either PM or PM + AECM. Further study showed that soy peptide, collagen peptide, and AECM also inhibited the activities of mushroom tyrosinase with IC50 values of 82.3, 28.2, and 1.6 μg/ml, respectively. Additionally, PM + AECM reduced melanogenesis by 46.2% at the concentration of 10 mg/ml in B16 mouse melanoma cells. Meanwhile, the UV-induced increase of antioxidative indicators, including glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and malondialdehyde (MDA), was reduced significantly after treatment with 1.83 g/kg/dbw of PM + AECM. This evidence supported the synergistic antioxidative effect of AECM with PM. These results demonstrated that oral intake of PM and AECM had synergistic antimelanogenic and antioxidative effects in UV-irradiated mice.

  10. A conjugate of the lytic peptide Hecate and gallic acid: structure, activity against cervical cancer, and toxicity.

    PubMed

    Sanches, Paulo R S; Carneiro, Bruno M; Batista, Mariana N; Braga, Ana Cláudia S; Lorenzón, Esteban N; Rahal, Paula; Cilli, Eduardo Maffud

    2015-07-01

    Conjugate compounds constitute a new class of molecules of important biological interest mainly for the treatment of diseases such as cancer. The N-terminus region of cationic peptides has been described as important for their biological activity. The aim of this study was to evaluate the lytic peptide Hecate (FALALKALKKALKKLKKALKKAL) and the effect of conjugating this macromolecule with gallic acid (C7H6O5) in terms of structure, anti-cancer activity, and toxicity. An N-terminus GA-Hecate peptide conjugate was synthesized to provide information regarding the relationship between the amino-terminal region and its charge and the secondary structure and biological activity of the peptide; and the effects of gallic acid on these parameters. Peptide secondary structure was confirmed using circular dichroism (CD). The CD measurements showed that the peptide has a high incidence of α-helical structures in the presence of SDS and LPC, while GA-Hecate presented lower incidence of α-helical structures in the same chemical environment. An evaluation of the anti-cancer activity in HeLa cancer cells indicated that both peptides are active, but that coupling gallic acid at the N-terminus decreased the activity of the free peptide. GA-Hecate showed lower activity in non-tumor keratinocyte cells but higher hemolytic activity. Our findings suggest that the N-terminus of Hecate plays an important role in its activity against cervical cancer by affecting it secondary structure, toxicity, and hemolytic activity. This study highlights the importance of the N-terminus in antitumor activity and could provide an important tool for developing new anti-cancer drugs.

  11. In vitro activities of lytic peptides against the sporozoites of Cryptosporidium parvum.

    PubMed Central

    Arrowood, M J; Jaynes, J M; Healey, M C

    1991-01-01

    Cryptosporidium parvum is a protozoan parasite that causes mild to severe diarrheal disease in animals and humans. There are currently no effective chemotherapeutic agents available for the treatment of cryptosporidiosis. Recent studies have described small, naturally occurring antimicrobial lytic peptides with antiprotozoal activities. In the present study, the anticryptosporidial activities of three synthetic lytic peptides were determined in an in vitro sporozoite susceptibility assay. Sporozoite viability was assessed microscopically by the uptake of the vital dyes fluorescein diacetate and propidium iodide. Sporozoite viability was reduced by 93.5% following a 60-min exposure to 10 microM Hecate-1 at 37 degrees C. Shiva-10 reduced sporozoite viability by approximately 74.0% after a 60-min exposure at 100 microM and 37 degrees C. The cecropin-b analog SB-37 reduced sporozoite viability by 6.0% following a 60-min exposure at 100 microM and 37 degrees C. A control peptide showed no anticryptosporidial activity. PMID:1708975

  12. RV-23, a Melittin-Related Peptide with Cell-Selective Antibacterial Activity and High Hemocompatibility.

    PubMed

    Zhang, Shi-Kun; Ma, Qian; Li, Su-Bo; Gao, Hong-Wei; Tan, Ying-Xia; Gong, Feng; Ji, Shou-Ping

    2016-06-28

    RV-23 is a melittin-related antibacterial peptide (MRP) with lower cytotoxicity than either melittin or AR-23, another MRP. The aim of this study was to explore the mechanism of RV- 23's antibacterial selectivity and its hemocompatibility. The results showed that all the peptides exhibited lytic activity against Staphylococcus aureus and Escherichia coli, with RV-23 showing the highest potency. Moreover, RV-23 had lower cytotoxicity than melittin or AR-23 at their minimal inhibitory concentration. In addition, CD experiments showed that melittin, RV-23, and AR-23 all had a typical α-helical structure, and RV-23 had the lowest α-helix content. The structural information showed that RV-23 has the lowest hydrophobicity and highest hydrophobic moment. Because hydrophobicity and α-helix content are believed to correlate with hemolysis, the results indicate that the selective lytic activity against bacteria of RV-23 may be due to its low hydrophobicity and α-helicity, which lead to low cytotoxicity without affecting antibacterial activity. Furthermore, RV-23 did not affect the structure and function of blood components such as red blood cells, platelets, albumin, and the blood coagulation system. In conclusion, RV-23 is a cell-selective antibacterial peptide with high hemocompatibility due to its unique structure. PMID:26975766

  13. Trypanocidal and leishmanicidal activities of different antimicrobial peptides (AMPs) isolated from aquatic animals.

    PubMed

    Löfgren, S E; Miletti, L C; Steindel, M; Bachère, E; Barracco, M A

    2008-02-01

    Most of the available animal antimicrobial peptides (AMPs) have been tested against bacteria and fungi, but very few against protozoan parasites. In the present study, we investigated the antiparasitic activity of different AMPs isolated from aquatic animals: tachyplesin (Tach, from Tachypleus tridentatus), magainin (Mag, from Xenopus laevis), clavanin (Clav, from Styela clava), penaeidin (Pen, from Litopenaeus vannamei), mytilin (Myt, from Mytilus edulis) and anti-lipopolysaccharide factor (ALF, from Penaeus monodon). The antiparasitic activity was evaluated against the promastigote form of Leishmania braziliensis and epi and trypomastigote forms of Trypanosoma cruzi, through the MTT method. Tach was the most potent peptide, killing completely L. braziliensis and trypomastigote T. cruzi from 12.5microM, whereas Pen and Clav were weakly active against trypomastigotes and Myt against L. braziliensis, only at a high concentration (100microM). Tach and Mag were markedly hemolytic at high concentrations, whereas the other peptides caused only a slight hemolysis (<10% up to 50microM). Our results point to Tach as the only potential candidate for further investigation and potential application as a therapeutic agent. PMID:17888907

  14. Helical 1:1 α/Sulfono-γ-AA Heterogeneous Peptides with Antibacterial Activity.

    PubMed

    She, Fengyu; Nimmagadda, Alekhya; Teng, Peng; Su, Ma; Zuo, Xiaobing; Cai, Jianfeng

    2016-05-01

    As one of the greatest threats facing the 21st century, antibiotic resistance is now a major public health concern. Host-defense peptides (HDPs) offer an alternative approach to combat emerging multi-drug-resistant bacteria. It is known that helical HDPs such as magainin 2 and its analogs adopt cationic amphipathic conformations upon interaction with bacterial membranes, leading to membrane disruption and subsequent bacterial cell death. We have previously shown that amphipathic sulfono-γ-AApeptides could mimic magainin 2 and exhibit bactericidal activity. In this article, we demonstrate for the first time that amphipathic helical 1:1 α/sulfono-γ-AA heterogeneous peptides, in which regular amino acids and sulfono-γ-AApeptide building blocks are alternatively present in a 1:1 pattern, display potent antibacterial activity against both Gram-positive and Gram-negative bacterial pathogens. Small angle X-ray scattering (SAXS) suggests that the lead sequences adopt defined helical structures. The subsequent studies including fluorescence microscopy and time-kill experiments indicate that these hybrid peptides exert antimicrobial activity by mimicking the mechanism of HDPs. Our findings may lead to the development of HDP-mimicking antimicrobial peptidomimetics that combat drug-resistant bacterial pathogens. In addition, our results also demonstrate the effective design of a new class of helical foldamer, which could be employed to interrogate other important biological targets such as protein-protein interactions in the future. PMID:27030636

  15. Structure-activity relationship of lipid core peptide-based Group A Streptococcus vaccine candidates.

    PubMed

    Chan, Amy; Hussein, Waleed M; Ghaffar, Khairunnisa Abdul; Marasini, Nirmal; Mostafa, Ahmed; Eskandari, Sharareh; Batzloff, Michael R; Good, Michael F; Skwarczynski, Mariusz; Toth, Istvan

    2016-07-15

    Infection with Group A Streptococcus (GAS) can result in a range of different illnesses, some of which are fatal. Currently, our efforts to develop a vaccine against GAS focuses on the lipid core peptide (LCP) system, a subunit vaccine containing a lipoamino acid (LAA) moiety which allows the stimulation of systemic antibody activity. In the present study, a peptide (J14) representing the B-cell epitope from the GAS M protein was incorporated alongside a universal T-helper epitope (P25) in four LCP constructs of different spatial orientation or LAA lengths. Through structure-activity studies, it was discovered that while the alteration of the LCP orientation had a weaker effect on immunostimulation, increasing the LAA side chain length within the construct increased antibody responses in murine models. Furthermore, the mice immunised with the lead LCP construct were also able to maintain antibody activity throughout the course of five months. These findings highlight the importance of LAA moieties in the development of intranasal peptide vaccines and confirmed that its side chain length has an effect on the immunogenicity of the structure. PMID:27246859

  16. Transformable Peptide Nanocarriers for Expeditious Drug Release and Effective Cancer Therapy via Cancer‐Associated Fibroblast Activation

    PubMed Central

    Ji, Tianjiao; Zhao, Ying; Ding, Yanping; Wang, Jing; Zhao, Ruifang; Lang, Jiayan; Qin, Hao; Liu, Xiaoman; Shi, Jian; Tao, Ning; Qin, Zhihai; Nie, Guangjun

    2015-01-01

    Abstract A novel cleavable amphiphilic peptide (CAP) was designed to be specifically responsive to fibroblast activation protein‐α (FAP‐α), a protease specifically expressed on the surface of cancer‐associated fibroblasts. The CAP self‐assembled into fiber‐like nanostructures in solution, while the presence of hydrophobic chemotherapeutic drugs readily transformed the assemblies into drug‐loaded spherical nanoparticles. The disassembly of these nanoparticles (CAP‐NPs) upon FAP‐α cleavage resulted in rapid and efficient release of the encapsulated drugs specifically at tumor sites. This Transformers‐like drug delivery strategy could allow them to disrupt the stromal barrier and enhance local drug accumulation. Therapeutic results suggested that drug‐loaded CAP‐NPs hold promising tumor specificity and therapeutic efficacy for various solid tumor models, confirming its potential utility and versatility in antitumor therapy. PMID:26283097

  17. Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity-A Structural and Biochemical Approach.

    PubMed

    Dutta, Sruti; Choudhury, Debi; Roy, Sumana; Dattagupta, Jiban Kanti; Biswas, Sampa

    2016-01-01

    Papain-like proteases contain an N-terminal pro-peptide in their zymogen form that is important for correct folding and spatio-temporal regulation of the proteolytic activity of these proteases. Catalytic removal of the pro-peptide is required for the protease to become active. In this study, we have generated three different mutants of papain (I86F, I86L and I86A) by replacing the residue I86 in its pro-peptide region, which blocks the specificity determining S2-subsite of the catalytic cleft of the protease in its zymogen form with a view to investigate the effect of mutation on the catalytic activity of the protease. Steady-state enzyme kinetic analyses of the corresponding mutant proteases with specific peptide substrates show significant alteration of substrate specificity-I86F and I86L have 2.7 and 29.1 times higher kcat/Km values compared to the wild-type against substrates having Phe and Leu at P2 position, respectively, while I86A shows lower catalytic activity against majority of the substrates tested. Far-UV CD scan and molecular mass analyses of the mature form of the mutant proteases reveal similar CD spectra and intact masses to that of the wild-type. Crystal structures of zymogens of I86F and I86L mutants suggest that subtle reorganization of active site residues, including water, upon binding of the pro-peptide may allow the enzyme to achieve discriminatory substrate selectivity and catalytic efficiency. However, accurate and reliable predictions on alteration of substrate specificity require atomic resolution structure of the catalytic domain after zymogen activation, which remains a challenging task. In this study we demonstrate that through single amino acid substitution in pro-peptide, it is possible to modify the substrate specificity of papain and hence the pro-peptide of a protease can also be a useful target for altering its catalytic activity/specificity. PMID:27352302

  18. Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity—A Structural and Biochemical Approach

    PubMed Central

    Dutta, Sruti; Choudhury, Debi; Roy, Sumana; Dattagupta, Jiban Kanti; Biswas, Sampa

    2016-01-01

    Papain-like proteases contain an N-terminal pro-peptide in their zymogen form that is important for correct folding and spatio-temporal regulation of the proteolytic activity of these proteases. Catalytic removal of the pro-peptide is required for the protease to become active. In this study, we have generated three different mutants of papain (I86F, I86L and I86A) by replacing the residue I86 in its pro-peptide region, which blocks the specificity determining S2-subsite of the catalytic cleft of the protease in its zymogen form with a view to investigate the effect of mutation on the catalytic activity of the protease. Steady-state enzyme kinetic analyses of the corresponding mutant proteases with specific peptide substrates show significant alteration of substrate specificity—I86F and I86L have 2.7 and 29.1 times higher kcat/Km values compared to the wild-type against substrates having Phe and Leu at P2 position, respectively, while I86A shows lower catalytic activity against majority of the substrates tested. Far-UV CD scan and molecular mass analyses of the mature form of the mutant proteases reveal similar CD spectra and intact masses to that of the wild-type. Crystal structures of zymogens of I86F and I86L mutants suggest that subtle reorganization of active site residues, including water, upon binding of the pro-peptide may allow the enzyme to achieve discriminatory substrate selectivity and catalytic efficiency. However, accurate and reliable predictions on alteration of substrate specificity require atomic resolution structure of the catalytic domain after zymogen activation, which remains a challenging task. In this study we demonstrate that through single amino acid substitution in pro-peptide, it is possible to modify the substrate specificity of papain and hence the pro-peptide of a protease can also be a useful target for altering its catalytic activity/specificity. PMID:27352302

  19. The Spider Venom Peptide Lycosin-II Has Potent Antimicrobial Activity against Clinically Isolated Bacteria

    PubMed Central

    Wang, Yongjun; Wang, Ling; Yang, Huali; Xiao, Haoliang; Farooq, Athar; Liu, Zhonghua; Hu, Min; Shi, Xiaoliu

    2016-01-01

    Antimicrobial peptides have been accepted as excellent candidates for developing novel antibiotics against drug-resistant bacteria. Recent studies indicate that spider venoms are the source for the identification of novel antimicrobial peptides. In the present study, we isolated and characterized an antibacterial peptide named lycosin-II from the venom of the spider Lycosa singoriensis. It contains 21 amino acid residue lacking cysteine residues and forms a typical linear amphipathic and cationic α-helical conformation. Lycosin-II displays potent bacteriostatic effect on the tested drug-resistant bacterial strains isolated from hospital patients, including multidrug-resistant A. baumannii, which has presented a huge challenge for the infection therapy. The inhibitory ability of lycosin-II might derive from its binding to cell membrane, because Mg2+ could compete with the binding sites to reduce the bacteriostatic potency of lycosin-II. Our data suggest that lycosin-II might be a lead in the development of novel antibiotics for curing drug-resistant bacterial infections. PMID:27128941

  20. The Spider Venom Peptide Lycosin-II Has Potent Antimicrobial Activity against Clinically Isolated Bacteria.

    PubMed

    Wang, Yongjun; Wang, Ling; Yang, Huali; Xiao, Haoliang; Farooq, Athar; Liu, Zhonghua; Hu, Min; Shi, Xiaoliu

    2016-04-26

    Antimicrobial peptides have been accepted as excellent candidates for developing novel antibiotics against drug-resistant bacteria. Recent studies indicate that spider venoms are the source for the identification of novel antimicrobial peptides. In the present study, we isolated and characterized an antibacterial peptide named lycosin-II from the venom of the spider Lycosa singoriensis. It contains 21 amino acid residue lacking cysteine residues and forms a typical linear amphipathic and cationic α-helical conformation. Lycosin-II displays potent bacteriostatic effect on the tested drug-resistant bacterial strains isolated from hospital patients, including multidrug-resistant A. baumannii, which has presented a huge challenge for the infection therapy. The inhibitory ability of lycosin-II might derive from its binding to cell membrane, because Mg(2+) could compete with the binding sites to reduce the bacteriostatic potency of lycosin-II. Our data suggest that lycosin-II might be a lead in the development of novel antibiotics for curing drug-resistant bacterial infections.

  1. Short, Synthetic Cationic Peptides Have Antibacterial Activity against Mycobacterium smegmatis by Forming Pores in Membrane and Synergizing with Antibiotics

    PubMed Central

    Gupta, Kajal; Singh, Sameer; van Hoek, Monique L.

    2015-01-01

    Multicellular organisms are constantly exposed to a multitude of pathogenic microbes. Infection is inhibited in vivo by the innate and adaptive immune system. Mycobacterium species have emerged that are resistant to most antibiotics. We identified several naturally occurring cationic antimicrobial peptides that were active at low micromolar concentrations against Mycobacterium smegmatis. Human-derived cathelicidin LL-37 is well characterized and studied against M. smegmatis; we compared LL-37 with Chinese cobra-derived cathelicidin NA-CATH and mouse cathelicidin (mCRAMP). Two synthetic 11-residue peptides (ATRA-1A and ATRA-2) containing variations of a repeated motif within NA-CATH were tested for their activity against M. smegmatis along with a short synthetic peptide derivative from the human beta-defensin hBD3 (hBD3-Pep4). We hypothesized that these smaller synthetic peptides may demonstrate antimicrobial effectiveness with shorter length (and at less cost), making them strong potential candidates for development into broad-spectrum antimicrobial compounds or use in combination with antibiotics. These peptides have antimicrobial activity with EC50 ranging from 0.05 to 1.88 μg/mL against Mycobacterium smegmatis. The ATRA-1A short peptide was found to be the most effective antimicrobial peptide (AMP) (EC50 = 0.05 μg/mL). High bactericidal activity correlated with bacterial membrane depolarization and permeabilization activities. The efficacy of the peptides was further analyzed through Minimal Inhibitory Concentration (MIC) assays. The MICs were determined by the microdilution method. The peptide mCRAMP showed the best MIC activity at 15.6 μg/mL. Neither of the effective short synthetic peptides demonstrated synergy with the antibiotic rifampicin, although both demonstrated synergy with the cyclic peptide antibiotic polymyxin B. The peptides LL-37 and mCRAMP displayed synergism with rifampicin in MIC assays, whereas antibiotic polymyxin B displayed synergism

  2. Structural analysis and insertion study reveal the ideal sites for surface displaying foreign peptides on a betanodavirus-like particle.

    PubMed

    Xie, Junfeng; Li, Kunpeng; Gao, Yuanzhu; Huang, Runqing; Lai, Yuxiong; Shi, Yan; Yang, Shaowei; Zhu, Guohua; Zhang, Qinfen; He, Jianguo

    2016-01-01

    Betanodavirus infection causes fatal disease of viral nervous necrosis in many cultured marine and freshwater fish worldwide and the virus-like particles (VLP) are effective vaccines against betanodavirus. But vaccine and viral vector designs of betanodavirus VLP based on their structures remain lacking. Here, the three-dimensional structure of orange-spotted grouper nervous necrosis virus (OGNNV) VLP (RBS) at 3.9 Å reveals the organization of capsid proteins (CP). Based on the structural results, seven putative important sites were selected to genetically insert a 6× histidine (His)-tag for VLP formation screen, resulting in four His-tagged VLP (HV) at positions N-terminus, Ala220, Pro292 and C-terminus. The His-tags of N-terminal HV (NHV) were concealed inside virions while those of 220HV and C-terminal HV (CHV) were displayed at the outer surface. NHV, 220HV and CHV maintained the same cell entry ability as RBS in the Asian sea bass (SB) cell line, indicating that their similar surface structures can be recognized by the cellular entry receptor(s). For application of vaccine design, chromatography-purified CHV could provoke NNV-specific antibody responses as strong as those of RBS in a sea bass immunization assay. Furthermore, in carrying capacity assays, N-terminus and Ala220 can only carry short peptides and C-terminus can even accommodate large protein such as GFP to generate fluorescent VLP (CGV). For application of a viral vector, CGV could be real-time visualized to enter SB cells in invasion study. All the results confirmed that the C-terminus of CP is a suitable site to accommodate foreign peptides for vaccine design and viral vector development. PMID:26754256

  3. Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies

    DOE PAGES

    Donaldson, Joshua M.; Zer, Cindy; Avery, Kendra N.; Bzymek, Krzysztof P.; Horne, David A.; Williams, John C.

    2013-10-07

    Capitalizing on their extraordinary specificity, monoclonal antibodies (mAbs) have become one of the most reengineered classes of biological molecules. A major goal in many of these engineering efforts is to add new functionality to the parental mAb, including the addition of cytotoxins and imaging agents for medical applications. Herein, we present a unique peptide-binding site within the central cavity of the fragment antigen binding framework region of the chimeric, anti-epidermal growth factor receptor mAb cetuximab. We demonstrate through diffraction methods, biophysical studies, and sequence analysis that this peptide, a meditope, has moderate affinity for the Fab, is specific to cetuximabmore » (i.e., does not bind to human IgGs), and has no significant effect on antigen binding. We further demonstrate by diffraction studies and biophysical methods that the meditope binding site can be grafted onto the anti-human epidermal growth factor receptor 2 mAb trastuzumab, and that the antigen binding affinity of the grafted trastuzumab is indistinguishable from the parental mAb. Lastly, we demonstrate a bivalent meditope variant binds specifically and stably to antigen-bearing cells only in the presence of the meditope-enabled mAbs. Collectively, this finding and the subsequent characterization and engineering efforts indicate that this unique interface could serve as a noncovalent “linker” for any meditope-enabled mAb with applications in multiple mAb-based technologies including diagnostics, imaging, and therapeutic delivery.« less

  4. Activity Based High-Throughput Screening for Novel O-GlcNAc Transferase Substrates Using a Dynamic Peptide Microarray

    PubMed Central

    Shi, Jie; Sharif, Suhela; Ruijtenbeek, Rob; Pieters, Roland J.

    2016-01-01

    O-GlcNAcylation is a reversible and dynamic protein post-translational modification in mammalian cells. The O-GlcNAc cycle is catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays important role in many vital cellular events including transcription, cell cycle regulation, stress response and protein degradation, and altered O-GlcNAcylation has long been implicated in cancer, diabetes and neurodegenerative diseases. Recently, numerous approaches have been developed to identify OGT substrates and study their function, but there is still a strong demand for highly efficient techniques. Here we demonstrated the utility of the peptide microarray approach to discover novel OGT substrates and study its specificity. Interestingly, the protein RBL-2, which is a key regulator of entry into cell division and may function as a tumor suppressor, was identified as a substrate for three isoforms of OGT. Using peptide Ala scanning, we found Ser 420 is one possible O-GlcNAc site in RBL-2. Moreover, substitution of Ser 420, on its own, inhibited OGT activity, raising the possibility of mechanism-based development for selective OGT inhibitors. This approach will prove useful for both discovery of novel OGT substrates and studying OGT specificity. PMID:26960196

  5. In Vitro and In Vivo Imaging of Peptide-Encapsulated Polymer Nanoparticles for Cancer Biomarker Activated Drug Delivery

    PubMed Central

    Lee, Matthew B.; Cheng, Felice; Haque, Munima; Choi, Hyungsoo; Kim, Kyekyoon; O’Brien, William D.; Liu, G. Logan

    2014-01-01

    Gelatin nanoparticles coated with Cathepsin D-specific peptides were developed as a vehicle for the targeted delivery of the cancer drug doxorubicin (DOX) to treat breast malignancy. Cathepsin D, a breast cancer cell secretion enzyme, triggered the release of DOX by digesting the protective peptide-coating layer of nanoparticles. Fabricated nanoparticles were successfully detected with ultrasound imaging in both in vitro conditions and in vivo mouse cancer models. Cell viability experiments were conducted to determine the efficacy of biomarker activation specific to breast cancer cell lines. These experimental results were compared with the outcome of a viability experiment conducted on noncancerous cells. Viability decreased in human MCF7 mammary adenocarcinoma and mouse 4T1 mammary carcinoma cells, while that of noncancerous 3T3 fibroblast cells remained unaffected. Next, a real-time video of nanoparticle flow in mouse models was obtained using in vivo ultrasound imaging. The fluorescent profile of DOX was used as a means to examine nanoparticle localization in vivo. Results show the distribution of nanoparticles concentrated primarily within bladder and tumor sites of subject mice bodies. These findings support the use of biomarker coated nanoparticles in target specific therapy for breast cancer treatment. PMID:23955780

  6. EPR Studies of Functionally Active, Nitroxide Spin-Labeled Peptide Analogs of the C-terminus of a G-Protein Alpha Subunit

    PubMed Central

    Van Eps, Ned; Anderson, Lori L.; Kisselev, Oleg G.; Baranski, Thomas J.; Hubbell, Wayne L.; Marshall, Garland R.

    2010-01-01

    The C-terminal tail of the transducin alpha subunit, Gtα(340–350), is known to bind and stabilize the active conformation of rhodopsin upon photoactivation (R*). Five spin-labeled analogs of Gtα(340–350) demonstrated native-like activity in their ability to bind and stabilize R*. The spin label 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) was employed at interior sites within the peptide, whereas a Proxyl (3-carboxyl-2,2,5,5-tetramethyl-pyrrolidinyloxy) spin label was employed at the amino terminus of the peptide. Upon binding to R*, the electron paramagnetic resonance spectrum of TOAC343-Gtα(340–350) revealed greater immobilization of the nitroxide when compared to that of the N-terminal modified Proxyl-Gtα(340–350) analog. A double-labeled Proxyl/TOAC348-Gtα(340–350) was examined by DEER spectroscopy to determine the distribution of distances between the two nitroxides in the peptides when in solution and when bound to R*. TOAC and Proxyl spin labels in this GPCR-G-protein α-peptide system provide unique biophysical probes that can be used to explore the structure and conformational changes at the rhodopsin-G-protein interface. PMID:20695526

  7. Immune regulatory activities of fowlicidin-1, a cathelicidin host defense peptide.

    PubMed

    Bommineni, Yugendar R; Pham, Giang H; Sunkara, Lakshmi T; Achanta, Mallika; Zhang, Guolong

    2014-05-01

    Appropriate modulation of immunity is beneficial in antimicrobial therapy and vaccine development. Host defense peptides (HDPs) constitute critically important components of innate immunity with both antimicrobial and immune regulatory activities. We previously showed that a chicken HDP, namely fowlicidin-1(6-26), has potent antibacterial activities in vitro and in vivo. Here we further revealed that fowl-1(6-26) possesses strong immunomodulatory properties. The peptide is chemotactic specifically to neutrophils, but not monocytes or lymphocytes, after injected into the mouse peritoneum. Fowl-1(6-26) also has the capacity to activate macrophages by inducing the expression of inflammatory mediators including IL-1β, CCL2, and CCL3. However, unlike bacterial lipopolysaccharide that triggers massive production of inflammatory cytokines and chemokines, fowl-1(6-26) only marginally increased their expression in mouse RAW264.7 macrophages. Additionally, fowl-1(6-26) enhanced the surface expression of MHC II and CD86 on RAW264.7 cells, suggesting that it may facilitate development of adaptive immune response. Indeed, co-immunization of mice with chicken ovalbumin (OVA) and fowl-1(6-26) augmented both OVA-specific IgG1 and IgG2a titers, relative to OVA alone. We further showed that fowl-1(6-26) is capable of preventing a methicillin-resistant Staphylococcus aureus (MRSA) infection due to its enhancement of host defense. All mice survived from an otherwise lethal infection when the peptide was administered 1-2 days prior to MRSA infection, and 50% mice were protected if receiving the peptide 4 days before infection. Taken together, with a strong capacity to stimulate innate and adaptive immunity, fowl-1(6-26) may have potential to be developed as a novel antimicrobial and a vaccine adjuvant.

  8. Antibacterial Activity of Novel Cationic Peptides against Clinical Isolates of Multi-Drug Resistant Staphylococcus pseudintermedius from Infected Dogs

    PubMed Central

    Mohamed, Mohamed F.; Hammac, G. Kenitra; Guptill, Lynn; Seleem, Mohamed N.

    2014-01-01

    Staphylococcus pseudintermedius is a major cause of skin and soft tissue infections in companion animals and has zoonotic potential. Additionally, methicillin-resistant S. pseudintermedius (MRSP) has emerged with resistance to virtually all classes of antimicrobials. Thus, novel treatment options with new modes of action are required. Here, we investigated the antimicrobial activity of six synthetic short peptides against clinical isolates of methicillin-susceptible and MRSP isolated from infected dogs. All six peptides demonstrated potent anti-staphylococcal activity regardless of existing resistance phenotype. The most effective peptides were RRIKA (with modified C terminus to increase amphipathicity and hydrophobicity) and WR-12 (α-helical peptide consisting exclusively of arginine and tryptophan) with minimum inhibitory concentration50 (MIC50) of 1 µM and MIC90 of 2 µM. RR (short anti-inflammatory peptide) and IK8 “D isoform” demonstrated good antimicrobial activity with MIC50 of 4 µM and MIC90 of 8 µM. Penetratin and (KFF)3K (two cell penetrating peptides) were the least effective with MIC50 of 8 µM and MIC90 of 16 µM. Killing kinetics revealed a major advantage of peptides over conventional antibiotics, demonstrating potent bactericidal activity within minutes. Studies with propidium iodide and transmission electron microscopy revealed that peptides damaged the bacterial membrane leading to leakage of cytoplasmic contents and consequently, cell death. A potent synergistic increase in the antibacterial effect of the cell penetrating peptide (KFF)3K was noticed when combined with other peptides and with antibiotics. In addition, all peptides displayed synergistic interactions when combined together. Furthermore, peptides demonstrated good therapeutic indices with minimal toxicity toward mammalian cells. Resistance to peptides did not evolve after 10 passages of S. pseudintermedius at sub-inhibitory concentration. However, the MICs of amikacin and

  9. Enabling the Discovery and Virtual Screening of Potent and Safe Antimicrobial Peptides. Simultaneous Prediction of Antibacterial Activity and Cytotoxicity.

    PubMed

    Kleandrova, Valeria V; Ruso, Juan M; Speck-Planche, Alejandro; Dias Soeiro Cordeiro, M Natália

    2016-08-01

    Antimicrobial peptides (AMPs) represent promising alternatives to fight against bacterial pathogens. However, cellular toxicity remains one of the main concerns in the early development of peptide-based drugs. This work introduces the first multitasking (mtk) computational model focused on performing simultaneous predictions of antibacterial activities, and cytotoxicities of peptides. The model was created from a data set containing 3592 cases, and it displayed accuracy higher than 96% for classifying/predicting peptides in both training and prediction (test) sets. The technique known as alanine scanning was computationally applied to illustrate the calculation of the quantitative contributions of the amino acids (in their respective positions of the sequence) to the biological effects of a defined peptide. A small library formed by 10 peptides was generated, where peptides were designed by considering the interpretations of the different descriptors in the mtk-computational model. All the peptides were predicted to exhibit high antibacterial activities against multiple bacterial strains, and low cytotoxicity against various cell types. The present mtk-computational model can be considered a very useful tool to support high throughput research for the discovery of potent and safe AMPs.

  10. Enabling the Discovery and Virtual Screening of Potent and Safe Antimicrobial Peptides. Simultaneous Prediction of Antibacterial Activity and Cytotoxicity.

    PubMed

    Kleandrova, Valeria V; Ruso, Juan M; Speck-Planche, Alejandro; Dias Soeiro Cordeiro, M Natália

    2016-08-01

    Antimicrobial peptides (AMPs) represent promising alternatives to fight against bacterial pathogens. However, cellular toxicity remains one of the main concerns in the early development of peptide-based drugs. This work introduces the first multitasking (mtk) computational model focused on performing simultaneous predictions of antibacterial activities, and cytotoxicities of peptides. The model was created from a data set containing 3592 cases, and it displayed accuracy higher than 96% for classifying/predicting peptides in both training and prediction (test) sets. The technique known as alanine scanning was computationally applied to illustrate the calculation of the quantitative contributions of the amino acids (in their respective positions of the sequence) to the biological effects of a defined peptide. A small library formed by 10 peptides was generated, where peptides were designed by considering the interpretations of the different descriptors in the mtk-computational model. All the peptides were predicted to exhibit high antibacterial activities against multiple bacterial strains, and low cytotoxicity against various cell types. The present mtk-computational model can be considered a very useful tool to support high throughput research for the discovery of potent and safe AMPs. PMID:27280735

  11. The active site of ribulose-bisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.

    1991-01-01

    The active site of ribulose-bisphosphate carboxylase/oxygenase requires interacting domains of adjacent, identical subunits. Most active-site residues are located within the loop regions of an eight-stranded {beta}/{alpha}-barrel which constitutes the larger C-terminal domain; additional key residues are located within a segment of the smaller N-terminal domain which partially covers the mouth of the barrel. Site-directed mutagenesis of the gene encoding the enzyme from Rhodospirillum rubrum has been used to delineate functions of active-site residues. 6 refs., 2 figs.

  12. Analysis of the immune response against mixotope peptide libraries from a main antigenic site of foot-and-mouth disease virus.

    PubMed

    Oliveira, Eliandre de; Jiménez-Clavero, Miguel Angel; Núñez, José Ignacio; Sobrino, Francisco; Andreu, David

    2005-04-01

    The design of vaccines for RNA viral diseases is complicated by the high genetic variability of the viruses, which favors the selection of escape mutants. A case in point is foot-and-mouth disease virus (FMDV), for which only limited protection has been observed in vaccination with single peptides. We have explored the potential of immunogens of higher sequence diversity, covering a broad range of field or culture-induced mutations at the immunodominant site A of FMDV, serotype C. Four mixotope-type peptide libraries, containing ca. 3 x 10(3) or ca. 3 x 10(5) peptides each, in either linear or cyclic form, and combining most significant mutations found or induced at site A have been synthesized and used to immunize guinea-pigs. Substantial levels of serum conversion have been observed for all four mixotope libraries, as well as for single peptides, linear or cyclic, corresponding to the consensus site A sequence. The specificity and neutralizing ability of the anti-mixotope and -peptide antibodies have been evaluated by direct ELISA and by plaque reduction and micro-neutralization assays, respectively. Challenge experiments with an infectious, guinea-pig-adapted FMDV strain, have shown higher protection rates in animals immunized with the cyclic versions, either in single sequence or in combinatorial mixotope form. PMID:15780448

  13. A novel bioactive peptide from wasp venom

    PubMed Central

    Chen, Lingling; Chen, Wenlin; Yang, Hailong; Lai, Ren

    2010-01-01

    Wasp venoms contain a number of pharmacologically active biomolecules, undertaking a wide range of functions necessary for the wasp's survival. We purified and characterized a novel bioactive peptide (vespin) from the venoms of Vespa magnifica (Smith) wasps with unique primary structure. Its amino acid sequence was determined to be CYQRRVAITAGGLKHRLMSSLIIIIIIRINYLRDNSVIILESSY. It has 44 residues including 15 leucines or isoleucines (32%) in the sequence. Vespin showed contractile activity on isolated ileum smooth muscle. The cDNA encoding vespin precursor was cloned from the cDNA library of the venomous glands. The precursor consists of 67 amino acid residues including the predicted signal peptide and mature vespin. A di-basic enzymatic processing site (-KR-) is located between the signal peptide and the mature peptide. Vespin did not show similarity with any known proteins or peptides by BLAST search, suggesting it is a novel bioactive peptide from wasp venoms. PMID:21544181

  14. Quantitative site-specific reactivity profiling of S-nitrosylation in mouse skeletal muscle using cysteinyl peptide enrichment coupled with mass spectrometry

    SciTech Connect

    Su, Dian; Shukla, Anil K.; Chen, Baowei; Kim, Jong-Seo; Nakayasu, Ernesto; Qu, Yi; Aryal, Uma; Weitz, Karl; Clauss, Therese R. W.; Monroe, Matthew E.; Camp II, David G.; Bigelow, Diana J.; Smith, Richard D.; Kulkarni, Rohit N.; Qian, Wei-Jun

    2013-04-01

    S-nitrosylation (SNO) is an important reversible thiol oxidation event that has been increasingly recognized for its role in cell signaling. While many proteins susceptible to S-nitrosylation have been reported, site-specific identification of physiologically relevant SNO modifications remains an analytical challenge due to the low-abundance and labile nature of the modification. Herein we present further improvement and optimization of the recently reported, resin-assisted cysteinyl peptide enrichment protocol for SNO identification and the extension of this application to mouse skeletal muscle to identify specific sites sensitive to S-nitrosylation by quantitative reactivity profiling. The results of our data indicate that the protein- and peptide-level enrichment protocols provide comparable specificity and coverage of SNO-peptide identifications. S-nitrosylation reactivity profiling was performed by quantitatively comparing the site-specific SNO modification levels in samples treated with S-nitrosoglutathione (GSNO), an NO donor, at two different physiologically relevant concentrations (i.e., 10 μM and 100 μM). The reactivity profiling experiments overall identified 489 SNO-modified cysteine sites from 197 proteins with the specificity of 95.2% at the unique-peptide-level based on the percentage of Cys-peptides. Among these sites, 260 sites from 135 proteins were observed with relatively high reactivity to S-nitrosylation; such SNO-sensitive sites are more likely to be physiologically relevant. Many of the SNO-sensitive proteins are preferentially localized in mitochondria, contractile fiber and actin cytoskeleton, suggesting the susceptibility of these subcellular compartments to redox regulation. Moreover, the SNO-sensitive proteins seem to be primarily involved in metabolic pathways, including TCA cycle, glycolysis/gluconeogenesis, glutathione metabolism, and fatty acid metabolism, suggesting the importance of redox regulation in muscle metabolism and

  15. Quantitative site-specific reactivity profiling of S-nitrosylation in mouse skeletal muscle using cysteinyl peptide enrichment coupled with mass spectrometry

    PubMed Central

    Su, Dian; Shukla, Anil K.; Chen, Baowei; Kim, Jong-Seo; Nakayasu, Ernesto; Qu, Yi; Aryal, Uma; Weitz, Karl; Clauss, Therese R.W.; Monroe, Matthew E.; Camp, David G.; Bigelow, Diana J.; Smith, Richard D.; Kulkarni, Rohit N.; Qian, Wei-Jun

    2013-01-01

    S-nitrosylation, the formation of S-nitrosothiol (SNO), is an important reversible thiol oxidation event that has been increasingly recognized for its role in cell signaling. Although many proteins susceptible to S-nitrosylation have been reported, site-specific identification of physiologically relevant SNO modifications remains an analytical challenge because of the low abundance and labile nature of this modification. Herein we present further improvement and optimization of the recently reported resin-assisted cysteinyl peptide enrichment protocol for SNO identification and its application to mouse skeletal muscle to identify specific cysteine sites sensitive to S-nitrosylation by a quantitative reactivity profiling strategy. Our results indicate that the protein- and peptide-level enrichment protocols provide comparable specificity and coverage of SNO-peptide identifications. S-nitrosylation reactivity profiling was performed by quantitatively comparing the site-specific SNO modification levels in samples treated with S-nitrosoglutathione, an NO donor, at two different concentrations (i.e., 10 and 100 μM). The reactivity profiling experiments led to the identification of 488 SNO-modified sites from 197 proteins with specificity of ~95% at the unique peptide level, i.e., ~95% of enriched peptides contain cysteine residues as the originally SNO-modified sites. Among these sites, 281 from 145 proteins were considered more sensitive to S-nitrosylation based on the ratios of observed SNO levels between the two treatments. These SNO-sensitive sites are more likely to be physiologically relevant. Many of the SNO-sensitive proteins are localized in mitochondria, contractile fiber, and actin cytoskeleton, suggesting the susceptibility of these subcellular compartments to redox regulation. Moreover, these observed SNO-sensitive proteins are primarily involved in metabolic pathways, including the tricarboxylic acid cycle, glycolysis/gluconeogenesis, glutathione

  16. A free cysteine prolongs the half-life of a homing peptide and improves its tumor-penetrating activity.

    PubMed

    Pang, Hong-Bo; Braun, Gary B; She, Zhi-Gang; Kotamraju, Venkata R; Sugahara, Kazuki N; Teesalu, Tambet; Ruoslahti, Erkki

    2014-02-10

    The accessibility of extravascular tumor tissue to drugs is critical for therapeutic efficacy. We previously described a tumor-targeting peptide (iRGD) that elicits active transport of drugs and macromolecules (covalently coupled or co-administered) across the vascular wall into tumor tissue. Short peptides (iRGD is a 9-amino acid cyclic peptide) generally have a plasma half-life measured in minutes. Since short half-life limits the window of activity obtained with a bolus injection of iRGD, we explored to extend the half-life of the peptide. We show here that addition of a cysteine residue prolongs the plasma half-life of iRGD and increases the accumulation of the peptide in tumors. This modification prolongs the activity of iRGD in inducing macromolecular extravasation and leads to greater drug accumulation in tumors than is obtained with the unmodified peptide. This effect is mediated by covalent binding of iRGD to plasma albumin through a disulfide bond. Our study provides a simple strategy to improve peptide pharmacokinetics and activity. Applied to RGD, it provides a means to increase the entry of therapeutic agents into tumors.

  17. Savannah River Site prioritization of transition activities

    SciTech Connect

    Finley, R.H.

    1993-11-01

    Effective management of SRS conversion from primarily a production facility to other missions (or Decontamination and Decommissioning (D&D)) requires a systematic and consistent method of prioritizing the transition activities. This report discusses the design of a prioritizing method developed to achieve systematic and consistent methods of prioritizing these activities.

  18. Solution Structures, Dynamics, and Ice Growth Inhibitory Activity of Peptide Fragments Derived from an Antarctic Yeast Protein

    PubMed Central

    Asmawi, Azren A.; Rahman, Mohd Basyaruddin A.; Murad, Abdul Munir A.; Mahadi, Nor M.; Basri, Mahiran; Rahman, Raja Noor Zaliha A.; Salleh, Abu B.; Chatterjee, Subhrangsu; Tejo, Bimo A.; Bhunia, Anirban

    2012-01-01

    Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities. PMID:23209600

  19. DOE site performance assessment activities. Radioactive Waste Technical Support Program

    SciTech Connect

    Not Available

    1990-07-01

    Information on performance assessment capabilities and activities was collected from eight DOE sites. All eight sites either currently dispose of low-level radioactive waste (LLW) or plan to dispose of LLW in the near future. A survey questionnaire was developed and sent to key individuals involved in DOE Order 5820.2A performance assessment activities at each site. The sites surveyed included: Hanford Site (Hanford), Idaho National Engineering Laboratory (INEL), Los Alamos National Laboratory (LANL), Nevada Test Site (NTS), Oak Ridge National Laboratory (ORNL), Paducah Gaseous Diffusion Plant (Paducah), Portsmouth Gaseous Diffusion Plant (Portsmouth), and Savannah River Site (SRS). The questionnaire addressed all aspects of the performance assessment process; from waste source term to dose conversion factors. This report presents the information developed from the site questionnaire and provides a comparison of site-specific performance assessment approaches, data needs, and ongoing and planned activities. All sites are engaged in completing the radioactive waste disposal facility performance assessment required by DOE Order 5820.2A. Each site has achieved various degrees of progress and have identified a set of critical needs. Within several areas, however, the sites identified common needs and questions.

  20. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

    The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive material contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites.

  1. Plasminogen activator inhibitor-1 fused with erythropoietin (EPO) mimetic peptide (EMP) enhances the EPO activity of EMP.

    PubMed

    Kuai, L; Wu, C; Qiu, Q; Zhang, J; Zhou, A; Wang, S; Zhang, H; Song, Q; Liao, S; Han, Y; Liu, J; Ma, Z

    2000-08-01

    Erythropoietin (EPO) mimetic peptide (EMP) encoding sequence was inserted into the gene of plasminogen activator inhibitor-1 (PAI-1) between Ala348 and Pro349 (P2'-P3'), generating a novel gene, PAI-1/EMP (PMP). This was cloned into pET32a expression vector, fused with TrxA peptide in the vector, and a 63-kDa protein was expressed in inclusion bodies with an expression level >50%. The TrxA/PMP protein was purified by Ni-NTA-agarose metal-ligand affinity chromatography to a purity >90%, showing a single, silver-stained band on SDS-PAGE. Using a reticulocyte counting assay, the EPO activity of PMP was determined to be 5,000 IU/mg, 2,500-fold that of EMP.

  2. Chemical Synthesis of Arabidopsis CLV3 Glycopeptide Reveals the Impact of Hydroxyproline Arabinosylation on Peptide Conformation and Activity

    PubMed Central

    Shinohara, Hidefumi; Matsubayashi, Yoshikatsu

    2013-01-01

    Arabinosylation of hydroxyproline (Hyp) is a post-translational modification often found in secreted peptide signals in plants. The physiological importance of this modification was highlighted by the finding that CLAVATA3 (CLV3), a key peptide signal for regulating the fate of stem cells in the shoot apical meristem in Arabidopsis, contains three l-arabinose residues linked via linear β-1,2-linkages. However, understanding the functions and properties of arabinosylated peptides has been hindered by difficulties in synthesizing the complex arabinose chain. Here we report the stereoselective total synthesis of β-1,2-linked triarabinosylated CLV3 peptide ([Ara3]CLV3). Chemically synthesized [Ara3]CLV3 restricted stem cell activity more effectively than did unmodified CLV3 peptide. Comparison of mono-, di- and triarabinosylated CLV3 glycopeptides revealed that the biological activity increased progressively as the arabinose chain length increased. Thus, the arabinose chain length of CLV3 is important for its biological activity. Nuclear magnetic resonance spectroscopy and nuclear Overhauser effect-based structure calculations further revealed the structural impact of the arabinose chain on peptide conformation. The arabinose chain of [Ara3]CLV3 extends toward the C-terminal end of the peptide, and its non-reducing end is positioned proximal to the peptide backbone. Consequently, the arabinose chain causes distinct distortion in the C-terminal half of the peptide in a highly directional manner. The established synthetic route of [Ara3]CLV3 will greatly contribute to our