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Sample records for peptide derivative targeting

  1. Targeting diverse protein–protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold

    SciTech Connect

    Checco, James W.; Kreitler, Dale F.; Thomas, Nicole C.; Belair, David G.; Rettko, Nicholas J.; Murphy, William L.; Forest, Katrina T.; Gellman, Samuel H.

    2015-04-14

    Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α- and β-amino acid residues ("α/β-peptides") that mimic several peptides derived from the three-helix bundle "Z-domain" scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF₁₆₅-induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain–mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.

  2. Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

    PubMed

    Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

    2016-01-01

    The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling. PMID:25410289

  3. Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine.

    PubMed

    Riedl, Sabrina; Leber, Regina; Rinner, Beate; Schaider, Helmut; Lohner, Karl; Zweytick, Dagmar

    2015-11-01

    Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9-11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro-repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20 μM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2-3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non-selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity. PMID:26239537

  4. Tumor Imaging and Targeting Potential of an Hsp70-Derived 14-Mer Peptide

    PubMed Central

    Oellinger, Rupert; Breuninger, Stephanie; Rad, Roland; Pockley, Alan G.; Multhoff, Gabriele

    2014-01-01

    Background We have previously used a unique mouse monoclonal antibody cmHsp70.1 to demonstrate the selective presence of a membrane-bound form of Hsp70 (memHsp70) on a variety of leukemia cells and on single cell suspensions derived from solid tumors of different entities, but not on non-transformed cells or cells from corresponding ’healthy‘ tissue. This antibody can be used to image tumors in vivo and target them for antibody-dependent cellular cytotoxicity. Tumor-specific expression of memHsp70 therefore has the potential to be exploited for theranostic purposes. Given the advantages of peptides as imaging and targeting agents, this study assessed whether a 14-mer tumor penetrating peptide (TPP; TKDNNLLGRFELSG), the sequence of which is derived from the oligomerization domain of Hsp70 which is expressed on the cell surface of tumor cells, can also be used for targeting membrane Hsp70 positive (memHsp70+) tumor cells, in vitro. Methodology/Principal Findings The specificity of carboxy-fluorescein (CF-) labeled TPP (TPP) to Hsp70 was proven in an Hsp70 knockout mammary tumor cell system. TPP specifically binds to different memHsp70+ mouse and human tumor cell lines and is rapidly taken up via endosomes. Two to four-fold higher levels of CF-labeled TPP were detected in MCF7 (82% memHsp70+) and MDA-MB-231 (75% memHsp70+) cells compared to T47D cells (29% memHsp70+) that exhibit a lower Hsp70 membrane positivity. After 90 min incubation, TPP co-localized with mitochondrial membranes in memHsp70+ tumors. Although there was no evidence that any given vesicle population was specifically localized, fluorophore-labeled cmHsp70.1 antibody and TPP preferentially accumulated in the proximity of the adherent surface of cultured cells. These findings suggest a potential association between membrane Hsp70 expression and cytoskeletal elements that are involved in adherence, the establishment of intercellular synapses and/or membrane reorganization. Conclusions

  5. Targeting Leishmania major parasite with peptides derived from a combinatorial phage display library.

    PubMed

    Rhaiem, Rafik Ben; Houimel, Mehdi

    2016-07-01

    Cutaneous leishmaniasis (CL) is a global problem caused by intracellular protozoan pathogens of the genus Leishmania for which there are no suitable vaccine or chemotherapy options. Thus, de novo identification of small molecules binding to the Leishmania parasites by direct screening is a promising and appropriate alternative strategy for the development of new drugs. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select binding peptides to metacyclic promastigotes from a highly virulent strain of Leishmania major (Zymodeme MON-25; MHOM/TN/94/GLC94). After four rounds of stringent selection and amplification, polyclonal and monoclonal phage-peptides directed against L. major metacyclic promastigotes were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to L. major by monoclonal phage ELISA. The DNA of 42 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences deduced. Six different peptide sequences were obtained with frequencies of occurrence ranging from 2.3% to 85.7%. The biological effect of the peptides was assessed in vitro on human monocytes infected with L. major metacyclic promastigotes, and in vivo on susceptible parasite-infected BALB/c mice. The development of cutaneous lesions in the right hind footpads of infected mice after 13 weeks post-infection showed a protection rate of 81.94% with the injected peptide P2. Moreover, Western blots revealed that the P2 peptide interacted with the major surface protease gp63, a protein of 63kDa molecular weight. Moreover, bioinformatics were used to predict the interaction between peptides and the major surface molecule of the L. major. The molecular docking showed that the P2 peptide has the minimum interaction energy and maximum shape complimentarity with the L. major gp63 active site. Our study demonstrated that the P2 peptide occurs at high frequency

  6. Synthesis, characterization, and biological activity of poly(arginine)-derived cancer-targeting peptides in HepG2 liver cancer cells.

    PubMed

    Joseph, Stesha C; Blackman, Brittany A; Kelly, Megan L; Phillips, Mariana; Beaury, Michael W; Martinez, Ivonne; Parronchi, Christopher J; Bitsaktsis, Constantine; Blake, Allan D; Sabatino, David

    2014-09-01

    The solid-phase synthesis, structural characterization, and biological evaluation of a small library of cancer-targeting peptides have been determined in HepG2 hepatoblastoma cells. These peptides are based on the highly specific Pep42 motif, which has been shown to target the glucose-regulated protein 78 receptors overexpressed and exclusively localized on the cell surface of tumors. In this study, Pep42 was designed to contain varying lengths (3-12) of poly(arginine) sequences to assess their influence on peptide structure and biology. Peptides were effectively synthesized by 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis, in which the use of a poly(ethylene glycol) resin provided good yields (14-46%) and crude purities >95% as analyzed by liquid chromatography-mass spectrometry. Peptide structure and biophysical properties were investigated using circular dichroism spectroscopy. Interestingly, peptides displayed secondary structures that were contingent on solvent and length of the poly(arginine) sequences. Peptides exhibited helical and turn conformations, while retaining significant thermal stability. Structure-activity relationship studies conducted by flow cytometry and confocal microscopy revealed that the poly(arginine) derived Pep42 sequences maintained glucose-regulated protein 78 binding on HepG2 cells while exhibiting cell translocation activity that was contingent on the length of the poly(arginine) strand. In single dose (0.15 mM) and dose-response (0-1.5 mM) cell viability assays, peptides were found to be nontoxic in human HepG2 liver cancer cells, illustrating their potential as safe cancer-targeting delivery agents. PMID:24931620

  7. Targeting TLR4 Signaling by TLR4 TIR-derived Decoy Peptides: Identification of the TLR4 TIR Dimerization Interface

    PubMed Central

    Toshchakov, Vladimir Y.; Szmacinski, Henryk; Couture, Leah A.; Lakowicz, Joseph R.; Vogel, Stefanie N.

    2011-01-01

    Agonist-induced dimerization of TLR4 TIR domains initiates intracellular signaling. Therefore, identification of the TLR4 TIR dimerization interface is one key to the rational design of therapeutics that block TLR4 signaling. A library of cell-permeating “decoy peptides,” each of which represents a non-fragmented patch of the TLR4 TIR surface, was designed such that the peptides entirely encompass the TLR4 TIR surface. Each peptide was synthesized in tandem with a cell-permeating Antennapedia homeodomain sequence and tested for the ability to inhibit early cytokine mRNA expression and MAPK activation in LPS-stimulated primary murine macrophages. Five peptides, 4R1, 4R3, 4BB, 4R9, and 4αE, potently inhibited all manifestations of TLR4, but not TLR2 signaling. When tested for their ability to bind directly to TLR4 TIR by FRET using time-resolved fluorescence spectroscopy, Bodipy-TMR-X (BTX)-labeled 4R1, 4BB, and 4αE quenched fluorescence of TLR4-Cerulean (Cer) expressed in HeLa or HEK293T cells, while 4R3 was partially active and 4R9 was least active. These findings suggest that the area between BB loop of TLR4 and its fifth helical region mediates TLR4 TIR dimerization. Moreover, our data provide direct evidence for the utility of the “decoy peptide approach,” in which peptides representing various surface-exposed segments of a protein are initially probed for the ability to inhibit protein function and then their specific targets are identified by FRET, to define recognition sites in signaling proteins that may be targeted therapeutically to disrupt functional transient protein interactions. PMID:21402890

  8. Specific interaction between Mycobacterium tuberculosis lipoprotein-derived peptides and target cells inhibits mycobacterial entry in vitro

    PubMed Central

    Ocampo, Marisol; Curtidor, Hernando; Vanegas, Magnolia; Patarroyo, Manuel Alfonso; Patarroyo, Manuel Elkin

    2014-01-01

    Summary Tuberculosis (TB) continues being one of the diseases having the greatest mortality rates around the world, 8.7 million cases having been reported in 2011. An efficient vaccine against TB having a great impact on public health is an urgent need. Usually, selecting antigens for vaccines has been based on proteins having immunogenic properties for patients suffering TB and having had promising results in mice and non-human primates. Our approach has been based on a functional approach involving the pathogen–host interaction in the search for antigens to be included in designing an efficient, minimal, subunit-based anti-tuberculosis vaccine. This means that Mycobacterium tuberculosis has mainly been involved in studies and that lipoproteins represent an important kind of protein on the cell envelope which can also contribute towards this pathogen's virulence. This study has assessed the expression of four lipoproteins from M. tuberculosis H37Rv, i.e. Rv1411c (LprG), Rv1911c (LppC), Rv2270 (LppN) and Rv3763 (LpqH), and the possible biological activity of peptides derived from these. Five peptides were found for these proteins which had high specific binding to both alveolar A549 epithelial cells and U937 monocyte-derived macrophages which were able to significantly inhibit mycobacterial entry to these cells in vitro. PMID:25041568

  9. Food-derived immunomodulatory peptides.

    PubMed

    Santiago-López, Lourdes; Hernández-Mendoza, Adrián; Vallejo-Cordoba, Belinda; Mata-Haro, Verónica; González-Córdova, Aarón F

    2016-08-01

    Food proteins contain specific amino acid sequences within their structures that may positively impact bodily functions and have multiple immunomodulatory effects. The functional properties of these specific sequences, also referred to as bioactive peptides, are revealed only after the degradation of native proteins during digestion processes. Currently, milk proteins have been the most explored source of bioactive peptides, which presents an interesting opportunity for the dairy industry. However, plant- and animal-derived proteins have also been shown to be important sources of bioactive peptides. This review summarizes the in vitro and in vivo evidence of the role of various food proteins as sources of immunomodulatory peptides and discusses the possible pathways involving these properties. © 2016 Society of Chemical Industry. PMID:26940008

  10. Therapeutic targets for olive pollen allergy defined by gene markers modulated by Ole e 1-derived peptides.

    PubMed

    Calzada, David; Aguerri, Miriam; Baos, Selene; Montaner, David; Mata, Manuel; Dopazo, Joaquín; Quiralte, Joaquín; Florido, Fernando; Lahoz, Carlos; Cárdaba, Blanca

    2015-04-01

    Two regions of Ole e 1, the major olive-pollen allergen, have been characterized as T-cell epitopes, one as immunodominant region (aa91-130) and the other, as mainly recognized by non-allergic subjects (aa10-31). This report tries to characterize the specific relevance of these epitopes in the allergic response to olive pollen by analyzing the secreted cytokines and the gene expression profiles induced after specific stimulation of peripheral blood mononuclear cells (PBMCs). PBMCs from olive pollen-allergic and non-allergic control subjects were stimulated with olive-pollen extract and Ole e 1 dodecapeptides containing relevant T-cell epitopes. Levels of cytokines were measured in cellular supernatants and gene expression was determined by microarrays, on the RNAs extracted from PBMCs. One hundred eighty-nine differential genes (fold change >2 or <-2, P<0.05) were validated by qRT-PCR in a large population. It was not possible to define a pattern of response according the overall cytokine results but interesting differences were observed, mainly in the regulatory cytokines. Principal component (PCA) gene-expression analysis defined clusters that correlated with the experimental conditions in the group of allergic subjects. Gene expression and functional analyses revealed differential genes and pathways among the experimental conditions. A set of 51 genes (many essential to T-cell tolerance and homeostasis) correlated with the response to aa10-31 of Ole e 1. In conclusion, two peptides derived from Ole e 1 could regulate the immune response in allergic patients, by gene-expression modification of several regulation-related genes. These results open new research ways to the regulation of allergy by Oleaceae family members. PMID:25553522

  11. Inhibition of lethal inflammatory responses through the targeting of membrane-associated Toll-like receptor 4 signaling complexes with a Smad6-derived peptide.

    PubMed

    Lee, Youn Sook; Park, Jin Seok; Jung, Su Myung; Kim, Sang-Doo; Kim, Jun Hwan; Lee, Jae Young; Jung, Kyeong Cheon; Mamura, Mizuko; Lee, Sangho; Kim, Seong-Jin; Bae, Yoe-Sik; Park, Seok Hee

    2015-05-01

    We have previously reported that Smad6, one of the inhibitory Smads of transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP) signaling, inhibits Toll-like receptor (TLR) 4 signaling by disrupting the Pellino-1-mediated TLR4 signaling complex. Here, we developed Smaducin-6, a novel membrane-tethered palmitic acid-conjugated Smad6-derived peptide composed of amino acids 422-441 of Smad6. Smaducin-6 interacted with Pellino-1, located in the inner membrane, thereby disrupting the formation of IRAK1-, RIP1-, IKKε-mediated TLR4 signaling complexes. Systemic administration of Smaducin-6 showed a significant therapeutic effect on mouse TLR4-mediated inflammatory disease models, cecal-ligation-puncture (CLP)-induced sepsis, and lipopolysaccharide-induced endotoxemia, by inhibiting pro-inflammatory cytokine production and apoptosis while enhancing neutrophil migration and bacterial clearance. Our findings provide clues to develop new peptide-based drugs to target Pellino-1 protein in TLR4 signaling pathway for the treatment of sepsis. PMID:25766838

  12. Identification of the C3a Receptor (C3AR1) as the Target of the VGF-derived Peptide TLQP-21 in Rodent Cells

    PubMed Central

    Hannedouche, Sebastien; Beck, Valerie; Leighton-Davies, Juliet; Beibel, Martin; Roma, Guglielmo; Oakeley, Edward J.; Lannoy, Vincent; Bernard, Jerome; Hamon, Jacques; Barbieri, Samuel; Preuss, Inga; Lasbennes, Marie-Christine; Sailer, Andreas W.; Suply, Thomas; Seuwen, Klaus; Parker, Christian N.; Bassilana, Frederic

    2013-01-01

    TLQP-21, a peptide derived from VGF (non-acronymic) by proteolytic processing, has been shown to modulate energy metabolism, differentiation, and cellular response to stress. Although extensively investigated, the receptor for this endogenous peptide has not previously been described. This study describes the use of a series of studies that show G protein-coupled receptor-mediated biological activity of TLQP-21 signaling in CHO-K1 cells. Unbiased genome-wide sequencing of the transcriptome from responsive CHO-K1 cells identified a prioritized list of possible G protein-coupled receptors bringing about this activity. Further experiments using a series of defined receptor antagonists and siRNAs led to the identification of complement C3a receptor-1 (C3AR1) as a target for TLQP-21 in rodents. We have not been able to demonstrate so far that this finding is translatable to the human receptor. Our results are in line with a large number of physiological observations in rodent models of food intake and metabolic control, where TLQP-21 shows activity. In addition, the sensitivity of TLQP-21 signaling to pertussis toxin is consistent with the known signaling pathway of C3AR1. The binding of TLQP-21 to C3AR1 not only has effects on signaling but also modulates cellular functions, as TLQP-21 was shown to have a role in directing migration of mouse RAW264.7 cells. PMID:23940034

  13. Membrane damage as first and DNA as the secondary target for anti-candidal activity of antimicrobial peptide P7 derived from cell-penetrating peptide ppTG20 against Candida albicans.

    PubMed

    Li, Lirong; Song, Fengxia; Sun, Jin; Tian, Xu; Xia, Shufang; Le, Guowei

    2016-06-01

    P7, a peptide analogue derived from cell-penetrating peptide ppTG20, possesses antibacterial and antitumor activities without significant hemolytic activity. In this study, we investigated the antifungal effect of P7 and its anti-Candida acting mode in Candida albicans. P7 displayed antifungal activity against the reference C. albicans (MIC = 4 μM), Aspergilla niger (MIC = 32 μM), Aspergillus flavus (MIC = 8 μM), and Trichopyton rubrum (MIC = 16 μM). The effect of P7 on the C. albicans cell membrane was examined by investigating the calcein leakage from fungal membrane models made of egg yolk l-phosphatidylcholine/ergosterol (10 : 1, w/w) liposomes. P7 showed potent leakage effects against fungal liposomes similar to Melittin-treated cells. C. albicans protoplast regeneration assay demonstrated that P7 interacted with the C. albicans plasma membrane. Flow cytometry of the plasma membrane potential and integrity of C. albicans showed that P7 caused 60.9 ± 1.8% depolarization of the membrane potential of intact C. albicans cells and caused 58.1 ± 3.2% C. albicans cell membrane damage. Confocal laser scanning microscopy demonstrated that part of FITC-P7 accumulated in the cytoplasm. DNA retardation analysis was also performed, which showed that P7 interacted with C. albicans genomic DNA after penetrating the cell membrane, completely inhibiting the migration of genomic DNA above the weight ratio (peptide : DNA) of 6. Our results indicated that the plasma membrane was the primary target, and DNA was the secondary intracellular target of the mode of action of P7 against C. albicans. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27197902

  14. Solid Phase Synthesis and Application of Labeled Peptide Derivatives: Probes of Receptor-Opioid Peptide Interactions

    PubMed Central

    Aldrich, Jane V.; Kumar, Vivek; Dattachowdhury, Bhaswati; Peck, Angela M.; Wang, Xin; Murray, Thomas F.

    2009-01-01

    Solid phase synthetic methodology has been developed in our laboratory to incorporate an affinity label (a reactive functionality such as isothiocyanate or bromoacetamide) into peptides (Leelasvatanakij, L. and Aldrich, J. V. (2000) J. Peptide Res. 56, 80), and we have used this synthetic strategy to prepare affinity label derivatives of a variety of opioid peptides. To date side reactions have been detected only in two cases, both involving intramolecular cyclization. We have identified several peptide-based affinity labels for δ opioid receptors that exhibit wash-resistant inhibition of binding to these receptors and are valuable pharmacological tools to study opioid receptors. Even in cases where the peptide derivatives do not bind covalently to their target receptor, studying their binding has revealed subtle differences in receptor interactions with particular opioid peptide residues, especially Phe residues in the N-terminal “message” sequences. Solid phase synthetic methodology for the incorporation of other labels (e.g. biotin) into the C-terminus of peptides has also been developed in our laboratory (Kumar, V. and Aldrich, J. V. (2003) Org. Lett. 5, 613). These two synthetic approaches have been combined to prepare peptides containing multiple labels that can be used as tools to study peptide ligand-receptor interactions. These solid phase synthetic methodologies are versatile strategies that are applicable to the preparation of labeled peptides for a variety of targets in addition to opioid receptors. PMID:19956785

  15. Targeting B-cell neoplasia with T-cell receptors recognizing a CD20-derived peptide on patient-specific HLA.

    PubMed

    Mensali, Nadia; Ying, Fan; Sheng, Vincent Oei Yi; Yang, Weiwen; Walseng, Even; Kumari, Shraddha; Fallang, Lars-Egil; Kolstad, Arne; Uckert, Wolfgang; Malmberg, Karl Johan; Wälchli, Sébastien; Olweus, Johanna

    2016-05-01

    T cells engineered to express chimeric antigen receptors (CARs) targeted to CD19 are effective in treatment of B-lymphoid malignancies. However, CARs recognize all CD19 positive (pos) cells, and durable responses are linked to profound depletion of normal B cells. Here, we designed a strategy to specifically target patient B cells by utilizing the fact that T-cell receptors (TCRs), in contrast to CARs, are restricted by HLA. Two TCRs recognizing a peptide from CD20 (SLFLGILSV) in the context of foreign HLA-A*02:01 (CD20p/HLA-A2) were expressed as 2A-bicistronic constructs. T cells re-directed with the A23 and A94 TCR constructs efficiently recognized malignant HLA-A2(pos) B cells endogenously expressing CD20, including patient-derived follicular lymphoma and chronic lymphocytic leukemia (CLL) cells. In contrast, a wide range of HLA-A2(pos)CD20(neg) cells representing different tissue origins, and HLA-A2(neg)CD20(pos) cells, were not recognized. Cytotoxic T cells re-directed with CD20p/HLA-A2-specific TCRs or CD19 CARs responded with similar potencies to cells endogenously expressing comparable levels of CD20 and CD19. The CD20p/HLA-A2-specific TCRs recognized CD20p bound to HLA-A2 with high functional avidity. The results show that T cells expressing CD20p/HLA-A2-specific TCRs efficiently and specifically target B cells. When used in context of an HLA-haploidentical allogeneic stem cell transplantation where the donor is HLA-A2(neg) and the patient HLA-A2(pos), these T cells would selectively kill patient-derived B cells and allow reconstitution of the B-cell compartment with HLA-A2(neg) donor cells. These results should pave the way for clinical testing of T cells genetically engineered to target malignant B cells without permanent depletion of normal B cells. PMID:27467957

  16. Molecular imaging probes derived from natural peptides.

    PubMed

    Charron, C L; Hickey, J L; Nsiama, T K; Cruickshank, D R; Turnbull, W L; Luyt, L G

    2016-06-01

    Covering: up to the end of 2015.Peptides are naturally occurring compounds that play an important role in all living systems and are responsible for a range of essential functions. Peptide receptors have been implicated in disease states such as oncology, metabolic disorders and cardiovascular disease. Therefore, natural peptides have been exploited as diagnostic and therapeutic agents due to the unique target specificity for their endogenous receptors. This review discusses a variety of natural peptides highlighting their discovery, endogenous receptors, as well as their derivatization to create molecular imaging agents, with an emphasis on the design of radiolabelled peptides. This review also highlights methods for discovering new and novel peptides when knowledge of specific targets and endogenous ligands are not available. PMID:26911790

  17. Peptide mediated cancer targeting of nanoconjugates

    PubMed Central

    Raha, Sumita; Paunesku, Tatjana; Woloschak, Gayle

    2013-01-01

    Targeted use of nanoparticles in vitro, in cells and in vivo requires nanoparticle surface functionalization. Moieties that can be used for such a purpose include small molecules as well as polymers made of different biological and organic materials. Short amino acid polymers--peptides can often rival target binding avidity of much larger molecules. At the same time, peptides are smaller than most nanoparticles and thus allow for multiple nanoparticle modifications and creation of pluripotent nanoparticles. Most nanoparticles provide multiple binding sites for different cargo and targeting peptides which can be used for development of novel approaches for cancer targeting, diagnostics and therapy. In this review, we will focus on peptides which have been used for preparation of different nanoparticles designed for cancer research. PMID:21046660

  18. Computer-based prediction of mitochondria-targeting peptides.

    PubMed

    Martelli, Pier Luigi; Savojardo, Castrense; Fariselli, Piero; Tasco, Gianluca; Casadio, Rita

    2015-01-01

    Computational methods are invaluable when protein sequences, directly derived from genomic data, need functional and structural annotation. Subcellular localization is a feature necessary for understanding the protein role and the compartment where the mature protein is active and very difficult to characterize experimentally. Mitochondrial proteins encoded on the cytosolic ribosomes carry specific patterns in the precursor sequence from where it is possible to recognize a peptide targeting the protein to its final destination. Here we discuss to which extent it is feasible to develop computational methods for detecting mitochondrial targeting peptides in the precursor sequences and benchmark our and other methods on the human mitochondrial proteins endowed with experimentally characterized targeting peptides. Furthermore, we illustrate our newly implemented web server and its usage on the whole human proteome in order to infer mitochondrial targeting peptides, their cleavage sites, and whether the targeting peptide regions contain or not arginine-rich recurrent motifs. By this, we add some other 2,800 human proteins to the 124 ones already experimentally annotated with a mitochondrial targeting peptide. PMID:25631024

  19. Peptide-targeted radionuclide therapy for melanoma.

    PubMed

    Miao, Yubin; Quinn, Thomas P

    2008-09-01

    Melanocortin-1 receptor (MC1-R) and melanin are two attractive melanoma-specific targets for peptide-targeted radionuclide therapy for melanoma. Radiolabeled peptides targeting MC1-R/melanin can selectively and specifically target cytotoxic radiation generated from therapeutic radionuclides to melanoma cells for cell killing, while sparing the normal tissues and organs. This review highlights the recent advances of peptide-targeted radionuclide therapy of melanoma targeting MC1-R and melanin. The promising therapeutic efficacies of 188Re-(Arg(11))CCMSH (188Re-[Cys(3,4,10), D-Phe(7),Arg(11)]-alpha-MSH(3-13)), 177Lu- and 212Pb-labeled DOTA-Re(Arg(11))CCMSH (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-[ReO-(Cys(3,4,10), D-Phe(7), Arg(11))]-alpha-MSH(3-13)) and 188Re-HYNIC-4B4 (188Re-hydrazinonicotinamide-Tyr-Glu-Arg-Lys-Phe-Trp-His-Gly-Arg-His) in preclinical melanoma-bearing models demonstrate an optimistic outlook for peptide-targeted radionuclide therapy for melanoma. Peptide-targeted radionuclide therapy for melanoma will likely contribute in an adjuvant setting, once the primary tumor has been surgically removed, to treat metastatic deposits and for treatment of end-stage disease. The lack of effective treatments for metastatic melanoma and end-stage disease underscores the necessity to develop and implement new treatment strategies, such as peptide-targeted radionuclide therapy. PMID:18387816

  20. Human anti-Aβ IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

    PubMed

    Welzel, Alfred T; Williams, Angela D; McWilliams-Koeppen, Helen P; Acero, Luis; Weber, Alfred; Blinder, Veronika; Mably, Alex; Bunk, Sebastian; Hermann, Corinna; Farrell, Michael A; Ehrlich, Hartmut J; Schwarz, Hans P; Walsh, Dominic M; Solomon, Alan; O'Nuallain, Brian

    2012-01-01

    Soluble non-fibrillar assemblies of amyloid-beta (Aβ) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer's disease (AD). Anti-Aβ immunotherapy is a promising and advanced therapeutic strategy, but the precise Aβ species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aβ conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aβ dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aβ extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aβ's conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody's nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aβ monomers and peptide fragments. The antibody's lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aβ NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aβ NAbs are warranted. PMID:23209707

  1. Human Anti-Aβ IgGs Target Conformational Epitopes on Synthetic Dimer Assemblies and the AD Brain-Derived Peptide

    PubMed Central

    Welzel, Alfred T.; Williams, Angela D.; McWilliams-Koeppen, Helen P.; Acero, Luis; Weber, Alfred; Blinder, Veronika; Mably, Alex; Bunk, Sebastian; Hermann, Corinna; Farrell, Michael A.; Ehrlich, Hartmut J.; Schwarz, Hans P.; Walsh, Dominic M.; Solomon, Alan; O’Nuallain, Brian

    2012-01-01

    Soluble non-fibrillar assemblies of amyloid-beta (Aβ) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer’s disease (AD). Anti-Aβ immunotherapy is a promising and advanced therapeutic strategy, but the precise Aβ species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aβ conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aβ dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aβ extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aβ’s conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody’s nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aβ monomers and peptide fragments. The antibody’s lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aβ NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aβ NAbs are warranted. PMID:23209707

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

    PubMed Central

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

    2015-01-01

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

  3. A peptide derived from phage display library exhibits anti-tumor activity by targeting GRP78 in gastric cancer multidrug resistance cells.

    PubMed

    Kang, Jianqin; Zhao, Guohong; Lin, Tao; Tang, Shanhong; Xu, Guanghui; Hu, Sijun; Bi, Qian; Guo, Changcun; Sun, Li; Han, Shuang; Xu, Qian; Nie, Yongzhan; Wang, Biaoluo; Liang, Shuhui; Ding, Jie; Wu, Kaichun

    2013-10-10

    Multidrug resistance (MDR) remains a significant challenge to the clinical treatment of gastric cancer (GC). In the present study, using a phage display approach combined with MTT assays, we screened a specific peptide GMBP1 (Gastric cancer MDR cell-specific binding peptide), ETAPLSTMLSPY, which could bind to the surface of GC MDR cells specifically and reverse their MDR phenotypes. Immunocytochemical staining showed that the potential receptor of GMBP1 was located at the membrane and cytoplasm of MDR cells. In vitro and in vivo drug sensitivity assays, FACS analysis and Western blotting confirmed that GMBP1 was able to re-sensitize MDR cells to chemical drugs. Western blotting and proteomic approaches were used to screen the receptor of GMBP1, and GRP78, a MDR-related protein, was identified as a receptor of GMBP1. This result was further supported by immunofluoresence microscopy and Western blot. Additionally, Western blotting demonstrated that pre-incubation of GMBP1 in MDR cells greatly diminished MDR1, Bcl-2 and GRP78 expression but increased the expression of Bax, whereas downregulation of GRP78, function as a receptor and directly target for GMBP1, only inhibited MDR1 expression. Our findings suggest that GMBP1 could re-sensitize GC MDR cells to a variety of chemotherapeutic agents and this role might be mediated partly through down-regulating GRP78 expression and then inhibiting MDR1 expression. These findings indicate that peptide GMBP1 likely recognizes a novel GRP78 receptor and mediates cellular activities associated with the MDR phenotype, which provides new insight into research on the management of MDR in gastric cancer cells. PMID:23792224

  4. Anticoagulant activity of original synthetic peptide derivatives.

    PubMed

    Drozd, N N; Tolstenkov, A S; Makarov, V A; Miphtakhova, N T; Voyushina, T L; Sergeev, M E

    2008-01-01

    Original synthetic peptide derivatives exhibit anticoagulant activity in vitro and in vivo. They delayed fibrin clot formation from human blood plasma in tests for the intrinsic coagulation pathway (activated partial thromboplastin time) and final stage of plasma coagulation (thrombin time) and inhibited amidolytic activity of thrombin. We determined the minimum effective dose of the most active compound providing a 2-fold lengthening of blood clotting time (activated partial thromboplastin time test and thrombin time test), which persisted for 2-3 h. PMID:19024001

  5. The proteome targets of intracellular targeting antimicrobial peptides.

    PubMed

    Shah, Pramod; Hsiao, Felix Shih-Hsiang; Ho, Yu-Hsuan; Chen, Chien-Sheng

    2016-04-01

    Antimicrobial peptides have been considered well-deserving candidates to fight the battle against microorganisms due to their broad-spectrum antimicrobial activities. Several studies have suggested that membrane disruption is the basic mechanism of AMPs that leads to killing or inhibiting microorganisms. Also, AMPs have been reported to interact with macromolecules inside the microbial cells such as nucleic acids (DNA/RNA), protein synthesis, essential enzymes, membrane septum formation and cell wall synthesis. Proteins are associated with many intracellular mechanisms of cells, thus protein targets may be specifically involved in mechanisms of action of AMPs. AMPs like pyrrhocoricin, drosocin, apidecin and Bac 7 are documented to have protein targets, DnaK and GroEL. Moreover, the intracellular targeting AMPs are reported to influence more than one protein targets inside the cell, suggesting for the multiple modes of actions. This complex mechanism of intracellular targeting AMPs makes them more difficult for the development of resistance. Herein, we have summarized the current status of AMPs in terms of their mode of actions, entry to cytoplasm and inhibition of macromolecules. To reveal the mechanism of action, we have focused on AMPs with intracellular protein targets. We have also included the use of high-throughput proteome microarray to determine the unidentified AMP protein targets in this review. PMID:26648572

  6. Targeting B16 tumors in vivo with peptide-conjugated gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Poon, Wilson; Zhang, Xuan; Bekah, Devesh; Teodoro, Jose G.; Nadeau, Jay L.

    2015-07-01

    This study examines the effects of polyethylene glycol (PEG) and peptide conjugation on the biodistribution of ultrasmall (2.7 nm) gold nanoparticles in mice bearing B16 melanoma allografts. Nanoparticles were delivered intravenously, and biodistribution was measured at specific timepoints by organ digestion and inductively coupled plasma mass spectrometry. All major organs were examined. Two peptides were tested: the cyclic RGD peptide (cRGD, which targets integrins); and a recently described peptide derived from the myxoma virus. We found the greatest specific tumor delivery using the myxoma peptide, with or without PEGylation. Un-PEGylated cRGD performed poorly, but PEGylated RGD showed a significant transient collection in the tumor. Liver and kidney were the primary targets of all constructs. None of the particles were able to cross the blood-brain barrier. Although it was able to deliver Au to B16 cells, the myxoma peptide did not show any cytotoxic activity against these cells, in contrast to previous reports. These results indicate that the effect of passive targeting by PEGylation and active targeting by peptides can be independent or combined, and that they should be evaluated on a case-by-case basis when designing new nanosystems for targeted therapies. Both myxoma peptide and cRGD should be considered for specific targeting to melanoma, but a thorough investigation of the cytotoxicity of the myxoma peptide to different cell lines remains to be performed.

  7. Designer interface peptide grafts target estrogen receptor alpha dimerization.

    PubMed

    Chakraborty, S; Asare, B K; Biswas, P K; Rajnarayanan, R V

    2016-09-01

    The nuclear transcription factor estrogen receptor alpha (ERα), triggered by its cognate ligand estrogen, regulates a variety of cellular signaling events. ERα is expressed in 70% of breast cancers and is a widely validated target for anti-breast cancer drug discovery. Administration of anti-estrogen to block estrogen receptor activation is still a viable anti-breast cancer treatment option but anti-estrogen resistance has been a significant bottle-neck. Dimerization of estrogen receptor is required for ER activation. Blocking ERα dimerization is therefore a complementary and alternative strategy to combat anti-estrogen resistance. Dimer interface peptide "I-box" derived from ER residues 503-518 specifically blocks ER dimerization. Recently using a comprehensive molecular simulation we studied the interaction dynamics of ERα LBDs in a homo-dimer. Based on this study, we identified three interface recognition peptide motifs LDKITDT (ERα residues 479-485), LQQQHQRLAQ (residues 497-506), and LSHIRHMSNK (residues 511-520) and reported the suitability of using LQQQHQRLAQ (ER 497-506) as a template to design inhibitors of ERα dimerization. Stability and self-aggregation of peptide based therapeutics poses a significant bottle-neck to proceed further. In this study utilizing peptide grafted to preserve their pharmacophoric recognition motif and assessed their stability and potential to block ERα mediated activity in silico and in vitro. The Grafted peptides blocked ERα mediated cell proliferation and viability of breast cancer cells but did not alter their apoptotic fate. We believe the structural clues identified in this study can be used to identify novel peptidometics and small molecules that specifically target ER dimer interface generating a new breed of anti-cancer agents. PMID:27462021

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

    PubMed

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

    2016-12-15

    Encapsulation of peptides can be used to enhance their stability, delivery and bioavailability. This study focused on the effect of the molecular weight range of whey peptides on their encapsulation within soy lecithin-derived nanoliposomes. Peptide molecular weight did not have a major impact on encapsulation efficiency or liposome size. However, it influenced peptide distribution amongst the surface, core, and bilayer regions of the liposomes, as determined by electrical charge (ζ-potential) and FTIR analysis. The liposome ζ-potential depended on peptide molecular weight, suggesting that the peptide charged groups were in different locations relative to the liposome surfaces. FTIR analysis indicated that the least hydrophobic peptide fractions interacted more strongly with choline on the liposome surfaces. The results suggested that the peptides were unequally distributed within the liposomes, even at the same encapsulation efficiency. These findings are important for designing delivery systems for commercial production of encapsulated peptides with improved functional attributes. PMID:27451165

  9. Targeting DNA vaccines to myeloid cells using a small peptide.

    PubMed

    Ye, Chunting; Choi, Jang Gi; Abraham, Sojan; Shankar, Premlata; Manjunath, N

    2015-01-01

    Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein Ags to DCs, currently there is no method that targets DNA vaccines directly to DCs. Here, we show that a small peptide derived from the rabies virus glycoprotein fused to protamine residues (RVG-P) can target DNA to myeloid cells, including DCs, which results in enhanced humoral and T-cell responses. DCs targeted with a DNA vaccine encoding the immunodominant vaccinia B8R gene via RVG-P were able to restimulate vaccinia-specific memory T cells in vitro. Importantly, a single i.v. injection of B8R gene bound to RVG-P was able to prime a vaccinia-specific T-cell response that was able to rapidly clear a subsequent vaccinia challenge in mice. Moreover, delivery of DNA in DCs was enough to induce DC maturation and efficient Ag presentation without the need for adjuvants. Finally, immunization of mice with a DNA-vaccine encoding West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WNV-neutralizing Abs that protected mice from lethal WNV challenge. Thus, RVG-P provides a reagent to target DNA vaccines to myeloid cells and elicit robust T-cell and humoral immune responses. PMID:25270431

  10. Role of signal peptides in targeting of proteins in cyanobacteria.

    PubMed Central

    Mackle, M M; Zilinskas, B A

    1994-01-01

    Proteins of cyanobacteria may be transported across one of two membrane systems: the typical eubacterial cell envelope (consisting of an inner membrane, periplasmic space, and an outer membrane) and the photosynthetic thylakoids. To investigate the role of signal peptides in targeting in cyanobacteria, Synechococcus sp. strain PCC 7942 was transformed with vectors carrying the chloramphenicol acetyltransferase reporter gene fused to coding sequences for one of four different signal peptides. These included signal peptides of two proteins of periplasmic space origin (one from Escherichia coli and the other from Synechococcus sp. strain PCC 7942) and two other signal peptides of proteins located in the thylakoid lumen (one from a cyanobacterium and the other from a higher plant). The location of the gene fusion products expressed in Synechococcus sp. strain PCC 7942 was determined by a chloramphenicol acetyltransferase enzyme-linked immunosorbent assay of subcellular fractions. The distribution pattern for gene fusions with periplasmic signal peptides was different from that of gene fusions with thylakoid lumen signal peptides. Primary sequence analysis revealed conserved features in the thylakoid lumen signal peptides that were absent from the periplasmic signal peptides. These results suggest the importance of the signal peptide in protein targeting in cyanobacteria and point to the presence of signal peptide features conserved between chloroplasts and cyanobacteria for targeting of proteins to the thylakoid lumen. Images PMID:8144451

  11. Antimicrobial and Immunomodulatory Activities of PR-39 Derived Peptides

    PubMed Central

    Veldhuizen, Edwin J. A.; Schneider, Viktoria A. F.; Agustiandari, Herfita; van Dijk, Albert; Tjeerdsma-van Bokhoven, Johanna L. M.; Bikker, Floris J.; Haagsman, Henk P.

    2014-01-01

    The porcine cathelicidin PR-39 is a host defence peptide that plays a pivotal role in the innate immune defence of the pig against infections. Besides direct antimicrobial activity, it is involved in immunomodulation, wound healing and several other biological processes. In this study, the antimicrobial- and immunomodulatory activity of PR-39, and N- and C-terminal derivatives of PR-39 were tested. PR-39 exhibited an unexpected broad antimicrobial spectrum including several Gram positive strains such as Bacillus globigii and Enterococcus faecalis. Of organisms tested, only Staphylococcus aureus was insensitive to PR-39. Truncation of PR-39 down to 15 (N-terminal) amino acids did not lead to major loss of activity, while peptides corresponding to the C-terminal part of PR-39 were hampered in their antimicrobial activity. However, shorter peptides were all much more sensitive to inhibition by salt. Active peptides induced ATP leakage and loss of membrane potential in Bacillus globigii and Escherichia coli, indicating a lytic mechanism of action for these peptides. Finally, only the mature peptide was able to induce IL-8 production in porcine macrophages, but some shorter peptides also had an effect on TNF-α production showing differential regulation of cytokine induction by PR-39 derived peptides. None of the active peptides showed high cytotoxicity highlighting the potential of these peptides for use as an alternative to antibiotics. PMID:24755622

  12. New antibacterial peptide derived from bovine hemoglobin.

    PubMed

    Daoud, Rachid; Dubois, Veronique; Bors-Dodita, Loredana; Nedjar-Arroume, Naima; Krier, Francois; Chihib, Nour-Eddine; Mary, Patrice; Kouach, Mostafa; Briand, Gilbert; Guillochon, Didier

    2005-05-01

    Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields an intermediate peptide fraction exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis after separation by reversed-phase HPLC. From this fraction a pure peptide was isolated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). This peptide correspond to the 107-136 fragment of the alpha chain of bovine hemoglobin. The minimum inhibitory concentrations (MIC) towards the four strains and its hemolytic activity towards bovine erythrocytes were determined. A MIC of 38 microM was reported against L. innocua and 76 microM for other various bacterial species. This peptide had no hemolytic activity up to 380 microM concentration. PMID:15808900

  13. Targeting and Therapeutic Peptides in Nanomedicine for Atherosclerosis

    PubMed Central

    Chung, Eun Ji

    2016-01-01

    Peptides in atherosclerosis nanomedicine provide structural, targeting, and therapeutic functionality, and can assist in overcoming delivery barriers of traditional pharmaceuticals. Moreover, their inherent biocompatibility and biodegradability make them especially attractive as materials intended for use in vivo. In this review, an overview of nanoparticle-associated targeting and therapeutic peptides for atherosclerosis are provided, including peptides designed for cellular targets such as endothelial cells, monocytes, and macrophages as well as for plaque components such as collagen and fibrin. An emphasis is placed on recent advances in multimodal strategies and a discussion on current challenges and barriers for clinical applicability is presented. PMID:27022138

  14. Laminin-111-derived peptides and cancer

    PubMed Central

    Kikkawa, Yamato; Hozumi, Kentaro; Katagiri, Fumihiko; Nomizu, Motoyoshi; Kleinman, Hynda K.; Koblinski, Jennifer E.

    2013-01-01

    Laminin-111 is a large trimeric basement membrane glycoprotein with many active sites. In particular, four peptides active in tumor malignancy studies have been identified in laminin-111 using a systematic peptide screening method followed by various assays. Two of the peptides (IKVAV and AG73) are found on the α1 chain, one (YIGSR) of the β1 chain and one (C16) on the γ1 chain. The four peptides have distinct activities and receptors. Since three of the peptides (IKVAV, AG73 and C16) strongly promote tumor growth, this may explain the potent effects laminin-111 has on malignant cells. The peptide, YIGSR, decreases tumor growth and experimental metastasis via a 32/67 kD receptor while IKVAV increases tumor growth, angiogenesis and protease activity via integrin receptors. AG73 increases tumor growth and metastases via syndecan receptors. C16 increases tumor growth and angiogenesis via integrins. Identification of such sites on laminin-111 will have use in defining strategies to develop therapeutics for cancer. PMID:23263633

  15. Constrained peptides with target-adapted cross-links as inhibitors of a pathogenic protein-protein interaction.

    PubMed

    Glas, Adrian; Bier, David; Hahne, Gernot; Rademacher, Christoph; Ottmann, Christian; Grossmann, Tom N

    2014-02-24

    Bioactive conformations of peptides can be stabilized by macrocyclization, resulting in increased target affinity and activity. Such macrocyclic peptides proved useful as modulators of biological functions, in particular as inhibitors of protein-protein interactions (PPI). However, most peptide-derived PPI inhibitors involve stabilized α-helices, leaving a large number of secondary structures unaddressed. Herein, we present a rational approach towards stabilization of an irregular peptide structure, using hydrophobic cross-links that replace residues crucially involved in target binding. The molecular basis of this interaction was elucidated by X-ray crystallography and isothermal titration calorimetry. The resulting cross-linked peptides inhibit the interaction between human adaptor protein 14-3-3 and virulence factor exoenzyme S. Taking into consideration that irregular peptide structures participate widely in PPIs, this approach provides access to novel peptide-derived inhibitors. PMID:24504455

  16. Label-Free, In-Solution Screening of Peptide Libraries for Binding to Protein Targets Using Hydrogen Exchange Mass Spectrometry.

    PubMed

    Maaty, Walid S; Weis, David D

    2016-02-01

    There is considerable interest in the discovery of peptide ligands that bind to protein targets. Discovery of such ligands is usually approached by screening large peptide libraries. However, the individual peptides must be tethered to a tag that preserves their individual identities (e.g., phage display or one-bead one-compound). To overcome this limitation, we have developed a method for screening libraries of label-free peptides for binding to a protein target in solution as a single batch. The screening is based on decreased amide hydrogen exchange by peptides that bind to the target. Hydrogen exchange is measured by mass spectrometry. We demonstrate the approach using a peptide library derived from the Escherichia coli proteome that contained 6664 identifiable features. The library was spiked separately with a peptide spanning the calmodulin binding domain of endothelial nitric oxide synthase (eNOS, 494-513) and a peptide spanning the N-terminal 20 residues of bovine ribonuclease A (S peptide). Human calmodulin and bovine ribonuclease S (RNase S) were screened against the library. Using a novel data analysis workflow, we identified the eNOS peptide as the only calmodulin binding peptide and S peptide as the only ribonuclease S binding peptide in the library. PMID:26741284

  17. Decreased outer membrane permeability protects mycobacteria from killing by ubiquitin-derived peptides.

    PubMed

    Purdy, Georgiana E; Niederweis, Michael; Russell, David G

    2009-09-01

    Ubiquitin-derived peptides are bactericidal in vitro and contribute to the mycobactericidal activity of the lysosome. To further define interactions of ubiquitin-derived peptides with mycobacteria, we screened for mutants with increased resistance to the bactericidal activity of the synthetic ubiquitin-derived peptide Ub2. The four Ub2-resistant Mycobacterium smegmatis mutants were also resistant to the bactericidal action of other antimicrobial peptides and macrophages. Two mutants were in the mspA gene encoding the main M. smegmatis porin. Using a translocation-deficient MspA point mutant, we showed that susceptibility of M. smegmatis to Ub2 was independent of MspA channel activity. Instead, the M. smegmatis Ub2-resistant mutants shared a common phenotype of decreased cell wall permeability compared with wild-type bacteria. Expression of mspA rendered Mycobacterium tuberculosis CDC1551 more susceptible both to ubiquitin-derived peptides in vitro and to lysosomal killing in macrophages. Finally, biochemical assays designed to assess membrane integrity indicated that Ub2 treatment impairs membrane function of M. smegmatis and M. tuberculosis cells. The M. smegmatis Ub2-resistant mutants were more resistant than wild-type M. smegmatis to this damage. We conclude that Ub2 targets mycobacterial membranes and that reduced membrane permeability provides mycobacteria intrinsic resistance against antimicrobial compounds including bactericidal ubiquitin-derived peptides. PMID:19682257

  18. Peptidic Tumor Targeting Agents: The Road from Phage Display Peptide Selections to Clinical Applications

    PubMed Central

    Brown, Kathlynn C.

    2014-01-01

    Cancer has become the number one cause of death amongst Americans, killing approximately 1,600 people per day. Novel methods for early detection and the development of effective treatments are an eminent priority in medicine. For this reason, isolation of tumor-specific ligands is a growing area of research. Tumor-specific binding agents can be used to probe the tumor cell surface phenotype and customize treatment accordingly by conjugating the appropriate cell-targeting ligand to an anticancer drug. This refines the molecular diagnosis of the tumor and creates guided drugs that can target the tumor while sparing healthy tissues. Additionally, these targeting agents can be used as in vivo imaging agents that allow for earlier detection of tumors and micrometastasis. Phage display is a powerful technique for the isolation of peptides that bind to a particular target with high affinity and specificity. The biopanning of intact cancer cells or tumors in animals can be used to isolate peptides that bind to cancer-specific cell surface biomarkers. Over the past 10 years, unbiased biopanning of phage-displayed peptide libraries has generated a suite of cancer targeting peptidic ligands. This review discusses the recent advances in the isolation of cancer-targeting peptides by unbiased biopanning methods and highlights the use of the isolated peptides in clinical applications. PMID:20030617

  19. Targeted Proapoptotic Peptides Depleting Adipose Stromal Cells Inhibit Tumor Growth.

    PubMed

    Daquinag, Alexes C; Tseng, Chieh; Zhang, Yan; Amaya-Manzanares, Felipe; Florez, Fernando; Dadbin, Ali; Zhang, Tao; Kolonin, Mikhail G

    2016-02-01

    Progression of many cancers is associated with tumor infiltration by mesenchymal stromal cells (MSC). Adipose stromal cells (ASC) are MSC that serve as adipocyte progenitors and endothelium-supporting cells in white adipose tissue (WAT). Clinical and animal model studies indicate that ASC mobilized from WAT are recruited by tumors. Direct evidence for ASC function in tumor microenvironment has been lacking due to unavailability of approaches to specifically inactivate these cells. Here, we investigate the effects of a proteolysis-resistant targeted hunter-killer peptide D-WAT composed of a cyclic domain CSWKYWFGEC homing to ASC and of a proapoptotic domain KLAKLAK2. Using mouse bone marrow transplantation models, we show that D-WAT treatment specifically depletes tumor stromal and perivascular cells without directly killing malignant cells or tumor-infiltrating leukocytes. In several mouse carcinoma models, targeted ASC cytoablation reduced tumor vascularity and cell proliferation resulting in hemorrhaging, necrosis, and suppressed tumor growth. We also validated a D-WAT derivative with a proapoptotic domain KFAKFAK2 that was found to have an improved cytoablative activity. Our results for the first time demonstrate that ASC, recruited as a component of tumor microenvironment, support cancer progression. We propose that drugs targeting ASC can be developed as a combination therapy complementing conventional cancer treatments. PMID:26316391

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

    PubMed

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

    2016-08-01

    Animal venoms represent a rich source of pharmacologically active peptides that interact with ion channels. However, a challenge to discovering drugs remains because of the slow pace at which venom peptides are discovered and refined. An efficient autocrine-based high-throughput selection system was developed to discover and refine venom peptides that target ion channels. The utility of this system was demonstrated by the discovery of novel Kv1.3 channel blockers from a natural venom peptide library that was formatted for autocrine-based selection. We also engineered a Kv1.3 blocker peptide (ShK) derived from sea anemone to generate a subtype-selective Kv1.3 blocker with a long half-life in vivo. PMID:27197631

  1. Inhibition of inflammation by a p38 MAP kinase targeted cell permeable peptide.

    PubMed

    Fu, Jing; Meng, Xianmei; He, Junyun; Gu, Jun

    2008-11-01

    p38 MAPK has been the key therapeutic target for multiple inflammation diseases. However, the clinical applications of p38 inhibitors, most of which target on the ATP binding groove in the kinase, have been held back, largely because of their limited specificity and severe side-effects. An alternative strategy to generate highly selective p38 inhibitor is to block the specific interaction in the p38 signal pathway. Based on the hypothesis that specific binding peptides targeting on the docking groove would interfere the intrinsic interaction between p38 and its partners, we have designed a fusion peptide containing 12aa p38 docking sequence derived from MKK3b and 11aa HIV-TAT transmembrane sequence to form a cell permeable peptide. The peptide specifically binds to p38, and aborts its interaction with upstream kinase as well as downstream substrates, and thus to inhibit p38 phosphorylation and its signaling. Furthermore, the induction and secretion of TNFalpha and other inflammatory factors by LPS are blocked in peptide treated cells and mice. Finally the peptide has been shown to significantly inhibit ear oedema in mice. Therefore, the peptide holds great potential as an anti-inflammation agent for the treatment of inflammation and its related diseases. PMID:18991745

  2. The problem with peptide presumption and the downfall of target-decoy false discovery rates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In proteomics, peptide-tandem mass spectrum match scores and target-decoy database derived false discovery rates (FDR) are confidence indicators describing the quality of individual and sets of tandem mass spectrum matches. A user can impose a standard by prescribing a limit to these values, equival...

  3. Novel alpha-MSH peptide analogs for melanoma targeting

    NASA Astrophysics Data System (ADS)

    Flook, Adam Michael

    Skin cancer is the one of the most diagnosed cancers in the United States with increasing incidence over the past two decades. There are three major forms of skin cancer but melanoma is the deadliest. It is estimated that 76,690 new diagnoses of melanoma and 9,480 deaths will occur in 2013. Melanoma accounts for approximately 1.6% of all cancer related deaths and is the 5 th leading diagnosed cancer in the United States. The mean survival rate of patients diagnosed with metastatic melanoma is six months, with five year survival rates of less than 5%. In this project, we describe the design and characterization of novel melanoma-targeting peptide analogs for use in diagnostic imaging of both primary and metastatic melanoma lesions. Novel alpha-MSH peptide conjugates were designed to target the melanocortin-1 receptor present and over-expressed on melanoma cells. These peptides were synthesized and their in-vitro melanocortin-1 receptor binding affinities were established in murine melanoma cells. Once binding affinities were determined, the peptides were radiolabeled with 99mTc utilizing a novel direct radiolabeling technique developed in our laboratory. The peptides were purified via reverse-phase high performance liquid chromatography and in-vivo melanoma targeting and pharmacokinetic properties were determined in B16/F1 melanoma-bearing female C57BL/6 mice. Biodistribution and SPECT/CT imaging studies were performed with the promising 99m Tc-labeled peptide conjugates. All alpha-MSH peptide conjugates tested showed low nanomolar binding affinity for the melanocortin-1 receptor. All peptides were readily radiolabeld with 99mTc with greater than 95% radiochemical purity. All 99mTc-labeled peptides displayed high specific in-vivo melanoma tumor uptake while maintaining low normal organ accumulation, and were excreted through the urinary system in a timely fashion. In addition, all tested 99mTc-labeld alpha-MSH peptides demonstrated clear visualization of in

  4. Affinity Peptide for Targeted Detection of Dysplasia in Barrett's Esophagus

    PubMed Central

    Li, Meng; Anastassiades, Costas P.; Joshi, Bishnu; Komarck, Chris M.; Piraka, Cyrus; Elmunzer, Badih J.; Turgeon, Danielle K.; Johnson, Timothy D.; Appelman, Henry; Beer, David G.; Wang, Thomas D.

    2012-01-01

    Background & Aims Dysplasia is a pre-malignant condition in Barrett's esophagus that is difficult to detect on screening endoscopy because of its flat architecture and patchy distribution. Peptides are promising for use as novel molecular probes that identify cell surface targets unique to disease, and can be fluorescence-labeled for detection. We aim to select and validate an affinity peptide that binds to esophageal dysplasia for future clinical studies. Methods Peptide selection was performed using phage display by removing non-specific binders using Q-hTERT (intestinal metaplasia) cells and achieving specific binding against OE33 (esophageal adenocarcinoma) cells. Selective binding was confirmed on bound phage counts, ELISA, flow cytometry, competitive inhibition, and fluorescence microscopy. On stereomicroscopy, specific peptide binding to dysplasia on endoscopically resected specimens was assessed by rigorous registration of fluorescence intensity to histology in 1 mm intervals. Results The peptide sequence SNFYMPL was selected and demonstrated preferential binding to target cells on bound phage counts, ELISA, and flow cytometry. Reducing binding was observed on competition with unlabeled peptide in a dose dependent manner, an affinity of Kd = 164 nM was measured, and peptide binding to the surface of OE33 cells was validated on fluorescence microscopy. On esophageal specimens (n=12), the fluorescence intensity (mean±SEM) in 1 mm intervals classified histologically as squamous (n=145), intestinal metaplasia (n=83), dysplasia (n=61) and gastric mucosa (n=69) was 46.5±1.6, 62.3±5.8, 100.0±9.0, and 42.4±3.0 arb units, respectively. Conclusions The peptide sequence SNFYMPL binds specifically to dysplasia in Barrett's esophagus, and can be fluorescence-labeled to target pre-malignant mucosa on imaging. PMID:20637198

  5. Milk derived bioactive peptides and their impact on human health - A review.

    PubMed

    Mohanty, D P; Mohapatra, S; Misra, S; Sahu, P S

    2016-09-01

    Milk-derived bioactive peptides have been identified as potential ingredients of health-promoting functional foods. These bioactive peptides are targeted at diet-related chronic diseases especially the non-communicable diseases viz., obesity, cardiovascular diseases and diabetes. Peptides derived from the milk of cow, goat, sheep, buffalo and camel exert multifunctional properties, including anti-microbial, immune modulatory, anti-oxidant, inhibitory effect on enzymes, anti-thrombotic, and antagonistic activities against various toxic agents. Majority of those regulate immunological, gastrointestinal, hormonal and neurological responses, thereby playing a vital role in the prevention of cancer, osteoporosis, hypertension and other disorders as discussed in this review. For the commercial production of such novel bioactive peptides large scale technologies based on membrane separation and ion exchange chromatography methods have been developed. Separation and identification of those peptides and their pharmacodynamic parameters are necessary to transfer their potent functional properties into food applications. The present review summarizes the preliminary classes of bioactive milk-derived peptides along with their physiological functions, general characteristics and potential applications in health-care. PMID:27579006

  6. Phage Peptide Libraries As a Source of Targeted Ligands

    PubMed Central

    Nemudraya, A. A.; Richter, V. A.; Kuligina, E. V.

    2016-01-01

    One of the dominant trends in modern pharmacology is the creation of drugs that act directly on the lesion focus and have minimal toxicity on healthy tissues and organs. This problem is particularly acute in relation to oncologic diseases. Short tissue- and organ-specific peptides capable of delivering drugs to the affected organ or tissue are considered promising targeted agents that can be used in the diagnosis and therapy of diseases, including cancer. The review discusses in detail the technology of phage display as a method for obtaining specific targeted peptide agents and offers examples of their use in diagnostic and clinical practice. PMID:27099784

  7. Autophagy as a target for therapeutic uses of multifunctional peptides.

    PubMed

    Muciño, Gabriel; Castro-Obregón, Susana; Hernandez-Pando, Rogelio; Del Rio, Gabriel

    2016-04-01

    The emergence of complex diseases is promoting a change from one-target to multitarget drugs and peptides are ideal molecules to fulfill this polypharmacologic role. Here we review current status in the design of polypharmacological peptides aimed to treat complex diseases, focusing on tuberculosis. In this sense, combining multiple activities in single molecules is a two-sided sword, as both positive and negative side effects might arise. These polypharmacologic compounds may be directed to regulate autophagy, a catabolic process that enables cells to eliminate intracellular microbes (xenophagy), such as Mycobacterium tuberculosis (MBT). Here we review some strategies to control MBT infection and propose that a peptide combining both antimicrobial and pro-autophagic activities would have a greater potential to limit MBT infection. This endeavor may complement the knowledge gained in understanding the mechanism of action of antibiotics and may lead to the design of better polypharmacological peptides to treat complex diseases such as tuberculosis. PMID:26968336

  8. Peptiderive server: derive peptide inhibitors from protein-protein interactions.

    PubMed

    Sedan, Yuval; Marcu, Orly; Lyskov, Sergey; Schueler-Furman, Ora

    2016-07-01

    The Rosetta Peptiderive protocol identifies, in a given structure of a protein-protein interaction, the linear polypeptide segment suggested to contribute most to binding energy. Interactions that feature a 'hot segment', a linear peptide with significant binding energy compared to that of the complex, may be amenable for inhibition and the peptide sequence and structure derived from the interaction provide a starting point for rational drug design. Here we present a web server for Peptiderive, which is incorporated within the ROSIE web interface for Rosetta protocols. A new feature of the protocol also evaluates whether derived peptides are good candidates for cyclization. Fast computation times and clear visualization allow users to quickly assess the interaction of interest. The Peptiderive server is available for free use at http://rosie.rosettacommons.org/peptiderive. PMID:27141963

  9. Chemokine-Derived Peptides: Novel Antimicrobial and Antineoplasic Agents.

    PubMed

    Valdivia-Silva, Julio; Medina-Tamayo, Jaciel; Garcia-Zepeda, Eduardo A

    2015-01-01

    Chemokines are a burgeoning family of chemotactic cytokines displaying a broad array of functions such as regulation of homeostatic leukocyte traffic and development, as well as activating the innate immune system. Their role in controlling early and late inflammatory stages is now well recognized. An improper balance either in chemokine synthesis or chemokine receptor expression contributes to various pathological disorders making chemokines and their receptors a useful therapeutic target. Research in this area is progressing rapidly, and development of novel agents based on chemokine/ chemokine receptors antagonist functions are emerging as attractive alternative drugs. Some of these novel agents include generation of chemokine-derived peptides (CDP) with potential agonist and antagonist effects on inflammation, cancer and against bacterial infections. CDP have been generated mainly from N- and C-terminus chemokine sequences with subsequent modifications such as truncations or elongations. In this review, we present a glimpse of the different pharmacological actions reported for CDP and our current understanding regarding the potential use of CDP alone or as part of the novel therapies proposed in the treatment of microbial infections and cancer. PMID:26062132

  10. Chemokine-Derived Peptides: Novel Antimicrobial and Antineoplasic Agents

    PubMed Central

    Valdivia-Silva, Julio; Medina-Tamayo, Jaciel; Garcia-Zepeda, Eduardo A.

    2015-01-01

    Chemokines are a burgeoning family of chemotactic cytokines displaying a broad array of functions such as regulation of homeostatic leukocyte traffic and development, as well as activating the innate immune system. Their role in controlling early and late inflammatory stages is now well recognized. An improper balance either in chemokine synthesis or chemokine receptor expression contributes to various pathological disorders making chemokines and their receptors a useful therapeutic target. Research in this area is progressing rapidly, and development of novel agents based on chemokine/chemokine receptors antagonist functions are emerging as attractive alternative drugs. Some of these novel agents include generation of chemokine-derived peptides (CDP) with potential agonist and antagonist effects on inflammation, cancer and against bacterial infections. CDP have been generated mainly from N- and C-terminus chemokine sequences with subsequent modifications such as truncations or elongations. In this review, we present a glimpse of the different pharmacological actions reported for CDP and our current understanding regarding the potential use of CDP alone or as part of the novel therapies proposed in the treatment of microbial infections and cancer. PMID:26062132

  11. DMP1-derived peptides promote remineralization of human dentin.

    PubMed

    Padovano, J D; Ravindran, S; Snee, P T; Ramachandran, A; Bedran-Russo, A K; George, A

    2015-04-01

    Remineralization of dentin during dental caries is of considerable clinical interest. Dentin matrix protein 1 (DMP1) is a non-collagenous calcium-binding protein that plays a critical role in biomineralization. In the present study, we tested if peptides derived from DMP1 can be used for dentin remineralization. Peptide pA (pA, MW = 1.726 kDa) and peptide pB (pB, MW = 2.185), containing common collagen-binding domains and unique calcium-binding domains, were synthesized by solid-phase chemistry. An extreme caries lesion scenario was created by collagenase digestion, and the biomineral-nucleating potential of these peptides was ascertained when coated on collagenase-treated dentin matrix and control, native human dentin matrix under physiological levels of calcium and phosphate. Scanning electron microscopy analysis suggests that peptide pB was an effective nucleator when compared with pA. However, a 1:4 ratio of pA to pB was determined to be ideal for dentin remineralization, based on hydroxyapatite (HA) morphology and calcium/phosphorus ratios. Interestingly, HA was nucleated on collagenase-challenged dentin with as little as 20 min of 1:4 peptide incubation. Electron diffraction confirmed the presence of large HA crystals that produced a diffraction pattern indicative of a rod-like crystal structure. These findings suggest that DMP1-derived peptides may be useful to modulate mineral deposition and subsequent formation of HA when exposed to physiological concentrations of calcium and phosphate. PMID:25694469

  12. Photoperiod Regulates vgf-Derived Peptide Processing in Siberian Hamsters

    PubMed Central

    Noli, Barbara; Brancia, Carla; Pilleri, Roberta; D’Amato, Filomena; Messana, Irene; Manconi, Barbara; Ebling, Francis J. P.; Ferri, Gian-Luca; Cocco, Cristina

    2015-01-01

    VGF mRNA is induced in specific hypothalamic areas of the Siberian hamster upon exposure to short photoperiods, which is associated with a seasonal decrease in appetite and weight loss. Processing of VGF generates multiple bioactive peptides, so the objective of this study was to determine the profile of the VGF-derived peptides in the brain, pituitary and plasma from Siberian hamsters, and to establish whether differential processing might occur in the short day lean state versus long day fat. Antisera against short sequences at the C- or N- termini of proVGF, as well as against NERP-1, TPGH and TLQP peptides, were used for analyses of tissues, and both immunohistochemistry and enzyme linked immunosorbent assay (ELISA) coupled with high-performance liquid (HPLC) or gel chromatography were carried out. VGF peptide immunoreactivity was found within cortex cholinergic perikarya, in multiple hypothalamic nuclei, including those containing vasopressin, and in pituitary gonadotrophs. ELISA revealed that exposure to short day photoperiod led to a down-regulation of VGF immunoreactivity in the cortex, and a less pronounced decrease in the hypothalamus and pituitary, while the plasma VGF levels were not affected by the photoperiod. HPLC and gel chromatography both confirmed the presence of multiple VGF-derived peptides in these tissues, while gel chromatography showed the presence of the VGF precursor in all tissues tested except for the cortex. These observations are consistent with the view that VGF-derived peptides have pleiotropic actions related to changing photoperiod, possibly by regulating cholinergic systems in the cortex, vasopressin hypothalamic pathways, and the reproductive axis. PMID:26555143

  13. A chirality change in XPC- and Sfi1-derived peptides affects their affinity for centrin.

    PubMed

    Grecu, Dora; Irudayaraj, Victor Paul Raj; Martinez-Sanz, Juan; Mallet, Jean-Maurice; Assairi, Liliane

    2016-04-01

    The Ca(2+)-binding protein centrin binds to a hydrophobic motif (W(1)xxL(4)xxxL(8)) included in the sequence of several cellular targets: XPC (xeroderma pigmentosum group C protein), Sfi1 (suppressor of fermentation-induced loss of stress resistance protein1), and Sac3 [the central component of the transcription and mRNA export (TREX-2) complex]. However, centrin binding occurs in a reversed orientation (L(8)xxxL(4)xxW(1)) for Sfi1 and Sac3 compared with XPC. Because D-peptides have been investigated for future therapeutic use, we analyzed their centrin-binding properties. Their affinity for centrin was measured using isothermal titration calorimetry. The chirality change in the target-derived peptides affected their ability to bind centrin in a specific manner depending on the sequence orientation of the centrin-binding motif. In contrast to L-XPC-P10, D-XPC-P10 bound C-HsCen1 in a Ca(2+)-dependent manner and to a lesser extent. D-XPC-P10 exhibited a reduced affinity for C-HsCen1 (Ka=0.064 × 10(6) M(-1)) by a factor of 2000 compared with L-XPC-P10 (Ka=132 × 10(6) M(-1)). D-peptides have a lower affinity than L-peptides for centrin, and the strength of this affinity depends on the sequence orientation of the target-derived peptides. The residual affinity observed for D-XPC suggests that the use of d-peptides represents a promising strategy for inhibiting centrin binding to its targets. PMID:26923803

  14. Food Derived Bioactive Peptides and Intestinal Barrier Function

    PubMed Central

    Martínez-Augustin, Olga; Rivero-Gutiérrez, Belén; Mascaraque, Cristina; Sánchez de Medina, Fermín

    2014-01-01

    A wide range of food-derived bioactive peptides have been shown to exert health-promoting actions and are therefore considered functional foods or nutraceuticals. Some of these actions are related to the maintenance, reinforcement or repairment of the intestinal barrier function (IBF) whose role is to selectively allow the absorption of water, nutrients and ions while preventing the influx of microorganisms from the intestinal lumen. Alterations in the IBF have been related to many disorders, such as inflammatory bowel disease or metabolic syndrome. Components of IBF are the intestinal epithelium, the mucus layer, secretory immunoglobulin A and cells of the innate and adaptive immune systems. Here we review the effects of food derived bioactive peptides on these IBF components. In vitro and in vivo effects, both in healthy and disease states, have been reviewed. Although limited, the available information indicates a potential for food-derived peptides to modify IBF and to contribute to disease treatment, but further research is needed to better isolate responsible peptides, and to help define their mode of action. PMID:25501338

  15. Uptake and cellular distribution of nucleolar targeting peptides (NrTPs) in different cell types.

    PubMed

    Rodrigues, Margarida; Andreu, David; Santos, Nuno C

    2015-03-01

    Nucleolar targeting peptides (NrTPs) are a family of cell penetrating peptides (CPPs) derived from crotamine, a rattlesnake venom toxin. They were named NrTPs for their remarkable nucleolus-homing properties and have been studied for their potential as drug delivery vehicles. Live cell microscopy experiments were conducted to monitor NrTP uptake and distribution in different cell types, including primary cells (PBMCs and erythrocytes) and different immortalized cell lines (HeLa, BHK21, BV-173, and MOLT-4). Uptake dependence on cell type (primary vs. immortalized, suspension vs. adherent, cancer vs. healthy cells), peptide concentration and cell viability were evaluated. To gain further insight on the internalization mechanism, uptake kinetics was also monitored. Results showed the uptake and distribution pattern as strongly dependent on peptide sequence, peptide concentration and membrane constituents. Under similar conditions, NrTP6 is more internalized than NrTP1, NrTP2 and NrTP5. Additionally, while internalization of NrTP7 and NrTP8 may cause cytotoxicity, NrTP6 is noncytotoxic. Higher peptide concentrations can be correlated to nucleolar targeting, although even at low concentrations a residual number of cells reveal positive nucleolar labeling. NrTPs were successfully internalized into all cell types tested except erythrocytes. PMID:25620660

  16. Intrinsically disordered amphiphilic peptides as potential targets in drug delivery vehicles.

    PubMed

    Vincenzi, Marian; Accardo, Antonella; Costantini, Susan; Scala, Stefania; Portella, Luigi; Trotta, Annamaria; Ronga, Luisa; Guillon, Jean; Leone, Marilisa; Colonna, Giovanni; Rossi, Filomena; Tesauro, Diego

    2015-11-01

    Intrinsically disordered proteins/peptides play a crucial role in many physiological and pathological events and may assume a precise conformation upon binding to a specific target. Recently, we have described the conformational and functional properties of two linear ester peptides provided with the following sequences: Y-G-E-C-P-C-K-OAllyl (PepK) and Y-G-E-C-P-C-E-OAllyl (PepE). Both peptides are characterized by the presence of the "CPC" motif together with a few amino acids able to promote disorder. The CPC sequence is a binding motif for the CXCR4 receptor that represents a well-known target for cancer therapies. In this paper, we report on synthetic amphiphilic peptides that consist of lipophilic derivatives of PepE and PepK bearing two stearic alkyl chains and/or an ethoxylic spacer. These peptide amphiphiles form stable supramolecular aggregates; they present conformational features that are typical of intrinsically disordered molecules as shown by CD spectroscopy. Solution fluorescence and DLS studies have been performed to evaluate Critical Micellar Concentrations and the dimension of supramolecular aggregates. Moreover, preliminary in vitro cell-based assays have been conducted to investigate the molecular recognition processes involving the CXCR4 receptor. In the end, the results obtained have been compared with the previous data generated by the corresponding non-amphiphilic peptides (PepE and PepK). PMID:26263446

  17. Identification of an HLA-A2-Restricted Epitope Peptide Derived from Hypoxia-Inducible Protein 2 (HIG2)

    PubMed Central

    Yoshimura, Sachiko; Tsunoda, Takuya; Osawa, Ryuji; Harada, Makiko; Watanabe, Tomohisa; Hikichi, Tetsuro; Katsuda, Masahiro; Miyazawa, Motoki; Tani, Masaji; Iwahashi, Makoto; Takeda, Kazuyoshi; Katagiri, Toyomasa; Nakamura, Yusuke; Yamaue, Hiroki

    2014-01-01

    We herein report the identification of an HLA-A2 supertype-restricted epitope peptide derived from hypoxia-inducible protein 2 (HIG2), which is known to be a diagnostic marker and a potential therapeutic target for renal cell carcinoma. Among several candidate peptides predicted by the HLA-binding prediction algorithm, HIG2-9-4 peptide (VLNLYLLGV) was able to effectively induce peptide-specific cytotoxic T lymphocytes (CTLs). The established HIG2-9-4 peptide-specific CTL clone produced interferon-γ (IFN-γ) in response to HIG2-9-4 peptide-pulsed HLA-A*02:01-positive cells, as well as to cells in which HLA-A*02:01 and HIG2 were exogenously introduced. Moreover, the HIG2-9-4 peptide-specific CTL clone exerted cytotoxic activity against HIG2-expressing HLA-A*02:01-positive renal cancer cells, thus suggesting that the HIG2-9-4 peptide is naturally presented on HLA-A*02:01 of HIG-2-expressing cancer cells and is recognized by CTLs. Furthermore, we found that the HIG2-9-4 peptide could also induce CTLs under HLA-A*02:06 restriction. Taken together, these findings indicate that the HIG2-9-4 peptide is a novel HLA-A2 supertype-restricted epitope peptide that could be useful for peptide-based immunotherapy against cancer cells with HIG2 expression. PMID:24416375

  18. Progressive Structuring of a Branched Antimicrobial Peptide on the Path to the Inner Membrane Target*

    PubMed Central

    Bai, Yang; Liu, Shouping; Li, Jianguo; Lakshminarayanan, Rajamani; Sarawathi, Padmanabhan; Tang, Charles; Ho, Duncun; Verma, Chandra; Beuerman, Roger W.; Pervushin, Konstantin

    2012-01-01

    In recent years, interest has grown in the antimicrobial properties of certain natural and non-natural peptides. The strategy of inserting a covalent branch point in a peptide can improve its antimicrobial properties while retaining host biocompatibility. However, little is known regarding possible structural transitions as the peptide moves on the access path to the presumed target, the inner membrane. Establishing the nature of the interactions with the complex bacterial outer and inner membranes is important for effective peptide design. Structure-activity relationships of an amphiphilic, branched antimicrobial peptide (B2088) are examined using environment-sensitive fluorescent probes, electron microscopy, molecular dynamics simulations, and high resolution NMR in solution and in condensed states. The peptide is reconstituted in bacterial outer membrane lipopolysaccharide extract as well as in a variety of lipid media mimicking the inner membrane of Gram-negative pathogens. Progressive structure accretion is observed for the peptide in water, LPS, and lipid environments. Despite inducing rapid aggregation of bacteria-derived lipopolysaccharides, the peptide remains highly mobile in the aggregated lattice. At the inner membranes, the peptide undergoes further structural compaction mediated by interactions with negatively charged lipids, probably causing redistribution of membrane lipids, which in turn results in increased membrane permeability and bacterial lysis. These findings suggest that peptides possessing both enhanced mobility in the bacterial outer membrane and spatial structure facilitating its interactions with the membrane-water interface may provide excellent structural motifs to develop new antimicrobials that can overcome antibiotic-resistant Gram-negative pathogens. PMID:22700968

  19. Self-Assembling Peptide Amphiphiles for Targeted Drug Delivery

    NASA Astrophysics Data System (ADS)

    Moyer, Tyson

    The systemic delivery of therapeutics is currently limited by off-target side effects and poor drug uptake into the cells that need to be treated. One way to circumvent these issues is to target the delivery and release of therapeutics to the desired location while limiting systemic toxicity. Using self-assembling peptide amphiphiles (PAs), this work has investigated supramolecular nanostructures for the development of targeted therapies. Specifically, the research has focused on the interrelationships between presentation of targeting moeities and the control of nanostructure morphology in the context of systemic delivery for targeting cancer and vascular injuries. The self-assembly region of the PA was systematically altered to achieve control of nanostructure widths, from 100 nm to 10 nm, by the addition of valine-glutamic acid dimers into the chemical structure, subsequently increasing the degree of nanostructure twist. For the targeting of tumors, a homing PA was synthesized to include a dimeric, cyclic peptide sequence known to target the cancer-specific, death receptor 5 (DR5) and initiate apoptosis through the oligomerization of DR5. This PA presented a multivalent display of DR5-binding peptides, resulting in improved binding affinity measured by surface plasmon resonance. The DR5-targeting PA also showed enhanced efficacy in both in vitro and in vivo tumor models relative to non-targeted controls. Alternative modifications to the PA-based antitumor therapies included the use of a cytotoxic, membrane-lytic PA coassembled with a pegylated PA, which showed enhanced biodistribution and in vivo activity after coassembly. The functionalization of the hydrophobic core was also accomplished through the encapsulation of the chemotherapy camptothecin, which was shown to be an effective treatment in vivo. Additionally, a targeted PA nanostructure was designed to bind to the site of vascular intervention by targeting collagen IV. Following balloon angioplasty

  20. Reactive Center Loop (RCL) Peptides Derived from Serpins Display Independent Coagulation and Immune Modulating Activities.

    PubMed

    Ambadapadi, Sriram; Munuswamy-Ramanujam, Ganesh; Zheng, Donghang; Sullivan, Colin; Dai, Erbin; Morshed, Sufi; McFadden, Baron; Feldman, Emily; Pinard, Melissa; McKenna, Robert; Tibbetts, Scott; Lucas, Alexandra

    2016-02-01

    Serpins regulate coagulation and inflammation, binding serine proteases in suicide-inhibitory complexes. Target proteases cleave the serpin reactive center loop scissile P1-P1' bond, resulting in serpin-protease suicide-inhibitory complexes. This inhibition requires a near full-length serpin sequence. Myxomavirus Serp-1 inhibits thrombolytic and thrombotic proteases, whereas mammalian neuroserpin (NSP) inhibits only thrombolytic proteases. Both serpins markedly reduce arterial inflammation and plaque in rodent models after single dose infusion. In contrast, Serp-1 but not NSP improves survival in a lethal murine gammaherpesvirus68 (MHV68) infection in interferon γ-receptor-deficient mice (IFNγR(-/-)). Serp-1 has also been successfully tested in a Phase 2a clinical trial. We postulated that proteolytic cleavage of the reactive center loop produces active peptide derivatives with expanded function. Eight peptides encompassing predicted protease cleavage sites for Serp-1 and NSP were synthesized and tested for inhibitory function in vitro and in vivo. In engrafted aorta, selected peptides containing Arg or Arg-Asn, not Arg-Met, with a 0 or +1 charge, significantly reduced plaque. Conversely, S-6 a hydrophobic peptide of NSP, lacking Arg or Arg-Asn with -4 charge, induced early thrombosis and mortality. S-1 and S-6 also significantly reduced CD11b(+) monocyte counts in mouse splenocytes. S-1 peptide had increased efficacy in plasminogen activator inhibitor-1 serpin-deficient transplants. Plaque reduction correlated with mononuclear cell activation. In a separate study, Serp-1 peptide S-7 improved survival in the MHV68 vasculitis model, whereas an inverse S-7 peptide was inactive. Reactive center peptides derived from Serp-1 and NSP with suitable charge and hydrophobicity have the potential to extend immunomodulatory functions of serpins. PMID:26620556

  1. Ligand-Based Peptide Design and Combinatorial Peptide Libraries to Target G Protein-Coupled Receptors

    PubMed Central

    Gruber, Christian W.; Muttenthaler, Markus; Freissmuth, Michael

    2016-01-01

    G protein-coupled receptors (GPCRs) are considered to represent the most promising drug targets; it has been repeatedly said that a large fraction of the currently marketed drugs elicit their actions by binding to GPCRs (with cited numbers varying from 30–50%). Closer scrutiny, however, shows that only a modest fraction of (~60) GPCRs are, in fact, exploited as drug targets, only ~20 of which are peptide-binding receptors. The vast majority of receptors in the humane genome have not yet been explored as sites of action for drugs. Given the drugability of this receptor class, it appears that opportunities for drug discovery abound. In addition, GPCRs provide for binding sites other than the ligand binding sites (referred to as the “orthosteric site”). These additional sites include (i) binding sites for ligands (referred to as “allosteric ligands”) that modulate the affinity and efficacy of orthosteric ligands, (ii) the interaction surface that recruits G proteins and arrestins, (iii) the interaction sites of additional proteins (GIPs, GPCR interacting proteins that regulate G protein signaling or give rise to G protein-independent signals). These sites can also be targeted by peptides. Combinatorial and natural peptide libraries are therefore likely to play a major role in identifying new GPCR ligands at each of these sites. In particular the diverse natural peptide libraries such as the venom peptides from marine cone-snails and plant cyclotides have been established as a rich source of drug leads. High-throughput screening and combinatorial chemistry approaches allow for progressing from these starting points to potential drug candidates. This will be illustrated by focusing on the ligand-based drug design of oxytocin (OT) and vasopressin (AVP) receptor ligands using natural peptide leads as starting points. PMID:20687879

  2. In Silico Design of Novel Anticoagulant Peptides targeting Blood Coagulation Factor VIIa

    PubMed Central

    Al-Amri, Manal S Q; Alrasadi, Khalid; Bayoumi, Riad; Banerjee, Yajnavalka

    2011-01-01

    Objectives: The coagulation cascade initiated during vascular injury prevents bleeding. Unwanted clot formation is however detrimental and requires the use of anticoagulants for prophylaxis and treatment. Anticoagulants targeting a specific step or an enzyme in the clotting process are most preferred as they minimise disadvantageous side-effects. A principal step in the discovery of novel anticoagulants encompasses the in silico design of potential leads. This study depicts the in silico design of peptide anticoagulants targeting coagulation factor VIIa. Methods: Applying the proline bracket rule and using various bioinformatics tools: the basic alignment search tool (BLAST) of National Center for Biotechnology Information; the T-coffee module provided by European Molecular Biology Laboratory-European Bioinformatics Institute, and several modules available on the ExPASy server, we designed five bivalent chimeric anticoagulants targeting factor VIIa, using factor VIIa inhibitors – hemextin A from Hemachatus haemachatus (African Ringhals cobra) venom and factor VIIa exosite-inhibitor peptide as templates. Six peptides were derived from hemextin A, which were concomitantly fused with factor VIIa exosite-inhibitor peptide intermediated by a polyalanine spacer, and analysed for structural stability using the SWISS-MODEL software developed at the Swiss Institute of Bioinformatics and WebLab ViewerPro (Version 4.2). Results: Twelve chimeric peptides were obtained; only five exhibited stable structures in silico. Conclusion: The five peptides obtained are probable anticoagulant leads that should be further evaluated using suitable in vitro and in vivo assays. Further, this study shows how simple web-based modules can be used for the rational design of probable leads targeting specific physiological molecular targets. PMID:21509213

  3. Peptides derived from the dependence receptor ALK are proapoptotic for ALK-positive tumors

    PubMed Central

    Aubry, A; Galiacy, S; Ceccato, L; Marchand, C; Tricoire, C; Lopez, F; Bremner, R; Racaud-Sultan, C; Monsarrat, B; Malecaze, F; Allouche, M

    2015-01-01

    ALK is a receptor tyrosine kinase with an oncogenic role in various types of human malignancies. Despite constitutive activation of the kinase through gene alterations, such as chromosomal translocation, gene amplification or mutation, treatments with kinase inhibitors invariably lead to the development of resistance. Aiming to develop new tools for ALK targeting, we took advantage of our previous demonstration identifying ALK as a dependence receptor, implying that in the absence of ligand the kinase-inactive ALK triggers or enhances apoptosis. Here, we synthesized peptides mimicking the proapoptotic domain of ALK and investigated their biological effects on tumor cells. We found that an ALK-derived peptide of 36 amino acids (P36) was cytotoxic for ALK-positive anaplastic large-cell lymphoma and neuroblastoma cell lines. In contrast, ALK-negative tumor cells and normal peripheral blood mononuclear cells were insensitive to P36. The cytotoxic effect was due to caspase-dependent apoptosis and required N-myristoylation of the peptide. Two P36-derived shorter peptides as well as a cyclic peptide also induced apoptosis. Surface plasmon resonance and mass spectrometry analysis of P36-interacting proteins from two responsive cell lines, Cost lymphoma and SH-SY5Y neuroblastoma, uncovered partners that could involve p53-dependent signaling and pre-mRNA splicing. Furthermore, siRNA-mediated knockdown of p53 rescued these cells from P36-induced apoptosis. Finally, we observed that a treatment combining P36 with the ALK-specific inhibitor crizotinib resulted in additive cytotoxicity. Therefore, ALK-derived peptides could represent a novel targeted therapy for ALK-positive tumors. PMID:25950466

  4. Retention of Conformational Entropy upon Calmodulin Binding to Target Peptides is Driven by Transient Salt Bridges

    SciTech Connect

    Smith, Dayle MA; Straatsma, TP; Squier, Thomas C.

    2012-10-03

    Calmodulin (CaM) is a highly flexible calcium-binding protein that mediates signal transduction through an ability to differentially bind to highly variable binding sequences in target proteins. To identify how binding affects CaM motions, and its relationship to conformational entropy and target peptide sequence, we have employed fully atomistic, explicit solvent molecular dynamics simulations of unbound CaM and CaM bound to five different target peptides. The calculated CaM conformational binding entropies correlate with experimentally derived conformational entropies with a correlation coefficient R2 of 0.95. Selected side-chain interactions with target peptides restrain interhelical loop motions, acting to tune the conformational entropy of the bound complex via widely distributed CaM motions. In the complex with the most conformational entropy retention (CaM in complex with the neuronal nitric oxide synthase binding sequence), Lys-148 at the C-terminus of CaM forms transient salt bridges alternating between Glu side chains in the N-domain, the central linker, and the binding target. Additional analyses of CaM structures, fluctuations, and CaM-target interactions illuminate the interplay between electrostatic, side chain, and backbone properties in the ability of CaM to recognize and discriminate against targets by tuning its conformational entropy, and suggest a need to consider conformational dynamics in optimizing binding affinities.

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

    SciTech Connect

    Imai, Masaki; Baranyi, Lajos; Okada, Noriko; Okada, Hidechika; E-mail: hiokada@med.nagoya-cu.ac.jp

    2007-02-23

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

  6. Transepithelial transport of milk derived bioactive peptide VLPVPQK.

    PubMed

    Vij, Rishika; Reddi, Srinu; Kapila, Suman; Kapila, Rajeev

    2016-01-01

    The transepithelial transport of an antioxidative and ACE inhibitory peptide, VLPVPQK (named peptide C) derived from casein hydrolysates was investigated along with extensively studied opioid peptide β-casomorphin using a human intestinal cell (Caco-2) monolayer. The susceptibility to the brush-border peptidases and route of transepithelial transport were observed to be the primary factors influencing the transport of these peptides. The apical to basal transport mechanism was studied using bradykinin as control as it shows resistance to cellular peptidases and its route of transepithelial transport had been established. VLPVPQK and BCM 5 were hydrolyzed by cellular peptidases while bradykinin was found intact. The transport of VLPVPQK (1.0%) was found to be relatively much higher than BCM 5 (0.03%) and bradykinin (0.1%). Interestingly the effect of some inhibitors on the transport of VLPVPQK suggested involvement of PepT1 like transporters/SOPT2 while BCM 5, its hydrolytic product and bradykinin were suggested to be transported mainly via the intracellular transcytosis pathway. PMID:26213026

  7. Forward Vaccinology: CTL Targeting Based upon Physical Detection of HLA-Bound Peptides

    PubMed Central

    Reinherz, Ellis L.; Keskin, Derin B.; Reinhold, Bruce

    2014-01-01

    Vaccine-elicited cytotoxic T lymphocytes (CTL) recognizing conserved fragments of a pathogen’s proteome could greatly impact infectious diseases and cancers. Enabling this potential are recent advances in mass spectrometry that identify specific target peptides among the myriad HLA-bound peptides on altered cells. Ultrasensitivity of these physical detection methods allows for the direct assessment of peptide presentation on small numbers of tissue-derived cells. In addition, concurrent advances in immunobiology suggest ways to induce CTLs with requisite functional avidity and tissue deployment. Elicitation of high-avidity resident-memory T cells through vaccination may shift the vaccinology paradigm both for preventive and therapeutic approaches to human disease control. PMID:25237310

  8. LHRH-Conjugated Lytic Peptides Directly Target Prostate Cancer Cells

    PubMed Central

    Yates, Clayton; Sharp, Starlette; Jones, Jacqueline; Topps, Daphne; Coleman, Mathew; Aneja, Ritu; Jaynes, Jesse; Turner, Timothy

    2010-01-01

    Prostate cancer is the second leading cause of cancer deaths among men. For patients with hormone-refractory disease, few treatments are available once the tumor has metastasized beyond the prostate. In the present study, two conjugated lytic peptide sequences (named JCHLHRH and JC21LHRH) were designed to target luteinizing hormone-releasing hormone receptors (LHRH-R). Our results indicate that human prostate cancer cell lines were sensitive to both LHRH-conjugated and non-conjugated lytic peptides, with IC50 concentrations for LNCaP cells, 4.4 and 9.1 µM; for DU-145 cells, 4.8 and 5.7 µM; and for PC-3 cells, 4.4 and 8.2 µM, respectively. JCHLHRH and JC21LHRH were nontoxic to normal primary human prostate epithelial cells or to bone marrow stromal cells in co-culture. There were morphological changes in PC-3 cells after 3 hr of exposure to either peptide; after 6 hr, there were significant reductions in cell numbers. Exposure of PC-3 cells for 24 hr to either JCHLHRH or JC21LHRH blocked their growth over 3 days. Since JCHLHRH and JC21LHRH have specificity for and anti-proliferative activity against tumor cells, and low toxicity for normal prostate cells, these peptides could serve as a new type of therapy for prostate cancer. PMID:20869347

  9. Peptide-based carbon nanotubes for mitochondrial targeting.

    PubMed

    Battigelli, Alessia; Russier, Julie; Venturelli, Enrica; Fabbro, Chiara; Petronilli, Valeria; Bernardi, Paolo; Da Ros, Tatiana; Prato, Maurizio; Bianco, Alberto

    2013-10-01

    In the present study, we report the design and synthesis of peptide-based-multi-walled carbon nanotubes (MWCNTs) to target mitochondria. Targeting these intracellular organelles might open the way to develop alternative systems to address diseases related to genetic mutations in mitochondrial (mt)-DNA, by delivering therapeutic oligonucleotides. The first step towards mitochondrial delivery of this type of nucleic acid was to target MWCNTs to mitochondria by covalent functionalization with a well-known endogenous mitochondrial targeting sequence (MTS). The subcellular localization of the conjugates, which were fluorescently labeled, in murine RAW 264.7 macrophages and human HeLa cells was then studied using different microscopy techniques, such as wide-field epifluorescence microscopy, confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). The localization of the MTS-MWCNT conjugates into mitochondria was further confirmed by analyzing the isolated organelles using TEM. PMID:23903095

  10. Peptide-based carbon nanotubes for mitochondrial targeting

    NASA Astrophysics Data System (ADS)

    Battigelli, Alessia; Russier, Julie; Venturelli, Enrica; Fabbro, Chiara; Petronilli, Valeria; Bernardi, Paolo; da Ros, Tatiana; Prato, Maurizio; Bianco, Alberto

    2013-09-01

    In the present study, we report the design and synthesis of peptide-based-multi-walled carbon nanotubes (MWCNTs) to target mitochondria. Targeting these intracellular organelles might open the way to develop alternative systems to address diseases related to genetic mutations in mitochondrial (mt)-DNA, by delivering therapeutic oligonucleotides. The first step towards mitochondrial delivery of this type of nucleic acid was to target MWCNTs to mitochondria by covalent functionalization with a well-known endogenous mitochondrial targeting sequence (MTS). The subcellular localization of the conjugates, which were fluorescently labeled, in murine RAW 264.7 macrophages and human HeLa cells was then studied using different microscopy techniques, such as wide-field epifluorescence microscopy, confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). The localization of the MTS-MWCNT conjugates into mitochondria was further confirmed by analyzing the isolated organelles using TEM.In the present study, we report the design and synthesis of peptide-based-multi-walled carbon nanotubes (MWCNTs) to target mitochondria. Targeting these intracellular organelles might open the way to develop alternative systems to address diseases related to genetic mutations in mitochondrial (mt)-DNA, by delivering therapeutic oligonucleotides. The first step towards mitochondrial delivery of this type of nucleic acid was to target MWCNTs to mitochondria by covalent functionalization with a well-known endogenous mitochondrial targeting sequence (MTS). The subcellular localization of the conjugates, which were fluorescently labeled, in murine RAW 264.7 macrophages and human HeLa cells was then studied using different microscopy techniques, such as wide-field epifluorescence microscopy, confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). The localization of the MTS-MWCNT conjugates into mitochondria was further confirmed by analyzing the

  11. Designing Human m1 Muscarinic Receptor-Targeted Hydrophobic Eigenmode Matched Peptides as Functional Modulators

    PubMed Central

    Selz, Karen A.; Mandell, Arnold J.; Shlesinger, Michael F.; Arcuragi, Vani; Owens, Michael J.

    2004-01-01

    A new proprietary de novo peptide design technique generated ten 15-residue peptides targeting and containing the leading nontransmembrane hydrophobic autocorrelation wavelengths, “modes”, of the human m1 muscarinic cholinergic receptor, m1AChR. These modes were also shared by the m4AChR subtype (but not the m2, m3, or m5 subtypes) and the three-finger snake toxins that pseudoirreversibly bind m1AChR. The linear decomposition of the hydrophobically transformed m1AChR amino acid sequence yielded ordered eigenvectors of orthogonal hydrophobic variational patterns. The weighted sum of two eigenvectors formed the peptide design template. Amino acids were iteratively assigned to template positions randomly, within hydrophobic groups. One peptide demonstrated significant functional indirect agonist activity, and five produced significant positive allosteric modulation of atropine-reversible, direct-agonist-induced cellular activation in stably m1AChR-transfected Chinese hamster ovary cells, reflected in integrated extracellular acidification responses. The peptide positive allosteric ligands produced left-shifts and peptide concentration-response augmentation in integrated extracellular acidification response asymptotic sigmoidal functions and concentration-response behavior in Hill number indices of positive cooperativity. Peptide mode specificity was suggested by negative crossover experiments with human m2ACh and D2 dopamine receptors. Morlet wavelet transformation of the leading eigenvector-derived, m1AChR eigenfunctions locates seven hydrophobic transmembrane segments and suggests possible extracellular loop locations for the peptide-receptor mode-matched, modulatory hydrophobic aggregation sites. PMID:14990463

  12. Molecular Targets of Antihypertensive Peptides: Understanding the Mechanisms of Action Based on the Pathophysiology of Hypertension

    PubMed Central

    Majumder, Kaustav; Wu, Jianping

    2014-01-01

    There is growing interest in using functional foods or nutraceuticals for the prevention and treatment of hypertension or high blood pressure. Although numerous preventive and therapeutic pharmacological interventions are available on the market, unfortunately, many patients still suffer from poorly controlled hypertension. Furthermore, most pharmacological drugs, such as inhibitors of angiotensin-I converting enzyme (ACE), are often associated with significant adverse effects. Many bioactive food compounds have been characterized over the past decades that may contribute to the management of hypertension; for example, bioactive peptides derived from various food proteins with antihypertensive properties have gained a great deal of attention. Some of these peptides have exhibited potent in vivo antihypertensive activity in both animal models and human clinical trials. This review provides an overview about the complex pathophysiology of hypertension and demonstrates the potential roles of food derived bioactive peptides as viable interventions targeting specific pathways involved in this disease process. This review offers a comprehensive guide for understanding and utilizing the molecular mechanisms of antihypertensive actions of food protein derived peptides. PMID:25547491

  13. Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts

    PubMed Central

    Kmiec, Beata; Teixeira, Pedro F.; Berntsson, Ronnie P.-A.; Murcha, Monika W.; Branca, Rui M. M.; Radomiljac, Jordan D.; Regberg, Jakob; Svensson, Linda M.; Bakali, Amin; Langel, Ülo; Lehtiö, Janne; Whelan, James; Stenmark, Pål; Glaser, Elzbieta

    2013-01-01

    Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptide-degrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N- and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8–1.9 Å, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 Å3. The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts. PMID:24043784

  14. Targeting DNA G-Quadruplex Structures with Peptide Nucleic Acids

    PubMed Central

    Panyutin, Igor G.; Onyshchenko, Mykola I.; Englund, Ethan A.; Appella, Daniel H.; Neumann, Ronald D.

    2012-01-01

    Regulation of genetic functions based on targeting DNA or RNA sequences with complementary oligonucleotides is especially attractive in the post-genome era. Oligonucleotides can be rationally designed to bind their targets based on simple nucleic acid base pairing rules. However, the use of natural DNA and RNA oligonucleotides as targeting probes can cause numerous off-target effects. In addition, natural nucleic acids are prone to degradation in vivo by various nucleases. To address these problems, nucleic acid mimics such as peptide nucleic acids (PNA) have been developed. They are more stable, show less off-target effects, and, in general, have better binding affinity to their targets. However, their high affinity to DNA can reduce their sequence-specificity. The formation of alternative DNA secondary structures, such as the G-quadruplex, provides an extra level of specificity as targets for PNA oligomers. PNA probes can target the loops of G-quadruplex, invade the core by forming PNA-DNA guanine-tetrads, or bind to the open bases on the complementary cytosine-rich strand. Not only could the development of such G-quadruplex-specific probes allow regulation of gene expression, but it will also provide a means to clarify the biological roles G-quadruplex structures may possess. PMID:22376112

  15. The potential of food protein-derived anti-inflammatory peptides against various chronic inflammatory diseases.

    PubMed

    Majumder, Kaustav; Mine, Yoshinori; Wu, Jianping

    2016-05-01

    Inflammation is considered as one of the major causes for the initiation of various chronic diseases such as asthma, cancer, cardiovascular disease, diabetes, obesity, inflammatory bowel disease, osteoporosis and neurological diseases like Parkinson's disease. Increasing scientific evidence has delineated that inflammatory markers such as TNF-α, IL-1, IL-6, IL-8 and CRP and different transcription factors such as NF-κB and STAT are the major key factors that regulate these inflammatory diseases. Food protein-derived bioactive peptides have been shown to exhibit anti-inflammatory activity by inhibiting or reducing the expression of these inflammatory biomarkers and/or by modulating the activity of these transcription factors. This review aims to discuss various molecular targets and underlying mechanisms of food protein-derived anti-inflammatory peptides and to explore their potential against various chronic inflammatory diseases. © 2015 Society of Chemical Industry. PMID:26711001

  16. Importance of Tryptophan in Transforming an Amphipathic Peptide into a Pseudomonas aeruginosa-Targeted Antimicrobial Peptide

    PubMed Central

    Zhu, Xin; Ma, Zhi; Wang, Jiajun; Chou, Shuli; Shan, Anshan

    2014-01-01

    Here, we found that simple substitution of amino acids in the middle position of the hydrophobic face of an amphipathic peptide RI16 with tryptophan (T9W) considerably transformed into an antimicrobial peptide specifically targeting Pseudomonas aeruginosa. Minimal inhibitory concentration (MIC) results demonstrated that T9W had a strong and specifically antimicrobial activity against P. aeruginosa, including antibiotic-resistant strains, but was not active against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Staphyfococcus epidermidis. Fluorescent spectroscopic assays indicated that T9W interacted with the membrane of P. aeruginosa, depolarizing the outer and the inner membrane of bacterial cells. Salt susceptibility assay showed that T9W still maintained its strong anti-pseudomonas activity in the presence of salts at physiological concentrations, and in hemolytic and MTT assays T9W also showed no toxicity against human blood cells and macrophages. In vivo assay demonstrated that T9W also displayed no toxicity to Chinese Kun Ming (KM) mice. Furthermore, the strong antibiofilm activity was also observed with the peptide T9W, which decreased the percentage of biomass formation in a dose-dependent manner. Overall, these findings indicated that design of single-pathogen antimicrobial agents can be achieved by simple amino acid mutation in naturally occurring peptide sequences and this study suggested a model of optimization/design of anti-pseudomonas drugs in which the tryptophan residue was a conserved element. PMID:25494332

  17. Novel harmine derivatives for tumor targeted therapy

    PubMed Central

    Gu, Fan; Wang, Zhaohui; Tian, Caiping; Qian, Zhiyu; Tang, Liping; Gu, Yueqing

    2015-01-01

    Harmine is a beta-carboline alkaloid found in medicinal plant PeganumHarmala, which has served as a folk anticancer medicine. However, clinical applications of harmine were limited by its low pharmacological effects and noticeable neurotoxicity. In this study, we modified harmine to increase the therapeutic efficacy and to decrease the systemic toxicity. Specifically, two tumor targeting harmine derivatives 2DG-Har-01 and MET-Har-02 were synthesized by modifying substituent in position-2, -7 and -9 of harmine ring with two different targeting group2-amino-2-deoxy-D-glucose (2DG) and Methionine (Met), respectively. Their therapeutic efficacy and toxicity were investigated both in vitro and in vivo. Results suggested that the two newharmine derivatives displayed much higher therapeutic effects than non-modified harmine. In particular, MET-Har-02 was more potent than 2DG-Har-01 with promising potential for targeted cancer therapy. PMID:25940702

  18. Chromogranin A and Derived Peptides in Health and Disease

    PubMed Central

    Loh, Y. Peng; Cheng, Yong; Mahata, Sushil K.; Corti, Angelo; Tota, Bruno

    2012-01-01

    Chromogranin A (CgA) is a member of the granins, a family of acidic proteins found in abundance in (neuro)endocrine cells (e.g. in chromaffin cells) and in some tumors. Like other granins, CgA has a granulogenic role in secretory granule biogenesis and is stored in these organelles. CgA is partially processed differentially in various cell types to yield biologically active peptides, such as vasostatin, pancreastatin, catestatin and serpinins. In this review we describe the roles of CgA and several of its derived peptides. CgA, which is elevated in the blood of cancer patients, inhibits angiogenesis and exerts protective effects on the endothelial barrier function in tumors, thus affecting response to chemotherapy. Recent studies indicate that the serpinins promote cell survival and myocardial contractility and relaxation. Other peptides such as pancreastatin was found to have significant effects on inhibition of glucose stimulated insulin secretion and glucose up-take, induction of glycogenolysis in hepatocytes and inhibition of lipogenesis. In contrast, catestatin has opposite effects to that of pancreastatin in glucose metabolism and lipogenesis. Catestatin appears to also play a significant role in cardiac function, blood pressure regulation, and mutations in the catestatin domain of the CgA gene are associated with hypertension in humans. PMID:22388654

  19. Anti-Hemagglutinin Antibody Derived Lead Peptides for Inhibitors of Influenza Virus Binding.

    PubMed

    Memczak, Henry; Lauster, Daniel; Kar, Parimal; Di Lella, Santiago; Volkmer, Rudolf; Knecht, Volker; Herrmann, Andreas; Ehrentreich-Förster, Eva; Bier, Frank F; Stöcklein, Walter F M

    2016-01-01

    Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/Mute Swan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing. PMID:27415624

  20. Anti-Hemagglutinin Antibody Derived Lead Peptides for Inhibitors of Influenza Virus Binding

    PubMed Central

    Kar, Parimal; Di Lella, Santiago; Volkmer, Rudolf; Knecht, Volker; Herrmann, Andreas; Ehrentreich-Förster, Eva; Bier, Frank F.; Stöcklein, Walter F. M.

    2016-01-01

    Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/Mute Swan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing. PMID:27415624

  1. PFR peptide, one of the antimicrobial peptides identified from the derivatives of lactoferrin, induces necrosis in leukemia cells

    PubMed Central

    Lu, Yan; Zhang, Teng-Fei; Shi, Yue; Zhou, Han-Wei; Chen, Qi; Wei, Bu-Yun; Wang, Xi; Yang, Tian-Xin; Chinn, Y. Eugene; Kang, Jian; Fu, Cai-Yun

    2016-01-01

    LF11-322 (PFWRIRIRR-NH2) (PFR peptide), a nine amino acid-residue peptide fragment derived from human lactoferricin, possesses potent cytotoxicity against bacteria. We report here the discovery and characterization of its antitumor activity in leukemia cells. PFR peptide inhibited the proliferation of MEL and HL-60 leukemia cells by inducing cell death in the absence of the classical features of apoptosis, including chromatin condensation, Annexin V staining, Caspase activation and increase of abundance of pro-apoptotic proteins. Instead, necrotic cell death as evidenced by increasing intracellular PI staining and LDH release, inducing membrane disruption and up-regulating intracellular calcium level, was observed following PFR peptide treatment. In addition to necrotic cell death, PFR peptide also induced G0/G1 cell cycle arrest. Moreover, PFR peptide exhibited favorable antitumor activity and tolerability in vivo. These findings thus provide a new clue of antimicrobial peptides as a potential novel therapy for leukemia. PMID:26860588

  2. Investigation into the mechanism of action of the antimicrobial peptides Os and Os-C derived from a tick defensin.

    PubMed

    Taute, Helena; Bester, Megan J; Neitz, Albert W H; Gaspar, Anabella R M

    2015-09-01

    Os and Os-C are two novel antimicrobial peptides, derived from a tick defensin, which have been shown to have a larger range of antimicrobial activity than the parent peptide, OsDef2. The aim of this study was to determine whether the peptides Os and Os-C are mainly membrane acting, or if these peptides have possible additional intracellular targets in Escherichia coli and Bacillus subtilis. Transmission electron microscopy revealed that both peptides adversely affected intracellular structure of both bacteria causing different degrees of granulation of the intracellular contents. At the minimum bactericidal concentrations, permeabilization as determined with the SYTOX green assay seemed not to be the principle mode of killing when compared to melittin. However, fluorescent triple staining indicated that the peptides caused permeabilization of stationary phase bacteria and TEM indicated membrane effects. Studies using fluorescently labeled peptides revealed that the membrane penetrating activity of Os and Os-C was similar to buforin II. Os-C was found to associate with the septa of B. subtilis. Plasmid binding studies showed that Os and Os-C binds E. coli plasmid DNA at a similar charge ratio as melittin. These studies suggest membrane activity for Os and Os-C with possible intracellular targets such as DNA. The differences in permeabilization at lower concentrations and binding to DNA between Os and Os-C, suggest that the two peptides have dissimilar modes of action. PMID:26215047

  3. Targeted Type 1 phototherapeutic agents using azido-peptide bioconjugates

    NASA Astrophysics Data System (ADS)

    Rajagopalan, Raghavan; Achilefu, Samuel I.; Jimenez, Hermo N.; Webb, Elizabeth G.; Schmidt, Michelle A.; Bugaj, Joseph E.; Dorshow, Richard B.

    2001-07-01

    Five peptides binding to somatostatin and bombesin receptors were conjugated to 4-azido-2,3,4,6-tetrafluorophenylbenzoic acid, a Type 1 photosensitizer, at the N-terminal position. The receptor affinities were determined by competition binding assay using two different pancreatic tumor cell lines, CA20948 and AR42-J, that expresses somatostatin-2 (SST-2) and bombesin receptors receptively. All compounds exhibited high receptor specificity, i.e., the IC50 values ranged between 1.0 to 64.0 nM. These conjugates may be useful for targeted Type 1 phototherapy via the generation of nitrenes at the cell surfaces expressing these receptors.

  4. Target Promiscuity and Heterogeneous Effects of Tarantula Venom Peptides Affecting Na+ and K+ Ion Channels*

    PubMed Central

    Redaelli, Elisa; Cassulini, Rita Restano; Silva, Deyanira Fuentes; Clement, Herlinda; Schiavon, Emanuele; Zamudio, Fernando Z.; Odell, George; Arcangeli, Annarosa; Clare, Jeffrey J.; Alagón, Alejandro; de la Vega, Ricardo C. Rodríguez; Possani, Lourival D.; Wanke, Enzo

    2010-01-01

    Venom-derived peptide modulators of ion channel gating are regarded as essential tools for understanding the molecular motions that occur during the opening and closing of ion channels. In this study, we present the characterization of five spider toxins on 12 human voltage-gated ion channels, following observations about the target promiscuity of some spider toxins and the ongoing revision of their “canonical” gating-modifying mode of action. The peptides were purified de novo from the venom of Grammostola rosea tarantulas, and their sequences were confirmed by Edman degradation and mass spectrometry analysis. Their effects on seven tetrodotoxin-sensitive Na+ channels, the three human ether-à-go-go (hERG)-related K+ channels, and two human Shaker-related K+ channels were extensively characterized by electrophysiological techniques. All the peptides inhibited ion conduction through all the Na+ channels tested, although with distinctive patterns. The peptides also affected the three pharmaceutically relevant hERG isoforms differently. At higher concentrations, all peptides also modified the gating of the Na+ channels by shifting the activation to more positive potentials, whereas more complex effects were recorded on hERG channels. No effects were evident on the two Shaker-related K+ channels at concentrations well above the IC50 value for the affected channels. Given the sequence diversity of the tested peptides, we propose that tarantula toxins should be considered both as multimode and target-promiscuous ion channel modulators; both features should not be ignored when extracting mechanistic interpretations about ion channel gating. Our observations could also aid in future structure-function studies and might help the development of novel ion channel-specific drugs. PMID:19955179

  5. pHLIP peptide targets nanogold particles to tumors.

    PubMed

    Yao, Lan; Daniels, Jennifer; Danniels, Jennifer; Moshnikova, Anna; Kuznetsov, Sergey; Ahmed, Aftab; Engelman, Donald M; Reshetnyak, Yana K; Andreev, Oleg A

    2013-01-01

    Progress in nanomedicine depends on the development of nanomaterials and targeted delivery methods. In this work, we describe a method for the preferential targeting of gold nanoparticles to a tumor in a mouse model. The method is based on the use of the pH Low Insertion Peptide (pHLIP), which targets various imaging agents to acidic tumors. We compare tumor targeting by nonfunctionalized nanogold particles with nanogold-pHLIP conjugates, where nanogold is covalently attached to the N terminus of pHLIP. Our most important finding is that both intratumoral and i.v. administration demonstrated a significant enhancement of tumor uptake of gold nanoparticles conjugated with pHLIP. Statistically significant reduction of gold accumulation was observed in acidic tumors and kidney when pH-insensitive K-pHLIP was used as a vehicle, suggesting an important role of pH in the pHLIP-mediated targeting of gold nanoparticles. The pHLIP technology can substantially improve the delivery of gold nanoparticles to tumors by providing specificity of targeting, enhancing local concentration in tumors, and distributing nanoparticles throughout the entire tumor mass where they remain for an extended period (several days), which is beneficial for radiation oncology and imaging. PMID:23267062

  6. FKBPL and Peptide Derivatives: Novel Biological Agents That Inhibit Angiogenesis by a CD44-Dependent Mechanism

    PubMed Central

    Valentine, Andrea; O’Rourke, Martin; Yakkundi, Anita; Worthington, Jenny; Hookham, Michelle; Bicknell, Roy; McCarthy, Helen O.; McClelland, Keeva; McCallum, Lynn; Dyer, Hayder; McKeen, Hayley; Waugh, David; Roberts, Jennifer; McGregor, Joanne; Cotton, Graham; James, Iain; Harrison, Timothy; Hirst, David G.; Robson, Tracy

    2011-01-01

    Purpose Anti-angiogenic therapies can be an important adjunct to the management of many malignancies. Here we investigated a novel protein, FKBPL, and peptide derivative for their anti-angiogenic activity and mechanism of action. Experimental Design Recombinant FKBPL (rFKBPL) and its peptide derivative were assessed in a range of human microvascular endothelial cell (HMEC-1) assays in vitro. Their ability to inhibit proliferation, migration and Matrigel dependent tubule formation was determined. They were further evaluated in an ex-vivo rat model of neo-vascularisation and in two in vivo mouse models of angiogenesis; the sponge implantation and the intra-vital microscopy models. Anti-tumor efficacy was determined in two human tumor xenograft models grown in SCID mice. Finally, the dependence of peptide on CD44 was determined using a CD44 targeted siRNA approach or in cell lines of differing CD44 status. Results rFKBPL inhibited endothelial cell migration, tubule formation and microvessel formation in vitro and in vivo. The region responsible for FKBPL’s anti-angiogenic activity was identified and a 24 amino acid peptide (AD-01) spanning this sequence was synthesised. It was potently anti-angiogenic and inhibited growth in two human tumor xenograft models (DU145 and MDA-231) when administered systemically, either on its own, or in combination with docetaxel. The anti-angiogenic activity of FKBPL and AD-01 was dependent on the cell surface receptor CD44 and signalling downstream of this receptor promoted an anti-migratory phenotype. Conclusion FKBPL and its peptide derivative AD-01 have potent anti-angiogenic activity. Thus, these agents offer the potential of an attractive new approach to anti-angiogenic therapy. PMID:21364036

  7. Biologically and diagenetically derived peptide modifications in moa collagens.

    PubMed

    Cleland, Timothy P; Schroeter, Elena R; Schweitzer, Mary H

    2015-06-01

    The modifications that occur on proteins in natural environments over time are not well studied, yet characterizing them is vital to correctly interpret sequence data recovered from fossils. The recently extinct moa (Dinornithidae) is an excellent candidate for investigating the preservation of proteins, their post-translational modifications (PTMs) and diagenetic alterations during degradation. Moa protein extracts were analysed using mass spectrometry, and peptides from collagen I, collagen II and collagen V were identified. We also identified biologically derived PTMs (i.e. methylation, di-methylation, alkylation, hydroxylation, fucosylation) on amino acids at locations consistent with extant proteins. In addition to these in vivo modifications, we detected novel modifications that are probably diagenetically derived. These include loss of hydroxylation/glutamic semialdehyde, carboxymethyllysine and peptide backbone cleavage, as well as previously noted deamidation. Moa collagen sequences and modifications provide a baseline by which to evaluate proteomic studies of other fossils, and a framework for defining the molecular relationship of moa to other closely related taxa. PMID:25972464

  8. Biologically and diagenetically derived peptide modifications in moa collagens

    PubMed Central

    Cleland, Timothy P.; Schroeter, Elena R.; Schweitzer, Mary H.

    2015-01-01

    The modifications that occur on proteins in natural environments over time are not well studied, yet characterizing them is vital to correctly interpret sequence data recovered from fossils. The recently extinct moa (Dinornithidae) is an excellent candidate for investigating the preservation of proteins, their post-translational modifications (PTMs) and diagenetic alterations during degradation. Moa protein extracts were analysed using mass spectrometry, and peptides from collagen I, collagen II and collagen V were identified. We also identified biologically derived PTMs (i.e. methylation, di-methylation, alkylation, hydroxylation, fucosylation) on amino acids at locations consistent with extant proteins. In addition to these in vivo modifications, we detected novel modifications that are probably diagenetically derived. These include loss of hydroxylation/glutamic semialdehyde, carboxymethyllysine and peptide backbone cleavage, as well as previously noted deamidation. Moa collagen sequences and modifications provide a baseline by which to evaluate proteomic studies of other fossils, and a framework for defining the molecular relationship of moa to other closely related taxa. PMID:25972464

  9. Formyl peptide receptors and the regulation of ACTH secretion: targets for annexin A1, lipoxins, and bacterial peptides

    PubMed Central

    John, C. D.; Sahni, V.; Mehet, D.; Morris, J. F.; Christian, H. C.; Perretti, M.; Flower, R. J.; Solito, E.; Buckingham, J. C.

    2007-01-01

    The N-formyl peptide receptors (FPRs) are a family of G-protein coupled receptors that respond to proinflammatory N-formylated bacterial peptides (e.g., formyl-Met-Leu-Phe, fMLF) and, thus, contribute to the host response to bacterial infection. Paradoxically, a growing body of evidence suggests that some members of this receptor family may also be targets for certain anti-inflammatory molecules, including annexin A1 (ANXA1), which is an important mediator of glucocorticoid (GC) action. To explore further the potential role of FPRs in mediating ANXA1 actions, we have focused on the pituitary gland, where ANXA1 has a well-defined role as a cell-cell mediator of the inhibitory effects of GCs on the secretion of corticotrophin (ACTH), and used molecular, genetic, and pharmacological approaches to address the question in well-established rodent models. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis identified mRNAs for four FPR family members in the mouse anterior pituitary gland, Fpr-rs1, Fpr-rs2, Fpr-rs6, and Fpr-rs7. Functional studies confirmed that, like dexamethasone, ANXA1 and two ANXA1-derived peptides (ANXA11-188 and ANXA1Ac2-26) inhibit the evoked release of ACTH from rodent anterior pituitary tissue in vitro. Fpr1 gene deletion failed to modify the pituitary responses to dexamethasone or ANXA1Ac2-26. However, lipoxin A4 (LXA4, 0.02-2μM, a lipid mediator with high affinity for Fpr-rs1) mimicked the inhibitory effects of ANXA1 on ACTH release as also did fMLF in high (1-100 μM) but not lower (10-100 nM) concentrations. Additionally, a nonselective FPR antagonist (Boc1, 100 μM) overcame the effects of dexamethasone, ANXA11-188, ANXA1Ac2-26, fMLF, and LXA4 on ACTH release, although at a lower concentration (50 μM), it was without effect. Together, the results suggest that the actions of ANXA1 in the pituitary gland are independent of Fpr1 but may involve other FPR family members, in particular, Fpr-rs1 or a closely related receptor. They

  10. RGD based peptide amphiphiles as drug carriers for cancer targeting

    NASA Astrophysics Data System (ADS)

    Saraf, Poonam S.

    Specific interactions of ligands with receptors is one of the approaches for active targeting of anticancer drugs to cancer cells. Over expression of integrin receptors is a physiological manifestation in several cancers and is associated with cancer progression and metastasis, which makes it an attractive target for cancer chemotherapy. The peptide sequence for this integrin recognition is the Arg-Gly-Asp (RGD). Self-assembly offers a unique way of presenting ligands to target receptors for recognition and binding. This study focuses on development of integrin specific peptide amphiphile self-assemblies as carriers for targeted delivery of paclitaxel to αvbeta 3 integrin overexpressing cancers. Amphiphiles composed of conjugates of different analogs of RGD (linear, cyclic or glycosylated) and aliphatic fatty acid with or without 8-amino-3,6-dioxaoctanoic acid (ADA) as linker were synthesized and characterized. The amphiphiles exhibited Critical Micellar Concentration in the range of 7-30 μM. Transmission electron microscopy images revealed the formation of spherical micelles in the size range of 10-40 nm. Forster Resonance Energy Transfer studies revealed entrapment of hydrophobic dyes within a tight micellar core and provided information regarding the cargo exchange within micelles. The RGD micelles exhibited competitive binding with 55% displacement of a bound fluorescent probe by the cyclic RGD micelles. The internalization of fluorescein isothiocynate (FITC) loaded RGD micelles was significantly higher in A2058 melanoma cells compared to free FITC within 20 minutes of incubation at 37°C. The same micelles showed significantly lower internalization at 4°C and on pretreatment with 0.45M sucrose confirming endocytotic uptake of the RGD micellar carriers. The IC50 of paclitaxel in A2058 melanoma cells was lower when treated within RGD micelles as compared to treatment of free drug. On the other hand, IC50 values increased by 2 to 9 fold for micellar treatment

  11. Lipid-Targeting Peptide Probes for Extracellular Vesicles.

    PubMed

    Flynn, Aaron D; Yin, Hang

    2016-11-01

    Extracellular vesicles released from cells are under intense investigation for their roles in cell-cell communication and cancer progression. However, individual vesicles have been difficult to probe as their small size renders them invisible by conventional light microscopy. However, as a consequence of their small size these vesicles possess highly curved lipid membranes that offer an unconventional target for curvature-sensing probes. In this article, we present a strategy for using peptide-based biosensors to detect highly curved membranes and the negatively charged membrane lipid phosphatidylserine, we delineate several assays used to validate curvature- and lipid-targeting mechanisms, and we explore potential applications in probing extracellular vesicles released from sources such as apoptotic cells, cancer cells, or activated platelets. J. Cell. Physiol. 231: 2327-2332, 2016. © 2016 Wiley Periodicals, Inc. PMID:26909741

  12. Focal Targeting of the Bacterial Envelope by Antimicrobial Peptides.

    PubMed

    Rashid, Rafi; Veleba, Mark; Kline, Kimberly A

    2016-01-01

    Antimicrobial peptides (AMPs) are utilized by both eukaryotic and prokaryotic organisms. AMPs such as the human beta defensins, human neutrophil peptides, human cathelicidin, and many bacterial bacteriocins are cationic and capable of binding to anionic regions of the bacterial surface. Cationic AMPs (CAMPs) target anionic lipids [e.g., phosphatidylglycerol (PG) and cardiolipins (CL)] in the cell membrane and anionic components [e.g., lipopolysaccharide (LPS) and lipoteichoic acid (LTA)] of the cell envelope. Bacteria have evolved mechanisms to modify these same targets in order to resist CAMP killing, e.g., lysinylation of PG to yield cationic lysyl-PG and alanylation of LTA. Since CAMPs offer a promising therapeutic alternative to conventional antibiotics, which are becoming less effective due to rapidly emerging antibiotic resistance, there is a strong need to improve our understanding about the AMP mechanism of action. Recent literature suggests that AMPs often interact with the bacterial cell envelope at discrete foci. Here we review recent AMP literature, with an emphasis on focal interactions with bacteria, including (1) CAMP disruption mechanisms, (2) delocalization of membrane proteins and lipids by CAMPs, and (3) CAMP sensing systems and resistance mechanisms. We conclude with new approaches for studying the bacterial membrane, e.g., lipidomics, high resolution imaging, and non-detergent-based membrane domain extraction. PMID:27376064

  13. Focal Targeting of the Bacterial Envelope by Antimicrobial Peptides

    PubMed Central

    Rashid, Rafi; Veleba, Mark; Kline, Kimberly A.

    2016-01-01

    Antimicrobial peptides (AMPs) are utilized by both eukaryotic and prokaryotic organisms. AMPs such as the human beta defensins, human neutrophil peptides, human cathelicidin, and many bacterial bacteriocins are cationic and capable of binding to anionic regions of the bacterial surface. Cationic AMPs (CAMPs) target anionic lipids [e.g., phosphatidylglycerol (PG) and cardiolipins (CL)] in the cell membrane and anionic components [e.g., lipopolysaccharide (LPS) and lipoteichoic acid (LTA)] of the cell envelope. Bacteria have evolved mechanisms to modify these same targets in order to resist CAMP killing, e.g., lysinylation of PG to yield cationic lysyl-PG and alanylation of LTA. Since CAMPs offer a promising therapeutic alternative to conventional antibiotics, which are becoming less effective due to rapidly emerging antibiotic resistance, there is a strong need to improve our understanding about the AMP mechanism of action. Recent literature suggests that AMPs often interact with the bacterial cell envelope at discrete foci. Here we review recent AMP literature, with an emphasis on focal interactions with bacteria, including (1) CAMP disruption mechanisms, (2) delocalization of membrane proteins and lipids by CAMPs, and (3) CAMP sensing systems and resistance mechanisms. We conclude with new approaches for studying the bacterial membrane, e.g., lipidomics, high resolution imaging, and non-detergent-based membrane domain extraction. PMID:27376064

  14. Functional evaluation of fluorescein-labeled derivatives of a peptide inhibitor of the EGF receptor dimerization.

    PubMed

    Toyama, Kei; Mizuguchi, Takaaki; Nomura, Wataru; Tamamura, Hirokazu

    2016-08-15

    A cyclic decapeptide (1, ), which acts on the extracellular region of the EGF receptor, preventing it from dimerizing, has been developed. Peptide 2, which was labeled with fluorescein at the N-terminus of peptide 1, was synthesized based on structure-activity relationship studies. Peptide 2 essentially retained the inhibitory activity of peptide 1 against the receptor autophosphorylation. Confocal microscopy studies revealed that in carcinoma cells, the fluorescence of peptide 2 was localized inside some vesicles. Treatment of intact cells by peptide 1 in combination with peptide 2 decreased the fluorescence intensity significantly compared to treatment with only peptide 2. These results indicate that peptide 2 competes with peptide 1 for binding to the cellular surface. Six derivatives of peptide 2, in which constituent amino acids, with the exception of two cysteines and proline were randomized, were synthesized and used to treat the cells. Peptides 6 and 9 showed the highest fluorescence intensity in cells. From the results of the EGF receptor autophosphorylation assay, these two derivatives were proven to have higher inhibitory activity than peptide 2, which would therefore be a useful delivery peptide and fluorescent probe to find new inhibitors against the EGF receptor. Peptides 6 and 9 are promising leads for EGF receptor inhibitors. PMID:27283787

  15. An approach to the construction of tailor-made amphiphilic peptides that strongly and selectively bind to hairpin RNA targets.

    PubMed

    Lee, Su Jin; Hyun, Soonsil; Kieft, Jeffrey S; Yu, Jaehoon

    2009-02-18

    The hairpin RNA motif is one of the most frequently observed secondary structures and is often targeted by therapeutic agents. An amphiphilic peptide with seven lysine and eight leucine residues and its derivatives were designed for use as ligands against RNA hairpin motifs. We hypothesized that variations in both the hydrophobic leucine-rich and hydrophilic lysine-rich spheres of these amphiphilic peptides would create extra attractive interactions with hairpin RNA targets. A series of alanine-scanned peptides were probed to identify the most influential lysine residues in the hydrophilic sphere. The binding affinities of these modified peptides with several hairpins, such as RRE, TAR from HIV, a short hairpin from IRES of HCV, and a hairpin from the 16S A-site stem from rRNA, were determined. Since the hairpin from IRES of HCV was the most susceptible to the initial series of alanine-scanned peptides, studies investigating how further variations in the peptides effect binding employed the IRES hairpin. Next, the important Lys residues were substituted by shorter chain amines, such as ornithine, to place the peptide deeper into the hairpin groove. In a few cases, a 70-fold improved binding was observed for peptides that contained the specifically located shorter amine side chains. To further explore changes in binding affinities brought about by alterations in the hydrophobic sphere, tryptophan residues were introduced in place of leucine. A few peptides with tryptophan in specific positions also displayed 70-fold improved binding affinities. Finally, double mutant peptides incorporating both specifically located shorter amine side chains in the hydrophilic region and tryptophan residues in the hydrophobic region were synthesized. The binding affinities of peptides containing the simple double modification were observed to be 80 times lower, and their binding specificities were increased 40-fold. The results of this effort provide important information about

  16. Development of a peptide-based vaccine targeting TMPRSS2:ERG fusion positive prostate cancer

    PubMed Central

    Kissick, Haydn Thomas; Sanda, Martin George; Dunn, Laura Kathleen; Arredouani, Mohamed Simo

    2013-01-01

    Identification of novel vaccine targets is critical for the design and advancement of prostate cancer (PCa) immunotherapy. Ideal targets are proteins that are abundant in prostate tumors while absent in extra-prostatic tissues. The fusion of the androgen-regulated TMPRSS2 gene with the ETS transcription factor ERG occurs in approximately 50% of prostate cancer cases and results in aberrant ERG expression. Because expression of ERG is very low in peripheral tissue, we evaluated the suitability of this protein as an antigen target in PCa vaccines. ERG-derived HLA-A*0201-restricted immunogenic epitopes were identified through a 3-step strategy that included in silico, in vitro, and in vivo validation. Algorithms were used to predict potential HLA-A*0201-binding epitopes. High scoring epitopes were tested for binding to HLA-A*0201 using the T2-based stabilization assay in vitro. Five peptides were found to bind HLA-A*0201 and were subsequently tested for immunogenicity in humanized HLA-A*0201 transgenic mice. The in vivo screening identified three immunogenic peptides. One of these peptides, ERG295, overcame peripheral tolerance in HLA-A*0201 mice that expressed prostate restricted ERG. Also, this peptide induced an antigen specific response against ERG-expressing human prostate tumor cells. Finally, tetramer assay showed detectable and responsive ERG295-specific cytotoxic lymphocytes in peripheral blood of HLA-A*0201+ prostate cancer patients. Detection of ERG-specific CTLs in both mice and the blood of prostate cancer patients indicates that ERG-specific tolerance can be overcome. Additionally, these data suggest that ERG is a suitable target antigen for PCa immunotherapy. PMID:24149465

  17. Design of a novel microtubule targeted peptide vesicle for delivering different anticancer drugs.

    PubMed

    Adak, Anindyasundar; Mohapatra, Saswat; Mondal, Prasenjit; Jana, Batakrishna; Ghosh, Surajit

    2016-06-18

    A microtubule targeted peptide-based delivery vehicle has been designed using two oppositely charged peptides, which targets tubulin/microtubules, delivers both hydrophilic and hydrophobic drugs into their target site through lysosome at acidic pH. Drug loaded vesicles show a significant anticancer effect compared to control drugs in a 2D monolayer and a 3D spheroid cell. PMID:27153208

  18. Protective efficacy of a peptide derived from a potential adhesin of Pseudomonas aeruginosa against corneal infection.

    PubMed

    Yuan, Qing; Wu, Yuting; Wang, Yiqiang; Chen, Lin; Qu, Mingli; Duan, Kangmin; Zhao, Ge

    2016-02-01

    Dissecting the interactions between Pseudomonas aeruginosa and corneal cells is important to identify a novel target for prevention and treatment of Pseudomonas keratitis. The current study began with a peptide identified by phage display, and was to investigate the protective efficacy against P. aeruginosa infection in cornea. The original peptide Pc-E, with high homology to a hypothetical membrane protein (HmpA) in P. aeruginosa, and the derived peptide Pc-EP, with the same sequence as a region in HmpA, were synthesized. Peptide Pc-EP could directly bind to HCEC, stronger than Pc-E, and specifically activate toll-like receptor 5, and thereby significantly induce the production of pro-inflammatory factors, such as IL-1β, IL-6, IFN-γ and IL-17. Moreover, Pc-EP could act as an antagonist to inhibit the adhesion of wild-type P. aeruginosa to HCEC and mouse corneas. No inhibitory effect was observed on the adhesion of the strain loss of HmpA. When compared to the wild-type strain, the adhesion of the hmpA mutant to corneal cells was significantly decreased. Treatment of infected mouse corneas with Pc-EP before infection significantly decreased the bacterial load in the cornea and attenuated the corneal pathology. These results indicate that Pc-EP can be a useful prophylactic agent for P. aeruginosa keratitis. PMID:26500187

  19. Interactions of histatin 5 and histatin 5-derived peptides with liposome membranes: surface effects, translocation and permeabilization.

    PubMed Central

    Den Hertog, Alice L; Wong Fong Sang, Harro W; Kraayenhof, Ruud; Bolscher, Jan G M; Van't Hof, Wim; Veerman, Enno C I; Nieuw Amerongen, Arie V

    2004-01-01

    A number of cationic antimicrobial peptides, among which are histatin 5 and the derived peptides dhvar4 and dhvar5, enter their target cells and interact with internal organelles. There still are questions about the mechanisms by which antimicrobial peptides translocate across the membrane. We used a liposome model to study membrane binding, translocation and membrane-perturbing capacities of histatin 5, dhvar4 and dhvar5. Despite the differences in amphipathic characters of these peptides, they bound equally well to liposomes, whereas their membrane activities differed remarkably: dhvar4 translocated at the fastest rate, followed by dhvar5, whereas the histatin 5 translocation rate was much lower. The same pattern was seen for the extent of calcein release: highest with dhvar4, less with dhvar5 and almost none with histatin 5. The translocation and disruptive actions of dhvar5 did not seem to be coupled, because translocation occurred on a much longer timescale than calcein release, which ended within a few minutes. We conclude that peptide translocation can occur through peptide-phospholipid interactions, and that this is a possible mechanism by which antimicrobial peptides enter cells. However, the translocation rate was much lower in this model membrane system than that seen in yeast cells. Thus it is likely that, at least for some peptides, additional features promoting the translocation across biological membranes are involved as well. PMID:14733612

  20. Novel target for peptide-based imaging and treatment of brain tumors

    PubMed Central

    Hyvönen, Maija; Enbäck, Juulia; Huhtala, Tuulia; Lammi, Johanna; Sihto, Harri; Weisell, Janne; Joensuu, Heikki; Rosenthal-Aizman, Katri; El-Andaloussi, Samir; Langel, Ulo; Närvänen, Ale; Bergers, Gabriele; Laakkonen, Pirjo

    2014-01-01

    Malignant gliomas are associated with high mortality due to infiltrative growth, recurrence and malignant progression. Even with the most efficient therapy combinations, median survival of the glioblastoma multiforme (grade IV) patients is less than 15 months. Therefore, new treatment approaches are urgently needed. We describe here identification of a novel homing peptide that recognizes tumor vessels and invasive tumor satellites in glioblastomas. We demonstrate successful brain tumor imaging using radiolabeled peptide in whole-body SPECT/CT-imaging. Peptide-targeted delivery of chemotherapeutics prolonged the lifespan of mice bearing invasive brain tumors and significantly reduced the number of tumor satellites compared to the free drug. Moreover, we identified mammary-derived growth inhibitor (MDGI/H-FABP/FABP3), as the interacting partner for our peptide on brain tumor tissue. MDGI was expressed in human brain tumor specimens in a grade-dependent manner and its expression positively correlated with the histological grade of the tumor suggesting MDGI as a novel marker for malignant gliomas. PMID:24493698

  1. Multimerized HIV-gp41-derived peptides as fusion inhibitors and vaccines.

    PubMed

    Nomura, Wataru; Mizuguchi, Takaaki; Tamamura, Hirokazu

    2016-11-01

    To date, several antigens based on the amino-terminal leucine/isoleucine heptad repeat (NHR) region of an HIV-1 envelope protein gp41 and fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of gp41 have been reported. We have developed a synthetic antigen targeting the membrane-fusion mechanism of HIV-1. This uses a template designed with C3-symmetric linkers and mimics the trimeric form of the NHR-derived peptide N36. The antiserum obtained by immunization of the N36 trimeric antigen binds preferentially to the N36 trimer and blocks HIV-1 infection effectively, compared with the antiserum obtained by immunization of the N36 monomer. Using another template designed with different C3-symmetric linkers, we have also developed a synthetic peptide mimicking the trimeric form of the CHR-derived peptide C34, with ∼100 times the inhibitory activity against the HIV-1 fusion mechanism than that of the monomer C34 peptide. A dimeric derivative of C34 has potent inhibitory activity at almost the same levels as this C34 trimer mimic, suggesting that presence of a dimeric form of C34 is structurally critical for fusion inhibitors. As examples of rising mid-size drugs, this review describes an effective strategy for the design of HIV vaccines and fusion inhibitors based on a relationship with the native structure of proteins involved in HIV fusion mechanisms. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 622-628, 2016. PMID:26583370

  2. Applications of Cell-Penetrating Peptides for Tumor Targeting and Future Cancer Therapies

    PubMed Central

    Regberg, Jakob; Srimanee, Artita; Langel, Ülo

    2012-01-01

    Cell-penetrating peptides provide a highly promising strategy for intracellular drug delivery. One relevant clinical application of cell-penetrating peptides is cancer therapeutics. Peptide based delivery could increase the uptake of drugs in tumor cells and thereby increase the efficacy of the treatment, either of conventional small molecular drugs or oligonucleotide based therapeutics. This review is focused on the cancer applications of cell penetrating peptides as delivery systems; different aspects of drug loading, cargoes and delivery are discussed together with methods for targeted delivery, activatable cell-penetrating peptides and transducible agents coupled to cell-penetrating peptides. PMID:24280701

  3. Peptide Anchor for Folate-Targeted Liposomal Delivery.

    PubMed

    Nogueira, Eugénia; Mangialavori, Irene C; Loureiro, Ana; Azoia, Nuno G; Sárria, Marisa P; Nogueira, Patrícia; Freitas, Jaime; Härmark, Johan; Shimanovich, Ulyana; Rollett, Alexandra; Lacroix, Ghislaine; Bernardes, Gonçalo J L; Guebitz, Georg; Hebert, Hans; Moreira, Alexandra; Carmo, Alexandre M; Rossi, Juan Pablo F C; Gomes, Andreia C; Preto, Ana; Cavaco-Paulo, Artur

    2015-09-14

    Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol. PMID:26241560

  4. Melanoma Therapy via Peptide-Targeted a-Radiation

    SciTech Connect

    Miao, Yubin; Hylarides, Mark; Fisher, Darrell R.; Shelton, Tiffani; Moore, Herbert A.; Wester, Dennis W.; Fritzberg, Alan R.; Winkelmann, Christopher T.; Hoffman, Timothy J.; Quinn, Thomas P.

    2005-08-01

    Malignant melanoma is the most lethal form of skin cancer. Current chemotherapy and external beam radiation therapy regimens are ineffective agents against melanoma, as shown by a 10-year survival rate for patients with disseminated disease of approximately 5% (reference?). In this study, the unique combination of a melanoma targeting peptide and an in vivo generated a-particle emitting radioisotope was investigated for its melanoma therapy potential. Alpha-radiation is densely ionizing and energy is locally absorbed, resulting in high concentrations of destructive free radicals and irreparable DNA double strand breaks. This high linear-energy-transfer overcomes radiation resistant tumor cells and oxygen-enhancement effects. The melanoma targeting peptide DOTA-Re(Arg11)CCMSH was radiolabeled with 212Pb, the parent of 212Bi, which decays via alpha and beta decay. Biodistribution and therapy studies were performed in the B16/F1 melanoma bearing C57 mouse flank tumor model. 212Pb[DOTA]-R e(Arg11)CCMSH exhibited rapid tumor uptake and extended retention coupled with rapid whole body disappearance. Radiation dose delivered to the tumor was estimated to be 61 cGy/uCi 212Pb administered. Treatment of melanoma-bearing mice with 50, 100 and 200 uCi of 212Pb[DOTA]-Re(Arg11)CCMSH extended mean survival of mice to 22, 28, and 49.8 days, respectively, compared to the 14.6 day mean survival of the placebo control group. Forty-five percent of the mice receiving 200 uCi survived the study disease-free.

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

    PubMed Central

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

    2016-01-01

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

  6. Design of p53-derived peptides with cytotoxicity on breast cancer.

    PubMed

    Fang, Yi; Jin, Rongzhong; Gao, Yinqi; Gao, Jidong; Wang, Jing

    2014-08-01

    The tumor suppressor p53 plays essential role in conserving stability by preventing genome mutation, which is inactivated naturally by its negative regulator MDM2. Thus, targeting p53-MDM2 protein-protein interaction has been raised as a new cancer therapy in the medicinal community. In the current study, we report a successful application of an integrative protocol to design novel p53-derived peptides with cytotoxicity on human breast cancer cells. A quantitative structure-activity relationship-improved statistical potential was used to evaluate the binding potency of totally 24,054 single- and dual-point mutants of p53 peptide to MDM2 in a high-throughput manner, from which 46 peptide mutants with high predicted affinity and typical helical feature were involved in a rigorous modeling procedure that employed molecular dynamics simulations and post-binding energy analysis to systematically investigate the structural, energetic and dynamic aspects of peptide interactions with MDM2. Subsequently, a biological analysis was performed on a number of promising peptide candidates to determine their cytotoxic effects on human breast cancer cell line MDF-7. Six dual-point mutants were found to have moderate or high activities with their IC50 values ranging from 16.3 to 137.0 μM, which are better than that of wild-type p53 peptide (IC50 = 182.6 μM) and close to that of classical anticancer agent cis-platin (IC50 = 4.3 μM). Further, the most active peptide ETFSDWWKLLAE was selected as parent to further derive new mutants on the basis of the structural and energetic profile of its complex with MDM2. Consequently, three triple-point mutants (LTFSDWWKLLAE, ESFSDWWKLLAE and ETFADWWKLLAE) were obtained, and their biological activities (IC50 = 15.1, 27.0 and 8.7 μM, respectively) were determined to be comparable or better than the parent (IC50 = 16.3 μM). PMID:24830845

  7. Milk-derived proteins and peptides in clinical trials.

    PubMed

    Artym, Jolanta; Zimecki, Michał

    2013-01-01

    Clinical trials are reviewed, involving proteins and peptides derived from milk (predominantly bovine), with the exception of lactoferrin, which will be the subject of another article. The most explored milk fraction is α-lactalbumin (LA), which is often applied with glycomacropeptide (GMP) - a casein degradation product. These milk constituents are used in health-promoting infant and adult formulae as well as in a modified form (HAMLET) to treat cancer. Lactoperoxidase (LCP) is used as an additive to mouth hygiene products and as a salivary substitute. Casein derivatives are applied, in addition, in the dry mouth syndrome. On the other hand, casein hydrolysates, containing active tripeptides, found application in hypertension and in type 2 diabetes. Lysozyme is routinely used for food conservation and in pharmaceutical products. It was successfully used in premature infants with concomitant diseases to improve health parameters. When used as prophylaxis in patients with scheduled surgery, it significantly reduced the incidence of hepatitis resulting from blood transfusion. Lysozyme was also used in infected children as an antimicrobial agent showing synergistic effects in combination with different antibiotics. Proline-rich polypeptide (PRP) was introduced to therapy of Alzheimer's disease patients. The therapeutic value of PRP was proved in several clinical trials and supported by studies on its mechanism of action. Concentrated immunoglobulin preparations from colostrum and milk of hyperimmunized cows showed efficacy in prevention of infections by bacteria, viruses and protozoa. A nutrition formula with milk-derived TGF-β2 (Modulen IBD®) found application in treatment of pediatric Crohn's disease. In conclusion, the preparations containing milk-derived products are safe and effective measures in prevention and treatment of infections as well as autoimmune and neoplastic diseases. PMID:24018446

  8. Successful immunotherapy with matrix metalloproteinase-derived peptides in adjuvant arthritis depends on the timing of peptide administration

    PubMed Central

    van Bilsen, Jolanda HM; Wagenaar-Hilbers, Josée PA; van der Cammen, Maarten JF; van Dijk, Mariska EA; van Eden, Willem; Wauben, Marca HM

    2002-01-01

    We have recently found that matrix metalloproteinases (MMPs) are targets for T-cell and B-cell reactivity in experimental arthritis. In the present article, we investigate whether modulation of MMP-specific T-cell responses could influence the course of adjuvant arthritis (AA). Lewis rats were treated nasally with MMP peptides prior to or after AA induction. Administration of the MMP-10 or the MMP-16 peptide prior to AA induction reduced the arthritic symptoms. In contrast, administration of the MMP-10 peptide after AA induction aggravated the arthritic symptoms. The present study shows the possible usefulness of MMP peptides for immunotherapy. However, a clear understanding of proper timing of peptide administration is crucial for the development of such therapies. PMID:12106501

  9. Antimicrobial peptide scolopendrasin VII, derived from the centipede Scolopendra subspinipes mutilans, stimulates macrophage chemotaxis via formyl peptide receptor 1.

    PubMed

    Park, Yoo Jung; Lee, Ha Young; Jung, Young Su; Park, Joon Seong; Hwang, Jae Sam; Bae, Yoe-Sik

    2015-08-01

    In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses. PMID:26129676

  10. Peptide-conjugated nanoparticles for targeted imaging and therapy of prostate cancer.

    PubMed

    Yeh, Chen-Yun; Hsiao, Jong-Kai; Wang, Yi-Ping; Lan, Chun-Hsin; Wu, Han-Chung

    2016-08-01

    While there has been extensive development of anti-cancer drugs for treatment of prostate cancer, the therapeutic efficacy of such drugs remains inadequate in many cases. Here, we performed in vitro biopanning of the PC3 human prostate carcinoma cell line to select prostate cancer-specific peptides by phage display. We successfully identified specific peptides targeting prostate cancer cells, and their specificity was confirmed by cellular ELISA and flow cytometry. Moreover, we found that the phage clones also recognize other prostate cancer cell lines and surgical specimens from prostate cancer patients. The tumor targeting ability of these phages was validated in a xenograft model, in which high accumulation of targeting phage was observed. To investigate whether selected peptides are able to target tumors and enhance drug delivery into cancer cells, we synthesized peptide-PEGylated lipids and post-inserted them into preformed liposomal doxorubicin and vinorelbine. The results of our cellular uptake and MTT assays indicate that peptide-conjugated liposomes exhibit enhanced drug intracellular delivery and cytotoxicity. The conjugation of targeting peptide to imaging agents, such as quantum dots (QDs) and superparamagnetic iron oxide nanoparticles (SPIONs), results in more precise delivery of these agents to tumor sites. Furthermore, administration of liposomal doxorubicin and vinorelbine conjugated with targeting peptides was found to markedly increase the inhibition of human prostate tumor growth in mouse xenograft and orthotopic models. These results indicate that targeting peptide, SP204, has significant potential for targeted therapy and molecular imaging in prostate cancer. PMID:27209258

  11. Design and Evaluation of Antimalarial Peptides Derived from Prediction of Short Linear Motifs in Proteins Related to Erythrocyte Invasion

    PubMed Central

    Bianchin, Alessandra; Bell, Angus; Chubb, Anthony J.; Doolan, Nathalie; Leneghan, Darren; Stavropoulos, Ilias; Shields, Denis C.; Mooney, Catherine

    2015-01-01

    The purpose of this study was to investigate the blood stage of the malaria causing parasite, Plasmodium falciparum, to predict potential protein interactions between the parasite merozoite and the host erythrocyte and design peptides that could interrupt these predicted interactions. We screened the P. falciparum and human proteomes for computationally predicted short linear motifs (SLiMs) in cytoplasmic portions of transmembrane proteins that could play roles in the invasion of the erythrocyte by the merozoite, an essential step in malarial pathogenesis. We tested thirteen peptides predicted to contain SLiMs, twelve of them palmitoylated to enhance membrane targeting, and found three that blocked parasite growth in culture by inhibiting the initiation of new infections in erythrocytes. Scrambled peptides for two of the most promising peptides suggested that their activity may be reflective of amino acid properties, in particular, positive charge. However, one peptide showed effects which were stronger than those of scrambled peptides. This was derived from human red blood cell glycophorin-B. We concluded that proteome-wide computational screening of the intracellular regions of both host and pathogen adhesion proteins provides potential lead peptides for the development of anti-malarial compounds. PMID:26039561

  12. Marine algae-derived bioactive peptides for human nutrition and health.

    PubMed

    Fan, Xiaodan; Bai, Lu; Zhu, Liang; Yang, Li; Zhang, Xuewu

    2014-09-24

    Within the parent protein molecule, most peptides are inactive, and they are released with biofunctionalities after enzymatic hydrolysis. Marine algae have high protein content, up to 47% of the dry weight, depending on the season and the species. Recently, there is an increasing interest in using marine algae protein as a source of bioactive peptides due to their health promotion and disease therapy potentials. This review presents an overview of marine algae-derived bioactive peptides and especially highlights some key issues, such as in silico proteolysis and quantitative structure-activity relationship studies, in vivo fate of bioactive peptides, and novel technologies in bioactive peptides studies and production. PMID:25179496

  13. Enhancing tumor-specific intracellular delivering efficiency of cell-penetrating peptide by fusion with a peptide targeting to EGFR.

    PubMed

    Nguyen, Long The; Yang, Xu-Zhong; Du, Xuan; Wang, Jia-Wei; Zhang, Rui; Zhao, Jian; Wang, Fu-Jun; Dong, Yang; Li, Peng-Fei

    2015-05-01

    Cell-penetrating peptides (CPPs) are well known as intracellular delivery vectors. However, unsatisfactory delivery efficiency and poor specificity are challenging barriers to CPP applications at the clinical trial stage. Here, we showed that S3, an EGFR-binding domain derived from vaccinia virus growth factor, when fused to a CPP such as HBD or TAT can substantially enhance its internalization efficiency and tumor selectivity. The uptake of S3-HBD (S3H) recombinant molecule by tumor cells was nearly 80 folds increased compared to HBD alone. By contrast, the uptake of S3H by non-neoplastic cells still remained at a low level. The specific recognition between S3 and its receptor, EGFR, as well as between HBD and heparan sulfate proteoglycans on the cell surface was essential for these improvements, suggesting a syngeneic effect between the two functional domains in conjugation. This syngeneic effect is likely similar to that of the heparin-binding epidermal growth factor, which is highly abundant particularly in metastatic tumors. The process that S3H entered cells was dependent on time, dosage, and energy, via macropinocytosis pathway. With excellent cell-penetrating efficacy and a novel tumor-targeting ability, S3H appears as a promising candidate vector for targeted anti-cancer drug delivery. PMID:25655386

  14. Bactericidal Activity of Mammalian Cathelicidin-Derived Peptides

    PubMed Central

    Travis, Sue M.; Anderson, Norma N.; Forsyth, William R.; Espiritu, Cesar; Conway, Barbara D.; Greenberg, E. P.; McCray, Paul B.; Lehrer, Robert I.; Welsh, Michael J.; Tack, Brian F.

    2000-01-01

    Endogenous antimicrobial peptides of the cathelicidin family contribute to innate immunity. The emergence of widespread antibiotic resistance in many commonly encountered bacteria requires the search for new bactericidal agents with therapeutic potential. Solid-phase synthesis was employed to prepare linear antimicrobial peptides found in cathelicidins of five mammals: human (FALL39/LL37), rabbit (CAP18), mouse (mCRAMP), rat (rCRAMP), and sheep (SMAP29 and SMAP34). These peptides were tested at ionic strengths of 25 and 175 mM against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. Each peptide manifested activity against P. aeruginosa irrespective of the NaCl concentration. CAP18 and SMAP29 were the most effective peptides of the group against all test organisms under both low- and high-salt conditions. Select peptides of 15 to 21 residues, modeled on CAP18 (37 residues), retained activity against the gram-negative bacteria and methicillin-sensitive S. aureus, although the bactericidal activity was reduced compared to that of the parent peptide. In accordance with the behavior of the parent molecule, the truncated peptides adopted an α-helical structure in the presence of trifluoroethanol or lipopolysaccharide. The relationship between the bactericidal activity and several physiochemical properties of the cathelicidins was examined. The activities of the full-length peptides correlated positively with a predicted gradient of hydrophobicity along the peptide backbone and with net positive charge; they correlated inversely with relative abundance of anionic residues. The salt-resistant, antimicrobial properties of CAP18 and SMAP29 suggest that these peptides or congeneric structures have potential for the treatment of bacterial infections in normal and immunocompromised persons and individuals with cystic fibrosis. PMID:10768969

  15. Bactericidal activity of mammalian cathelicidin-derived peptides.

    PubMed

    Travis, S M; Anderson, N N; Forsyth, W R; Espiritu, C; Conway, B D; Greenberg, E P; McCray, P B; Lehrer, R I; Welsh, M J; Tack, B F

    2000-05-01

    Endogenous antimicrobial peptides of the cathelicidin family contribute to innate immunity. The emergence of widespread antibiotic resistance in many commonly encountered bacteria requires the search for new bactericidal agents with therapeutic potential. Solid-phase synthesis was employed to prepare linear antimicrobial peptides found in cathelicidins of five mammals: human (FALL39/LL37), rabbit (CAP18), mouse (mCRAMP), rat (rCRAMP), and sheep (SMAP29 and SMAP34). These peptides were tested at ionic strengths of 25 and 175 mM against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. Each peptide manifested activity against P. aeruginosa irrespective of the NaCl concentration. CAP18 and SMAP29 were the most effective peptides of the group against all test organisms under both low- and high-salt conditions. Select peptides of 15 to 21 residues, modeled on CAP18 (37 residues), retained activity against the gram-negative bacteria and methicillin-sensitive S. aureus, although the bactericidal activity was reduced compared to that of the parent peptide. In accordance with the behavior of the parent molecule, the truncated peptides adopted an alpha-helical structure in the presence of trifluoroethanol or lipopolysaccharide. The relationship between the bactericidal activity and several physiochemical properties of the cathelicidins was examined. The activities of the full-length peptides correlated positively with a predicted gradient of hydrophobicity along the peptide backbone and with net positive charge; they correlated inversely with relative abundance of anionic residues. The salt-resistant, antimicrobial properties of CAP18 and SMAP29 suggest that these peptides or congeneric structures have potential for the treatment of bacterial infections in normal and immunocompromised persons and individuals with cystic fibrosis. PMID:10768969

  16. A Heparan Sulfate-Binding Cell Penetrating Peptide for Tumor Targeting and Migration Inhibition

    PubMed Central

    Kuo, Ping-Hsueh; Chang, Pei-Lin; Wang, Wen-Ching; Chuang, Yung-Jen; Chang, Margaret Dah-Tsyr

    2015-01-01

    As heparan sulfate proteoglycans (HSPGs) are known as co-receptors to interact with numerous growth factors and then modulate downstream biological activities, overexpression of HS/HSPG on cell surface acts as an increasingly reliable prognostic factor in tumor progression. Cell penetrating peptides (CPPs) are short-chain peptides developed as functionalized vectors for delivery approaches of impermeable agents. On cell surface negatively charged HS provides the initial attachment of basic CPPs by electrostatic interaction, leading to multiple cellular effects. Here a functional peptide (CPPecp) has been identified from critical HS binding region in hRNase3, a unique RNase family member with in vitro antitumor activity. In this study we analyze a set of HS-binding CPPs derived from natural proteins including CPPecp. In addition to cellular binding and internalization, CPPecp demonstrated multiple functions including strong binding activity to tumor cell surface with higher HS expression, significant inhibitory effects on cancer cell migration, and suppression of angiogenesis in vitro and in vivo. Moreover, different from conventional highly basic CPPs, CPPecp facilitated magnetic nanoparticle to selectively target tumor site in vivo. Therefore, CPPecp could engage its capacity to be developed as biomaterials for diagnostic imaging agent, therapeutic supplement, or functionalized vector for drug delivery. PMID:26064887

  17. Targeted in vivo photodynamic therapy with epidermal growth factor receptor-specific peptide linked nanoparticles.

    PubMed

    Narsireddy, Amreddy; Vijayashree, Kurra; Irudayaraj, Joseph; Manorama, Sunkara V; Rao, Nalam M

    2014-08-25

    In targeted photodynamic therapy (tPDT), photosensitizers (PS) are targeted to disease tissue to reduce the dosage of PS and in addition to reduce the photo damage to the non-target tissue. We synthesized iron oxide nanoparticles (NP) armored with tumor targeting peptide and PS for targeted PDT. Chitosan covered Fe3O4 NPs (30 nm) were deposited with gold NPs to generate two distinct chemical surfaces. To the gold particles PS was attached with a lipoic acid linker. Human epidermal growth factor receptor (hEGFR)-specific peptide was also attached to the same particles via a nickel-nitrilotriacetic acid linker attached to the chitosan. Using these nanoparticles, peptide specific uptake and PDT mediated cell death of the SK-OV-3 cells (Her2(+) positive cells) were demonstrated by confocal microscopy, T2 imaging and viability assays. Peptide mediated preferential distribution of these NPs into tumor tissue was also shown in a xenograft tumor model. After one intravenous injection and one PDT dose, peptide bound NPs retarded tumor growth significantly compared to dark controls or treatments with NPs without peptide. The tumor retardation by targeted NPs was achieved at a PS concentration of 3.9 nmol/animal, whereas similar effect was seen with free PS at 220 nmol/animal. Therapeutic potential of these peptide containing NPs would be a useful in targeted PDT and in imaging the target tissue. PMID:24939618

  18. Assessment of RNA carrier function in peptide amphiphiles derived from the HIV fusion peptide.

    PubMed

    Pratumyot, Yaowalak; Torres, Oscar B; Bong, Dennis

    2016-05-01

    A small library of amphiphilic peptides has been evaluated for duplex RNA carrier function into A549 cells. We studied peptides in which a C-terminal 7-residue cationic domain is attached to a neutral/hydrophobic 23-residue domain that is based on the viral fusion peptide of HIV. We also examined peptides in which the cationic charge was evenly distributed throughout the peptide. Strikingly, subtle sequence variations in the hydrophobic domain that do not alter net hydrophobicity result in wide variation in RNA uptake. Additionally, cyclic cystine variants are much less active as RNA carriers than their open-chain cysteine analogs. With regard to electrostatic effects, we find that lysine is less effective than arginine in facilitating uptake, and that even distribution of cationic residues throughout the peptide sequence results in especially effective RNA carrier function. Overall, minor changes in peptide hydrophobicity, flexibility and charge distribution can significantly alter carrier function. We hypothesize this is due to altered properties of the peptide-RNA assembly rather than peptide secondary structure. PMID:26988874

  19. Enhanced Intracellular Hyperthermia Efficiency by Magnetic Nanoparticles Modified with Nucleus and Mitochondria Targeting Peptides.

    PubMed

    Wang, Xiaowen; Zhou, Jumei; Chen, Benke; Tang, Zhenghai; Zhang, Jieying; Li, Liya; Tang, Jintian

    2016-06-01

    In order to investigate whether cell organelle targeting peptide can transport magnetic nanoparticles (MNPs) into specific cell organelle, peptides bearing nuclear localization signal (NLS) or mitochondria targeting sequences were coagulated to MNPs. In vitro cytotoxicity study on the human liver cancer cells (HepG2) was tested by using MTT assay. Sub-cellular location of each peptide modified MNP (PEP-MNPs) was observed by transmission electron microscopy (TEM). The uptake of HepG2 cells growing in PEP-MNPs was measured by using ICP-OES. Magnetic induction heating efficacies of PEP-MNPs were analyzed by exposing the PEP-MNPs containing cells in an alternating magnetic field (AMF). It was demonstrated that PEP-MNPs were efficient agents for cancer nanothermotherapy with satisfactory biocompatibility. TEM showed that the fate of MNPs inside the cells depended on the peptide sequence attached to the particle surface. The uptake improvement was observed both in PEP-MNPs bearing NLS peptides and in PEP-MNPs bearing mitochondria targeting sequences. Virus original endocytosis sequence can enhance the uptake. MNP bearing mitochondria targeting sequence exerted a better magnetic induction hyperthermia performance comparing to that of NLS. Our investigation provides a strategy for fabrication cell organelle targeting magnetic nanoparticles. For instance, mitochondria targeting peptide conjugated MNPs for highly-efficiency magnetic nanothermotherapy and nuclear targeting peptides conjugated MNPs for gene magnetofection. PMID:27427753

  20. 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

  1. Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display.

    PubMed

    Writer, Michele J; Marshall, Barry; Pilkington-Miksa, Michael A; Barker, Susie E; Jacobsen, Marianne; Kritz, Angelika; Bell, Paul C; Lester, Douglas H; Tabor, Alethea B; Hailes, Helen C; Klein, Nigel; Hart, Stephen L

    2004-05-01

    Human airway epithelial cell targeting peptides were identified by biopanning on 1HAEo-cells, a well characterised epithelial cell line. Bound phage were recovered after three rounds of binding, high stringency washing and elution, leading to the production of an enriched phage peptide population. DNA sequencing of 56 clones revealed 14 unique sequences. Subsequent binding analysis revealed that 13 of these peptides bound 1HAEo-cells with high affinity. Three peptides, SERSMNF, YGLPHKF and PSGAARA were represented at high frequency. Three clearly defined families of peptide were identified on the basis of sequence motifs including (R/K)SM, L(P/Q)HK and PSG(A/T)ARA. Two peptides, LPHKSMP and LQHKSMP contained two motifs. Further detailed sequence analysis by comparison of peptide sequences with the SWISSPROT protein database revealed that some of the peptides closely resembled the cell binding proteins of viral and bacterial pathogens including Herpes Simplex Virus, rotavirus, Mycoplasma pneumoniae and rhinovirus, the latter two being respiratory pathogens, as well as peptide YGLPHKF having similarity to a protein of unknown function from the respiratory pathogen Legionella pneumophila. Peptides were incorporated into gene delivery formulations with the cationic lipid Lipofectin and plasmid DNA and shown to confer a high degree of transfection efficiency and specificity in 1HAEo-cells. Improved transfection efficiency and specificity was also observed in human endothelial cells, fibroblasts and keratinocytes. Therefore, on the basis of clone frequency after biopanning, cell binding affinity, peptide sequence conservation and pathogenic similarity, we have identified 3 novel peptide families and 5 specific peptides that have the potential for gene transfer to respiratory epithelium in vivo as well as providing useful in vitro transfection reagents for primary human cell types of scientific and commercial interest. PMID:15506167

  2. Targeting Cyclin-Dependent Kinases in Human Cancers: From Small Molecules to Peptide Inhibitors

    PubMed Central

    Peyressatre, Marion; Prével, Camille; Pellerano, Morgan; Morris, May C.

    2015-01-01

    Cyclin-dependent kinases (CDK/Cyclins) form a family of heterodimeric kinases that play central roles in regulation of cell cycle progression, transcription and other major biological processes including neuronal differentiation and metabolism. Constitutive or deregulated hyperactivity of these kinases due to amplification, overexpression or mutation of cyclins or CDK, contributes to proliferation of cancer cells, and aberrant activity of these kinases has been reported in a wide variety of human cancers. These kinases therefore constitute biomarkers of proliferation and attractive pharmacological targets for development of anticancer therapeutics. The structural features of several of these kinases have been elucidated and their molecular mechanisms of regulation characterized in depth, providing clues for development of drugs and inhibitors to disrupt their function. However, like most other kinases, they constitute a challenging class of therapeutic targets due to their highly conserved structural features and ATP-binding pocket. Notwithstanding, several classes of inhibitors have been discovered from natural sources, and small molecule derivatives have been synthesized through rational, structure-guided approaches or identified in high throughput screens. The larger part of these inhibitors target ATP pockets, but a growing number of peptides targeting protein/protein interfaces are being proposed, and a small number of compounds targeting allosteric sites have been reported. PMID:25625291

  3. 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

  4. Small Peptides Derived from Penetratin as Antibacterial Agents.

    PubMed

    Parravicini, Oscar; Somlai, Csaba; Andujar, Sebastián A; Garro, Adriana D; Lima, Beatriz; Tapia, Alejandro; Feresin, Gabriela; Perczel, Andras; Tóth, Gabor; Cascales, Javier López; Rodríguez, Ana M; Enriz, Ricardo D

    2016-04-01

    The synthesis, in vitro evaluation and conformational study of several small-size peptides acting as antibacterial agents are reported. Among the compounds evaluated, the peptides Arg-Gln-Ile-Lys-Ile-Trp-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH2 , Arg-Gln-Ile-Lys-Ile-Arg-Arg-Met-Lys-Trp-Arg-NH2 , and Arg-Gln-Ile-Trp-Trp-Trp-Trp-Gln-Arg-NH2 exhibited significant antibacterial activity. These were found to be very active antibacterial compounds, considering their small molecular size. In order to better understand the antibacterial activity obtained for these peptides, an exhaustive conformational analysis was performed, using both theoretical calculations and experimental measurements. Molecular dynamics simulations using two different media (water and trifluoroethanol/water) were employed. The results of these theoretical calculations were corroborated by experimental circular dichroism measurements. A brief discussion on the possible mechanism of action of these peptides at molecular level is also presented. Some of the peptides reported here constitute very interesting structures to be used as starting compounds for the design of new small-size peptides possessing antibacterial activity. PMID:26972341

  5. AVPdb: a database of experimentally validated antiviral peptides targeting medically important viruses.

    PubMed

    Qureshi, Abid; Thakur, Nishant; Tandon, Himani; Kumar, Manoj

    2014-01-01

    Antiviral peptides (AVPs) have exhibited huge potential in inhibiting viruses by targeting various stages of their life cycle. Therefore, we have developed AVPdb, available online at http://crdd.osdd.net/servers/avpdb, to provide a dedicated resource of experimentally verified AVPs targeting over 60 medically important viruses including Influenza, HCV, HSV, RSV, HBV, DENV, SARS, etc. However, we have separately provided HIV inhibiting peptides in 'HIPdb'. AVPdb contains detailed information of 2683 peptides, including 624 modified peptides experimentally tested for antiviral activity. In modified peptides a chemical moiety is attached for increasing their efficacy and stability. Detailed information include: peptide sequence, length, source, virus targeted, virus family, cell line used, efficacy (qualitative/quantitative), target step/protein, assay used in determining the efficacy and PubMed reference. The database also furnishes physicochemical properties and predicted structure for each peptide. We have provided user-friendly browsing and search facility along with other analysis tools to help the users. Entering of many synthetic peptide-based drugs in various stages of clinical trials reiterate the importance for the AVP resources. AVPdb is anticipated to cater to the needs of scientific community working for the development of antiviral therapeutics. PMID:24285301

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

    PubMed

    Duprez, Wilko; Bachu, Prabhakar; Stoermer, Martin J; Tay, Stephanie; McMahon, Róisín M; Fairlie, David P; Martin, Jennifer L

    2015-01-01

    Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately needed to address the growing problem of antibiotic resistance. The periplasmic oxidative folding system in Gram-negative bacteria represents a possible target for anti-virulence antibacterials. By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota) might be minimized. Here, we undertook the design of peptidomimetic inhibitors targeting the interaction between the two key enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence factor assembly. Structures of DsbB--or peptides--complexed with DsbA revealed key interactions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the active site. The present work aimed to discover peptidomimetics that target the hydrophobic groove to generate non-covalent DsbA inhibitors. The previously reported structure of a Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the heptapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomimetic fragment library. The highest scoring fragment compound and nine derivatives were synthesized and evaluated for DsbA binding and inhibition. These experiments discovered peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors. PMID:26225423

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

    PubMed Central

    Stoermer, Martin J.; Tay, Stephanie; McMahon, Róisín M.; Fairlie, David P.; Martin, Jennifer L.

    2015-01-01

    Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately needed to address the growing problem of antibiotic resistance. The periplasmic oxidative folding system in Gram-negative bacteria represents a possible target for anti-virulence antibacterials. By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota) might be minimized. Here, we undertook the design of peptidomimetic inhibitors targeting the interaction between the two key enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence factor assembly. Structures of DsbB - or peptides - complexed with DsbA revealed key interactions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the active site. The present work aimed to discover peptidomimetics that target the hydrophobic groove to generate non-covalent DsbA inhibitors. The previously reported structure of a Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the heptapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomimetic fragment library. The highest scoring fragment compound and nine derivatives were synthesized and evaluated for DsbA binding and inhibition. These experiments discovered peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors. PMID:26225423

  8. Mechanism of antibacterial action of a synthetic peptide with an Ala-peptoid residue based on the scorpion-derived antimicrobial peptide IsCT.

    PubMed

    Lim, Shin Saeng; Yoon, Sang-Pil; Park, Yoonkyoung; Zhu, Wan Long; Park, Il-Seon; Hahm, Kyung-Soo; Shin, Song Yub

    2006-09-01

    A novel bacterial cell-selective antimicrobial peptide, IsCT-P (ILKKIWKPIKKLF-NH(2)), was designed based on the scorpion-derived alpha-helical antimicrobial peptide, IsCT. Here, we investigated the effect of substituting Pro(8) of IsCT-P with the Ala-peptoid residue (N-methylglycine) on the peptide's structure and mechanism of action. Circular dichroism analysis revealed that the modified peptide, IsCT-a, has a much lower alpha-helicity than IsCT-P in membrane mimicking conditions, suggesting the peptoid residue provides much more structural flexibility than the proline residue. IsCT-a was also much less effective than IsCT-P at causing leakage of fluorescent dye entrapped within negatively charged vesicles and at dissipating the membrane potential of Staphylococcus aureus. Collectively, our results suggest that the antibacterial action of IsCT-a is due to the inhibition of intracellular targets rather than the disruption and depolarization of bacterial cell membranes. PMID:16871429

  9. [Derivatives of N-amidinoproline and their use in conventional and solid phase peptide synthesis].

    PubMed

    Burov, S V; Moskalenko, Iu E; Leko, M V; Dorosh, M Iu; Panarin, E F

    2006-01-01

    N-Amidinoproline, a hybrid structure modeling key features of the Arg-Pro sequence, was synthesized. The activation of carboxyl group of free N-amidinoproline was found to result in the formation of a cyclic side product, whose structure was confirmed by ESI MS, 1H NMR, and 13C NMR spectra. The preparation of N-(mesitylenesulfonylamidino)-L-proline using the mesitylenesulfonyl derivative of 2-methylisourea was demonstrated to be accompanied by partial racemization. The target product was synthesized by modification of N-amidinoproline by mesitylenesulfonyl chloride. The possibility of using N-amidinoproline in the N-terminal modification of a peptide chain was shown by the example of synthesis of an analogue of the 95-98 fragment of fibrinogen alpha chain. PMID:17180906

  10. Peptides in Cancer Nanomedicine: Drug Carriers, Targeting Ligands and Protease Substrates

    PubMed Central

    Zhang, Xiao-Xiang; Eden, Henry S.; Chen, Xiaoyuan

    2011-01-01

    Peptides are attracting increasing attention as therapeutic agents, as the technologies for peptide development and manufacture continue to mature. Concurrently, with booming researches in nanotechnology for biomedical applications, peptides have been studied as an important class of components in nanomedicine, and they have been used either alone or in combination with nanomaterials of every reported composition. Peptides possess many advantages, such as smallness, ease of synthesis and modification, and good biocompatibility. Their functions in cancer nanomedicine, discussed in this review, include serving as drug carriers; as targeting ligands; and as protease-responsive substrates for drug delivery. PMID:22056916

  11. Immunogenicity of HLA Class I and II Double Restricted Influenza A-Derived Peptides

    PubMed Central

    Pedersen, Sara Ram; Christensen, Jan Pravsgaard; Buus, Søren; Rasmussen, Michael; Korsholm, Karen Smith; Nielsen, Morten; Claesson, Mogens Helweg

    2016-01-01

    The aim of the present study was to identify influenza A-derived peptides which bind to both HLA class I and -II molecules and by immunization lead to both HLA class I and class II restricted immune responses. Eight influenza A-derived 9-11mer peptides with simultaneous binding to both HLA-A*02:01 and HLA-DRB1*01:01 molecules were identified by bioinformatics and biochemical technology. Immunization of transgenic HLA-A*02:01/HLA-DRB1*01:01 mice with four of these double binding peptides gave rise to both HLA class I and class II restricted responses by CD8 and CD4 T cells, respectively, whereas four of the double binding peptides did result in HLA-A*02:01 restricted responses only. According to their cytokine profile, the CD4 T cell responses were of the Th2 type. In influenza infected mice, we were unable to detect natural processing in vivo of the double restricted peptides and in line with this, peptide vaccination did not decrease virus titres in the lungs of intranasally influenza challenged mice. Our data show that HLA class I and class II double binding peptides can be identified by bioinformatics and biochemical technology. By immunization, double binding peptides can give rise to both HLA class I and class I restricted responses, a quality which might be of potential interest for peptide-based vaccine development. PMID:26731261

  12. Serum Stability and Affinity Optimization of an M2 Macrophage-Targeting Peptide (M2pep).

    PubMed

    Ngambenjawong, Chayanon; Gustafson, Heather H; Pineda, Julio M; Kacherovsky, Nataly A; Cieslewicz, Maryelise; Pun, Suzie H

    2016-01-01

    Tumor associated macrophages (TAMs) are a major stromal component of the tumor microenvironment in several cancers. TAMs are a potential target for adjuvant cancer therapies due to their established roles in promoting proliferation of cancer cells, angiogenesis, and metastasis. We previously discovered an M2 macrophage-targeting peptide (M2pep) which was successfully used to target and deliver a pro-apoptotic KLA peptide to M2-like TAMs in a CT-26 colon carcinoma model. However, the effectiveness of in vivo TAM-targeting using M2pep is limited by its poor serum stability and low binding affinity. In this study, we synthesized M2pep derivatives with the goals of increasing serum stability and binding affinity. Serum stability evaluation of M2pepBiotin confirmed its rapid degradation attributed to exolytic cleavage from the N-terminus and endolytic cleavages at the W10/W11 and S16/K17 sites. N-terminal acetylation of M2pepBiotin protected the peptide against the exolytic degradation while W10w and K(17,18,19)k substitutions were able to effectively protect endolytic degradation at their respective cleavage sites. However, no tested amino acid changes at the W10 position resulted in both protease resistance at that site and retention of binding activity. Therefore, cyclization of M2pep was investigated. Cyclized M2pep better resisted serum degradation without compromising binding activity to M2 macrophages. During the serum stability optimization process, we also discovered that K9R and W10Y substitutions significantly enhanced binding affinity of M2pep. In an in vitro binding study of different M2pep analogs pre-incubated in mouse serum, cyclic M2pep with K9R and W10Y modifications (cyclic M2pep(RY)) retained the highest binding activity to M2 macrophages over time due to its improved serum stability. Finally, we evaluated the in vivo accumulation of sulfo-Cy5-labeled M2pep and cyclic M2pep(RY) in both the CT-26 and 4T1 breast carcinoma models. Cyclic M2pep

  13. Serum Stability and Affinity Optimization of an M2 Macrophage-Targeting Peptide (M2pep)

    PubMed Central

    Ngambenjawong, Chayanon; Gustafson, Heather H.; Pineda, Julio M.; Kacherovsky, Nataly A.; Cieslewicz, Maryelise; Pun, Suzie H.

    2016-01-01

    Tumor associated macrophages (TAMs) are a major stromal component of the tumor microenvironment in several cancers. TAMs are a potential target for adjuvant cancer therapies due to their established roles in promoting proliferation of cancer cells, angiogenesis, and metastasis. We previously discovered an M2 macrophage-targeting peptide (M2pep) which was successfully used to target and deliver a pro-apoptotic KLA peptide to M2-like TAMs in a CT-26 colon carcinoma model. However, the effectiveness of in vivo TAM-targeting using M2pep is limited by its poor serum stability and low binding affinity. In this study, we synthesized M2pep derivatives with the goals of increasing serum stability and binding affinity. Serum stability evaluation of M2pepBiotin confirmed its rapid degradation attributed to exolytic cleavage from the N-terminus and endolytic cleavages at the W10/W11 and S16/K17 sites. N-terminal acetylation of M2pepBiotin protected the peptide against the exolytic degradation while W10w and K(17,18,19)k substitutions were able to effectively protect endolytic degradation at their respective cleavage sites. However, no tested amino acid changes at the W10 position resulted in both protease resistance at that site and retention of binding activity. Therefore, cyclization of M2pep was investigated. Cyclized M2pep better resisted serum degradation without compromising binding activity to M2 macrophages. During the serum stability optimization process, we also discovered that K9R and W10Y substitutions significantly enhanced binding affinity of M2pep. In an in vitro binding study of different M2pep analogs pre-incubated in mouse serum, cyclic M2pep with K9R and W10Y modifications (cyclic M2pep(RY)) retained the highest binding activity to M2 macrophages over time due to its improved serum stability. Finally, we evaluated the in vivo accumulation of sulfo-Cy5-labeled M2pep and cyclic M2pep(RY) in both the CT-26 and 4T1 breast carcinoma models. Cyclic M2pep

  14. Nonopiate active proenkephalin-derived peptides are secreted by T helper cells.

    PubMed

    Roth, K A; Lorenz, R G; Unanue, R A; Weaver, C T

    1989-10-01

    Recent investigations have shown that the neuroendocrine and immune systems profoundly affect each other. In part, these interactions occur via common chemical messengers and receptors. One possible shared chemical messenger is the opioid precursor preproenkephalin, for which high concentrations of messenger RNA are present in brain, adrenal, and activated T helper cells. Because the biologic action of most peptide messengers depends on the posttranslational processing of the precursor, we have examined T helper cell lines for the production of proenkephalin-derived peptides. These peptides were characterized by multiple radioimmunoassays, gel filtration chromatography, and opiate radioreceptor assays. We found that activated T helper cells secrete significant concentrations of high-molecular-weight, opiate-inactive peptides, which are distinct from the proenkephalin-derived peptides of the neuroendocrine system. These studies clearly indicate cell-specific processing of proenkephalin, and suggest that the T helper cell-secreted products may have nonopiate receptor-mediated actions. PMID:2529160

  15. Novel EGFR-targeted strategy with hybrid peptide against oesophageal squamous cell carcinoma

    PubMed Central

    Kikuchi, Osamu; Ohashi, Shinya; Horibe, Tomohisa; Kohno, Masayuki; Nakai, Yukie; Miyamoto, Shin’ichi; Chiba, Tsutomu; Muto, Manabu; Kawakami, Koji

    2016-01-01

    Epidermal growth factor receptor (EGFR) is a key molecule in the pathophysiology of oesophageal squamous cell carcinoma (OSCC). However, EGFR-targeted agents such as anti-EGFR antibody or tyrosine kinase inhibitors for OSCC have not demonstrated any clinical benefits. Recently, a novel chemotherapeutic agent, EGFR(2R)-lytic hybrid peptide, a composite of EGFR-binding peptide and lytic peptide fragments, has been shown to exhibit a potent anti-tumour effect against cancers that express high EGFR levels. In this study, we investigated the validity of employing EGFR(2R)-lytic hybrid peptide against OSCC cells both in vitro and in vivo. Additionally, the toxicity of this peptide was assessed in mice. We found high EGFR expression levels on the cell surface of OSCC cells, and the EGFR-binding peptide fragment showed high affinity for OSCC cells. A potent cytotoxic effect was induced within 30 minutes by the exposure of OSCC cells to EGFR(2R)-lytic hybrid peptide. Furthermore, EGFR(2R)-lytic hybrid peptide markedly suppressed the tumour growth of OSCC cells in a xenograft model. Moreover, it did not cause any identifiable adverse effects in mice. Taken together, EGFR(2R)-lytic hybrid peptide was shown to be a valid therapeutic agent against OSCC, providing a crucial rationale regarding novel EGFR-targeted therapies against OSCC. PMID:26956916

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

    PubMed

    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

  17. 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

  18. Peptides as targeting probes against tumor vasculature for diagnosis and drug delivery

    PubMed Central

    2012-01-01

    Tumor vasculature expresses a distinct set of molecule signatures on the endothelial cell surface different from the resting blood vessels of other organs and tissues in the body. This makes them an attractive target for cancer therapy and molecular imaging. The current technology using the in vivo phage display biopanning allows us to quickly isolate and identify peptides potentially homing to various tumor blood vessels. Tumor-homing peptides in conjugation with chemotherapeutic drugs or imaging contrast have been extensively tested in various preclinical and clinical studies. These tumor-homing peptides have valuable potential as targeting probes for tumor molecular imaging and drug delivery. In this review, we summarize the recent advances about the applications of tumor-homing peptides selected by in vivo phage display library screening against tumor vasculature. We also introduce the characteristics of the latest discovered tumor-penetrating peptides in their potential clinical applications. PMID:23046982

  19. A Novel Cell-Penetrating Peptide Derived from Human Eosinophil Cationic Protein

    PubMed Central

    Fang, Shun-lung; Fan, Tan-chi; Fu, Hua-Wen; Chen, Chien-Jung; Hwang, Chi-Shin; Hung, Ta-Jen; Lin, Lih-Yuan; Chang, Margaret Dah-Tsyr

    2013-01-01

    Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP32–41, derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP32–41 entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP32–41 to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP32–41 internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP32–41 attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP32–41 internalization routes. ECP32–41 was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases. PMID:23469189

  20. Therapeutic potential of a peptide targeting BCL-2 cell guardians in cancer.

    PubMed

    Adams, Jerry M

    2012-06-01

    A promising approach to cancer therapy is to elicit apoptosis with "BH3 mimetic" drugs, which target proteins of the BCL-2 family. As of yet, however, such drugs can target only certain BCL-2 family proteins. Hence, in this issue of the JCI, LaBelle et al. assess instead the therapeutic potential of a "stapled" BH3 peptide from the BIM protein, which inactivates all its prosurvival relatives. The peptide killed cultured hematologic tumor cells and abated growth of a leukemia xenograft, without perturbing the hematopoietic compartment. Hence, such peptides might eventually provide a new way to treat refractory leukemias. PMID:22622043

  1. A polyalanine peptide derived from polar fish with anti-infectious activities

    NASA Astrophysics Data System (ADS)

    Cardoso, Marlon H.; Ribeiro, Suzana M.; Nolasco, Diego O.; de La Fuente-Núñez, César; Felício, Mário R.; Gonçalves, Sónia; Matos, Carolina O.; Liao, Luciano M.; Santos, Nuno C.; Hancock, Robert E. W.; Franco, Octávio L.; Migliolo, Ludovico

    2016-02-01

    Due to the growing concern about antibiotic-resistant microbial infections, increasing support has been given to new drug discovery programs. A promising alternative to counter bacterial infections includes the antimicrobial peptides (AMPs), which have emerged as model molecules for rational design strategies. Here we focused on the study of Pa-MAP 1.9, a rationally designed AMP derived from the polar fish Pleuronectes americanus. Pa-MAP 1.9 was active against Gram-negative planktonic bacteria and biofilms, without being cytotoxic to mammalian cells. By using AFM, leakage assays, CD spectroscopy and in silico tools, we found that Pa-MAP 1.9 may be acting both on intracellular targets and on the bacterial surface, also being more efficient at interacting with anionic LUVs mimicking Gram-negative bacterial surface, where this peptide adopts α-helical conformations, than cholesterol-enriched LUVs mimicking mammalian cells. Thus, as bacteria present varied physiological features that favor antibiotic-resistance, Pa-MAP 1.9 could be a promising candidate in the development of tools against infections caused by pathogenic bacteria.

  2. Biophysical analysis of the interaction of granulysin-derived peptides with enterobacterial endotoxins

    PubMed Central

    Chen, Xi; Howe, Jörg; Andrä, Jörg; Rössle, Manfred; Richter, Walter; Silva, Ana Paula Galvão da; Krensky, Alan M.; Clayberger, Carol; Brandenburg, Klaus

    2009-01-01

    To combat infections by Gram-negative bacteria, it is not only necessary to kill the bacteria but also to neutralize pathogenicity factors such as endotoxin (lipopolysaccharide, LPS). The development of antimicrobial peptides based on mammalian endotoxin-binding proteins is a promising tool in the fight against bacterial infections, and septic shock syndrome. Here, synthetic peptides derived from granulysin (Gra-pep) were investigated in microbiological and biophysical assays to understand their interaction with LPS. We analyzed the influence of the binding of Gra-pep on (1) the acyl chain melting of the hydrophobic moiety of LPS, lipid A, by Fourier-transform spectroscopy, (2) the aggregate structure of LPS by small-angle X-ray scattering and cryo-transmission electron microscopy, and 3) the enthalpy change by isothermal titration calorimetry. In addition, the influence of Gra-pep on the incorporation of LPS and LPS-LBP (lipopolysaccharide-binding protein) complexes into negatively charged liposomes was monitored. Our findings demonstrate a characteristic change in the aggregate structure of LPS into multilamellar stacks in the presence of Gra-pep, but little or no change of acyl chain fluidity. Neutralization of LPS by Gra-pep is not due to a scavenging effect in solution, but rather proceeds after incorporation into target membranes, suggesting a requisite membrane-bound step. PMID:17555705

  3. A polyalanine peptide derived from polar fish with anti-infectious activities

    PubMed Central

    Cardoso, Marlon H.; Ribeiro, Suzana M.; Nolasco, Diego O.; de la Fuente-Núñez, César; Felício, Mário R.; Gonçalves, Sónia; Matos, Carolina O.; Liao, Luciano M.; Santos, Nuno C.; Hancock, Robert E. W.; Franco, Octávio L.; Migliolo, Ludovico

    2016-01-01

    Due to the growing concern about antibiotic-resistant microbial infections, increasing support has been given to new drug discovery programs. A promising alternative to counter bacterial infections includes the antimicrobial peptides (AMPs), which have emerged as model molecules for rational design strategies. Here we focused on the study of Pa-MAP 1.9, a rationally designed AMP derived from the polar fish Pleuronectes americanus. Pa-MAP 1.9 was active against Gram-negative planktonic bacteria and biofilms, without being cytotoxic to mammalian cells. By using AFM, leakage assays, CD spectroscopy and in silico tools, we found that Pa-MAP 1.9 may be acting both on intracellular targets and on the bacterial surface, also being more efficient at interacting with anionic LUVs mimicking Gram-negative bacterial surface, where this peptide adopts α-helical conformations, than cholesterol-enriched LUVs mimicking mammalian cells. Thus, as bacteria present varied physiological features that favor antibiotic-resistance, Pa-MAP 1.9 could be a promising candidate in the development of tools against infections caused by pathogenic bacteria. PMID:26916401

  4. Mapping the HLA ligandome landscape of acute myeloid leukemia: a targeted approach toward peptide-based immunotherapy.

    PubMed

    Berlin, C; Kowalewski, D J; Schuster, H; Mirza, N; Walz, S; Handel, M; Schmid-Horch, B; Salih, H R; Kanz, L; Rammensee, H-G; Stevanović, S; Stickel, J S

    2015-03-01

    Identification of physiologically relevant peptide vaccine targets calls for the direct analysis of the entirety of naturally presented human leukocyte antigen (HLA) ligands, termed the HLA ligandome. In this study, we implemented this direct approach using immunoprecipitation and mass spectrometry to define acute myeloid leukemia (AML)-associated peptide vaccine targets. Mapping the HLA class I ligandomes of 15 AML patients and 35 healthy controls, more than 25 000 different naturally presented HLA ligands were identified. Target prioritization based on AML exclusivity and high presentation frequency in the AML cohort identified a panel of 132 LiTAAs (ligandome-derived tumor-associated antigens), and 341 corresponding HLA ligands (LiTAPs (ligandome-derived tumor-associated peptides)) represented subset independently in >20% of AML patients. Functional characterization of LiTAPs by interferon-γ ELISPOT (Enzyme-Linked ImmunoSpot) and intracellular cytokine staining confirmed AML-specific CD8(+) T-cell recognition. Of note, our platform identified HLA ligands representing several established AML-associated antigens (e.g. NPM1, MAGED1, PRTN3, MPO, WT1), but found 80% of them to be also represented in healthy control samples. Mapping of HLA class II ligandomes provided additional CD4(+) T-cell epitopes and potentially synergistic embedded HLA ligands, allowing for complementation of a multipeptide vaccine for the immunotherapy of AML. PMID:25092142

  5. Degradable polyethylenimine derivate coupled to a bifunctional peptide R13 as a new gene-delivery vector

    PubMed Central

    Liu, Kehai; Wang, Xiaoyu; Fan, Wei; Zhu, Qing; Yang, Jingya; Gao, Jing; Gao, Shen

    2012-01-01

    Background To solve the efficiency versus cytotoxicity and tumor-targeting problems of polyethylenimine (PEI) used as a nonviral gene delivery vector, a degradable PEI derivate coupled to a bifunctional peptide R13 was developed. Methods First, we synthesized a degradable PEI derivate by crosslinking low-molecular-weight PEI with pluronic P123, then used tumor-targeting peptide arginine-glycine-aspartate-cysteine (RGDC), in conjunction with the cell-penetrating peptide Tat (49–57), to yield a bifunctional peptide RGDC-Tat (49–57) named R13, which can improve cell selection and increase cellular uptake, and, lastly, adopted R13 to modify the PEI derivates so as to prepare a new polymeric gene vector (P123-PEI-R13). The new gene vector was characterized in terms of its chemical structure and biophysical parameters. We also investigated the specificity, cytotoxicity, and gene transfection efficiency of this vector in αvβ3-positive human cervical carcinoma Hela cells and murine melanoma B16 cells in vitro. Results The vector showed controlled degradation, strong targeting specificity to αvβ3 receptor, and noncytotoxicity in Hela cells and B16 cells at higher doses, in contrast to PEI 25 KDa. The particle size of P123-PEI-R13/DNA complexes was around 100–250 nm, with proper zeta potential. The nanoparticles can protect plasmid DNA from being digested by DNase I at a concentration of 6 U DNase I/μg DNA. The nanoparticles were resistant to dissociation induced by 50% fetal bovine serum and 600 μg/mL sodium heparin. P123-PEI-R13 also revealed higher transfection efficiency in two cell lines as compared with PEI 25 KDa. Conclusion P123-PEI-R13 is a potential candidate as a safe and efficient gene-delivery carrier for gene therapy. PMID:22412301

  6. Computational Studies of Venom Peptides Targeting Potassium Channels.

    PubMed

    Chen, Rong; Chung, Shin-Ho

    2015-12-01

    Small peptides isolated from the venom of animals are potential scaffolds for ion channel drug discovery. This review article mainly focuses on the computational studies that have advanced our understanding of how various toxins interfere with the function of K⁺ channels. We introduce the computational tools available for the study of toxin-channel interactions. We then discuss how these computational tools have been fruitfully applied to elucidate the mechanisms of action of a wide range of venom peptides from scorpions, spiders, and sea anemone. PMID:26633507

  7. Computational Studies of Venom Peptides Targeting Potassium Channels

    PubMed Central

    Chen, Rong; Chung, Shin-Ho

    2015-01-01

    Small peptides isolated from the venom of animals are potential scaffolds for ion channel drug discovery. This review article mainly focuses on the computational studies that have advanced our understanding of how various toxins interfere with the function of K+ channels. We introduce the computational tools available for the study of toxin-channel interactions. We then discuss how these computational tools have been fruitfully applied to elucidate the mechanisms of action of a wide range of venom peptides from scorpions, spiders, and sea anemone. PMID:26633507

  8. Anti-tumor effects of peptide analogs targeting neuropeptide hormone receptors on mouse pheochromocytoma cells.

    PubMed

    Ziegler, C G; Ullrich, M; Schally, A V; Bergmann, R; Pietzsch, J; Gebauer, L; Gondek, K; Qin, N; Pacak, K; Ehrhart-Bornstein, M; Eisenhofer, G; Bornstein, S R

    2013-05-22

    Pheochromocytoma is a rare but potentially lethal chromaffin cell tumor with currently no effective treatment. Peptide hormone receptors are frequently overexpressed on endocrine tumor cells and can be specifically targeted by various anti-tumor peptide analogs. The present study carried out on mouse pheochromocytoma cells (MPCs) and a more aggressive mouse tumor tissue-derived (MTT) cell line revealed that these cells are characterized by pronounced expression of the somatostatin receptor 2 (sst2), growth hormone-releasing hormone (GHRH) receptor and the luteinizing hormone-releasing hormone (LHRH) receptor. We further demonstrated significant anti-tumor effects mediated by cytotoxic somatostatin analogs, AN-162 and AN-238, by LHRH antagonist, Cetrorelix, by the cytotoxic LHRH analog, AN-152, and by recently developed GHRH antagonist, MIA-602, on MPC and for AN-152 and MIA-602 on MTT cells. Studies of novel anti-tumor compounds on these mouse cell lines serve as an important basis for mouse models of metastatic pheochromocytoma, which we are currently establishing. PMID:23267837

  9. Patient-Derived Antibody Targets Tumor Cells

    Cancer.gov

    An NCI Cancer Currents blog on an antibody derived from patients that killed tumor cells in cell lines of several cancer types and slowed tumor growth in mouse models of brain and lung cancer without evidence of side effects.

  10. Peptide aptamer identified by molecular docking targeting translationally controlled tumor protein in leukemia cells.

    PubMed

    Kadioglu, Onat; Efferth, Thomas

    2016-08-01

    Bioinformatics screening and molecular docking analyses were utilized to select high affinity peptides targeting translationally controlled tumor protein (TCTP). Selected peptide aptamers were tested towards cancer cell lines with different levels of TCTP expression. One peptide (WGQWPYHC) revealed specific cytotoxicity according to the TCTP expression in tumor cells without affecting normal cells. Western blot analysis showed peptide-induced down-regulation of TCTP as primary target as well as of cell-cycle related downstream proteins (CDK2, CDK6, Cyclin D3) in MOLT-4 leukemia cells. "WGQWPYHC" deserves further analysis for targeted therapy of TCTP-expressing tumor cells. Graphical abstract Molecular docking on TCTP, cytotoxicity toward MOLT-4 leukemia cell line and downregulation of CDK2, CDK6, CyclinD3 and TCTP proteins. PMID:26972431

  11. Bioavailability of milk protein-derived bioactive peptides: a glycaemic management perspective.

    PubMed

    Horner, Katy; Drummond, Elaine; Brennan, Lorraine

    2016-06-01

    Milk protein-derived peptides have been reported to have potential benefits for reducing the risk of type 2 diabetes. However, what the active components are and whether intact peptides exert this bioactivity has received little investigation in human subjects. Furthermore, potentially useful bioactive peptides can be limited by low bioavailability. Various peptides have been identified in the gastrointestinal tract and bloodstream after milk-protein ingestion, providing valuable insights into their potential bioavailability. However, these studies are currently limited and the structure and sequence of milk peptides exerting bioactivity for glycaemic management has received little investigation in human subjects. The present article reviews the bioavailability of milk protein-derived peptides in human studies to date, and examines the evidence on milk proteins and glycaemic management, including potential mechanisms of action. Areas in need of advancement are identified. Only by establishing the bioavailability of milk protein-derived peptides, the active components and the mechanistic pathways involved can the benefits of milk proteins for the prevention or management of type 2 diabetes be fully realised in future. PMID:27109024

  12. Stability and cytotoxicity of angiotensin-I-converting enzyme inhibitory peptides derived from bovine casein*

    PubMed Central

    Wu, Wei; Yu, Pan-pan; Zhang, Feng-yang; Che, Hong-xia; Jiang, Zhan-mei

    2014-01-01

    This study investigated the effect of heat treatment combined with acid and alkali on the angiotensin-I-converting enzyme (ACE) inhibitory activity of peptides derived from bovine casein. The free amino group content, color, and cytotoxicity of the peptides were measured under different conditions. When heated at 100 °C in the pH range from 9.0 to 12.0, ACE inhibitory activity was reduced and the appearance of the peptides was significantly darkened. After thermal treatment in the presence of acid and alkali, the free amino group content of ACE inhibitory peptides decreased markedly. High temperature and prolonged heating also resulted in the loss of ACE inhibitory activity, the loss of free amino groups, and the darker coloration of bovine casein-derived peptides. However, ACE inhibitory peptides, within a concentration range of from 0.01 to 0.2 mg/ml, showed no cytotoxicity to Caco-2 and ECV-304 cell lines after heat treatment. This indicated that high temperature and alkaline heat treatment impaired the stability of bovine casein-derived ACE inhibitory peptides. PMID:24510707

  13. Reiterated Targeting Peptides on the Nanoparticle Surface Significantly Promote Targeted Vascular Endothelial Growth Factor Gene Delivery to Stem Cells.

    PubMed

    Wang, Dong-Dong; Yang, Mingying; Zhu, Ye; Mao, Chuanbin

    2015-12-14

    Nonviral gene delivery vectors hold great promise for gene therapy due to the safety concerns with viral vectors. However, the application of nonviral vectors is hindered by their low transfection efficiency. Herein, in order to tackle this challenge, we developed a nonviral vector integrating lipids, sleeping beauty transposon system and 8-mer stem cell targeting peptides for safe and efficient gene delivery to hard-to-transfect mesenchymal stem cells (MSCs). The 8-mer MSC-targeting peptides, when synthetically reiterated in three folds and chemically presented on the surface, significantly promoted the resultant lipid-based nanoparticles (LBNs) to deliver VEGF gene into MSCs with a high transfection efficiency (∼52%) and long-lasting gene expression (for longer than 170 h) when compared to nonreiterated peptides. However, the reiterated stem cell targeting peptides do not enable the highly efficient gene transfer to other control cells. This work suggests that the surface presentation of the reiterated stem cell-targeting peptides on the nonviral vectors is a promising method for improving the efficiency of cell-specific nonviral gene transfection in stem cells. PMID:26588028

  14. CTHRSSVVC Peptide as a Possible Early Molecular Imaging Target for Atherosclerosis.

    PubMed

    Silva, Rosemeire A; Giordano, Ricardo J; Gutierrez, Paulo S; Rocha, Viviane Z; Rudnicki, Martina; Kee, Patrick; Abdalla, Dulcinéia S P; Puech-Leão, Pedro; Caramelli, Bruno; Arap, Wadih; Pasqualini, Renata; Meneghetti, José C; Marques, Fabio L N; Khoobchandani, Menka; Katti, Kattesh V; Lugão, Ademar B; Kalil, Jorge

    2016-01-01

    The purpose of our work was to select phages displaying peptides capable of binding to vascular markers present in human atheroma, and validate their capacity to target the vascular markers in vitro and in low-density lipoprotein receptor knockout (LDLr(-/-)) mouse model of atherosclerosis. By peptide fingerprinting on human atherosclerotic tissues, we selected and isolated four different peptides sequences, which bind to atherosclerotic lesions and share significant similarity to known human proteins with prominent roles in atherosclerosis. The CTHRSSVVC-phage peptide displayed the strongest reactivity with human carotid atherosclerotic lesions (p < 0.05), when compared to tissues from normal carotid arteries. This peptide sequence shares similarity to a sequence present in the fifth scavenger receptor cysteine-rich (SRCR) domain of CD163, which appeared to bind to CD163, and subsequently, was internalized by macrophages. Moreover, the CTHRSSVVC-phage targets atherosclerotic lesions of a low-density lipoprotein receptor knockout (LDLr(-/-)) mouse model of atherosclerosis in vivo to High-Fat diet group versus Control group. Tetraazacyclododecane-1,4,7,10-tetraacetic acid-CTHRSSVVC peptide (DOTA-CTHRSSVVC) was synthesized and labeled with (111)InCl₃ in >95% yield as determined by high performance liquid chromatography (HPLC), to validate the binding of the peptide in atherosclerotic plaque specimens. The results supported our hypothesis that CTHRSSVVC peptide has a remarkable sequence for the development of theranostics approaches in the treatment of atherosclerosis and other diseases. PMID:27563889

  15. A novel peptide specifically targeting ovarian cancer identified by in vivo phage display.

    PubMed

    Ma, Chuying; Yin, Guangfu; Yan, Danhong; He, Xueling; Zhang, Li; Wei, Yan; Huang, Zhongbing

    2013-12-01

    Discovery of peptide ligands that can target human ovarian cancer and deliver chemotherapeutics offers new opportunity for cancer therapy. The advent of phage-displayed peptide library facilitated the screening of such peptides. In vivo screening that set in a microanatomic and functional context was applied in our study, and a novel peptide WSGPGVWGASVK targeting ovarian cancer was isolated. The phage clone PC3-1 displaying peptide WSGPGVWGASVK can gain effective access to accumulate in the tumor sites after intravenous injection while reducing its accumulation in normal organs. Positive immunostaining of PC3-1 was located in both sites of tumor cells and tumor blood vessels, which resulted in a diffuse binding pattern through the tumor. In vitro study results confirmed the capability of peptide WSGPGVWGASVK binding to and being internalized by both tumor cells and angiogenic endothelial cells. Flow cytometry analysis revealed that the peptide bound to SKOV3 cells with Kd value of 5.43 ± 0.4 μM. Taken together, it suggested that peptide WSGPGVWGASVK is a lead candidate for delivering therapeutics to penetrate into tumors. PMID:24105738

  16. Prevention of passively transferred experimental autoimmune myasthenia gravis by a phage library-derived cyclic peptide

    PubMed Central

    Venkatesh, Natarajan; Im, Sin-Heyog; Balass, Moshe; Fuchs, Sara; Katchalski-Katzir, Ephraim

    2000-01-01

    Many pathogenic antibodies in myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are directed against the main immunogenic region (MIR) of the acetylcholine receptor (AcChoR). These antibodies are highly conformation dependent; hence, linear peptides derived from native receptor sequences are poor candidates for their immunoneutralization. We employed a phage-epitope library to identify peptide-mimotopes capable of preventing the pathogenicity of the anti-MIR mAb 198. We identified a 15-mer peptide (PMTLPENYFSERPYH) that binds specifically to mAb 198 and inhibits its binding to AcChoR. A 10-fold increase in the affinity of this peptide was achieved by incorporating flanking amino acid residues from the coat protein as present in the original phage library. This extended peptide (AEPMTLPENYFSERPYHPPPP) was constrained by the addition of cysteine residues on both ends of the peptide, thus generating a cyclic peptide that inhibited the binding of mAb 198 to AcChoR with a potency that is three orders of magnitude higher when compared with the parent library peptide. This cyclic peptide inhibited the in vitro binding of mAb 198 to AcChoR and prevented the antigenic modulation of AcChoR caused by mAb 198 in human muscle cell cultures. The cyclic peptide also reacted with several other anti-MIR mAbs and the sera of EAMG rats. In addition, this peptide blocked the ability of mAb 198 to passively transfer EAMG in rats. Further derivatization of the cyclic peptide may aid in the design of suitable synthetic mimotopes for modulation of MG. PMID:10639153

  17. Precision-guided antimicrobial peptide as a targeted modulator of human microbial ecology

    PubMed Central

    Guo, Lihong; McLean, Jeffrey S.; Yang, Youngik; Eckert, Randal; Kaplan, Christopher W.; Kyme, Pierre; Sheikh, Omid; Varnum, Brian; Lux, Renate; Shi, Wenyuan; He, Xuesong

    2015-01-01

    One major challenge to studying human microbiome and its associated diseases is the lack of effective tools to achieve targeted modulation of individual species and study its ecological function within multispecies communities. Here, we show that C16G2, a specifically targeted antimicrobial peptide, was able to selectively kill cariogenic pathogen Streptococcus mutans with high efficacy within a human saliva-derived in vitro oral multispecies community. Importantly, a significant shift in the overall microbial structure of the C16G2-treated community was revealed after a 24-h recovery period: several bacterial species with metabolic dependency or physical interactions with S. mutans suffered drastic reduction in their abundance, whereas S. mutans’ natural competitors, including health-associated Streptococci, became dominant. This study demonstrates the use of targeted antimicrobials to modulate the microbiome structure allowing insights into the key community role of specific bacterial species and also indicates the therapeutic potential of C16G2 to achieve a healthy oral microbiome. PMID:26034276

  18. Rational design of YAP WW1 domain-binding peptides to target TGFβ/BMP/Smad-YAP interaction in heterotopic ossification.

    PubMed

    Chen, Dong; Liu, Shenghe; Zhang, Wen; Sun, Luyuan

    2015-11-01

    The transforming growth factor-β/bone morphogenic protein/Smad signaling pathway has been raised as a new and promising therapeutic target of heterotopic ossification, which is mediated by recruitment of transcription coactivator Yes-associated protein (YAP) to Smad. Here, we described a successful integration of computational modeling and experimental assay to rationally design novel peptide aptamers to disrupt YAP-Smad interaction by targeting YAP WW1 domain. In the protocol, a computational genetic evolution strategy was used to improve a population of potential YAP WW1-binding peptides generated from the YAP-recognition site in Smad protein, from which several promising peptides were selected and their affinities toward YAP WW1 domain were determined using binding assay. In addition, a high-activity peptide was further optimized based on its complex structure with YAP WW1 domain to derive a number of derivative peptides with higher binding potency to the domain. We also found that a strong YAP WW1 binder should have a negatively charged N-terminus, a positively charged C-terminus and a nonpolar core to match the electrostatic distribution pattern in peptide-binding pocket of YAP WW1 domain, which may also form additional nonbonded interactions such as hydrogen bond, salt bridge and π-π stacking to confer stability and specificity for the domain-peptide recognition. PMID:26435515

  19. K1K8: an Hp1404-derived antibacterial peptide.

    PubMed

    Li, Zhongjie; Liu, Gaomin; Meng, Lanxia; Yu, Weiwei; Xu, Xiaobo; Li, Wenxin; Wu, Yingliang; Cao, Zhijian

    2016-06-01

    As an alternative class of antimicrobial agents used to overcome drug-resistant infections, antimicrobial peptides (AMPs) have recently gained significant attention. In this study, we designed an improved antimicrobial peptide, K1K8, based on the molecular template of Hp1404. Compared to the wild-type Hp1404, K1K8 showed an improved antibacterial spectrum in vitro, a lower hemolytic activity, and an enhanced serum stability. Importantly, K1K8 also decreased methicillin-resistant Staphylococcus aureus (MRSA) bacterial counts in the wounded region in a mouse skin infection model. Interestingly, K1K8 did not induce bacterial resistance or non-specific immune response reactions. Moreover, the peptide killed bacterial cells mainly by disrupting the bacterial membrane. In summary, K1K8 has the potential to be used as an improved anti-infection agent for topical use, which opens an avenue that potential anti-infection drugs may be designed and developed from the molecular templates of AMPs. PMID:26952110

  20. An overview of antifungal peptides derived from insect.

    PubMed

    Faruck, Mohammad Omer; Yusof, Faridah; Chowdhury, Silvia

    2016-06-01

    Fungi are not classified as plants or animals. They resemble plants in many ways but do not produce chlorophyll or make their own food photosynthetically like plants. Fungi are useful for the production of beer, bread, medicine, etc. More complex than viruses or bacteria; fungi can be destructive human pathogens responsible for various diseases in humans. Most people have a strong natural immunity against fungal infection. However, fungi can cause diseases when this immunity breaks down. In the last few years, fungal infection has increased strikingly and has been accompanied by a rise in the number of deaths of cancer patients, transplant recipients, and acquired immunodeficiency syndrome (AIDS) patients owing to fungal infections. The growth rate of fungi is very slow and quite difficult to identify. A series of molecules with antifungal activity against different strains of fungi have been found in insects, which can be of great importance to tackle human diseases. Insects secrete such compounds, which can be peptides, as a part of their immune defense reactions. Active antifungal peptides developed by insects to rapidly eliminate infectious pathogens are considered a component of the defense munitions. This review focuses on naturally occurring antifungal peptides from insects and their challenges to be used as armaments against human diseases. PMID:26093218

  1. Immunization of cattle with synthetic peptides derived from the Boophilus microplus gut protein (Bm86).

    PubMed

    Patarroyo, J H; Portela, R W; De Castro, R O; Pimentel, J Couto; Guzman, F; Patarroyo, M E; Vargas, M I; Prates, A A; Mendes, M A Dias

    2002-09-25

    Three synthetic peptides (SBm4912, SBm7462 and SBm19733), derived from the Bm86 glycoprotein from Boophilus microplus gut, were constructed and used to immunize cattle from a tick-free area. The immunized animals received three subcutaneous doses of the peptides, with saponin as adjuvant, at 30-day intervals. The immune response was evaluated by IgG elicited against the peptides by the detection of anti-Bm86 specific antibodies in situ and by Western blotting analysis. After tick challenge, reduction in the number, weight and oviposition capacity of engorged females was observed in the tick population that had fed on immunized animals. The results pointed a high efficacy (81.05%) for the SBm7462 synthetic peptide in relation to the others (p<0.01), demonstrating the efficiency of the immune response elicited by synthetic peptides to control the cattle tick B. microplus. PMID:12127414

  2. Myostatin inhibition by a follistatin-derived peptide ameliorates the pathophysiology of muscular dystrophy model mice

    PubMed Central

    Tsuchida, K

    2008-01-01

    Summary Gene-targeted therapies, such as adeno-associated viral vector (AAV)-mediated gene therapy and cell-mediated therapy using myogenic stem cells, are hopeful molecular strategies for muscular dystrophy. In addition, drug therapies based on the pathophysiology of muscular dystrophy patients are desirable. Multidisciplinary approaches to drug design would offer promising therapeutic strategies. Myostatin, a member of the transforming growth factor-β superfamily, is predominantly produced by skeletal muscle and negatively regulates the growth and differentiation of cells of the skeletal muscle lineage. Myostatin inhibition would increase the skeletal muscle mass and prevent muscle degeneration, regardless of the type of muscular dystrophy. Myostatin inhibitors include myostatin antibodies, myostatin propeptide, follistatin and follistatin-related protein. Although follistatin possesses potent myostatin-inhibiting activity, it works as an efficient inhibitor of activins. Unlike myostatin, activins regulate the growth and differentiation of nearly all cell types, including cells of the gonads, pituitary gland and skeletal muscle. We have developed a myostatin-specific inhibitor derived from follistatin, designated FS I-I. Transgenic mice expressing this myostatin-inhibiting peptide under the control of a skeletal muscle-specific promoter showed increased skeletal muscle mass and strength. mdx mice were crossed with FS I-I transgenic mice and any improvement of the pathological signs was investigated. The resulting mdx/FS I-I mice exhibited increased skeletal muscle mass and reduced cell infiltration in muscles. Muscle strength was also recovered in mdx/FS I-I mice. Our data indicate that myostatin inhibition by this follistatin-derived peptide has therapeutic potential for muscular dystrophy. PMID:19108572

  3. Receptor binding peptides for target-selective delivery of nanoparticles encapsulated drugs

    PubMed Central

    Accardo, Antonella; Aloj, Luigi; Aurilio, Michela; Morelli, Giancarlo; Tesauro, Diego

    2014-01-01

    Active targeting by means of drug encapsulated nanoparticles decorated with targeting bioactive moieties represents the next frontier in drug delivery; it reduces drug side effects and increases the therapeutic index. Peptides, based on their chemical and biological properties, could have a prevalent role to direct drug encapsulated nanoparticles, such as liposomes, micelles, or hard nanoparticles, toward the tumor tissues. A considerable number of molecular targets for peptides are either exclusively expressed or overexpressed on both cancer vasculature and cancer cells. They can be classified into three wide categories: integrins; growth factor receptors (GFRs); and G-protein coupled receptors (GPCRs). Therapeutic agents based on nanovectors decorated with peptides targeting membrane receptors belonging to the GPCR family overexpressed by cancer cells are reviewed in this article. The most studied targeting membrane receptors are considered: somatostatin receptors; cholecystokinin receptors; receptors associated with the Bombesin like peptides family; luteinizing hormone-releasing hormone receptors; and neurotensin receptors. Nanovectors of different sizes and shapes (micelles, liposomes, or hard nanoparticles) loaded with doxorubicin or other cytotoxic drugs and externally functionalized with natural or synthetic peptides are able to target the overexpressed receptors and are described based on their formulation and in vitro and in vivo behaviors. PMID:24741304

  4. REDV Peptide Conjugated Nanoparticles/pZNF580 Complexes for Actively Targeting Human Vascular Endothelial Cells.

    PubMed

    Shi, Changcan; Li, Qian; Zhang, Wencheng; Feng, Yakai; Ren, Xiangkui

    2015-09-16

    Herein, we demonstrate that the REDV peptide modified nanoparticles (NPs) can serve as a kind of active targeting gene carrier to condensate pZNF580 for specific promotion of the proliferation of endothelial cells (ECs). First, we synthesized a series of biodegradable amphiphilic copolymers by ring-opening polymerization reaction and graft modification with REDV peptide. Second, we prepared active targeting NPs via self-assembly of the amphiphilic copolymers using nanoprecipitation technology. After condensation with negatively charged pZNF580, the REDV peptide modified NPs/pZNF580 complexes were formed finally. Due to the binding affinity toward ECs of the specific peptide, these REDV peptide modified NPs/pZNF580 complexes could be recognized and adhered specifically by ECs in the coculture system of ECs and human artery smooth muscle cells (SMCs) in vitro. After expression of ZNF580, as the key protein to promote the proliferation of ECs, the relative ZNF580 protein level increased from 15.7% to 34.8%. The specificity in actively targeting ECs of the REDV peptide conjugated NPs/pZNF580 complexes was still retained in the coculture system. These findings in the present study could facilitate the development of actively targeting gene carriers for the endothelialization of artificial blood vessels. PMID:26373583

  5. Targeting cancer cell invasiveness using homing peptide-nanocomplexes

    NASA Astrophysics Data System (ADS)

    Suarato, Giulia; Cathcart, Jillian; Li, Weiyi; Cao, Jian; Meng, Yizhi

    Matrix metalloproteinase-14 (MMP-14) plays critical roles in digesting the basement membrane and extracellular matrix and inducing cancer migration. We recently unraveled a unique role in cell invasion of the hemopexin (PEX) domain of MMP-14. The minimal motif located at the outmost strand of the fourth blade of the PEX domain was identified to form homodimers of MMP-14. A peptide (IVS4) mimicking the binding motif was shown to interrupt MMP-14 dimerization and decrease MMP-14-mediated functions. Since most invasive cancer cells express upregulated MMP-14 at the surface, IVS4 could be used as a cancer homing peptide to specifically deliver cytotoxic drugs for cancer therapy. We developed cancer homing nanocarriers by linking IVS4 to polysaccharide-based micellar nanoparticles (NPs). To determine if conjugation of IVS4 to NPs maintains the IVS4 inhibition of MMP-14 function, substrate degradation and cell migration assays were performed. IVS4-NPs efficiently prevented MMP-14-mediated substrate degradation and cell migration, and were minimally uptaken by non-cancer cells. Importantly, IVS4 confers an uptake advantage compared to the control peptide in MMP-14-expressing cells. Taken together, our findings demonstrate the potential use of IVS4-NPs as novel cancer nanotherapeutics.

  6. C-TYPE NATRIURETIC PEPTIDE (CNP): CARDIOVASCULAR ROLES AND POTENTIAL AS A THERAPEUTIC TARGET

    PubMed Central

    Lumsden, Natalie G.; Khambata, Rayomand S.; Hobbs, Adrian J.

    2012-01-01

    Natriuretic peptides play a fundamental role in cardiovascular homeostasis by modulation of fluid and electrolyte balance and vascular tone. C-type natriuretic peptide (CNP) represents the paracrine element of the natriuretic peptide axis which complements the endocrine actions of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). CNP is produced by the endothelium and the heart and appears to play a prominent role in vascular and cardiac function, both physiologically and pathologically. This provides a rationale for the therapeutic potential of pharmacological interventions targeted to CNP signalling. This article provides an overview of the biology and pharmacology of CNP, with emphasis on the cardiovascular system, and discusses pathologies in which drugs designed to manipulate CNP signalling maybe of clinical benefit. PMID:21247399

  7. HIV-1 drug discovery: targeting folded RNA structures with branched peptides.

    PubMed

    Wynn, Jessica E; Santos, Webster L

    2015-06-01

    Human immunodeficiency virus type 1 (HIV-1) is an RNA virus that is prone to high rates of mutation. While the disease is managed with current antiretroviral therapies, drugs with a new mode of action are needed. A strategy towards this goal is aimed at targeting the native three-dimensional fold of conserved RNA structures. This perspective highlights medium-sized peptides and peptidomimetics used to target two conserved RNA structures of HIV-1. In particular, branched peptides have the capacity to bind in a multivalent fashion, utilizing a large surface area to achieve the necessary affinity and selectivity toward the target RNA. PMID:25958855

  8. HIV-1 Drug Discovery: Targeting Folded RNA Structures With Branched Peptides

    PubMed Central

    Wynn, Jessica E.

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) is an RNA virus that is prone to high rates of mutation. While the disease is managed with current antiretroviral therapies, drugs with a new mode of action are needed. A strategy towards this goal is aimed at targeting the native three-dimensional fold of conserved RNA structures. This perspective highlights medium-sized peptides and peptidomimetics used to target two conserved RNA structures of HIV-1. In particular, branched peptides have the capacity to bind in a multivalent fashion, utilizing a large surface area to achieve the necessary affinity and selectivity toward the target RNA. PMID:25958855

  9. The development of peptide ligands that target helix 69 rRNA of bacterial ribosomes.

    PubMed

    Dremann, Danielle N; Chow, Christine S

    2016-09-15

    Antibiotic resistance prevents successful treatment of common bacterial infections, making it clear that new target locations and drugs are required to resolve this ongoing challenge. The bacterial ribosome is a common target for antibacterials due to its essential contribution to cell viability. The focus of this work is a region of the ribosome called helix 69 (H69), which was recently identified as a secondary target site for aminoglycoside antibiotics. H69 has key roles in essential ribosomal processes such as subunit association, ribosome recycling, and tRNA selection. Conserved across phylogeny, bacterial H69 also contains two pseudouridines and one 3-methylpseudouridine. Phage display revealed a heptameric peptide sequence that targeted H69. Using solid-phase synthesis, peptide variants with higher affinity and improved selectivity to modified H69 were generated. Electrospray ionization mass spectrometry was used to determine relative apparent dissociation constants of the RNA-peptide complexes. PMID:27492196

  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. PMID:27165480

  11. Smac-Derived Aza-Peptide As an Aminopeptidase-Resistant XIAP BIR3 Antagonist.

    PubMed

    Elsawy, Mohamed A; Tikhonova, Irina G; Martin, Lorraine; Walker, Brian

    2015-01-01

    The peptidic nature of anti-IAPs N-terminus Smac-derived peptides precludes their utilization as potential therapeutic anticancer agents. Recent advances in the development of novel Smacderived peptidomimetics and non-peptidic molecules with improved anti-IAPs activity and resistance to proteolytic cleavage have been reported and led to a number of candidates that are currently in clinical trials including LCL-161, SM-406/AT-406, GDC-0512/GDC-0917, and birinapant. As an attempt to improve the proteolytic stability of Smac peptides, we developed the Aza-peptide AzaAla- Val-Pro-Phe-Tyr-NH2 (2). Unlike unmodified peptide Ala-Val-Pro-Phe-Tyr-NH2 (1), analogue (2) exhibited resistance towards proteolytic cleavage by two aminopeptidases; LAP and DPP-IV, while retaining its IAP inhibitory activity. This was due to the altered planar geometry of the P1 residue side chain. Our findings showed that using aza-isosteres of bioactive peptide sequences imbue the residue with imperviousness to proteolysis; underscoring a potential approach for developing a new generation of Smac-derived Aza-peptidomimetics. PMID:26282728

  12. Specific interactions between amyloid-β peptide and curcumin derivatives: Ab initio molecular simulations

    NASA Astrophysics Data System (ADS)

    Ishimura, Hiromi; Kadoya, Ryushi; Suzuki, Tomoya; Murakawa, Takeru; Shulga, Sergiy; Kurita, Noriyuki

    2015-07-01

    Alzheimer's disease is caused by accumulation of amyloid-β (Aβ) peptides in a brain. To suppress the production of Aβ peptides, it is effective to inhibit the cleavage of amyloid precursor protein (APP) by secretases. However, because the secretases also play important roles to produce vital proteins for human body, inhibitors for the secretases may have side effects. To propose new agents for protecting the cleavage site of APP from the attacking of the γ-secretase, we have investigated here the specific interactions between a short APP peptide and curcumin derivatives, using protein-ligand docking as well as ab initio molecular simulations.

  13. 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

  14. A Phage Display Screening Derived Peptide with Affinity for the Adeninyl Moiety

    PubMed Central

    Elmlund, Louise; Söderberg, Pernilla; Suriyanarayanan, Subramanian; Nicholls, Ian A.

    2014-01-01

    Phage display screening of a surface-immobilized adenine derivative led to the identification of a heptameric peptide with selectivity for adenine as demonstrated through quartz crystal microbalance (QCM) studies. The peptide demonstrated a concentration dependent affinity for an adeninyl moiety decorated surface (KD of 968 ± 53.3 μM), which highlights the power of piezoelectric sensing in the study of weak interactions. PMID:25587414

  15. Development and Testing of a 212Pb/212Bi Peptide for Targeting Metastatic Melanoma

    SciTech Connect

    Fisher, Darrell R.

    2012-10-25

    The purpose of this project is to develop a new radiolabeled peptide for imaging and treating metastatic melanoma. The immunoconjugate consists of a receptor-specific peptide that targets melanoma cells. The beta-emitter lead-212 (half-life = 10.4 hours) is linked by coordination chemistry to the peptide. After injection, the peptide targets melanoma receptors on the surfaces of melanoma cells. Lead-212 decays to the alpha-emitter bismuth-212 (half-life = 60 minutes). Alpha-particles that hit melanoma cell nuclei are likely to kill the melanoma cell. For cancer cell imaging, the lead-212 is replaced by lead-203 (half-life = 52 hours). Lead-203 emits 279 keV photons (80.1% abundance) that can be imaged and measured for biodistribution analysis, cancer imaging, and quantitative dosimetry.

  16. Intravenous phage display identifies peptide sequences that target the burn-injured intestine

    PubMed Central

    Costantini, Todd W.; Eliceiri, Brian P.; Putnam, James G.; Bansal, Vishal; Baird, Andrew; Coimbra, Raul

    2015-01-01

    The injured intestine is responsible for significant morbidity and mortality after severe trauma and burn; however, targeting the intestine with therapeutics aimed at decreasing injury has proven difficult. We hypothesized that we could use intravenous phage display technology to identify peptide sequences that target the injured intestinal mucosa in a murine model, and then confirm the cross-reactivity of this peptide sequence with ex vivo human gut. Four hours following 30% TBSA burn we performed an in vivo, intravenous systemic administration of phage library containing 1012 phage in balb/c mice to biopan for gut-targeting peptides. In vivo assessment of the candidate peptide sequences identified after 4 rounds of internalization was performed by injecting 1 × 1012 copies of each selected phage clone into sham or burned animals. Internalization into the gut was assessed using quantitative polymerase chain reaction. We then incubated this gut-targeting peptide sequence with human intestine and visualized fluorescence using confocal microscopy. We identified 3 gut-targeting peptide sequences which caused collapse of the phage library (4–1: SGHQLLLNKMP, 4–5: ILANDLTAPGPR, 4–11: SFKPSGLPAQSL). Sequence 4–5 was internalized into the intestinal mucosa of burned animals 9.3-fold higher than sham animals injected with the same sequence (2.9 × 105 vs. 3.1 × 104 particles per mg tissue). Sequences 4–1 and 4–11 were both internalized into the gut, but did not demonstrate specificity for the injured mucosa. Phage sequence 4–11 demonstrated cross-reactivity with human intestine. In the future, this gut-targeting peptide sequence could serve as a platform for the delivery of biotherapeutics. PMID:22960048

  17. F2L, a peptide derived from heme-binding protein, inhibits formyl peptide receptor-mediated signaling

    SciTech Connect

    Lee, Ha Young; Lee, Sun Young; Shin, Eun Ha; Kim, Sang Doo; Kim, Jung Mo; Lee, Mi-Sook; Ryu, Sung Ho; Bae, Yoe-Sik . E-mail: yoesik@donga.ac.kr

    2007-08-10

    F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein. Very recently, F2L was identified as an endogenous chemoattractant peptide acting specifically through formyl peptide receptor-like (FPRL)2. In the present study, we report that F2L stimulates chemotactic migration in human neutrophils. However, F2L inhibits formyl peptide receptor (FPR) and FPRL1 activities, resulting in the complete inhibition of intracellular calcium increases, and superoxide generation induced by N-formyl-Met-Leu-Phe, MMK-1, or Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) in human neutrophils. In terms of the inhibitory role of F2L on FPR- and FPRL-mediated signaling, we found that F2L competitively inhibits the binding of {sup 125}I-WKYMVm to its specific receptors, FPR and FPRL1. F2L is the first endogenous molecule that inhibits FPR- and FPRL1-mediated signaling, and is expected to be useful in the study of FPR and FPRL1 signaling and in the development of drugs to treat diseases involving the FPR family of receptors.

  18. Proaleurain vacuolar targeting is mediated by short contiguous peptide interactions.

    PubMed Central

    Holwerda, B C; Padgett, H S; Rogers, J C

    1992-01-01

    Targeting of soluble proteins to the plant vacuole is mediated by determinants that reside in the polypeptide. We identified the vacuolar targeting determinant of aleurain, a plant vacuolar thiol protease, by incorporating different sequences from proaleurain into the secreted thiol protease, proendoproteinase B (proEP-B), and vice versa. The targeting fates of the chimeric proteins were analyzed by transient expression in electroporated tobacco protoplasts. The targeting determinant SSSSFADSNPIR is positioned at the N terminus of the aleurain propeptide, and its substitution into the propeptide of EP-B caused vacuolar targeting of the resulting chimeric protein. This determinant can be divided into two smaller determinants, SSSSFADS and SNPIR, each of which is sufficient to target proEP-B chimeras to the vacuole, but with lower efficiency. These smaller determinants interact in a positive manner because the combined determinant SSSSFADSNPIR targeted proEP-B with an efficiency greater than each of the smaller determinants alone. Accordingly, the efficiency of aleurain targeting was decreased when either of the smaller determinants was disrupted by replacement with similarly positioned proEP-B sequences. Further experiments on proaleurain identified an additional determinant, VTDRAAST, adjacent to the SSSSFADSNPIR determinant that is also necessary for efficient vacuolar targeting. Our results provide evidence that efficient vacuolar targeting of this thiol protease in plant cells is mediated by the combined action of smaller contiguous determinants; two of these alone are sufficient for vacuolar targeting. PMID:1498598

  19. Commercially available antibodies can be applied in quantitative multiplexed peptide immunoaffinity enrichment targeted mass spectrometry assays

    PubMed Central

    Schoenherr, Regine M.; Zhao, Lei; Ivey, Richard G.; Voytovich, Uliana J.; Kennedy, Jacob; Yan, Ping; Lin, Chenwei; Whiteaker, Jeffrey R.; Paulovich, Amanda G.

    2016-01-01

    Immunoaffinity enrichment of peptides coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM) enables highly specific, sensitive, and precise quantification of peptides and post-translational modifications. Major obstacles to developing a large number of immuno-MRM assays are the poor availability of monoclonal antibodies (mAbs) validated for immunoaffinity enrichment of peptides and the cost and lead time of developing the antibodies de novo. Although many thousands of mAbs are commercially offered, few have been tested for application to immunoaffinity enrichment of peptides. In this study we tested the success rate of using commercially available mAbs for peptide immuno-MRM assays. We selected 105 commercial mAbs (76 targeting non-modified “pan” epitopes, 29 targeting phosphorylation) to proteins associated with the DNA damage response network. We found that 8 of the 76 pan (11%) and 5 of the 29 phospho-specific mAbs (17%) captured tryptic peptides (detected by LC-MS/MS) of their protein targets from human cell lysates. Seven of these mAbs were successfully used to configure and analytically characterize immuno-MRM assays. By applying selection criteria upfront, the results indicate that a screening success rate of up to 24% is possible, establishing the feasibility of screening a large number of catalog antibodies to provide readily-available assay reagents. PMID:27094115

  20. NGR Tumor-Homing Peptides: Structural Requirements for Effective APN (CD13) Targeting.

    PubMed

    Graziadio, Alessandra; Zanda, Matteo; Frau, Simona; Fleming, Ian N; Musolino, Manuele; Dall'Angelo, Sergio; Baldassarre, Massimiliano; Piras, Monica

    2016-05-18

    Cyclic CNGRC (cCNGRC) peptides are very important targeting ligands for Aminopeptidase N (APN or CD13), which is overexpressed on the surface of many cancer cells. In this work we have (1) developed an efficient solid-phase synthesis and (2) tested on purified porcine APN and APN-expressing human cells two different classes of cCNGRC peptides: the first carrying a biotin affinity tag or a fluorescent tag attached to the carboxyl Arg-Cys-COOH terminus and the second with the tags attached to the amino H2N-Cys-Asn terminus. Carboxyl-terminus functionalized cCNGRC peptides 3, 6, and 8 showed good affinity for porcine APN and very good capacity to target and be internalized into APN-expressing cells. In contrast, amino-terminus functionalized cCNGRC peptides 4, 5, and 7 displayed significantly decreased affinity and targeting capacity. These results, which are in agreement with the recently reported X-ray structure of a cCNGRC peptide bound to APN showing important stabilizing interactions between the unprotected cCNGRC amino terminus and the APN active site, indicate that the carboxyl and not the amino-terminus of cCNGRC peptides should be used as a "handle" for the attachment of toxic payloads for therapy or isotopically labeled functions for imaging and nuclear medicine. PMID:27077642

  1. The effects of shortening lactoferrin derived peptides against tumour cells, bacteria and normal human cells.

    PubMed

    Yang, Nannan; Strøm, Morten B; Mekonnen, Seble M; Svendsen, John S; Rekdal, Oystein

    2004-01-01

    A number of shortened derivatives of the lactoferrin model peptide L12, PAWRKAFRWAKRMLKKAA, were designed in order to elucidate the structural basis for antitumour activity of lactoferrin derivatives. Three tumour cell lines were included in the study and toxicity determined by measuring lysis of human red blood cells and fibroblasts. The results demonstrated a strong correlation between antitumour activity and net positive charge, in which a net charge close to +7 was essential for a high antitumour activity. In order to increase the antitumour activity of the shortest peptide with a net charge less than +7, the hydrophobicity had to be increased by adding a bulky Trp residue. None of the peptides were haemolytic, but toxicity against fibroblasts was observed. However, modifications of the peptides had a higher effect on reducing fibroblast toxicity than antitumour activity and thereby resulted in peptides displaying an almost 7-fold selectivity for tumour cells compared with fibroblasts. The antimicrobial activity against the Gram-negative bacteria Escherichia coil and the Gram-positive bacteria Staphylococcus aureus was also included in order to compare the structural requirements for antitumour activity with those required for a high antimicrobial activity. The results showed that most of the peptides were highly active against both bacterial strains. Less modification by shortening the peptide sequences was tolerated for maintaining a high antitumour activity and selectivity compared with antimicrobial activity. The order of the amino acid residues and thereby the conformation of the peptides was highly essential for antitumour activity, whereas the antimicrobial activity was hardly influenced by changes in this parameter. Thus, in addition to a certain net positive charge and hydrophobicity, the ability to adopt an amphipathic conformation was a more critical structural parameter for antitumour activity than for antimicrobial activity, and implied that a

  2. Peptide-decorated chitosan derivatives enhance fibroblast adhesion and proliferation in wound healing.

    PubMed

    Patrulea, V; Hirt-Burri, N; Jeannerat, A; Applegate, L A; Ostafe, V; Jordan, O; Borchard, G

    2016-05-20

    RGD peptide sequences are known to regulate cellular activities by interacting with α5β1, αvβ5 and αvβ3 integrin, which contributes to the wound healing process. In this study, RGDC peptide was immobilized onto chitosan derivative 1,6-diaminohexane-O-carboxymethyl-N,N,N-trimethyl chitosan (DAH-CMTMC) to display RGDC-promoting adhesion for enhanced wound healing. The efficiency of N-methylation, O-carboxymethylation and spacer grafting was quantitatively and qualitatively analyzed by (1)H NMR and FTIR, yielding 0.38 degree of substitution for N-methylation and >0.85 for O-carboxymethylation. The glass transition temperatures for chitosan derivatives were also studied. Peptide immobilization was achieved through sulfhydryl groups using sulfosuccinimidyl (4-iodoacetyl)amino-benzoate (sulfo-SIAB method). RGDC immobilized peptide onto DAH-CMTMC was found to be about 15.3 μg/mg of chitosan derivative by amino acid analysis (AAA). The significant increase of human dermal fibroblast (HDF) viability in vitro over 7 days suggests that RGDC-functionalized chitosan may lead to enhanced wound healing (viability >140%). Moreover, bio-adhesion and proliferation assays confirmed that coatings of RGDC-functionalized chitosan derivatives exhibit in vitro wound healing properties by enhancing fibroblast proliferation and adhesion. These results showed that RGDC peptide-functionalized chitosan provides an optimal environment for fibroblast adhesion and proliferation. PMID:26917381

  3. LL-37-Derived Peptides Eradicate Multidrug-Resistant Staphylococcus aureus from Thermally Wounded Human Skin Equivalents

    PubMed Central

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

    2014-01-01

    Burn wound infections are often difficult to treat due to the presence of multidrug-resistant bacterial strains and biofilms. Currently, mupirocin is used to eradicate methicillin-resistant Staphylococcus aureus (MRSA) from colonized persons; however, mupirocin resistance is also emerging. Since we consider antimicrobial peptides to be promising candidates for the development of novel anti-infective agents, we studied the antibacterial activities of a set of synthetic peptides against different strains of S. aureus, including mupirocin-resistant MRSA strains. The peptides were derived from P60.4Ac, a peptide based on the human cathelicidin LL-37. The results showed that peptide 10 (P10) was the only peptide more efficient than P60.4Ac, which is better than LL-37, in killing MRSA strain LUH14616. All three peptides displayed good antibiofilm activities. However, both P10 and P60.4Ac were more efficient than LL-37 in eliminating biofilm-associated bacteria. No toxic effects of these three peptides on human epidermal models were detected, as observed morphologically and by staining for mitochondrial activity. In addition, P60.4Ac and P10, but not LL-37, eradicated MRSA LUH14616 and the mupirocin-resistant MRSA strain LUH15051 from thermally wounded human skin equivalents (HSE). Interestingly, P60.4Ac and P10, but not mupirocin, eradicated LUH15051 from the HSEs. None of the peptides affected the excretion of interleukin 8 (IL-8) by thermally wounded HSEs upon MRSA exposure. In conclusion, the synthetic peptides P60.4Ac and P10 appear to be attractive candidates for the development of novel local therapies to treat patients with burn wounds infected with multidrug-resistant bacteria. PMID:24841266

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

    PubMed

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

    2014-08-01

    Burn wound infections are often difficult to treat due to the presence of multidrug-resistant bacterial strains and biofilms. Currently, mupirocin is used to eradicate methicillin-resistant Staphylococcus aureus (MRSA) from colonized persons; however, mupirocin resistance is also emerging. Since we consider antimicrobial peptides to be promising candidates for the development of novel anti-infective agents, we studied the antibacterial activities of a set of synthetic peptides against different strains of S. aureus, including mupirocin-resistant MRSA strains. The peptides were derived from P60.4Ac, a peptide based on the human cathelicidin LL-37. The results showed that peptide 10 (P10) was the only peptide more efficient than P60.4Ac, which is better than LL-37, in killing MRSA strain LUH14616. All three peptides displayed good antibiofilm activities. However, both P10 and P60.4Ac were more efficient than LL-37 in eliminating biofilm-associated bacteria. No toxic effects of these three peptides on human epidermal models were detected, as observed morphologically and by staining for mitochondrial activity. In addition, P60.4Ac and P10, but not LL-37, eradicated MRSA LUH14616 and the mupirocin-resistant MRSA strain LUH15051 from thermally wounded human skin equivalents (HSE). Interestingly, P60.4Ac and P10, but not mupirocin, eradicated LUH15051 from the HSEs. None of the peptides affected the excretion of interleukin 8 (IL-8) by thermally wounded HSEs upon MRSA exposure. In conclusion, the synthetic peptides P60.4Ac and P10 appear to be attractive candidates for the development of novel local therapies to treat patients with burn wounds infected with multidrug-resistant bacteria. PMID:24841266

  5. A polystyrene binding target-unrelated peptide isolated in the screening of phage display library.

    PubMed

    Bakhshinejad, Babak; Sadeghizadeh, Majid

    2016-11-01

    Phage display is a powerful methodology for the identification of peptide ligands binding to any desired target. However, the selection of target-unrelated peptides (TUPs) appears as a huge problem in the screening of phage display libraries through biopanning. The phage-displayed peptide TLHPAAD has been isolated both in our laboratory and by another reserach group on completely different screening targets prompting us to hypothesize that it may be a potential TUP. In the current study, we analyzed the binding characteristics and propagation rate of phage clone displaying TLHPAAD peptide (SW-TUP clone). The results of ELISA experiment and phage recovery assay provided strong support for the notion that SW-TUP phage binds to polystyrene with a significantly higher affinity than control phage clones. Furthermore, this polystyrene binding was demonstrated to occur in a concentration- and pH-dependent mode. Characterization of the propagation profile of phage clones within a specified time course revealed no statistically significant difference between the amplification rate of SW-TUP and control phages. Our findings lead us to the conclusion that SW-TUP phage clone with the displayed peptide TLHPAAD is not a true target binder and its selection in biopanning experiments results from its bidning affinity to the polystyrene surface of the solid phase. PMID:27555439

  6. Variant antigenic peptide promotes cytotoxic T lymphocyte adhesion to target cells without cytotoxicity

    PubMed Central

    Shotton, David M.; Attaran, Amir

    1998-01-01

    Timelapse video microscopy has been used to record the motility and dynamic interactions between an H-2Db-restricted murine cytotoxic T lymphocyte clone (F5) and Db-transfected L929 mouse fibroblasts (LDb) presenting normal or variant antigenic peptides from human influenza nucleoprotein. F5 cells will kill LDb target cells presenting specific antigen (peptide NP68: ASNENMDAM) after “browsing” their surfaces for between 8 min and many hours. Cell death is characterized by abrupt cellular rounding followed by zeiosis (vigorous “boiling” of the cytoplasm and blebbing of the plasma membrane) for 10–20 min, with subsequent cessation of all activity. Departure of cytotoxic T lymphocytes from unkilled target cells is rare, whereas serial killing is sometimes observed. In the absence of antigenic peptide, cytotoxic T lymphocytes browse target cells for much shorter periods, and readily leave to encounter other targets, while never causing target cell death. Two variant antigenic peptides, differing in nonamer position 7 or 8, also act as antigens, albeit with lower efficiency. A third variant peptide NP34 (ASNENMETM), which differs from NP68 in both positions and yet still binds Db, does not stimulate F5 cytotoxicity. Nevertheless, timelapse video analysis shows that NP34 leads to a significant modification of cell behavior, by up-regulating F5–LDb adhesive interactions. These data extend recent studies showing that partial agonists may elicit a subset of the T cell responses associated with full antigen stimulation, by demonstrating that TCR interaction with variant peptide antigens can trigger target cell adhesion and surface exploration without activating the signaling pathway that results in cytotoxicity. PMID:9861010

  7. Evolution of potent and stable placental-growth-factor-1-targeting CovX-bodies from phage display peptide discovery.

    PubMed

    Bower, Kristen E; Lam, Son N; Oates, Bryan D; Del Rosario, Joselyn R; Corner, Emily; Osothprarop, Trina F; Kinhikar, Arvind G; Hoye, Julie A; Preston, R Ryan; Murphy, Robert E; Campbell, Lioudmila A; Huang, Hanhua; Jimenez, Judith; Cao, Xia; Chen, Gang; Ainekulu, Zemeda W; Datt, Aakash B; Levin, Nancy J; Doppalapudi, Venkata R; Pirie-Shepherd, Steven R; Bradshaw, Curt; Woodnutt, Gary; Lappe, Rodney W

    2011-03-10

    Novel phage-derived peptides are the first reported molecules specifically targeting human placental growth factor 1 (PlGF-1). Phage data enabled peptide modifications that decreased IC(50) values in PlGF-1/VEGFR-1 competition ELISA from 100 to 1 μM. Peptides exhibiting enhanced potency were bioconjugated to the CovX antibody scaffold 1 (CVX-2000), generating bivalent CovX-Bodies with 2 nM K(D) against PlGF-1. In vitro and in vivo peptide cleavage mapping studies enabled the identification of proteolytic hotspots that were subsequently chemically modified. These changes decreased IC(50) to 0.4 nM and increased compound stability from 5% remaining at 6 h after injection to 35% remaining at 24 h with a β phase half-life of 75 h in mice. In cynomolgus monkey, a 78 h β half-life was observed for lead compound 2. The pharmacological properties of 2 are currently being explored. PMID:21280651

  8. Interaction study between wheat-derived peptides and procyanidin B3 by mass spectrometry.

    PubMed

    Dias, Ricardo; Perez-Gregorio, Maria Rosa; Mateus, Nuno; De Freitas, Victor

    2016-03-01

    Tannins have the ability to complex and precipitate proteins, being particularly reactive towards the proline-rich ones. The main structural feature of the wheat peptides responsible for the onset of Celiac Disease (CD) is their high content in proline residues. The aim of this work was to characterize the binding between a common food tannin (procyanidin B3) and different wheat-derived peptidic fractions. For this, seven peptide mixtures were obtained after in vitro digestion of a wheat gliadins crude extract and further characterized by LC-ESI-MS/MS. Several soluble B3-peptide complexes were identified by ESI-MS. The peptides involved in complex formation varied in terms of their size and diversity in CD epitopes. Although binding selectivity of procyanidin B3 towards peptides containing CD epitopes was not found, the major complexes contained or could contain immunoreactive peptides. This study highlights the potential beneficial effects of food polyphenols as a nutritional approach in the modulation of CD. PMID:26471686

  9. Peptide derived from Pvfp-1 as bioadhesive on bio-inert surface.

    PubMed

    Jiang, Zhen; Yu, Yabiao; Du, Lina; Ding, Xiyu; Xu, Hui; Sun, Yanan; Zhang, Qiqing

    2012-02-01

    Surface property is one important characteristic of materials, especially for ones that are bio-inert but designed for bio-medical application. In this study, we designed a series of peptides and compared their capacities as bioadhesive to improve the surface bioactivity of bio-inert material. The peptides were designed according to the sequence of Perna viridis foot protein 1 (Pvfp-1), one of the Mfp-1s (mussel foot protein 1) which play key roles in wet adhesion of mussel byssus. And the Teflon (PTFE) was chosen as a model of bio-inert material. With adsorption, adhesion and coating analysis, it was found that peptide C2 (M) (derived from the non-repeating region of Pvfp-1, contains modified DOPA) has superior coating and adhesion abilities especially on the bio-inert surface of PTFE. After coating with peptide C2 (M), the cell adhesion and spreading of osteoblast MC3T3-E1 cells on PTFE were significantly improved compared with those on non-coated surface, and the peptide-coating did not show any cell toxicity. Therefore, peptide C2 (M) is effective for improving the bioactivity of bio-inert PTFE, and could be potentially used as a bioadhesive on other bio-inert materials for biomedical application. Moreover, this study also provided new insights in designing other peptide-based bioadhesive materials. PMID:22079698

  10. Solid-State NMR Characterization of Autofluorescent Fibrils Formed by the Elastin-Derived Peptide GVGVAGVG

    PubMed Central

    2011-01-01

    The characterization of the molecular structure and physical properties of self-assembling peptides is an important aspect of optimizing their utility as scaffolds for biomaterials and other applications. Here we report the formation of autofluorescent fibrils by an octapeptide (GVGVAGVG) derived via a single amino acid substitution in one of the hydrophobic repeat elements of human elastin. This is the shortest and most well-defined peptide so far reported to exhibit intrinsic fluorescence in the absence of a discrete fluorophore. Structural characterization by FTIR and solid-state NMR reveals a predominantly β-sheet conformation for the peptide in the fibrils, which are likely assembled in an amyloid-like cross-β structure. Investigation of dynamics and the effects of hydration on the peptide are consistent with a rigid, water excluded structure, which has implications for the likely mechanism of intrinsic fibril fluorescence. PMID:21456595

  11. Mycobacterium tuberculosis PE9 protein has high activity binding peptides which inhibit target cell invasion.

    PubMed

    Díaz, Diana P; Ocampo, Marisol; Pabón, Laura; Herrera, Chonny; Patarroyo, Manuel A; Munoz, Marina; Patarroyo, Manuel E

    2016-05-01

    PE/PPE proteins are involved in several processes during Mycobacterium tuberculosis (Mtb) infection of target cells; studying them is extremely interesting as they are the only ones from the Mycobacterium genus, they abound in pathogenic species such as Mtb and their function remains yet unknown. The PE9 protein (Rv1088) was characterised, the rv1088 gene was identified by PCR in Mtb complex strains and its expression and localisation on mycobacterial surface was confirmed by Western blot and immunoelectron microscopy. Bioinformatics tools were used for predicting PE9 protein structural aspects and experimental study involved the circular dichroism of synthetic peptides. The peptides were tested in binding assays involving U937 and A549 cells; two high activity binding peptides (HABPs) were found for both cell lines (39226-(1)MSYMIATPAALTAAATDIDGI(21) and 39232-(125)YQRHFGTGGQPEFRQHSEHRR(144)), one for U937 (39231-(104)YAGAGRRQRRRRSGDGQWRLRQ(124)) and one for A549 (39230-(83)YGTGVFRRRRGRQTVTAAEHRA(103)). HABP 39232 inhibited mycobacterial entry to A549 cells (∼70%) and U937 cells (∼50%), peptides 39226 and 39231 inhibited entry to U937 cells (∼60% and 80%, respectively) and peptide 39230 inhibited entry to A549 cells (∼60%). This emphasised HABPs' functional importance in recognition between Mtb H37Rv and target cell receptors. These peptide sequences could be involved in invasion and were recognised by the host's immune system, thereby highlighting their use when designing an efficient anti-tuberculosis multiantigenic vaccine. PMID:26851205

  12. A peptide antigen derived from EGFR T790M is immunogenic in non-small cell lung cancer

    PubMed Central

    OFUJI, KAZUYA; TADA, YOSHITAKA; YOSHIKAWA, TOSHIAKI; SHIMOMURA, MANAMI; YOSHIMURA, MAYUKO; SAITO, KEIGO; NAKAMOTO, YASUNARI; NAKATSURA, TETSUYA

    2015-01-01

    Lung cancer is the leading cause of cancer-related deaths worldwide. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, have demonstrated marked clinical activity against non-small cell lung cancer (NSCLC) harboring activating epidermal growth factor receptor (EGFR) mutations. However, in most cases, patients develop acquired resistance to EGFR-TKI therapy. The threonine to methionine change at codon 790 of EGFR (EGFR T790M) mutation is the most common acquired resistance mutation, and is present in ~50% cases of TKI resistance. New treatment strategies for NSCLC patients harboring the EGFR T790M mutation are required. We evaluated the immunogenicity of an antigen derived from EGFR with the T790M mutation. Using BIMAS we selected several EGFR T790M-derived peptides bound to human leukocyte antigen (HLA)-A*02:01. T790M-A peptide (789–797) (IMQLMPFGC)-specific cytotoxic T lymphocytes (CTLs) were induced from peripheral blood mononuclear cells (PBMCs) of HLA-A2+ healthy donors. An established T790M-A-specific CTL line showed reactivity against the NCSLC cell line, H1975-A2 (HLA-A2+, T790M+), but not H1975 (HLA-A2−, T790M+), and the corresponding wild-type peptide (ITQLMPFGC)-pulsed T2 cells using an interferon-γ (IFN-γ) enzyme-linked immuno spot (ELISPOT) assay. This CTL line also demonstrated peptide-specific cytotoxicity against H1975-A2 cells. This finding suggests that the EGFR T790M mutation-derived antigen could be a new target for cancer immunotherapy. PMID:25532027

  13. Killer Bee Molecules: Antimicrobial Peptides as Effector Molecules to Target Sporogonic Stages of Plasmodium

    PubMed Central

    Carter, Victoria; Underhill, Ann; Baber, Ibrahima; Sylla, Lakamy; Baby, Mounirou; Larget-Thiery, Isabelle; Zettor, Agnès; Bourgouin, Catherine; Langel, Ülo; Faye, Ingrid; Otvos, Laszlo; Wade, John D.; Coulibaly, Mamadou B.; Traore, Sekou F.; Tripet, Frederic; Eggleston, Paul; Hurd, Hilary

    2013-01-01

    A new generation of strategies is evolving that aim to block malaria transmission by employing genetically modified vectors or mosquito pathogens or symbionts that express anti-parasite molecules. Whilst transgenic technologies have advanced rapidly, there is still a paucity of effector molecules with potent anti-malaria activity whose expression does not cause detrimental effects on mosquito fitness. Our objective was to examine a wide range of antimicrobial peptides (AMPs) for their toxic effects on Plasmodium and anopheline mosquitoes. Specifically targeting early sporogonic stages, we initially screened AMPs for toxicity against a mosquito cell line and P. berghei ookinetes. Promising candidate AMPs were fed to mosquitoes to monitor adverse fitness effects, and their efficacy in blocking rodent malaria infection in Anopheles stephensi was assessed. This was followed by tests to determine their activity against P. falciparum in An. gambiae, initially using laboratory cultures to infect mosquitoes, then culminating in preliminary assays in the field using gametocytes and mosquitoes collected from the same area in Mali, West Africa. From a range of 33 molecules, six AMPs able to block Plasmodium development were identified: Anoplin, Duramycin, Mastoparan X, Melittin, TP10 and Vida3. With the exception of Anoplin and Mastoparan X, these AMPs were also toxic to an An. gambiae cell line at a concentration of 25 µM. However, when tested in mosquito blood feeds, they did not reduce mosquito longevity or egg production at concentrations of 50 µM. Peptides effective against cultured ookinetes were less effective when tested in vivo and differences in efficacy against P. berghei and P. falciparum were seen. From the range of molecules tested, the majority of effective AMPs were derived from bee/wasp venoms. PMID:24278025

  14. MEPE-Derived ASARM Peptide Inhibits Odontogenic Differentiation of Dental Pulp Stem Cells and Impairs Mineralization in Tooth Models of X-Linked Hypophosphatemia

    PubMed Central

    Khaddam, Mayssam; Naji, Jiar; Coyac, Benjamin R.; Baroukh, Brigitte; Letourneur, Franck; Lesieur, Julie; Decup, Franck; Le Denmat, Dominique; Nicoletti, Antonino; Poliard, Anne; Rowe, Peter S.; Huet, Eric; Vital, Sibylle Opsahl; Linglart, Agnès; McKee, Marc D.; Chaussain, Catherine

    2013-01-01

    Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) cause X-linked familial hypophosphatemic rickets (XLH), a disorder having severe bone and tooth dentin mineralization defects. The absence of functional PHEX leads to abnormal accumulation of ASARM (acidic serine- and aspartate-rich motif) peptide − a substrate for PHEX and a strong inhibitor of mineralization − derived from MEPE (matrix extracellular phosphoglycoprotein) and other matrix proteins. MEPE-derived ASARM peptide accumulates in tooth dentin of XLH patients where it may impair dentinogenesis. Here, we investigated the effects of ASARM peptides in vitro and in vivo on odontoblast differentiation and matrix mineralization. Dental pulp stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into a 3D collagen scaffold, and induced towards odontogenic differentiation. Cultures were treated with synthetic ASARM peptides (phosphorylated and nonphosphorylated) derived from the human MEPE sequence. Phosphorylated ASARM peptide inhibited SHED differentiation in vitro, with no mineralized nodule formation, decreased odontoblast marker expression, and upregulated MEPE expression. Phosphorylated ASARM peptide implanted in a rat molar pulp injury model impaired reparative dentin formation and mineralization, with increased MEPE immunohistochemical staining. In conclusion, using complementary models to study tooth dentin defects observed in XLH, we demonstrate that the MEPE-derived ASARM peptide inhibits both odontogenic differentiation and matrix mineralization, while increasing MEPE expression. These results contribute to a partial mechanistic explanation of XLH pathogenesis: direct inhibition of mineralization by ASARM peptide leads to the mineralization defects in XLH teeth. This process appears to be positively reinforced by the increased MEPE expression induced by ASARM. The MEPE-ASARM system can therefore be considered as a potential therapeutic

  15. Salivary gland derived peptides as a new class of anti-inflammatory agents: review of preclinical pharmacology of C-terminal peptides of SMR1 protein

    PubMed Central

    2010-01-01

    The limitations of steroidal and non steroidal anti-inflammatory drugs have prompted investigation into other biologically based therapeutics, and identification of immune selective anti-inflammatory agents of salivary origin. The traditional view of salivary glands as accessory digestive structures is changing as their importance as sources of systemically active immunoregulatory and anti-inflammatory factors is recognized. Salivary gland involvement in maintenance of whole body homeostasis is regulated by the nervous system and thus constitutes a "neuroendocrine axis". The potent anti-inflammatory activities, both in vivo and in vitro, of the tripeptide Phe-Glu-Gly (FEG) are reviewed. FEG is a carboxyl terminal peptide of the prohormone SMR1 identified in the rat submandibular salivary gland, The D-isomeric form (feG) mimics the activity of its L-isomer FEG. Macropharmacologically, feG attenuates the cardiovascular and inflammatory effects of endotoxemia and anaphylaxis, by inhibition of hypotension, leukocyte migration, vascular leak, and disruption of pulmonary function and intestinal motility. Mechanistically, feG affects activated inflammatory cells, especially neutrophils, by regulating integrins and inhibiting intracellular production of reactive oxygen species. Pharmacodynamically, feG is active at low doses (100 μg/kg) and has a long (9-12 hour) biological half life. As a therapeutic agent, feG shows promise in diseases characterized by over exuberant inflammatory responses such as systemic inflammatory response syndrome and other acute inflammatory diseases. Arthritis, sepsis, acute pancreatitis, asthma, acute respiratory inflammation, inflammatory bowel disease, and equine laminitis are potential targets for this promising therapeutic peptide. The term "Immune Selective Anti-Inflammatory Derivatives" (ImSAIDs) is proposed for salivary-derived peptides to distinguish this class of agents from corticosteroids and nonsteroidal anti-inflammatory drugs

  16. Targeting lipopolyplexes using bifunctional peptides incorporating hydrophobic spacer amino acids: synthesis, transfection, and biophysical studies.

    PubMed

    Pilkington-Miksa, Michael A; Writer, Michele J; Sarkar, Supti; Meng, Qing-Hai; Barker, Suzie E; Shamlou, Parviz Ayazi; Hailes, Helen C; Hart, Stephen L; Tabor, Alethea B

    2007-01-01

    We have developed efficient synthetic routes to two hydrophobic amino acids, suitably protected for solid-phase peptide synthesis, and have successfully synthesized peptides containing these or other hydrophobic amino acids as spacers between a Lys16 moiety and an integrin-targeting motif. These peptides have in turn been used to formulate a range of lipopolyplex vectors with Lipofectin and plasmid DNA. The transfection efficiencies of these vectors and their aggregation behavior in buffers and in serum have been studied. We have shown that vectors containing peptides incorporating long linkers that are entirely hydrophobic are less efficient transfection agents. However, linkers of equivalent length that are in part hydrophobic show improved transfection properties, which is probably due to the improved accessibility of the integrin-binding motif. PMID:17915956

  17. Challenging the catechism of therapeutics for chronic neuropathic pain: targeting CaV2.2 interactions with CRMP2 peptides

    PubMed Central

    Feldman, Polina; Khanna, Rajesh

    2013-01-01

    Chronic neuropathic pain management is a worldwide concern. Pharmaceutical companies globally have historically targeted ion channels as the therapeutic catechism with many blockbuster successes. Remarkably, no new pain therapeutic has been approved by European or American regulatory agencies over the last decade. This article will provide an overview of an alternative approach to ion channel drug discovery: targeting regulators of ion channels, specifically focusing on voltage-gated calcium channels. We will highlight the discovery of an anti-nociceptive peptide derived from a novel calcium channel interacting partner – the collapsin response mediator protein 2 (CRMP2). In vivo administration of this peptide reduces pain behavior in a number of models of neuropathic pain without affecting sympathetic-associated cardiovascular activity, memory retrieval, sensorimotor function, or depression. A CRMP2-derived peptide analgesic, with restricted access to the CNS, represents a completely novel approach to the treatment of severe pain with an improved safety profile. As peptides now represent one of the fastest growing classes of new drugs, it is expected that peptide targeting of protein interactions within the calcium channel complex may be a paradigm shift in ion channel drug discovery. PMID:23831344

  18. Antimicrobial properties of two novel peptides derived from Theobroma cacao osmotin.

    PubMed

    Falcao, Loeni L; Silva-Werneck, Joseilde O; Ramos, Alessandra de R; Martins, Natalia F; Bresso, Emmanuel; Rodrigues, Magali A; Bemquerer, Marcelo P; Marcellino, Lucilia H

    2016-05-01

    The osmotin proteins of several plants display antifungal activity, which can play an important role in plant defense against diseases. Thus, this protein can be useful as a source for biotechnological strategies aiming to combat fungal diseases. In this work, we analyzed the antifungal activity of a cacao osmotin-like protein (TcOsm1) and of two osmotin-derived synthetic peptides with antimicrobial features, differing by five amino acids residues at the N-terminus. Antimicrobial tests showed that TcOsm1 expressed in Escherichia coli inhibits the growth of Moniliophthora perniciosa mycelium and Pichia pastoris X-33 in vitro. The TcOsm1-derived peptides, named Osm-pepA (H-RRLDRGGVWNLNVNPGTTGARVWARTK-NH2), located at R23-K49, and Osm-pepB (H-GGVWNLNVNPGTTGARVWARTK-NH2), located at G28-K49, inhibited growth of yeasts (Saccharomyces cerevisiae S288C and Pichia pastoris X-33) and spore germination of the phytopathogenic fungi Fusarium f. sp. glycines and Colletotrichum gossypi. Osm-pepA was more efficient than Osm-pepB for S. cerevisiae (MIC=40μM and MIC=127μM, respectively), as well as for P. pastoris (MIC=20μM and MIC=127μM, respectively). Furthermore, the peptides presented a biphasic performance, promoting S. cerevisiae growth in doses around 5μM and inhibiting it at higher doses. The structural model for these peptides showed that the five amino acids residues, RRLDR at Osm-pepA N-terminus, significantly affect the tertiary structure, indicating that this structure is important for the peptide antimicrobial potency. This is the first report of development of antimicrobial peptides from T. cacao. Taken together, the results indicate that the cacao osmotin and its derived peptides, herein studied, are good candidates for developing biotechnological tools aiming to control phytopathogenic fungi. PMID:26996966

  19. Click conjugation of peptide to hydrogel nanoparticles for tumor-targeted drug delivery.

    PubMed

    Qin, Ming; Zong, Hong; Kopelman, Raoul

    2014-10-13

    Here we introduce a modified peptide-decorated polymeric nanoparticle (NP) for cancer cell targeting, which can deliver drugs, such as doxorubicin (Dox), to several kinds of cancer cells. Specifically, we employ a nucleolin-targeting NP, with a matrix based on a copolymer of acrylamide (AAm) and 2-carboxyethyl acrylate (CEA). The negatively charged co(CEA-AAm) NP was conjugated with a nucleolin-targeting F3 peptide using a highly efficient and specific copper(I) catalyzed azide-alkyne click reaction. F3 peptide binds to angiogenic tumor vasculatures and other nucleolin overexpressing tumor cells. Attaching F3 peptide onto the NP increases the NP uptake by the nucleolin-expressing glioma cell line 9L and the breast cancer cell line MCF-7. Notably, the F3-conjugated NPs show much higher uptake by the nucleolin-overexpressing glioma cell line 9L than that by the breast cancer cell line MCF-7, the latter having a lower expression of nucleolin on its plasma membrane surface. Moreover, the F3 peptide also dramatically enhances the uptake of co(CEA-AAm) NPs by the drug-resistant cell line NCI/ADR-RES. Also, with this F3-conjugated co(CEA-AAm) NP, a high loading and slow release of doxorubicin were achieved. PMID:25162488

  20. Boswellic acids target the human immune system-modulating antimicrobial peptide LL-37.

    PubMed

    Henkel, Arne; Tausch, Lars; Pillong, Max; Jauch, Johann; Karas, Michael; Schneider, Gisbert; Werz, Oliver

    2015-12-01

    The antimicrobial peptide LL-37 is the sole member of the human cathelicidin family with immune system-modulating properties and roles in autoimmune disease development. Small molecules able to interact with LL-37 and to modulate its functions have not been described yet. Boswellic acids (BAs) are pentacyclic triterpene acids that are bioactive principles of frankincense extracts used as anti-inflammatory remedies. Although various anti-inflammatory modes of action have been proposed for BAs, the pharmacological profile of these compounds is still incompletely understood. Here, we describe the identification of human LL-37 as functional target of BAs. In unbiased target fishing experiments using immobilized BAs as bait and human neutrophils as target source, LL-37 was identified as binding partner assisted by MALDI-TOF mass spectrometry. Thermal stability experiments using circular dichroism spectroscopy confirm direct interaction between BAs and LL-37. Of interest, this binding of BAs resulted in an inhibition of the functionality of LL-37. Thus, the LPS-neutralizing properties of isolated LL-37 were inhibited by 3-O-acetyl-β-BA (Aβ-BA) and 3-O-acetyl-11-keto-β-BA (AKβ-BA) in a cell-free limulus amoebocyte lysate assay with EC50=0.2 and 0.8 μM, respectively. Also, LL-37 activity was inhibited by these BAs in LL-37-enriched supernatants of stimulated neutrophils or human plasma derived from stimulated human whole blood. Together, we reveal BAs as inhibitors of LL-37, which might be a relevant mechanism underlying the anti-inflammatory properties of BAs and suggests BAs as suitable chemical tools or potential agents for intervention with LL-37 and related disorders. PMID:26361729

  1. 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

  2. Antidepressant-like effect of food-derived pyroglutamyl peptides in mice.

    PubMed

    Yamamoto, Yukako; Mizushige, Takafumi; Mori, Yukiha; Shimmura, Yuki; Fukutomi, Ruuta; Kanamoto, Ryuhei; Ohinata, Kousaku

    2015-06-01

    The N-terminal glutamine residue, exposed by enzymatic cleavage of precursor proteins, is known to be modified to a pyroglutamyl residue with a cyclic structure in not only endogenous but also food-derived peptides. We investigated the effects of wheat-derived pyroglutamyl peptides on emotional behaviors. Pyroglutamyl leucine (pyroGlu-Leu, pEL) and pyroglutamyl glutaminyl leucine (pyroGlu-Gln-Leu, pEQL) exhibited antidepressant-like activity in the tail suspension and forced swim tests in mice. pEQL exhibited more potent antidepressant-like activity than pEL after i.p. and i.c.v. administration. pEQL exhibited antidepressant-like activity at a lower dose than Gln-Gln-Leu, suggesting that pyroglutamyl peptide had more potent activity. To examine whether pyroglutamyl peptides increased hippocampus neurogenesis, associated with the effects of antidepressants, we measured 5-bromo-2'-deoxyuridine (BrdU) incorporation. pEL and pEQL increased BrdU-positive cells in the dentate gyrus of the hippocampus. Intriguingly, pEL did not increase hippocampal mRNA and protein expression of brain-derived neurotrophic factor (BDNF), which is a factor associated with both neuropoietic and antidepressive effects. Thus, pyroglutamyl peptides may enhance hippocampal neurogenesis via a pathway independent of BDNF. We also confirmed that pEL and pEQL were produced in the subtilisin digest of major wheat proteins, glutenin and gliadin, after heat treatment. pEL and pEQL are the first peptides derived from wheat proteins to be shown to exhibit an antidepressant-like activity. PMID:25957094

  3. Pharmacological characteristics of endokinin C/D-derived peptides in nociceptive and inflammatory processing in rats.

    PubMed

    Naono-Nakayama, Rumi; Sunakawa, Natsuki; Ikeda, Tetsuya; Matsushima, Osamu; Nishimori, Toshikazu

    2011-12-01

    Endokinins designated from the human TAC4 gene consist of endokinin A, endokinin B, endokinin C (EKC) and endokinin D (EKD). EKC/D is a peptide using the common carboxyl-terminal in EKC and EKD and consists of 12 amino acids, and exerts antagonistic effects on the induction of scratching behavior by substance P (SP). Some of SP-preferring receptor antagonists have several d-tryptophan (d-Trp); however, the pharmacological effect of EKC/D-derived peptides with d-Trp remains to be solved. Therefore, to clarify the pharmacological characteristics of EKC/D-derived peptides, effects of pretreatment with these peptides on SP-induced scratching and thermal hyperalgesia, formalin-induced flinching and carrageenan-induced inflammation were evaluated. Intrathecal administration of [d-Trp(8)]-EKC/D and [d-Trp(10)]-EKC/D showed a markedly long inhibitory effect, at least 14 h, whereas the antagonistic effects of [d-Trp(8,10)]-EKC/D and EKC/D without d-Trp disappeared after 1h. Furthermore, the inhibitory effect of [d-Trp(10)]-EKC/D-derived peptides was dependent on the number of amino acids from the amino-terminus, and the more numerous the amino acids, the more marked the antagonistic effect. Thus, these results indicate that the effective duration of EKC/D-derived peptides is dependent on the number of d-Trp in the carboxyl-terminal region and the amino-terminal region regulates the antagonistic effect of EKC/D. PMID:22074956

  4. Nutritional value of D-amino acids, D-peptides, and amino acid derivatives in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper describes a method for determining the nutritional value of D-amino acids, D-peptides, and amino acid derivatives using a growth assay in mice fed a synthetic all-amino acid diet. A large number of experiments were carried out in which a molar equivalent of the test compound replaced a n...

  5. A proline-derived transannular N-cap for nucleation of short α-helical peptides.

    PubMed

    Tian, Yuan; Wang, Dongyuan; Li, Jingxu; Shi, Chuan; Zhao, Hui; Niu, Xiaogang; Li, Zigang

    2016-07-28

    We report herein a proline-derived transannular N-cap as a helix nucleating template in diverse bio-related peptide sequences via macrolactamization on resin. This approach takes advantage of synergistic stabilization effects of both N-capping properties of proline and substitution of a main chain hydrogen bond with a covalent bond. PMID:27357119

  6. A Fly-Through Mission Strategy Targeting Peptide as a Signature of Chemical Evolution and Possible Life in Enceladus Plumes

    NASA Technical Reports Server (NTRS)

    Fujishima, Kosuke; Dziomba, Szymon; Takahagi, Wataru; Shibuya, Takazo; Takano, Yoshinori; Guerrouache, Mohamed; Carbonnier, Benjamin; Takai, Ken; Rothschild, Lynn J.; Yano, Hajime

    2016-01-01

    In situ detection of organic molecules in the extraterrestrial environment provides a key step towards better understanding the variety and the distribution of building blocks of life and it may ultimately lead to finding extraterrestrial life within the Solar System. Here we present combined results of two separate experiments that enable us to realize such in situ life signature detection from the deep habitats of the "Ocean World": a hydrothermal reactor experiment simulating complex organic synthesis and a simulated fly-through capture experiment of organic-bearing microparticles using silica aerogels, followed by subsequent analysis. Both experiments employ peptide as a plausible organics existing in Encleadus plume particles produced in its subsurface ocean. Recent laboratory hydrothermal experiments and a theoretical model on silica saturation indicated an on going hydrothermal reactions in subsurface Enceladus ocean. Given the porous chondritic origin of the core, it is likely that organic compounds originated by radiation chemistry such as amino acid precursors could have been provided, leached, and altered through widespread water-rock interactions. By using the same laboratory experimental setup from the latest water-rock interaction study, we performed amino acid polymerization experiments for 144 days and monitored the organic complexity changing over time. So far over 3,000 peaks up to the size of greater than 600 MW were observed through the analysis of capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) with an indication of amino acid derivatives and short peptides. Generally abiotic polymerization of enantiomeric amino acids results in forming stereoisomeric peptides with identical molecular weight and formula as opposed to homochiral biopolymers. Assuming Enceladus plume particles may contain a mixture of stereoisomeric peptides, we were able to distinguish 16 of the 17 stereoisomeric tripeptides as a test sample using

  7. Dipeptidyl Peptidase IV-Inhibitory Peptides Derived from Silver Carp (Hypophthalmichthys molitrix Val.) Proteins.

    PubMed

    Zhang, Ying; Chen, Ran; Chen, Xiling; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

    2016-02-01

    The dipeptidyl peptidase IV (DPP-IV)-inhibitory bioactivity of silver carp protein (SCP) hydrolysates were investigated, and their containing efficacious DPP-IV-inhibitory peptides were explored by in silico hydrolysis analysis, peptide separation combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification, and chemical synthesis. SCP hydrolysates generated by six proteases all showed efficient DPP-IV-inhibitory activities, and Neutrase-generated hydrolysates had the greatest DPP-IV inhibition (IC50 of 1.12 mg/mL). In silico Neutrase hydrolysis revealed hundreds of fragments released from myosin, actin, and collagen of SCPs, which include different Pro-motif peptides but only three reported peptidic DPP-IV inhibitors with moderate or weak bioactivity. In addition, three new DPP-IV-inhibitory peptides were identified using LC-MS/MS; in particular, LPIIDI and APGPAGP showed high DPP-IV-inhibitory activity with IC50 of 105.44 and 229.14 μM, respectively, and behaved in competitive/non-competitive mixed-type DPP-IV inhibition mode. The results indicate that the SCP-derived DPP-IV-inhibitory peptides could be potential functional ingredients in the diabetic diet. PMID:26758401

  8. Purification of a novel nitric oxide inhibitory peptide derived from enzymatic hydrolysates of Mytilus coruscus.

    PubMed

    Kim, Eun-Kyung; Kim, Yon-Suk; Hwang, Jin-Woo; Kang, Seo Hee; Choi, Dong-Kug; Lee, Kwang-Ho; Lee, Jung Suck; Moon, Sang-Ho; Jeon, Byong-Tae; Park, Pyo-Jam

    2013-06-01

    Shellfish contain significant levels of high quality protein and are therefore a potential source for biofunctional high-value peptides. To purify a novel anti-inflammatory peptide from Mytilus coruscus (M. coruscus), we applied enzymatic hydrolysis and tangential flow filtration (TFF) and investigated its nitric oxide inhibitory property. To prepare the peptide, eight proteases were employed for enzymatic hydrolysis. Flavouzyme hydrolysates, which showed clearly superior nitric oxide inhibitory activity on lipopolysaccharide (LPS)-stimulated RAW264.7, were further purified using a TFF system and consecutive chromatographic methods. Finally, a novel anti-inflammatory peptide composed of 10 amino acid residues was obtained, and the sequence was identified as Gly-Val-Ser-Leu-Leu-Gln-Gln-Phe-Phe-Leu at N-terminal position. The peptide from M. coruscus effectively inhibited nitric oxide production on macrophage cells. This is the first report of an anti-inflammatory peptide derived from the hydrolysates of M. coruscus. PMID:23500953

  9. β-casein-derived peptides, produced by bacteria, stimulate cancer cell invasion and motility

    PubMed Central

    Oliveira, Maria José; Van Damme, Jozef; Lauwaet, Tineke; De Corte, Veerle; De Bruyne, Georges; Verschraegen, Gerda; Vaneechoutte, Mario; Goethals, Marc; Ahmadian, Mohammad Reza; Müller, Oliver; Vandekerckhove, Joël; Mareel, Marc; Leroy, Ancy

    2003-01-01

    In colon cancer, enteric bacteria and dietary factors are major determinants of the microenvironment but their effect on cellular invasion is not known. We therefore incubated human HCT-8/E11 colon cancer cells with bacteria or bacterial conditioned medium on top of collagen type I gels. Listeria monocytogenes stimulate cellular invasion through the formation of a soluble motility-promoting factor, identified as a 13mer β-casein-derived peptide (HKEMPFPKYPVEP). The peptide is formed through the combined action of Mpl, a Listeria thermolysin-like metalloprotease, and a collagen-associated trypsin-like serine protease. The 13mer peptide was also formed by tumour biopsies isolated from colon cancer patients and incubated with a β-casein source. The pro- invasive 13mer peptide-signalling pathway implicates activation of Cdc42 and inactivation of RhoA, linked to each other through the serine/threonine p21- activated kinase 1. Since both changes are necessary but not sufficient, another pathway might branch upstream of Cdc42 at phosphatidylinositol 3-kinase. Delta opioid receptor (δOR) is a candidate receptor for the 13mer peptide since naloxone, an δOR antagonist, blocks both δOR serine phosphorylation and 13mer peptide-mediated invasion. PMID:14609961

  10. PSP94 contributes to chemoresistance and its peptide derivative PCK3145 represses tumor growth in ovarian cancer.

    PubMed

    Yan, B-X; Ma, J-X; Zhang, J; Guo, Y; Riedel, H; Mueller, M D; Remick, S C; Yu, J J

    2014-11-01

    Tumor drug resistance remains a major challenge in the treatment of cancer. Here, we show that Prostatic secretory protein 94 (PSP94) levels are reduced in ovarian cancer patients with high levels of excision repair cross-complementing 1 (ERCC1), a marker for chemoresistance. We find that PSP94 is decreased in an ovarian cancer drug-resistant cell line, and plays an important role in the development of drug resistance in vitro. Our studies indicate that PSP94 can partially reverse drug resistance in mouse tumor models in vivo and that a PSP94 peptide derivative PCK3145 suppresses chemoresistant cancer cell and tumor growth in vitro and in vivo. Our investigation of the involved molecular mechanisms suggests that PSP94 may confer drug resistance by modulating the Lin28b/Let-7 signaling pathway. We introduce PSP94 and its peptide derivative PCK3145 as potential target to reverse chemoresistance in ovarian cancer and have begun to identify their relevant molecular targets in specific signaling pathways. PMID:24186202

  11. Bax-derived membrane-active peptides act as potent and direct inducers of apoptosis in cancer cells

    PubMed Central

    Valero, Juan Garcia; Sancey, Lucie; Kucharczak, Jérôme; Guillemin, Yannis; Gimenez, Diana; Prudent, Julien; Gillet, Germain; Salgado, Jesús; Coll, Jean-Luc; Aouacheria, Abdel

    2011-01-01

    SUMMARY Although many cancer cells are primed for apoptosis, they usually develop resistance to cell death at multiple levels. Permeabilization of the outer mitochondrial membrane, which is mediated by proapoptotic Bcl-2 family members like Bax, is considered as a point-of-no-return for initiating apoptotic cell death. This crucial role has placed Bcl-2 family proteins as recurrent targets for anticancer drug development. Here, we propose and demonstrate a new concept based on using minimal active version of Bax to induce cell death independently of endogenous Bcl-2 proteins. We show that membrane-active segments of Bax can directly induce the release of mitochondria-residing apoptogenic factors and commit tumor cells promptly and irreversibly to caspase-dependent apoptosis. On this basis, we designed a peptide encompassing part of the Bax pore-forming domain, able to target mitochondria, induce cytochrome c release and trigger caspase-dependent apoptosis. Moreover, this Bax-derived poropeptide produced effective tumor regression after peritumoral injection in a nude mouse xenograft model. Thus, peptides derived from proteins evolutionary functionalized to form pores in the mitochondrial outer membrane represent novel templates for anticancer agents. PMID:21245196

  12. Tuftsin-derived T-peptide prevents cellular immunosuppression and improves survival rate in septic mice

    PubMed Central

    Gao, Yu-Lei; Chai, Yan-Fen; Dong, Ning; Han, Su; Zhu, Xiao-Mei; Zhang, Qing-Hong; Yao, Yong-Ming

    2015-01-01

    The primary mechanisms of sepsis induced cellular immunesuppression involve immune dysfunction of T lymphocytes and negative immunoregulation of regulatory T cells (Tregs). It has been found that tuftsin is an immune modulating peptide derived from IgG in spleen. T-peptide is one of tuftsin analogs. Herein, we examined the effect of T-peptide on cell-mediated immunity in the presence of lipopolysaccharide (LPS) and the survival rate in septic mice. T-peptide regulated the proliferative ability of CD4+CD25− T cells in dual responses. Meanwhile, 10 and 100 μg/ml T-peptides were able to enhance the apoptotic rate of CD4+CD25− T cells compared with 1 μg/ml T-peptide, but markedly lowered interleukin (IL)-2 levels. When CD4+CD25+ Tregs were treated with T-peptide for 24 hours, and co-cultured with normal CD4+CD25− T cells, the suppressive ability of CD4+CD25+ Tregs on CD4+CD25− T cells was significantly lowered, along with decreased expression in forkhead/winged helix transcription factor p-3 (Foxp-3) as well as cytotoxic T lymphocyte-associated antigen (CTLA)-4, and secretion of transforming growth factor (TGF)-β. Moreover, T-peptide has the ability to improve outcome of septic mice in a dose- and time- dependent manner, and associated with improvement in the microenvironment of cellular immunosuppression in septic mice. PMID:26577833

  13. Evaluation of the effect of Sm28GST-derived peptides in murine hepatosplenic schistosomiasis: interest of the lipopeptidic form of the C-terminal peptide.

    PubMed

    Pancré, V; Wolowczuk, I; Bossus, M; Gras-Masse, H; Guerret, S; Delanoye, A; Capron, A; Auriault, C

    1994-11-01

    Among the synthetic peptides derived from the 28-kDa Schistosoma mansoni glutathione S-transferase (Sm28GST), immunization with the C-terminal peptide comprising amino acid residues 190-211 induced a reduction in splenomegaly, in the number of hepatic eggs and in hepatic fibrosis in mice infected by Schistosoma mansoni. The absence of antibodies specific for the Sm28GST or for the 190-211 peptide observed in our conditions of immunization with this peptide argued in favour of the involvement of cellular-dependent mechanisms in the reduction in hepatic pathology. This was confirmed by the passive transfer of 190-211 peptide-specific T-cell enriched spleen cells which reproduced the protective effect conferred by immunization with the 190-211 peptide. These 190-211 peptide-specific cells produced little IL4 and high levels of IFN-gamma, a potent inhibitor of collagen synthesis. Furthermore, the use of a lipopeptidic form of the 190-211 peptide significantly improved the reduction in hepatic pathology obtained with the uncoupled peptide and induced a durable protective response. These results provide encouraging information for the possible use of synthetic peptides in the immunoprophylaxis of Schistosomiasis. PMID:7969186

  14. Human antimicrobial peptide histatin 5 is a cell-penetrating peptide targeting mitochondrial ATP synthesis in Leishmania.

    PubMed

    Luque-Ortega, Juan Román; van't Hof, Wim; Veerman, Enno C I; Saugar, José M; Rivas, Luis

    2008-06-01

    Histatin 5 (Hst5) is a human salivary antimicrobial peptide that targets fungal mitochondria. In the human parasitic protozoa Leishmania, the mitochondrial ATP production is essential, as it lacks the bioenergetic switch between glycolysis and oxidative phosphorylation described in some yeasts. On these premises, Hst5 activity was assayed on both stages of its life cycle, promastigotes and amastigotes (LC(50)=7.3 and 14.4 microM, respectively). In a further step, its lethal mechanism was studied. The main conclusions drawn were as follows: 1) Hst5 causes limited and temporary damage to the plasma membrane of the parasites, as assessed by electron microscopy, depolarization, and entrance of the vital dye SYTOX Green; 2) Hst5 translocates into the cytoplasm of Leishmania in an achiral receptor-independent manner with accumulation into the mitochondrion, as shown by confocal microscopy; and 3) Hst5 produces a bioenergetic collapse of the parasite, caused essentially by the decrease of mitochondrial ATP synthesis through inhibition of F(1)F(0)-ATPase, with subsequent fast ATP exhaustion. By using the Hst5 enantiomer, it was found that the key steps of its lethal mechanism involved no chiral recognition. Hst5 thus constitutes the first leishmanicidal peptide with a defined nonstereospecific intracellular target. The prospects of its development, by its own or as a carrier molecule for other leishmanicidal molecules, into a novel anti-Leishmania drug with a preferential subcellular accumulation are discussed. PMID:18230684

  15. Molecular design and structural optimization of potent peptide hydroxamate inhibitors to selectively target human ADAM metallopeptidase domain 17.

    PubMed

    Wang, Zhengting; Wang, Lei; Fan, Rong; Zhou, Jie; Zhong, Jie

    2016-04-01

    Human ADAMs (a disintegrin and metalloproteinases) have been established as an attractive therapeutic target of inflammatory disorders such as inflammatory bowel disease (IBD). The ADAM metallopeptidase domain 17 (ADAM17 or TACE) and its close relative ADAM10 are two of the most important ADAM members that share high conservation in sequence, structure and function, but exhibit subtle difference in regulation of downstream cell signaling events. Here, we described a systematic protocol that combined computational modeling and experimental assay to discover novel peptide hydroxamate derivatives as potent and selective inhibitors for ADAM17 over ADAM10. In the procedure, a virtual combinatorial library of peptide hydroxamate compounds was generated by exploiting intermolecular interactions involved in crystal and modeled structures. The library was examined in detail to identify few promising candidates with both high affinity to ADAM17 and low affinity to ADAM10, which were then tested in vitro with enzyme inhibition assay. Consequently, two peptide hydroxamates Hxm-Phe-Ser-Asn and Hxm-Phe-Arg-Gln were found to exhibit potent inhibition against ADAM17 (Ki=92 and 47nM, respectively) and strong selectivity for ADAM17 over ADAM10 (∼7-fold and ∼5-fold, S=0.86 and 0.71, respectively). The structural basis and energetic property of ADAM17 and ADAM10 interactions with the designed inhibitors were also investigated systematically. It is found that the exquisite network of nonbonded interactions involving the side chains of peptide hydroxamates is primarily responsible for inhibitor selectivity, while the coordination interactions and hydrogen bonds formed by the hydroxamate moiety and backbone of peptide hydroxamates confer high affinity to inhibitor binding. PMID:26709988

  16. Initial characterization of a dually radiolabeled peptide for simultaneous monitoring of protein targets and enzymatic activity

    PubMed Central

    Mebrahtu, Efrem; Zheleznyak, Alexander; Hur, Minjun A.; Laforest, Richard; Lapi, Suzanne E.

    2016-01-01

    Objective The goal of this study was to develop dually radiolabeled peptides for simultaneous imaging of cancer cell localization by targeting the αvβ3 integrin and their pathophysiology by targeting the activity of the proteolytic enzyme MMP2, involved in the metastatic process. Methods A hybrid peptide c(RGDfE)K(DOTA)PLGVRY containing a RGD motif for binding to the αvβ3 integrin, a metal chelator (DOTA) for radiolabeling with [64Cu], and the MMP2 substrate cleavage sequence PLGVRY with terminal tyrosine for labeling with [123I] was synthesized, labeled with [64Cu] and [123I], and evaluated in vitro as a potential imaging agent. Results The peptide was synthesized and labeled with [64Cu] and [123I] with 300 and 40 μCi/μg (542 and 72.2 mCi/μmol) specific activities, respectively, and radiochemical purity of>98%.c(RGDfE)K(DOTA)PLGVRY demonstrated high affinity for αvβ3 integrins(Kd = 83.4 ± 13.2 nM) in both substrate competition and cell binding assays. c(RGDfE)K(DOTA)PLGVRY peptide, but not the scrambled version, c(RGDfE)K(DOTA)GRPLVY was specifically cleaved by MMP2. Conclusions These results demonstrate the feasibility of developing dually radiolabeled peptides for the simultaneous imaging of cancer cells and their pathophysiologic activity. PMID:23154178

  17. New Peptide-Conjugated Chlorin-Type Photosensitizer Targeting Neuropilin-1 for Anti-Vascular Targeted Photodynamic Therapy.

    PubMed

    Kamarulzaman, Ezatul Ezleen; Gazzali, Amirah Mohd; Acherar, Samir; Frochot, Céline; Barberi-Heyob, Muriel; Boura, Cédric; Chaimbault, Patrick; Sibille, Estelle; Wahab, Habibah A; Vanderesse, Régis

    2015-01-01

    Photodynamic therapy (PDT) is a cancer treatment modality that requires three components, namely light, dioxygen and a photosensitizing agent. After light excitation, the photosensitizer (PS) in its excited state transfers its energy to oxygen, which leads to photooxidation reactions. In order to improve the selectivity of the treatment, research has focused on the design of PS covalently attached to a tumor-targeting moiety. In this paper, we describe the synthesis and the physico-chemical and photophysical properties of six new peptide-conjugated photosensitizers designed for targeting the neuropilin-1 (NRP-1) receptor. We chose a TPC (5-(4-carboxyphenyl)-10,15, 20-triphenyl chlorine as photosensitizer, coupled via three different spacers (aminohexanoic acid, 1-amino-3,6-dioxaoctanoic acid, and 1-amino-9-aza-3,6,12,15-tetraoxa-10-on-heptadecanoic acid) to two different peptides (DKPPR and TKPRR). The affinity towards the NRP-1 receptor of the conjugated chlorins was evaluated along with in vitro and in vivo stability levels. The tissue concentration of the TPC-conjugates in animal model shows good distribution, especially for the DKPPR conjugates. The novel peptide-PS conjugates proposed in this study were proven to have potential to be further developed as future NRP-1 targeting photodynamic therapy agent. PMID:26473840

  18. A targeted proteomics approach to the identification of peptides modified by reactive metabolites.

    PubMed

    Tzouros, Manuel; Pähler, Axel

    2009-05-01

    Covalent binding of reactive metabolites is generally accepted as one underlying mechanism of drug-induced toxicity. However, identification of protein targets by reactive metabolites still remains a challenge due to their low abundance. Here, we report the development of a highly selective proteomics workflow for the targeted identification of peptides modified by reactive metabolites. An equimolar mixture of non- and radiolabeled furan containing 2-amino-pyrimidine drug candidate (1 and 14C(1)-1) along with rat liver microsomes were used for the in vitro generation of reactive metabolites. Liver microsomal proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, modified protein bands were highlighted by autoradiography and in-gel digested, and peptides were fractionated by strong cation exchange chromatography. Fractions enriched in modified peptides, as determined by radioactivity levels, were subjected to nanoLC-MS/MS and unambiguously detected based on their unique 12C/14C MS isotope pattern fingerprint. The peptide detection step could be automated using isotope pattern recognition software. Peptide sequencing, identification of the site of modification, and reactive metabolite characterization were achieved by MS2 and MS3 experiments using high-resolution and accurate mass detection. This approach led to the identification of four modified peptides originating from three drug-metabolizing enzymes, MGST1, FMO1, and P450 2C11. These revealed modifications by five different metabolite structures. This approach is generally suitable for the identification and characterization of modified proteins and metabolite structures involved in covalent binding and may serve as a valuable tool to link protein targets with clinically relevant toxicities. PMID:19317514

  19. Targeting of peptide conjugated magnetic nanoparticles to urokinase plasminogen activator receptor (uPAR) expressing cells

    NASA Astrophysics Data System (ADS)

    Hansen, Line; Unmack Larsen, Esben Kjær; Nielsen, Erik Holm; Iversen, Frank; Liu, Zhuo; Thomsen, Karen; Pedersen, Michael; Skrydstrup, Troels; Nielsen, Niels Chr.; Ploug, Michael; Kjems, Jørgen

    2013-08-01

    Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific targeting peptide onto polyethylene glycol (PEG) coated USPIO nanoparticles by click chemistry resulted in a five times higher uptake in vitro in a uPAR positive cell line compared to nanoparticles carrying a non-binding control peptide. In accordance with specific receptor-mediated recognition, a low uptake was observed in the presence of an excess of ATF, a natural ligand for uPAR. The uPAR specific magnetic nanoparticles can potentially provide a useful supplement for tumor patient management when combined with MRI and drug delivery.Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific

  20. Antibody-independent identification of bovine milk-derived peptides in breast-milk.

    PubMed

    Picariello, Gianluca; Addeo, Francesco; Ferranti, Pasquale; Nocerino, Rita; Paparo, Lorella; Passariello, Annalisa; Dallas, David C; Robinson, Randall C; Barile, Daniela; Canani, Roberto Berni

    2016-08-10

    Exclusively breast-fed infants can exhibit clear signs of IgE or non IgE-mediated cow's milk allergy. However, the definite characterization of dietary cow's milk proteins (CMP) that survive the maternal digestive tract to be absorbed into the bloodstream and secreted into breast milk remains missing. Herein, we aimed at assessing possible CMP-derived peptides in breast milk. Using high performance liquid chromatography (HPLC)-high resolution mass spectrometry (MS), we compared the peptide fraction of breast milk from 12 donors, among which 6 drank a cup of milk daily and 6 were on a strict dairy-free diet. We identified two bovine β-lactoglobulin (β-Lg, 2 out 6 samples) and one αs1-casein (1 out 6 samples) fragments in breast milk from mothers receiving a cup of bovine milk daily. These CMP-derived fragments, namely β-Lg (f42-54), (f42-57) and αs1-casein (f180-197), were absent in milk from mothers on dairy-free diet. In contrast, neither intact nor hydrolyzed β-Lg was detected by western blot and competitive ELISA in any breast milk sample. Eight additional bovine milk-derived peptides identified by software-assisted MS were most likely false positive. The results of this study demonstrate that CMP-derived peptides rather than intact CMP may sensitize or elicit allergic responses in the neonate through mother's milk. Immunologically active peptides from the maternal diet could be involved in priming the newborn's immune system, driving a tolerogenic response. PMID:27396729

  1. An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics

    PubMed Central

    Vandemoortele, Giel; Staes, An; Gonnelli, Giulia; Samyn, Noortje; De Sutter, Delphine; Vandermarliere, Elien; Timmerman, Evy; Gevaert, Kris; Martens, Lennart; Eyckerman, Sven

    2016-01-01

    The use of protein tagging to facilitate detailed characterization of target proteins has not only revolutionized cell biology, but also enabled biochemical analysis through efficient recovery of the protein complexes wherein the tagged proteins reside. The endogenous use of these tags for detailed protein characterization is widespread in lower organisms that allow for efficient homologous recombination. With the recent advances in genome engineering, tagging of endogenous proteins is now within reach for most experimental systems, including mammalian cell lines cultures. In this work, we describe the selection of peptides with ideal mass spectrometry characteristics for use in quantification of tagged proteins using targeted proteomics. We mined the proteome of the hyperthermophile Pyrococcus furiosus to obtain two peptides that are unique in the proteomes of all known model organisms (proteotypic) and allow sensitive quantification of target proteins in a complex background. By combining these ’Proteotypic peptides for Quantification by SRM’ (PQS peptides) with epitope tags, we demonstrate their use in co-immunoprecipitation experiments upon transfection of protein pairs, or after introduction of these tags in the endogenous proteins through genome engineering. Endogenous protein tagging for absolute quantification provides a powerful extra dimension to protein analysis, allowing the detailed characterization of endogenous proteins. PMID:27264994

  2. Conformational ensembles explored dynamically from disordered peptides targeting chemokine receptor CXCR4.

    PubMed

    Vincenzi, Marian; Costantini, Susan; Scala, Stefania; Tesauro, Diego; Accardo, Antonella; Leone, Marilisa; Colonna, Giovanni; Guillon, Jean; Portella, Luigi; Trotta, Anna Maria; Ronga, Luisa; Rossi, Filomena

    2015-01-01

    This work reports on the design and the synthesis of two short linear peptides both containing a few amino acids with disorder propensity and an allylic ester group at the C-terminal end. Their structural properties were firstly analyzed by means of experimental techniques in solution such as CD and NMR methods that highlighted peptide flexibility. These results were further confirmed by MD simulations that demonstrated the ability of the peptides to assume conformational ensembles. They revealed a network of transient and dynamic H-bonds and interactions with water molecules. Binding assays with a well-known drug-target, i.e., the CXCR4 receptor, were also carried out in an attempt to verify their biological function and the possibility to use the assays to develop new specific targets for CXCR4. Moreover, our data indicate that these peptides represent useful tools for molecular recognition processes in which a flexible conformation is required in order to obtain an interaction with a specific target. PMID:26030674

  3. Conformational Ensembles Explored Dynamically from Disordered Peptides Targeting Chemokine Receptor CXCR4

    PubMed Central

    Vincenzi, Marian; Costantini, Susan; Scala, Stefania; Tesauro, Diego; Accardo, Antonella; Leone, Marilisa; Colonna, Giovanni; Guillon, Jean; Portella, Luigi; Trotta, Anna Maria; Ronga, Luisa; Rossi, Filomena

    2015-01-01

    This work reports on the design and the synthesis of two short linear peptides both containing a few amino acids with disorder propensity and an allylic ester group at the C-terminal end. Their structural properties were firstly analyzed by means of experimental techniques in solution such as CD and NMR methods that highlighted peptide flexibility. These results were further confirmed by MD simulations that demonstrated the ability of the peptides to assume conformational ensembles. They revealed a network of transient and dynamic H-bonds and interactions with water molecules. Binding assays with a well-known drug-target, i.e., the CXCR4 receptor, were also carried out in an attempt to verify their biological function and the possibility to use the assays to develop new specific targets for CXCR4. Moreover, our data indicate that these peptides represent useful tools for molecular recognition processes in which a flexible conformation is required in order to obtain an interaction with a specific target. PMID:26030674

  4. Screening and identification of a specific peptide for targeting hypoxic hepatoma cells.

    PubMed

    Liu, Yiming; Xia, Xiangwen; Wang, Yong; Li, Xin; Zhou, Guofeng; Liang, Huiming; Feng, Gansheng; Zheng, Chuansheng

    2016-08-01

    The biological behaviors of residual hepatoma cells after transarterial embolization therapy, which exist in a hypoxic or even anaerobic tumor microenvironment, differ from the tumor cells under normoxic conditions. This study aimed to use a phage display peptide library for in vivo and in vitro screening to obtain a peptide which could specifically bind to hypoxic hepatoma cells, allowing further targeted diagnosis and treatment for liver cancer. In this study, hypoxic hepatoma cells HepG2 (targeted cells), and normal liver cells HL-7702 (control cells), were utilized to perform three rounds of in vitro screening using a phage-displayed 7-mer peptide library. In addition, hypoxic HepG2 were subcutaneously injected into nude mice to establish a hepatocarcinoma model, followed by performing three rounds of in vivo screening on the phages identified from the in vitro screening. The products from the screening were further identified using ELISA and immunofluorescence staining on cells and tissues. The results indicated that the P11 positive clone had the highest binding effect with hypoxic hepatoma cells. The sequence of the exogenous insert fragment of P11 positive clone was obtained by sequencing: GSTSFSK. The binding assay indicated that GSTSFSK could specifically bind to hypoxic hepatoma cells and hepatocarcinoma tissues. This 7-mer peptide has the potential to be developed as an useful molecular to the targeting diagnosis and treatment of residual hepatoma cells after transarterial chemoembolization. PMID:27381416

  5. In Vivo Detection of Human TRPV6-Rich Tumors with Anti-Cancer Peptides Derived from Soricidin

    PubMed Central

    Bowen, Chris V.; DeBay, Drew; Ewart, H. Stephen; Gallant, Pamela; Gormley, Sean; Ilenchuk, T. Toney; Iqbal, Umar; Lutes, Tyler; Martina, Marzia; Mealing, Geoffrey; Merkley, Nadine; Sperker, Sandra; Moreno, Maria J.; Rice, Christopher; Syvitski, Raymond T.; Stewart, John M.

    2013-01-01

    Soricidin is a 54-amino acid peptide found in the paralytic venom of the northern short-tailed shrew (Blarina brevicauda) and has been found to inhibit the transient receptor potential of vallinoid type 6 (TRPV6) calcium channels. We report that two shorter peptides, SOR-C13 and SOR-C27, derived from the C-terminus of soricidin, are high-affinity antagonists of human TRPV6 channels that are up-regulated in a number of cancers. Herein, we report molecular imaging methods that demonstrate the in vivo diagnostic potential of SOR-C13 and SOR-C27 to target tumor sites in mice bearing ovarian or prostate tumors. Our results suggest that these novel peptides may provide an avenue to deliver diagnostic and therapeutic reagents directly to TRPV6-rich tumors and, as such, have potential applications for a range of carcinomas including ovarian, breast, thyroid, prostate and colon, as well as certain leukemia's and lymphomas. PMID:23554944

  6. In vivo detection of human TRPV6-rich tumors with anti-cancer peptides derived from soricidin.

    PubMed

    Bowen, Chris V; DeBay, Drew; Ewart, H Stephen; Gallant, Pamela; Gormley, Sean; Ilenchuk, T Toney; Iqbal, Umar; Lutes, Tyler; Martina, Marzia; Mealing, Geoffrey; Merkley, Nadine; Sperker, Sandra; Moreno, Maria J; Rice, Christopher; Syvitski, Raymond T; Stewart, John M

    2013-01-01

    Soricidin is a 54-amino acid peptide found in the paralytic venom of the northern short-tailed shrew (Blarina brevicauda) and has been found to inhibit the transient receptor potential of vallinoid type 6 (TRPV6) calcium channels. We report that two shorter peptides, SOR-C13 and SOR-C27, derived from the C-terminus of soricidin, are high-affinity antagonists of human TRPV6 channels that are up-regulated in a number of cancers. Herein, we report molecular imaging methods that demonstrate the in vivo diagnostic potential of SOR-C13 and SOR-C27 to target tumor sites in mice bearing ovarian or prostate tumors. Our results suggest that these novel peptides may provide an avenue to deliver diagnostic and therapeutic reagents directly to TRPV6-rich tumors and, as such, have potential applications for a range of carcinomas including ovarian, breast, thyroid, prostate and colon, as well as certain leukemia's and lymphomas. PMID:23554944

  7. VY6, a β-lactoglobulin-derived peptide, altered metabolic lipid pathways in the zebra fish liver.

    PubMed

    Mohammed-Geba, K; Arrutia, F; Do-Huu, H; Borrell, Y J; Galal-Khallaf, A; Ardura, A; Riera, Francisco A; Garcia-Vazquez, Eva

    2016-04-20

    Today enormous research efforts are being focused on alleviating the massive, adverse effects of obesity. Short peptides are key targets for research as they can be generated from natural proteins, like milk. Here we conducted trypsinogen digestion of beta-lactoglobulin (β-lg), the major mammalian milk protein, to release the hexamer VY6. It was assayed in vivo for its activities on lipid metabolism using zebra fish as a vertebrate model. Zebra fish juveniles were injected with two different doses of the peptide: 100 and 800 μg per g fish and left for 5 days before sacrificing. Lipid measurements showed significant reduction in liver triglycerides and free cholesterol, as well as increased liver HDL cholesterol. Dose-dependent increases of the mRNA levels of the genes coding for the enzymes acyl coenzyme A oxidase 1 (acox1) and lipoprotein lipase (lpl) were also found. The complete results suggest significant anti-obesity activity of the β-lg-derived VY6 peptide. Its use as a nutraceutical has been discussed. PMID:26983953

  8. Heat shock protein-mediated cell penetration and cytosolic delivery of macromolecules by a telomerase-derived peptide vaccine.

    PubMed

    Lee, Seoung-Ae; Kim, Bo-Ram; Kim, Bu-Kyung; Kim, Dong-Won; Shon, Won-Jun; Lee, Na-Rae; Inn, Kyung-Soo; Kim, Bum-Joon

    2013-10-01

    A reverse-transcriptase-subunit of telomerase (hTERT) derived peptide, GV1001, has been developed as a vaccine against various cancers. Here, we report an unexpected function of GV1001 as a cell-penetrating peptide (CPP). GV1001 was delivered into a variety of cells including various cancer cell lines and primary blood cells. Moreover, the delivered GV1001 was predominantly located in the cytoplasm of the cells, while a significantly higher proportion of TAT peptide was localized in the nucleus. Macromolecules such as proteins, DNA and siRNA, which were linked to GV1001 by direct covalent conjugation or non-covalent complexation through poly-lysine, were successfully delivered into cells, indicating that GV1001 can be used as a carrier for macromolecules. Expression of the delivered DNA, and lowered expression of the target gene by the delivered siRNA, suggest the potential therapeutic use of GV1001. Pull-down analysis identified Heat Shock Protein 90 (HSP90) and 70 (HSP70) as GV1001 interacting proteins. Treatment of Anti-HSP90 and HSP70 antibodies lowered the internalization of GV1001, indicating that the interaction is critical for the efficient internalization of GV1001. Collectively, the results of this study suggest the pharmaceutical potential of GV1001, already proven safe in clinical trials, as a carrier for the delivery of macromolecular therapeutics into cells, in addition to its own anti-cancer activity. PMID:23827187

  9. Scanning of 16S Ribosomal RNA for Peptide Nucleic Acid Targets.

    PubMed

    Górska, Anna; Markowska-Zagrajek, Agnieszka; Równicki, Marcin; Trylska, Joanna

    2016-08-25

    We have designed a protocol and server to aid in the search for putative binding sites in 16S rRNA that could be targeted by peptide nucleic acid oligomers. Various features of 16S rRNA were considered to score its regions as potential targets for sequence-specific binding that could result in inhibition of ribosome function. Specifically, apart from the functional importance of a particular rRNA region, we calculated its accessibility, flexibility, energetics of strand invasion by an oligomer, as well as similarity to human rRNA. To determine 16S rRNA flexibility in the ribosome context, we performed all-atom molecular dynamics simulations of the 30S subunit in explicit solvent. We proposed a few 16S RNA target sites, and one of them was tested experimentally to verify inhibition of bacterial growth by a peptide nucleic acid oligomer. PMID:27105576

  10. 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.

  11. Amyloid beta peptides do not form peptide-derived free radicals spontaneously, but can enhance metal-catalyzed oxidation of hydroxylamines to nitroxides.

    PubMed

    Dikalov, S I; Vitek, M P; Maples, K R; Mason, R P

    1999-04-01

    Amyloid beta (Abeta) peptides play an important role in the pathogenesis of Alzheimer's disease. Free radical generation by Abeta peptides was suggested to be a key mechanism of their neurotoxicity. Reports that neurotoxic free radicals derived from Abeta-(1-40) and Abeta-(25-35) peptides react with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) to form a PBN/.Abeta peptide radical adduct with a specific triplet ESR signal assert that the peptide itself was the source of free radicals. We now report that three Abeta peptides, Abeta-(1-40), Abeta-(25-35), and Abeta-(40-1), do not yield radical adducts with PBN from the Oklahoma Medical Research Foundation (OMRF). In contrast to OMRF PBN, incubation of Sigma PBN in phosphate buffer without Abeta peptides produced a three-line ESR spectrum. It was shown that this nitroxide is di-tert-butylnitroxide and is formed in the Sigma PBN solution as a result of transition metal-catalyzed auto-oxidation of the respective hydroxylamine present as an impurity in the Sigma PBN. Under some conditions, incubation of PBN from Sigma with Abeta-(1-40) or Abeta-(25-35) can stimulate the formation of di-tert-butylnitroxide. It was shown that Abeta peptides enhanced oxidation of cyclic hydroxylamine 1-hydroxy-4-oxo-2,2,6, 6-tetramethylpiperidine (TEMPONE-H), which was strongly inhibited by the treatment of phosphate buffer with Chelex-100. It was shown that ferric and cupric ions are effective oxidants of TEMPONE-H. The data obtained allow us to conclude that under some conditions toxic Abeta peptides Abeta-(1-40) and Abeta-(25-35) enhance metal-catalyzed oxidation of hydroxylamine derivatives, but do not spontaneously form peptide-derived free radicals. PMID:10092619

  12. Angiotensin I Converting Enzyme Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata

    PubMed Central

    Furuta, Tomoe; Miyabe, Yoshikatsu; Yasui, Hajime; Kinoshita, Yasunori; Kishimura, Hideki

    2016-01-01

    We examined the inhibitory activity of angiotensin I converting enzyme (ACE) in protein hydrolysates from dulse, Palmaria palmata. The proteins extracted from dulse were mainly composed of phycoerythrin (PE) followed by phycocyanin (PC) and allophycocyanin (APC). The dulse proteins showed slight ACE inhibitory activity, whereas the inhibitory activity was extremely enhanced by thermolysin hydrolysis. The ACE inhibitory activity of hydrolysates was hardly affected by additional pepsin, trypsin and chymotrypsin treatments. Nine ACE inhibitory peptides (YRD, AGGEY, VYRT, VDHY, IKGHY, LKNPG, LDY, LRY, FEQDWAS) were isolated from the hydrolysates by reversed-phase high-performance liquid chromatography (HPLC), and it was demonstrated that the synthetic peptide LRY (IC50: 0.044 μmol) has remarkably high ACE inhibitory activity. Then, we investigated the structural properties of dulse phycobiliproteins to discuss the origin of dulse ACE inhibitory peptides. Each dulse phycobiliprotein possesses α-subunit (Mw: 17,477–17,638) and β-subunit (Mw: 17,455–18,407). The sequences of YRD, AGGEY, VYRT, VDHY, LKNPG and LDY were detected in the primary structure of PE α-subunit, and the LDY also exists in the APC α- and β-subunits. In addition, the LRY sequence was found in the β-subunits of PE, PC and APC. From these results, it was suggested that the dulse ACE inhibitory peptides were derived from phycobiliproteins, especially PE. To make sure the deduction, we carried out additional experiment by using recombinant PE. We expressed the recombinant α- and β-subunits of PE (rPEα and rPEβ, respectively), and then prepared their peptides by thermolysin hydrolysis. As a result, these peptides showed high ACE inhibitory activities (rPEα: 94.4%; rPEβ: 87.0%). Therefore, we concluded that the original proteins of dulse ACE inhibitory peptides were phycobiliproteins. PMID:26861357

  13. Angiotensin I Converting Enzyme Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata.

    PubMed

    Furuta, Tomoe; Miyabe, Yoshikatsu; Yasui, Hajime; Kinoshita, Yasunori; Kishimura, Hideki

    2016-02-01

    We examined the inhibitory activity of angiotensin I converting enzyme (ACE) in protein hydrolysates from dulse, Palmaria palmata. The proteins extracted from dulse were mainly composed of phycoerythrin (PE) followed by phycocyanin (PC) and allophycocyanin (APC). The dulse proteins showed slight ACE inhibitory activity, whereas the inhibitory activity was extremely enhanced by thermolysin hydrolysis. The ACE inhibitory activity of hydrolysates was hardly affected by additional pepsin, trypsin and chymotrypsin treatments. Nine ACE inhibitory peptides (YRD, AGGEY, VYRT, VDHY, IKGHY, LKNPG, LDY, LRY, FEQDWAS) were isolated from the hydrolysates by reversed-phase high-performance liquid chromatography (HPLC), and it was demonstrated that the synthetic peptide LRY (IC50: 0.044 μmol) has remarkably high ACE inhibitory activity. Then, we investigated the structural properties of dulse phycobiliproteins to discuss the origin of dulse ACE inhibitory peptides. Each dulse phycobiliprotein possesses α-subunit (Mw: 17,477-17,638) and β-subunit (Mw: 17,455-18,407). The sequences of YRD, AGGEY, VYRT, VDHY, LKNPG and LDY were detected in the primary structure of PE α-subunit, and the LDY also exists in the APC α- and β-subunits. In addition, the LRY sequence was found in the β-subunits of PE, PC and APC. From these results, it was suggested that the dulse ACE inhibitory peptides were derived from phycobiliproteins, especially PE. To make sure the deduction, we carried out additional experiment by using recombinant PE. We expressed the recombinant α- and β-subunits of PE (rPEα and rPEβ, respectively), and then prepared their peptides by thermolysin hydrolysis. As a result, these peptides showed high ACE inhibitory activities (rPEα: 94.4%; rPEβ: 87.0%). Therefore, we concluded that the original proteins of dulse ACE inhibitory peptides were phycobiliproteins. PMID:26861357

  14. Cementomimetics—constructing a cementum-like biomineralized microlayer via amelogenin-derived peptides

    PubMed Central

    Gungormus, Mustafa; Oren, Ersin E; Horst, Jeremy A; Fong, Hanson; Hnilova, Marketa; Somerman, Martha J; Snead, Malcolm L; Samudrala, Ram; Tamerler, Candan; Sarikaya, Mehmet

    2012-01-01

    Cementum is the outer-, mineralized-tissue covering the tooth root and an essential part of the system of periodontal tissue that anchors the tooth to the bone. Periodontal disease results from the destructive behavior of the host elicited by an infectious biofilm adhering to the tooth root and left untreated, may lead to tooth loss. We describe a novel protocol for identifying peptide sequences from native proteins with the potential to repair damaged dental tissues by controlling hydroxyapatite biomineralization. Using amelogenin as a case study and a bioinformatics scoring matrix, we identified regions within amelogenin that are shared with a set of hydroxyapatite-binding peptides (HABPs) previously selected by phage display. One 22-amino acid long peptide regions referred to as amelogenin-derived peptide 5 (ADP5) was shown to facilitate cell-free formation of a cementum-like hydroxyapatite mineral layer on demineralized human root dentin that, in turn, supported attachment of periodontal ligament cells in vitro. Our findings have several implications in peptide-assisted mineral formation that mimic biomineralization. By further elaborating the mechanism for protein control over the biomineral formed, we afford new insights into the evolution of protein–mineral interactions. By exploiting small peptide domains of native proteins, our understanding of structure–function relationships of biomineralizing proteins can be extended and these peptides can be utilized to engineer mineral formation. Finally, the cementomimetic layer formed by ADP5 has the potential clinical application to repair diseased root surfaces so as to promote the regeneration of periodontal tissues and thereby reduce the morbidity associated with tooth loss. PMID:22743342

  15. Peptide IDR-1018: modulating the immune system and targeting bacterial biofilms to treat antibiotic-resistant bacterial infections.

    PubMed

    Mansour, Sarah C; de la Fuente-Núñez, César; Hancock, Robert E W

    2015-05-01

    Host defense (antimicrobial) peptides, produced by all complex organisms, typically contain an abundance of positively charged and hydrophobic amino acid residues. A small synthetic peptide termed innate defense regulator (IDR-)1018 was derived by substantial modification of the bovine neutrophil host defense peptide bactenecin. Here, we review its intriguing properties that include anti-infective, anti-inflammatory, wound healing, and anti-biofilm activities. It was initially developed as an immune modulator with an ability to selectively enhance chemokine production and polarize cellular differentiation while suppressing/balancing the pro-inflammatory response. In this regard, it has demonstrated in vivo activity in murine models including enhancement of wound healing and an ability to protect against Staphylococcus aureus, multidrug resistant Mycobacterium tuberculosis, herpes virus, and inflammatory disorders, including cerebral malaria and neuronal damage in a pre-term birth model. More recently, IDR-1018 was shown, in a broad-spectrum fashion, to selectively target bacterial biofilms, which are adaptively resistant to many antibiotics and represent the most common growth state of bacteria in human infections. Furthermore, IDR-1018 demonstrated synergy with conventional antibiotics to both prevent biofilm formation and treat pre-existing biofilms. These data are consistent with a strong potential as an adjunctive therapy against antibiotic-resistant infections. PMID:25358509

  16. Prokaryotic expression and antimicrobial mechanism of XPF-St7-derived α-helical peptides.

    PubMed

    Yi, Tonghui; Huang, Yibing; Chen, Yuxin

    2015-01-01

    XPF-St7 (GLLSNVAGLLKQFAKGGVNAVLNPK) is an antimicrobial peptide isolated from Silurana tropicalis. We developed an α-helical segment of XPF-St7 termed as XPF2. Using the XPF2 as a framework, we increased the positive net charge of XPF2 by amino acid substitutions, and thus obtained two novel antimicrobial peptides XPF4 and XPF6. These were each fused with an ubiquitin tag and successfully expressed in Escherichia coli. This ubiquitin fusion system may present a viable alternative for industrial production of antimicrobial peptides. XPF4 and XPF6 showed much better overall antimicrobial activity against both Gram-negative and Gram-positive bacteria than XPF2. The therapeutic index of XPF4 and XPF6 was 5.6-fold and 6.7-fold of XPF2, respectively. Bacterial cell membrane permeabilization and genomic DNA interaction assays were utilized to explore the mechanism of action of XPF serial peptides. The results revealed that the target of these antimicrobial peptides was the bacterial cytoplasmic membrane. PMID:25421112

  17. 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

  18. Integrin-Targeting Knottin Peptide-Drug Conjugates Are Potent Inhibitors of Tumor Cell Proliferation.

    PubMed

    Cox, Nick; Kintzing, James R; Smith, Mark; Grant, Gerald A; Cochran, Jennifer R

    2016-08-16

    Antibody-drug conjugates (ADCs) offer increased efficacy and reduced toxicity compared to systemic chemotherapy. Less attention has been paid to peptide-drug delivery, which has the potential for increased tumor penetration and facile synthesis. We report a knottin peptide-drug conjugate (KDC) and demonstrate that it can selectively deliver gemcitabine to malignant cells expressing tumor-associated integrins. This KDC binds to tumor cells with low-nanomolar affinity, is internalized by an integrin-mediated process, releases its payload intracellularly, and is a highly potent inhibitor of brain, breast, ovarian, and pancreatic cancer cell lines. Notably, these features enable this KDC to bypass a gemcitabine-resistance mechanism found in pancreatic cancer cells. This work expands the therapeutic relevance of knottin peptides to include targeted drug delivery, and further motivates efforts to expand the drug-conjugate toolkit to include non-antibody protein scaffolds. PMID:27304709

  19. Purification and identification of lipolysis-stimulating peptides derived from enzymatic hydrolysis of soy protein.

    PubMed

    Tsou, May-June; Kao, Fuh-Juin; Lu, Hsi-Chi; Kao, Hao-Chun; Chiang, Wen-Dee

    2013-06-01

    The aim of this study was to purify and identify lipolysis-stimulating peptides derived from Flavourzyme®-soy protein isolate (SPI) hydrolysate (F-SPIH). Glycerol release was employed as a marker for lipolysis in 3T3-L1 adipocytes. A higher glycerol release represents a better lipolysis-stimulating activity. The peptide fraction with highest glycerol release obtained from F-SPIH fractionated by sequential ultrafiltration membranes was further purified using gel filtration chromatography and two steps of reverse-phase high-performance liquid chromatography. The peptides were identified using liquid chromatography-tandem mass spectrometry (LC/MS/MS). Three lipolysis-stimulating peptides were obtained, and the amino acid sequences were ILL, LLL and VHVV, respectively. The in vitro effect of gastrointestinal proteases on lipolysis-stimulating activity of synthetic ILL, LLL and VHVV, respectively, was also investigated. The result suggested that the gastrointestinal protease did not affect lipolysis-stimulating activity of the three novel peptides, which reveals their potential to act as anti-obesity ingredients. PMID:23411267

  20. Thiol-disulfide exchange in peptides derived from human growth hormone.

    PubMed

    Chandrasekhar, Saradha; Epling, Daniel E; Sophocleous, Andreas M; Topp, Elizabeth M

    2014-04-01

    Disulfide bonds stabilize proteins by cross-linking distant regions into a compact three-dimensional structure. They can also participate in hydrolytic and oxidative pathways to form nonnative disulfide bonds and other reactive species. Such covalent modifications can contribute to protein aggregation. Here, we present experimental data for the mechanism of thiol-disulfide exchange in tryptic peptides derived from human growth hormone in aqueous solution. Reaction kinetics was monitored to investigate the effect of pH (6.0-10.0), temperature (4-50°C), oxidation suppressants [ethylenediaminetetraacetic acid (EDTA) and N2 sparging], and peptide secondary structure (amide cyclized vs. open form). The concentrations of free thiol containing peptides, scrambled disulfides, and native disulfide-linked peptides generated via thiol-disulfide exchange and oxidation reactions were determined using reverse-phase HPLC and liquid chromatography-mass spectrometry. Concentration versus time data were fitted to a mathematical model using nonlinear least squares regression analysis. At all pH values, the model was able to fit the data with R(2) ≥ 0.95. Excluding oxidation suppressants (EDTA and N2 sparging) resulted in an increase in the formation of scrambled disulfides via oxidative pathways but did not influence the intrinsic rate of thiol-disulfide exchange. In addition, peptide secondary structure was found to influence the rate of thiol-disulfide exchange. PMID:24549831

  1. Characterization of Spontaneous Immune Responses against Long Peptides Derived from Bcl-X(L) in Cancer Patients Using Elispot

    PubMed Central

    Larsen, Stine Kiaer; Hansen, Morten; Svane, Inge Marie; Straten, Per Thor; Andersen, Mads Hald

    2012-01-01

    In recent years we and others have used the ELISPOT assay successfully to identify novel tumor antigens by the characterization of spontaneous HLA class I restricted immune responses against a number of minimal 9–10 amino acid long peptide epitopes. In the present study, we examined the capability of using longer peptides when scrutinizing Peripheral Blood Mononuclear Cells (PMBC) from melanoma patients for spontaneous immunity by means of ELISPOT IFN-γ secretion assay. To this end, we examined PBMC for the presence of specific T-cell responses against long peptides derived from the tumor associated antigen BCL-X(L). The protein product of the larger BCL-X(L) differs from Bcl-X(S) protein by an inserted region (amino acids 126–188). Thus, we scrutinized eight long peptides covering this inserted region for spontaneous immunity. The peptides were overlapping and consisted of 20–23 amino acids. PBMC were pre-stimulated with peptide-pulsed autologous dendritic cells (DC) and subjected to the IFN-γ ELISPOT assay. Four of the BCL-X(L) derived peptides elicited very frequent responses in several patients. Additionally, in all patients responses against more than one of the peptides could be detected. In conclusion several long BCL-X(L) derived peptide epitopes exist, which may be used in anti-cancer immunity. Furthermore, the ELISPOT assay offers an attractive and sensitive method for the characterization of spontaneous immune reactivity against long peptides. PMID:24710413

  2. The Polyomavirus BK Large T-Antigen-Derived Peptide Elicits an HLA-DR Promiscuous and Polyfunctional CD4+ T-Cell Response▿

    PubMed Central

    Ramaswami, Bala; Popescu, Iulia; Macedo, Camila; Luo, Chunqing; Shapiro, Ron; Metes, Diana; Chalasani, Geetha; Randhawa, Parmjeet S.

    2011-01-01

    BK virus (BKV) nephropathy and hemorrhagic cystitis are increasingly recognized causes of disease in renal and hematopoietic stem cell transplant recipients, respectively. Functional characterization of the immune response to BKV is important for clinical diagnosis, prognosis, and vaccine design. A peptide mix (PepMix) and overlapping (OPP) or random (RPP) peptide pools derived from BKV large T antigen (LTA) were used to restimulate 14-day-expanded peripheral blood mononuclear cells (PBMC) from 27 healthy control subjects in gamma interferon (IFN-γ)-specific enzyme-linked immunospot (ELISPOT) assays. A T-cell response to LTA PepMix was detected in 15/27 subjects. A response was frequently observed with peptides derived from the helicase domain (9/15 subjects), while the DNA binding and host range domains were immunologically inert (0/15 subjects). For all nine subjects who responded to LTA peptide pools, the immune response could be explained largely by a 15-mer peptide designated P313. P313-specific CD4+ T-cell clones demonstrated (i) stringent LTA peptide specificity; (ii) promiscuous recognition in the context of HLA-DR alleles; (iii) cross recognition of homologous peptides from the polyomavirus simian virus 40 (SV40); (iv) an effector memory phenotype, CD107a expression, and intracellular production of IFN-γ and tumor necrosis factor alpha (TNF-α); (v) cytotoxic activity in a chromium release assay; and (vi) the ability to directly present cognate antigen to autologous T cells. In conclusion, T-cell-mediated immunity to BKV in healthy subjects is associated with a polyfunctional population of CD4+ T cells with dual T-helper and T-cytotoxic properties. HLA class II promiscuity in antigen presentation makes the targeted LTA peptide sequence a suitable candidate for inclusion in immunotherapy protocols. PMID:21367979

  3. Targeted Melanoma Imaging and Therapy with Radiolabeled Alpha-Melanocyte Stimulating Hormone Peptide Analogues

    PubMed Central

    Quinn, Thomas; Zhang, Xiuli; Miao, Yubin

    2010-01-01

    Radiolabeled alpha-melanocyte stimulating hormone (α-MSH) analogues have been used to define the expression, affinity and function of the melanocortin-1 receptor (MC1-R). The MC1-R is one of a family of five G-protein linker receptors, which is primarily involved in regulation of skin pigmentation. Over-expression of the MC1-R on melanoma tumor cells has made it an attractive target for the development of α-MSH peptide based imaging and therapeutic agents. Initially, the native α-MSH peptide was radiolabeled directly, but it suffered from low specific activity and poor stability. The addition of non-natural amino acids yielded α-MSH analogues with greater MC-1R affinity and stability. Furthermore, peptide cyclization via disulfide and lactam bond formation as well as site-specific metal coordination resulted in additional gains in receptor affinity and peptide stability in vitro and in vivo. Radiochemical stability of the α-MSH analogues was improved through the conjugation of metal chelators to the peptide’s N-terminus or lysine residues for radionuclide coordination. In vitro cell binding studies demonstrated that the radiolabeled α-MSH analogues had low to subnanomolar affinities for the MC1-R. Biodistribution and imaging studies in the B16 mouse melanoma modeled showed rapid tumor uptake of the radiolabeled peptides, with the cyclic peptides demonstrating prolonged tumor retention. Cyclic α-MSH analogues labeled with beta and alpha emitting radionuclides demonstrated melanoma therapeutic efficacy in the B16 melanoma mouse model. Strong pre-clinical imaging and therapy data highlight the clinical potential use of radiolabeled α-MSH peptides for melanoma imaging and treatment of disseminated disease. PMID:20467398

  4. Identification of a peptide specifically targeting ovarian cancer by the screening of a phage display peptide library

    PubMed Central

    WANG, LEDAN; HU, YUE; LI, WENJU; WANG, FAN; LU, XIAOSHENG; HAN, XUEYING; LV, JIEQIANG; CHEN, JIE

    2016-01-01

    Ovarian cancer is the most common cause of cancer-associated mortality in terms of gynecological malignancies, and is difficult to diagnose due to the absence of reliable biomarkers. To identify ovarian cancer-specific biomarkers, the present study used a Ph.D.-7™ Phage Display Peptide Library to screen for ligands that selectively target HO-8910 ovarian cancer cells. Following 5 rounds of biopanning, the phage clone P2 was selected by enzyme-linked immunosorbent assay and DNA sequencing, and its characteristics were additionally validated by immunofluorescence and immunohistochemical assays. The results revealed the positive phage were enriched 92-fold following 5 rounds of biopanning, and the DNA sequence AAC CCG ATG ATT CGC CGC CAG (amino acid sequence, NPMIRRQ) was repeated most frequently (phage clones, P2, P3, P15, P30 and P54). Immunofluorescence and immunohistochemical assays revealed that the phage clone P2 was able to bind to ovarian cancer cells and tissues, and not those of cervical cancer. In conclusion, the peptide NPMIRRQ may be a potential agent for the diagnosis of ovarian cancer.

  5. Peptides derived from CXCL8 based on in silico analysis inhibit CXCL8 interactions with its receptor CXCR1

    NASA Astrophysics Data System (ADS)

    Jiang, Shinn-Jong; Liou, Je-Wen; Chang, Chun-Chun; Chung, Yi; Lin, Lee-Fong; Hsu, Hao-Jen

    2015-12-01

    Chemokine CXCL8 is crucial for regulation of inflammatory and immune responses via activating its cognate receptor CXCR1. In this study, molecular docking and binding free energy calculations were combined to predict the initial binding event of CXCL8 to CXCR1 for peptide drug design. The simulations reveal that in the initial binding, the N-loop of CXCL8 interacts with the N-terminus of CXCR1, which is dominated by electrostatic interactions. The derived peptides from the binding region of CXCL8 are synthesized for further confirmation. Surface plasmon resonance analyses indicate that the CXCL8 derived peptide with 14 residues is able to bind to the receptor CXCR1 derived peptide with equilibrium KD of 252 μM while the peptide encompassing a CXCL8 K15A mutation hardly binds to CXCR1 derived peptide (KD = 1553 μM). The cell experiments show that the designed peptide inhibits CXCL8-induced and LPS-activated monocytes adhesion and transmigration. However, when the peptides were mutated on two lysine residues (K15 and K20), the inhibition effects were greatly reduced indicating these two amino acids are key residues for the initial binding of CXCL8 to CXCR1. This study demonstrates that in silico prediction based functional peptide design can be effective for developing anti-inflammation drugs.

  6. Peptides derived from CXCL8 based on in silico analysis inhibit CXCL8 interactions with its receptor CXCR1.

    PubMed

    Jiang, Shinn-Jong; Liou, Je-Wen; Chang, Chun-Chun; Chung, Yi; Lin, Lee-Fong; Hsu, Hao-Jen

    2015-01-01

    Chemokine CXCL8 is crucial for regulation of inflammatory and immune responses via activating its cognate receptor CXCR1. In this study, molecular docking and binding free energy calculations were combined to predict the initial binding event of CXCL8 to CXCR1 for peptide drug design. The simulations reveal that in the initial binding, the N-loop of CXCL8 interacts with the N-terminus of CXCR1, which is dominated by electrostatic interactions. The derived peptides from the binding region of CXCL8 are synthesized for further confirmation. Surface plasmon resonance analyses indicate that the CXCL8 derived peptide with 14 residues is able to bind to the receptor CXCR1 derived peptide with equilibrium KD of 252 μM while the peptide encompassing a CXCL8 K15A mutation hardly binds to CXCR1 derived peptide (KD = 1553 μM). The cell experiments show that the designed peptide inhibits CXCL8-induced and LPS-activated monocytes adhesion and transmigration. However, when the peptides were mutated on two lysine residues (K15 and K20), the inhibition effects were greatly reduced indicating these two amino acids are key residues for the initial binding of CXCL8 to CXCR1. This study demonstrates that in silico prediction based functional peptide design can be effective for developing anti-inflammation drugs. PMID:26689258

  7. Peptides derived from CXCL8 based on in silico analysis inhibit CXCL8 interactions with its receptor CXCR1

    PubMed Central

    Jiang, Shinn-Jong; Liou, Je-Wen; Chang, Chun-Chun; Chung, Yi; Lin, Lee-Fong; Hsu, Hao-Jen

    2015-01-01

    Chemokine CXCL8 is crucial for regulation of inflammatory and immune responses via activating its cognate receptor CXCR1. In this study, molecular docking and binding free energy calculations were combined to predict the initial binding event of CXCL8 to CXCR1 for peptide drug design. The simulations reveal that in the initial binding, the N-loop of CXCL8 interacts with the N-terminus of CXCR1, which is dominated by electrostatic interactions. The derived peptides from the binding region of CXCL8 are synthesized for further confirmation. Surface plasmon resonance analyses indicate that the CXCL8 derived peptide with 14 residues is able to bind to the receptor CXCR1 derived peptide with equilibrium KD of 252 μM while the peptide encompassing a CXCL8 K15A mutation hardly binds to CXCR1 derived peptide (KD = 1553 μM). The cell experiments show that the designed peptide inhibits CXCL8-induced and LPS-activated monocytes adhesion and transmigration. However, when the peptides were mutated on two lysine residues (K15 and K20), the inhibition effects were greatly reduced indicating these two amino acids are key residues for the initial binding of CXCL8 to CXCR1. This study demonstrates that in silico prediction based functional peptide design can be effective for developing anti-inflammation drugs. PMID:26689258

  8. Chondroitin sulfate derived theranostic nanoparticles for targeted drug delivery.

    PubMed

    Varghese, Oommen P; Liu, Jianping; Sundaram, Karthi; Hilborn, Jöns; Oommen, Oommen P

    2016-08-16

    Glycosaminoglycan derived nanoparticles are a promising delivery system owing to their unique tumour targeting ability. Exploiting fluorescein for inducing amphiphilicity in these biopolymers provides inherent imaging and drug stabilization capabilities by π-π stacking interactions with aromatic antineoplastic agents. This offers a versatile and highly customizable nanocarrier with narrow size distribution and high drug loading efficiency (80%) with sustained drug release. PMID:27431007

  9. Inhibition of dengue virus entry into target cells using synthetic antiviral peptides.

    PubMed

    Alhoot, Mohammed Abdelfatah; Rathinam, Alwin Kumar; Wang, Seok Mui; Manikam, Rishya; Sekaran, Shamala Devi

    2013-01-01

    Despite the importance of DENV as a human pathogen, there is no specific treatment or protective vaccine. Successful entry into the host cells is necessary for establishing the infection. Recently, the virus entry step has become an attractive therapeutic strategy because it represents a barrier to suppress the onset of the infection. Four putative antiviral peptides were designed to target domain III of DENV-2 E protein using BioMoDroid algorithm. Two peptides showed significant inhibition of DENV when simultaneously incubated as shown by plaque formation assay, RT-qPCR, and Western blot analysis. Both DET4 and DET2 showed significant inhibition of virus entry (84.6% and 40.6% respectively) using micromolar concentrations. Furthermore, the TEM images showed that the inhibitory peptides caused structural abnormalities and alteration of the arrangement of the viral E protein, which interferes with virus binding and entry. Inhibition of DENV entry during the initial stages of infection can potentially reduce the viremia in infected humans resulting in prevention of the progression of dengue fever to the severe life-threatening infection, reduce the infected vector numbers, and thus break the transmission cycle. Moreover these peptides though designed against the conserved region in DENV-2 would have the potential to be active against all the serotypes of dengue and might be considered as Hits to begin designing and developing of more potent analogous peptides that could constitute as promising therapeutic agents for attenuating dengue infection. PMID:23630436

  10. Medicinal Chemistry of ATP Synthase: A Potential Drug Target of Dietary Polyphenols and Amphibian Antimicrobial Peptides

    PubMed Central

    Ahmad, Zulfiqar; Laughlin, Thomas F.

    2015-01-01

    In this review we discuss the inhibitory effects of dietary polyphenols and amphibian antimicrobial/antitumor peptides on ATP synthase. In the beginning general structural features highlighting catalytic and motor functions of ATP synthase will be described. Some details on the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it an interesting drug target with respect to dietary polyphenols and amphibian antimicrobial peptides will also be reviewed. ATP synthase is known to have distinct polyphenol and peptide binding sites at the interface of α/β subunits. Molecular interaction of polyphenols and peptides with ATP synthase at their respective binding sites will be discussed. Binding and inhibition of other proteins or enzymes will also be covered so as to understand the therapeutic roles of both types of molecules. Lastly, the effects of polyphenols and peptides on the inhibition of Escherichia coli cell growth through their action on ATP synthase will also be presented. PMID:20586714

  11. Inhibition of Influenza Virus Infection by a Novel Antiviral Peptide That Targets Viral Attachment to Cells▿

    PubMed Central

    Jones, Jeremy C.; Turpin, Elizabeth A.; Bultmann, Hermann; Brandt, Curtis R.; Schultz-Cherry, Stacey

    2006-01-01

    Influenza A viruses continue to cause widespread morbidity and mortality. There is an added concern that the highly pathogenic H5N1 influenza A viruses, currently found throughout many parts of the world, represent a serious public health threat and may result in a pandemic. Intervention strategies to halt an influenza epidemic or pandemic are a high priority, with an emphasis on vaccines and antiviral drugs. In these studies, we demonstrate that a 20-amino-acid peptide (EB, for entry blocker) derived from the signal sequence of fibroblast growth factor 4 exhibits broad-spectrum antiviral activity against influenza viruses including the H5N1 subtype in vitro. The EB peptide was protective in vivo, even when administered postinfection. Mechanistically, the EB peptide inhibits the attachment to the cellular receptor, preventing infection. Further studies demonstrated that the EB peptide specifically binds to the viral hemagglutinin protein. This novel peptide has potential value as a reagent to study virus attachment and as a future therapeutic. PMID:17005658

  12. Peptide-Metal Organic Framework Swimmers that Direct the Motion toward Chemical Targets.

    PubMed

    Ikezoe, Yasuhiro; Fang, Justin; Wasik, Tomasz L; Shi, Menglu; Uemura, Takashi; Kitagawa, Susumu; Matsui, Hiroshi

    2015-06-10

    Highly efficient and robust chemical motors are expected for the application in microbots that can selectively swim toward targets and accomplish their tasks in sensing, labeling, and delivering. However, one of major issues for such development is that current artificial swimmers have difficulty controlling their directional motion toward targets like bacterial chemotaxis. To program synthetic motors with sensing capability for the target-directed motion, we need to develop swimmers whose motions are sensitive to chemical gradients in environments. Here we create a new intelligent biochemical swimmer by integrating metal organic frameworks (MOFs) and peptides that can sense toxic heavy metals in solution and swim toward the targets. With the aid of Pb-binding enzymes, the peptide-MOF motor can directionally swim toward PbSe quantum dots (QD) by sensing pH gradient and eventually complete the motion as the swimmer reaches the highest gradient point at the target position in solution. This type of technology could be evolved to miniaturize chemical robotic systems that sense target chemicals and swim toward target locations. PMID:26010172

  13. Bladder tumor-targeted delivery of pro-apoptotic peptide for cancer therapy.

    PubMed

    Jung, Hyun-Kyung; Kim, Soyoun; Park, Rang-Woon; Park, Jae-Yong; Kim, In-San; Lee, Byungheon

    2016-08-10

    The overall prognosis of conventional chemotherapy for the treatment of bladder cancer is poor and reduction of its systemic side effects remains an unsolved issue. Targeted therapy for bladder cancer could improve therapeutic efficacy and reduce side effects. This study investigated a hybrid peptide (named Bld-1-KLA) composed of the CSNRDARRC peptide (Bld-1), which binds to bladder tumor cells, and the d-KLAKLAKKLAKLAK (KLA) peptide, which disrupts mitochondrial membrane and induces apoptotic cell death, as a bladder cancer-targeted therapeutic agent. Bld-1-KLA selectively bound to HT1376 bladder tumor cells and efficiently internalized into the cells but not to other types of tumor and normal cell lines. Bld-1-KLA exerted cytotoxic effects selectively to HT1376 cells (LC50=41.5μM), but not to other types of cells. Pretreatment of cells with Bld-1 inhibited the binding and cytotoxicity by Bld-1-KLA in HT1376 cells. It induced apoptosis of bladder tumor cells, while Bld-1 or KLA alone showed much lesser effect on apoptosis, and was co-localized in mitochondria. Bld-1-KLA was stable up to 24h in serum. In vivo fluorescence imaging showed that homing of Bld-1-KLA in the tumor in HT1376 tumor-bearing nude mice was greater than that of the control peptide-KLA after intravenous injection. Treatment of tumor-bearing mice with Bld-1-KLA, compared to the control peptide-KLA, induced apoptosis of tumor cells and inhibited tumor growth more efficiently. No significant side effects on body weight and the liver and myeloid function were observed in mice treated with Bld-1-KLA. These results suggest that Bld-1-KLA is a promising therapeutic agent for targeted therapy of bladder cancer. PMID:27282414

  14. Preparation of abiotic polymer nanoparticles for sequestration and neutralization of a target peptide toxin.

    PubMed

    Yoshimatsu, Keiichi; Koide, Hiroyuki; Hoshino, Yu; Shea, Kenneth J

    2015-04-01

    Synthetic polymer nanoparticles (NPs) with intrinsic affinity for target biomacromolecules hold great promise in the development of novel tools for biological and biomedical research. We recently reported the design and synthesis of abiotic, synthetic polymer NPs with high intrinsic affinity for a peptide toxin melittin. The NP was selected by screening a small library of NPs (∼100 nm) composed of various ratios of monomers that contain functional groups complementary to the peptide melittin. The selected polymer NP, a co-polymer of acrylic acid (AAc), N-tert-butylacrylamide (TBAm), N-isopropylacrylamide (NIPAm) and N,N'-methylenebisacrylamide (BIS), effectively captures and neutralizes the toxicity of the peptide through a combination of electrostatic and hydrophobic interactions. This protocol describes a step-by-step procedure for the preparation and evaluation of synthetic polymer NPs for sequestration and neutralization of the target peptide toxin. The polymer NPs can be synthesized in a one-step polymerization reaction using commercially available reagents. The polymerization reaction for the synthesis of polymer NPs takes several hours, and the total protocol including subsequent purification and characterization by dynamic light scattering, NMR and toxicity neutralization assays takes 1-2 weeks in total. PMID:25790112

  15. Targeted separation of antibacterial peptide from protein hydrolysate of anchovy cooking wastewater by equilibrium dialysis.

    PubMed

    Tang, Wenting; Zhang, Hui; Wang, Li; Qian, Haifeng; Qi, Xiguang

    2015-02-01

    Anchovy (Engraulis japonicus) cooking wastewater (ACWW) is a by-product resulted from the production of boiled-dried anchovies in the seafood processing industry. In this study, the protein hydrolysate of ACWW (ACWWPH) was found to have antimicrobial activity after enzymatic hydrolysis with Protamex. For the targeted screening of antibacterial peptides, liposomes constructed from Staphylococcus aureus membrane lipids were used in an equilibrium dialysis system. The hydrolysate was further purified by liposome equilibrium dialysis combined with high performance liquid chromatography. The purified antimicrobial peptide (ACWWP1) was determined to be GLSRLFTALK, with a molecular weight of 1104.6622Da. The peptide exhibited no haemolytic activity up to a concentration of 512μg/ml. It displayed a dose-dependent bactericidal effect in reconstituted milk. The change in cell surface hydrophobicity and membrane-permeable action of the purified ACWWP1 may have contributed to the antibacterial effect. This study suggests that liposome equilibrium dialysis can be used for the targeted screening of antimicrobial peptides. PMID:25172690

  16. Mitochondria-Targeted Peptide Reverses Mitochondrial Dysfunction and Cognitive Deficits in Sepsis-Associated Encephalopathy.

    PubMed

    Wu, Jing; Zhang, Mingqiang; Hao, Shuangying; Jia, Ming; Ji, Muhuo; Qiu, Lili; Sun, Xiaoyan; Yang, Jianjun; Li, Kuanyu

    2015-08-01

    Sepsis-associated encephalopathy (SAE) is associated with increased mortality, morbidity, and long-term cognitive impairments. Its pathophysiology remains to be determined and an effective pharmacologic treatment is lacking. The goal of this study was to investigate the effects of the mitochondria-targeted peptide SS-31 on mitochondrial function and cognitive deficits in SAE mice. C57BL/6 male mice were randomly divided into sham, sham + SS-31, cecal ligation and puncture (CLP), and CLP + SS-31 groups. Peptide SS-31 (5 mg/kg) was intraperitoneally administrated immediately after operation and afterwards once daily for six consecutive days. Surviving mice were subjected to behavioral tests and the hippocampus was collected for biochemical analysis 7 days after operation. The results showed that CLP resulted in high mortality rate and cognitive deficits, representative characteristics of SAE. A physiological mechanistic investigation revealed that mitochondrial function of hippocampus was severely impaired, coupled with reactive oxygen species (ROS) generation, triggering neuronal apoptosis and inflammation. Notably, administration of peptide SS-31 protected the integrity of mitochondria, reversed the mitochondrial dysfunction, inhibited the apoptosis resulting from the release of cytochrome c, diminished the response of inflammation, and ultimately reversed the behavior deficits in the SAE mice. In conclusion, our data demonstrate that daily treatment with mitochondria-targeted peptide SS-31 reduces mortality rate and ameliorates cognitive deficits, which is possibly through a mechanism of reversing mitochondrial dysfunction and partial inhibition of neuronal apoptosis and inflammation in the hippocampus of the SAE mice. PMID:25288156

  17. Targeted quantum dots fluorescence probes functionalized with aptamer and peptide for transferrin receptor on tumor cells

    NASA Astrophysics Data System (ADS)

    Zhang, Ming-Zhen; Yu, Rong-Na; Chen, Jun; Ma, Zhi-Ya; Zhao, Yuan-Di

    2012-12-01

    Quantum dots (QDs) fluorescent probes based on oligonucleotide aptamers and peptides with specific molecular recognition have attracted much attention. In this paper, CdSe/ZnS QDs probes for targeted delivery to mouse and human cells using aptamer GS24 and peptide T7 specific to mouse/human transferrin receptors were developed. Capillary electrophoresis analyses indicated that the optimal molar ratios of QDs to aptamer or peptide were 1:5. Fluorescence and confocal microscope imaging revealed QD-GS24 and QD-T7 probes were able to specifically recognize B16 cells and HeLa cells respectively. Quantitative flow cytometry analysis indicated the transportation of QD-GS24 or QD-T7 into cells could be promoted by corresponding free transferrin. Transmission electron microscopy confirmed the uptake of probes in cells and the effective intracellular delivery. MTT assay suggested the cytotoxicity of probes was related to the surface ligand, and aptamer GS24 (or peptide T7) could reduce the cytotoxicity of probes to a certain degree. The study has great significance for preparing QDs fluorescent probes using non-antibody target molecules.

  18. Peptides Targeting the Desmoglein 3 Adhesive Interface Prevent Autoantibody-induced Acantholysis in Pemphigus*

    PubMed Central

    Heupel, Wolfgang-Moritz; Müller, Thomas; Efthymiadis, Athina; Schmidt, Enno; Drenckhahn, Detlev; Waschke, Jens

    2009-01-01

    Pemphigus vulgaris (PV) autoantibodies directly inhibit desmoglein (Dsg) 3-mediated transinteraction. Because cellular signaling also seems to be required for PV pathogenesis, it is important to characterize the role of direct inhibition in pemphigus acantholysis to allow establishment of new therapeutic approaches. Therefore, we modeled the Dsg1 and Dsg3 sequences into resolved cadherin structures and predicted peptides targeting the adhesive interface of both Dsg3 and Dsg1. In atomic force microscopy single molecule experiments, the self-designed cyclic single peptide specifically blocked homophilic Dsg3 and Dsg1 transinteraction, whereas a tandem peptide (TP) consisting of two combined single peptides did not. TP did not directly block binding of pemphigus IgG to their target Dsg antigens but prevented PV-IgG-induced inhibition of Dsg3 transinteraction in cell-free (atomic force microscopy) and cell-based (laser tweezer) experiments, indicating stabilization of Dsg3 bonds. Similarly, PV-IgG-mediated acantholysis and disruption of Dsg3 localization in HaCaT keratinocytes was partially blocked by TP. This is the first evidence that direct inhibition of Dsg3 binding is important for PV pathogenesis and that peptidomimetics stabilizing Dsg transinteraction may provide a novel approach for PV treatment. PMID:19164289

  19. Mitochondria Targeted Peptides Protect Against 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Neurotoxicity

    PubMed Central

    Yang, Lichuan; Zhao, Kesheng; Calingasan, Noel Y.; Luo, Guoxiong; Szeto, Hazel H.

    2009-01-01

    Abstract A large body of evidence suggests that mitochondrial dysfunction and oxidative damage play a role in the pathogenesis of Parkinson's disease (PD). A number of antioxidants have been effective in animal models of PD. We have developed a family of mitochondria-targeted peptides that can protect against mitochondrial swelling and apoptosis (SS peptides). In this study, we examined the ability of two peptides, SS-31 and SS-20, to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice. SS-31 produced dose-dependent complete protection against loss of dopamine and its metabolites in striatum, as well as loss of tyrosine hydroxylase immunoreactive neurons in substantia nigra pars compacta. SS-20, which does not possess intrinsic ability in scavenging reactive oxygen species, also demonstrated significant neuroprotective effects on dopaminergic neurons of MPTP-treated mice. Both SS-31 and SS-20 were very potent (nM) in preventing MPP+ (1-methyl-4-phenylpyridinium)-induced cell death in cultured dopamine cells (SN4741). Studies with isolated mitochondria showed that both SS-31 and SS-20 prevented MPP+-induced inhibition of oxygen consumption and ATP production, and mitochondrial swelling. These findings provide strong evidence that these neuroprotective peptides, which target both mitochondrial dysfunction and oxidative damage, are a promising approach for the treatment of PD. Antioxid. Redox Signal. 11, 2095–2104. PMID:19203217

  20. Targeted therapy of colorectal neoplasia with rapamycin in peptide-labeled pegylated octadecyl lithocholate micelles.

    PubMed

    Khondee, Supang; Rabinsky, Emily F; Owens, Scott R; Joshi, Bishnu P; Qiu, Zhen; Duan, Xiyu; Zhao, Lili; Wang, Thomas D

    2015-02-10

    Many powerful drugs have limited clinical utility because of poor water solubility and high systemic toxicity. Here, we formulated a targeted nanomedicine, rapamycin encapsulated in pegylated octadecyl lithocholate micelles labeled with a new ligand for colorectal neoplasia, LTTHYKL peptide. CPC;Apc mice that spontaneously develop colonic adenomas were treated with free rapamycin, plain rapamycin micelles, and peptide-labeled rapamycin micelles via intraperitoneal injection for 35days. Endoscopy was performed to monitor adenoma regression in vivo. We observed complete adenoma regression at the end of therapy. The mean regression rate for peptide-labeled rapamycin micelles was significantly greater than that for plain rapamycin micelles, P<0.01. On immunohistochemistry, we observed a significant reduction in phospho-S6 but not β-catenin expression and reduced tumor cell proliferation, suggesting greater inhibition of downstream mTOR signaling. We observed significantly reduced renal toxicity for peptide-labeled rapamycin micelles compared to that of free drug, and no other toxicities were found on chemistries. Together, this unique targeted micelle represents a potential therapeutic for colorectal neoplasia with comparable therapeutic efficacy to rapamycin free drug and significantly less systemic toxicity. PMID:25483425

  1. Probing the Backbone Function of Tumor Targeting Peptides by an Amide-to-Triazole Substitution Strategy.

    PubMed

    Valverde, Ibai E; Vomstein, Sandra; Fischer, Christiane A; Mascarin, Alba; Mindt, Thomas L

    2015-09-24

    Novel backbone-modified radiolabeled analogs based on the tumor targeting peptide bombesin were synthesized and fully evaluated in vitro and in vivo. We have recently introduced the use of 1,4-disubstituted 1,2,3-triazoles as metabolically stable trans-amide bond surrogates in radiolabeled peptides in order to improve their tumor targeting. As an extension of our approach, we now report several backbone-modified analogs of the studied bombesin peptide bearing multiple triazole substitutions. We investigated the effect of the modifications on several biological parameters including the internalization of the radiopeptidomimetics into tumor cells, their affinity toward the gastrin releasing peptide receptor (GRPr), metabolic stability in blood plasma, and biodistribution in mice bearing GRPr-expressing xenografts. The backbone-modified radiotracers exhibited a significantly increased resistance to proteolytic degradation. In addition, some of the radiopeptidomimetics retained a nanomolar affinity toward GRPr, resulting in an up to 2-fold increased tumor uptake in vivo in comparison to a (all amide bond) reference compound. PMID:26309061

  2. Synthetic RGD peptides derived from the adhesive domains of snake-venom proteins: evaluation as inhibitors of platelet aggregation.

    PubMed Central

    Lu, X; Deadman, J J; Williams, J A; Kakkar, V V; Rahman, S

    1993-01-01

    Synthetic peptides based on the RGD domains of the potent platelet aggregation inhibitors kistrin and dendroaspin were generated. The 13-amino-acid peptides were synthesized as dicysteinyl linear and disulphide cyclic forms. In platelet-aggregation studies, the cyclic peptides showed 3-fold better inhibition than their linear equivalents and approx. 100-fold greater potency than synthetic linear RGDS peptides derived from fibronectin. An amino acid substitution, Asp10-->Ala, in the kistrin-based peptide gave a 4-fold decrease in potency in the linear peptide, but produced a 2-fold elevation in the inhibitory activity of the cyclic form, generating a peptide of potency comparable with that of the parent protein. PMID:8250845

  3. Interaction of PiB-derivative metal complexes with beta-amyloid peptides: selective recognition of the aggregated forms.

    PubMed

    Martins, André F; Dias, David M; Morfin, Jean-François; Lacerda, Sara; Laurents, Douglas V; Tóth, Éva; Geraldes, Carlos F G C

    2015-03-27

    Metal complexes are increasingly explored as imaging probes in amyloid peptide related pathologies. We report the first detailed study on the mechanism of interaction between a metal complex and both the monomer and the aggregated form of Aβ1-40 peptide. We have studied lanthanide(III) chelates of two PiB-derivative ligands (PiB=Pittsburgh compound B), L(1) and L(2), differing in the length of the spacer between the metal-complexing DO3A macrocycle (DO3A=1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) and the peptide-recognition PiB moiety. Surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR spectroscopy revealed that they both bind to aggregated Aβ1-40 (KD =67-160 μM), primarily through the benzothiazole unit. HSQC NMR spectroscopy on the (15) N-labeled, monomer Aβ1-40 peptide indicates nonsignificant interaction with monomeric Aβ. Time-dependent circular dichroism (CD), dynamic light scattering (DLS), and TEM investigations of the secondary structure and of the aggregation of Aβ1-40 in the presence of increasing amounts of the metal complexes provide coherent data showing that, despite their structural similarity, the two complexes affect Aβ fibril formation distinctly. Whereas GdL(1), at higher concentrations, stabilizes β-sheets, GdL(2) prevents aggregation by promoting α-helical structures. These results give insight into the behavior of amyloid-targeted metal complexes in general and contribute to a more rational design of metal-based diagnostic and therapeutic agents for amyloid- associated pathologies. PMID:25712142

  4. LDLR-mediated peptide-22-conjugated nanoparticles for dual-targeting therapy of brain glioma.

    PubMed

    Zhang, Bo; Sun, Xiyang; Mei, Heng; Wang, Yu; Liao, Ziwei; Chen, Jun; Zhang, Qizhi; Hu, Yu; Pang, Zhiqing; Jiang, Xinguo

    2013-12-01

    Chemotherapy for brain glioma has been of limited benefit due to the inability of drugs to penetrate the blood-brain barrier (BBB) and non-selective drug accumulation in the entire brain. To obviate these limitations, dual-targeting paclitaxel-loaded nanoparticles were developed by decoration with peptide-22 (PNP-PTX), a peptide with special affinity for low-density lipoprotein receptor (LDLR), to transport the drug across the BBB, and then target brain tumour cells. Enzyme-linked immune sorbent assay (ELISA) revealed that LDLR was over-expressed in C6 cells and brain capillary endothelial cells (BCECs), but low LDLR expression was observed in H92c(2-1) cells. Nanoparticle uptake demonstrated that peptide-22-decorated nanoparticles significantly increased the cellular uptake of nanoparticles by C6 cells and BCECs but not by H92c(2-1) cells, and excess free peptide-22 significantly inhibited the cellular uptake of PNP by C6 cells and BCECs. Cellular uptake mechanism experiments showed that PNP uptake by both BCECs and C6 cells was energy-dependant and caveolae- and clathrin-mediated endocytosis pathway other than macropinocytosis were involved. Dual-targeting effects in an in vitro BBB model showed that peptide-22 decoration on nanoparticles loaded with paclitaxel significantly increased the transport ratio of PTX across the BBB and induced apoptosis of C6 glioma cells below the BBB, and these effects were significantly inhibited by excess free peptide-22. Ex vivo and in vivo fluorescence imaging indicated that PNP labelled with a near-infrared dye could permeate the BBB and accumulate more in the glioma site than unmodified NP. Glioma section observed by fluorescence microscopy further demonstrated PNP distributed more extensively in both glioma bulk and infiltrative region around than unmodified NP. Pharmacodynamics results revealed that the median survival time of glioma-bearing mice administered with dual-targeting PNP-PTX was significantly prolonged compared

  5. Orthogonal ring-closing alkyne and olefin metathesis for the synthesis of small GTPase-targeting bicyclic peptides.

    PubMed

    Cromm, Philipp M; Schaubach, Sebastian; Spiegel, Jochen; Fürstner, Alois; Grossmann, Tom N; Waldmann, Herbert

    2016-01-01

    Bicyclic peptides are promising scaffolds for the development of inhibitors of biological targets that proved intractable by typical small molecules. So far, access to bioactive bicyclic peptide architectures is limited due to a lack of appropriate orthogonal ring-closing reactions. Here, we report chemically orthogonal ring-closing olefin (RCM) and alkyne metathesis (RCAM), which enable an efficient chemo- and regioselective synthesis of complex bicyclic peptide scaffolds with variable macrocycle geometries. We also demonstrate that the formed alkyne macrocycle can be functionalized subsequently. The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand. This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far. The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets. PMID:27075966

  6. Orthogonal ring-closing alkyne and olefin metathesis for the synthesis of small GTPase-targeting bicyclic peptides

    PubMed Central

    Cromm, Philipp M.; Schaubach, Sebastian; Spiegel, Jochen; Fürstner, Alois; Grossmann, Tom N.; Waldmann, Herbert

    2016-01-01

    Bicyclic peptides are promising scaffolds for the development of inhibitors of biological targets that proved intractable by typical small molecules. So far, access to bioactive bicyclic peptide architectures is limited due to a lack of appropriate orthogonal ring-closing reactions. Here, we report chemically orthogonal ring-closing olefin (RCM) and alkyne metathesis (RCAM), which enable an efficient chemo- and regioselective synthesis of complex bicyclic peptide scaffolds with variable macrocycle geometries. We also demonstrate that the formed alkyne macrocycle can be functionalized subsequently. The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand. This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far. The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets. PMID:27075966

  7. Dendritic-Tumor Fusion Cells Derived Heat Shock Protein70-Peptide Complex Has Enhanced Immunogenicity

    PubMed Central

    Chen, Jun; Liu, Yunyan; Luo, Wen

    2015-01-01

    Tumor-derived heat shock protein70-peptide complexes (HSP70.PC-Tu) have shown great promise in tumor immunotherapy due to numerous advantages. However, large-scale phase III clinical trials showed that the limited immunogenicity remained to be enhanced. In previous research, we demonstrated that heat shock protein 70-peptide complexes (HSP70.PC-Fc) derived from dendritic cell (DC)-tumor fusions exhibit enhanced immunogenicity compared with HSP70.PCs from tumor cells. However, the DCs used in our previous research were obtained from healthy donors and not from the patient population. In order to promote the clinical application of these complexes, HSP70.PC-Fc was prepared from patient-derived DC fused directly with patient-derived tumor cells in the current study. Our results showed that compared with HSP70.PC-Tu, HSP70.PC-Fc elicited much more powerful immune responses against the tumor from which the HSP70 was derived, including enhanced T cell activation, and CTL responses that were shown to be antigen specific and HLA restricted. Our results further indicated that the enhanced immunogenicity is related to the activation of CD4+ T cells and increased association with other heat shock proteins, such as HSP90. Therefore, the current study confirms the enhanced immunogenicity of HSP70.PC derived from DC-tumor fusions and may provide direct evidence promoting their future clinical use. PMID:25961716

  8. A peptide for targeted, systemic delivery of imaging and therapeutic compounds into acute brain injuries

    PubMed Central

    Mann, Aman P.; Scodeller, Pablo; Hussain, Sazid; Joo, Jinmyoung; Kwon, Ester; Braun, Gary B.; Mölder, Tarmo; She, Zhi-Gang; Kotamraju, Venkata Ramana; Ranscht, Barbara; Krajewski, Stan; Teesalu, Tambet; Bhatia, Sangeeta; Sailor, Michael J.; Ruoslahti, Erkki

    2016-01-01

    Traumatic brain injury (TBI) is a major health and socio-economic problem, but no pharmacological agent is currently approved for the treatment of acute TBI. Thus, there is a great need for advances in this field. Here, we describe a short peptide (sequence CAQK) identified by in vivo phage display screening in mice with acute brain injury. The CAQK peptide selectively binds to injured mouse and human brain, and systemically injected CAQK specifically homes to sites of brain injury in mouse models. The CAQK target is a proteoglycan complex upregulated in brain injuries. Coupling to CAQK increased injury site accumulation of systemically administered molecules ranging from a drug-sized molecule to nanoparticles. CAQK-coated nanoparticles containing silencing oligonucleotides provided the first evidence of gene silencing in injured brain parenchyma by systemically administered siRNA. These findings present an effective targeting strategy for the delivery of therapeutics in clinical management of acute brain injuries. PMID:27351915

  9. A peptide for targeted, systemic delivery of imaging and therapeutic compounds into acute brain injuries

    NASA Astrophysics Data System (ADS)

    Mann, Aman P.; Scodeller, Pablo; Hussain, Sazid; Joo, Jinmyoung; Kwon, Ester; Braun, Gary B.; Mölder, Tarmo; She, Zhi-Gang; Kotamraju, Venkata Ramana; Ranscht, Barbara; Krajewski, Stan; Teesalu, Tambet; Bhatia, Sangeeta; Sailor, Michael J.; Ruoslahti, Erkki

    2016-06-01

    Traumatic brain injury (TBI) is a major health and socio-economic problem, but no pharmacological agent is currently approved for the treatment of acute TBI. Thus, there is a great need for advances in this field. Here, we describe a short peptide (sequence CAQK) identified by in vivo phage display screening in mice with acute brain injury. The CAQK peptide selectively binds to injured mouse and human brain, and systemically injected CAQK specifically homes to sites of brain injury in mouse models. The CAQK target is a proteoglycan complex upregulated in brain injuries. Coupling to CAQK increased injury site accumulation of systemically administered molecules ranging from a drug-sized molecule to nanoparticles. CAQK-coated nanoparticles containing silencing oligonucleotides provided the first evidence of gene silencing in injured brain parenchyma by systemically administered siRNA. These findings present an effective targeting strategy for the delivery of therapeutics in clinical management of acute brain injuries.

  10. Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.

    PubMed

    Xian, Feng; Zi, Jin; Wang, Quanhui; Lou, Xiaomin; Sun, Haidan; Lin, Liang; Hou, Guixue; Rao, Weiqiao; Yin, Changcheng; Wu, Lin; Li, Shuwei; Liu, Siqi

    2016-08-01

    Because of its specificity and sensitivity, targeted proteomics using mass spectrometry for multiple reaction monitoring is a powerful tool to detect and quantify pre-selected peptides from a complex background and facilitates the absolute quantification of peptides using isotope-labeled forms as internal standards. How to generate isotope-labeled peptides remains an urgent challenge for accurately quantitative targeted proteomics on a large scale. Herein, we propose that isotope-labeled peptides fused with a quantitative tag could be synthesized through an expression system in vitro, and the homemade peptides could be enriched by magnetic beads with tag-affinity and globally quantified based on the corresponding multiple reaction monitoring signals provided by the fused tag. An Escherichia coli cell-free protein expression system, protein synthesis using recombinant elements, was adopted for the synthesis of isotope-labeled peptides fused with Strep-tag. Through a series of optimizations, we enabled efficient expression of the labeled peptides such that, after Strep-Tactin affinity enrichment, the peptide yield was acceptable in scale for quantification, and the peptides could be completely digested by trypsin to release the Strep-tag for quantification. Moreover, these recombinant peptides could be employed in the same way as synthetic peptides for multiple reaction monitoring applications and are likely more economical and useful in a laboratory for the scale of targeted proteomics. As an application, we synthesized four isotope-labeled glutathione S-transferase (GST) peptides and added them to mouse sera pre-treated with GST affinity resin as internal standards. A quantitative assay of the synthesized GST peptides confirmed the absolute GST quantification in mouse sera to be measurable and reproducible. PMID:27234506

  11. Targeted Delivery of Peptide-Tagged DNA Lipoplexes to Hepatocellular Carcinoma Cells.

    PubMed

    Ariatti, Mario

    2016-01-01

    The application of homing peptides to direct DNA and RNA lipoplexes to target cells is a rapidly evolving area of study, which may find application in corrective gene therapy for the treatment of neoplasms and other disorders of a genetic origin. Here, a step-wise account of the assembly and characterization of hepatocellular carcinoma cell-specific DNA lipoplexes and their cytotoxicity assessment in and delivery to the human hepatocellular carcinoma cell line HepG2 is given. PMID:27436315

  12. Targeted delivery of peptide-conjugated biocompatible gold nanoparticles into cancer cell nucleus

    NASA Astrophysics Data System (ADS)

    Qian, Wei; Curry, Taeyjuana; Che, Yong; Kopelman, Raoul

    2013-02-01

    Nucleus remains a significant target for nanoparticles with diagnostic and therapeutic applications because both genetic information of the cell and transcription machinery reside there. Novel therapeutic strategies (for example, gene therapy), enabled by safe and efficient delivery of nanoparticles and drug molecules into the nucleus, are heralded by many as the ultimate treatment for severe and intractable diseases. However, most nanomaterials and macromolecules are incapable of reaching the cell nucleus on their own, because of biological barriers carefully honed by evolution including cellular membrane and nuclear envelope. In this paper, we have demonstrated an approach of fabrication of biocompatible gold nanoparticle (Au NP)-based vehicles which can entering into cancer cell nucleus by modifying Au NPs with both PEG 5000 and two different peptides (RGD and nuclear localization signal (NLS) peptide). The Au NPs used were fabricated via femtosecond laser ablation of Au bulk target in deionized water. The Au NPs produced by this method provide chemical free, virgin surface, which allows us to carry out "Sequential Conjugation" to modify their surface with PEG 5000, RGD, and NLS. "Sequential Conjugation" described in this presentation is very critical for the fabrication of Au NP-based vehicles capable of entering into cancer cell nucleus as it enables the engineering and tuning surface chemistries of Au NPs by independently adjusting amounts of PEG and peptides bound onto surface of Au NPs so as to maximize their nuclear targeting performance and biocompatibility regarding the cell line of interest. Both optical microscopy and transmission electron microscopy (TEM) are used to confirm the in vitro targeted nuclear delivery of peptide-conjugated biocompatible Au NPs by showing their presence in the cancer cell nucleus.

  13. ZOT-derived peptide and chitosan functionalized nanocarrier for oral delivery of protein drug.

    PubMed

    Lee, Jong Hyun; Sahu, Abhishek; Choi, Won Il; Lee, Jae Young; Tae, Giyoong

    2016-10-01

    In this study, we developed a dual ligand functionalized pluronic-based nanocarrier (NC) for oral delivery of insulin. Chitosan and zonula occludins toxin (ZOT)-derived, tight junction opening peptide were conjugated to NC to increase the permeability of loaded insulin across the small intestine through the paracellular pathway. Surface functionalized NC, either by chitosan or peptide, could modulate the tight junction (TJ) integrity in contrast to no effect of unmodified NC, as evidenced by the change in transepithelial electrical resistance (TEER) and immunostaining of Claudin-4, a tight junction marker, in Caco-2 cell monolayer. On the other hand, dual ligand (chitosan and peptide) functionalized NC significantly further increased the permeation of insulin across Caco-2 cell monolayer. More importantly, insulin loaded, dual ligand functionalized NC could increase the plasma insulin level and efficiently regulate the glycemic response for a prolonged period of time (∼1 day) upon oral administration to diabetic rats, whereas delivery of insulin by single ligand functionalized NCs, either by chitosan or peptide, as well as by unmodified NC and free insulin, could not induce the effective regulation of the blood glucose level. The use of fluorescence dye labeled insulin (FITC-insulin) and Cy5.5 labeled NC revealed that both insulin and dual ligand functionalized NC were adequately penetrated across the whole intestine villi in contrast to limited adsorption of insulin and NC mainly onto the epithelial surface of the intestine for single ligand functionalized NCs. These results suggest that dual conjugation of ZOT-derived peptide and chitosan is a promising approach to functionalize the surface of nanocarrier for oral delivery of protein drugs. PMID:27380442

  14. Solution structure of a bacterial microcompartment targeting peptide and its application in the construction of an ethanol bioreactor

    PubMed Central

    Newnham, Sarah; Lee, Matthew J.; Brown, Ian R.; Xue, Wei-Feng; Rowe, Michelle L.; Mulvihill, Daniel P.; Prentice, Michael B; Howard, Mark J.; Warren, Martin J.

    2016-01-01

    The targeting of proteins to bacterial microcompartments (BMCs) is mediated by a short peptide sequence of 18 amino acids. Herein, we report the solution structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase associated with the 1,2-propanediol utilization metabolosome. The solution structure reveals the peptide to have a well defined-helical conformation along its whole length. Saturation transfer difference and transferred NOE NMR has highlighted the observed interaction surface on the peptide with its main interacting protein, PduK, a component of the outer shell of the microcompartment. By tagging both a pyruvate decarboxylase and an alcohol dehydrogenase with targeting peptides it has been possible to direct these enzymes to empty BMCs in vivo and to generate an ethanol bioreactor. Purification of the re-designed BMCs reveals that not only do they contain the ethanogenic enzymes but that they are able to transform pyruvate into ethanol efficiently. PMID:24933391

  15. Liver-targeted antiviral peptide nanocomplexes as potential anti-HCV therapeutics.

    PubMed

    Zhang, Jinjin; Garrison, Jered C; Poluektova, Larisa Y; Bronich, Tatiana K; Osna, Natalia A

    2015-11-01

    Great success in HCV therapy was achieved by the development of direct-acting antivirals (DAA). However, the unsolved issues such as high cost and genotype dependency drive us to pursue additional therapeutic agents to be used instead or in combination with DAA. The cationic peptide p41 is one of such candidates displaying submicromolar anti-HCV potency. By electrostatic coupling of p41 with anionic poly(amino acid)-based block copolymers, antiviral peptide nanocomplexes (APN) platform was developed to improve peptide stability and to reduce cytotoxicity associated with positive charge. Herein, we developed a facile method to prepare galactosylated Gal-APN and tested their feasibility as liver-specific delivery system. In vitro, Gal-APN displayed specific internalization in hepatoma cell lines. Even though liver-targeted and non-targeted APN displayed comparable antiviral activity, Gal-APN offered prominent advantages to prevent HCV association with lipid droplets and suppress intracellular expression of HCV proteins. Moreover, in vivo preferential liver accumulation of Gal-APN was revealed in the biodistribution study. Altogether, this work illustrates the potential of Gal-APN as a novel liver-targeted therapy against HCV. PMID:26298393

  16. Phthalocyanine-Peptide Conjugates for Epidermal Growth Factor Receptor Targeting1

    PubMed Central

    Ongarora, Benson G.; Fontenot, Krystal R.; Hu, Xiaoke; Sehgal, Inder; Satyanarayana-Jois, Seetharama D.; Vicente, M. Graça H.

    2012-01-01

    Four phthalocyanine (Pc)-peptide conjugates designed to target the epidermal growth factor receptor (EGFR) were synthesized and evaluated in vitro using four cell lines: human carcinoma A431 and HEp2, human colorectal HT-29, and kidney Vero (negative control) cells. Two peptide ligands for EGFR were investigated: EGFR-L1 and -L2, bearing 6 and 13 amino acid residues, respectively. The peptides and Pc-conjugates were shown to bind to EGFR using both theoretical (Autodock) and experimental (SPR) investigations. The Pc-EGFR-L1 conjugates 5a and 5b efficiently targeted EGFR and were internalized, in part due to their cationic charge, whereas the uncharged Pc-EGFR-L2 conjugates 4b and 6a poorly targeted EGFR maybe due to their low aqueous solubility. All conjugates were non-toxic (IC50 > 100 µM) to HT-29 cells, both in the dark and upon light activation (1 J/cm2). Intravenous (iv) administration of conjugate 5b into nude mice bearing A431 and HT-29 human tumor xenografts resulted in a near-IR fluorescence signal at ca. 700 nm, 24 h after administration. Our studies show that Pc-EGFR-L1 conjugates are promising near-IR fluorescent contrast agents for CRC, and potentially other EGFR over-expressing cancers. PMID:22468711

  17. Mechanisms of cell penetration and cytotoxicity of ultrasmall Au nanoparticles conjugated to doxorubicin and/or targeting peptides

    NASA Astrophysics Data System (ADS)

    Nadeau, Jay; Poon, Wilson; Zhang, Xuan

    2015-03-01

    The goals of this work were to determine whether conjugation of any of four selected peptides to Au nanoparticles improved their delivery to B16 melanoma in vitro and in vivo. In in vitro cytotoxicity assays, peptides and conjugates were endocytosed but did not escape from endosomes. None of the peptides showed any cytotoxicity, with or without conjugation to the nanoparticles. The combination of peptides and doxorubicin did not improve upon the cytotoxicity of gold-doxorubicin alone. We then tested targeting in vivo using inductively coupled plasma mass spectrometry to quantify the concentration of Au in the organs of B16 tumor-bearing mice 4, 24, and 72 h after intravenous Au nanoparticle injection. These experiments showed that in some cases, peptide conjugation improved upon the enhanced permeability and retention (EPR) effect. A peptide based upon the myxoma virus and the cyclic RGD peptide were both effective at tumor targeting; myxoma was more effective with un-PEGylated particles, and cRGD with PEGylated particles. The FREG and melanocyte stimulating hormone (MSH) peptides did not improve targeting. These results suggest that these peptides may improve delivery of Au particles to tumors, but also may prevent entry of particles into cell nuclei.

  18. Applications of Convertible Isonitriles in the Ligation and Macrocyclization of Multicomponent Reaction-Derived Peptides and Depsipeptides.

    PubMed

    Wessjohann, Ludger A; Morejón, Micjel C; Ojeda, Gerardo M; Rhoden, Cristiano R B; Rivera, Daniel G

    2016-08-01

    Peptide ligation and macrocyclization are among the most relevant approaches in the field of peptide chemistry. Whereas a variety of strategies relying on coupling reagents and native chemical ligation are available, there is a continuous need for efficient peptide ligation and cyclization methods. Herein we report on the utilization of convertible isonitriles as effective synthetic tools for the ligation and macrocyclization of peptides arising from isocyanide-based multicomponent reactions. The strategy relies on the use of convertible isonitriles-derived from Fukuyama amines-and peptide carboxylic acids in Ugi and Passerini reactions to afford N-alkylated peptides and depsipeptides, respectively, followed by conversion of the C-terminal amide onto either N-peptidoacyl indoles or pyrroles. Such activated peptides proved efficient in the ligation to peptidic, lipidic and fluorescently labeled amines and in macrocyclization protocols. As a result, a wide set of N-substituted peptides (with methyl, glycosyl and amino acids as N-substituents), cyclic N-methylated peptides and a depsipeptide were produced in good yields using conditions that involve either classical heating or microwave irradiation. This report improves the repertoire of peptide covalent modification methods by exploiting the synthetic potential of multicomponent reactions and convertible isonitriles. PMID:27390908

  19. The anti-cancer activity of a cationic anti-microbial peptide derived from monomers of polyhydroxyalkanoate.

    PubMed

    O'Connor, Stephen; Szwej, Emilia; Nikodinovic-Runic, Jasmina; O'Connor, Aisling; Byrne, Annette T; Devocelle, Marc; O'Donovan, Norma; Gallagher, William M; Babu, Ramesh; Kenny, Shane T; Zinn, Manfred; Zulian, Qun Ren; O'Connor, Kevin E

    2013-04-01

    The biodegradable polymer medium chain length polyhydroxyalkanoate (mclPHA), produced by Pseudomonas putida CA-3, was depolymerised and the predominant monomer (R)-3-hydroxydecanoic acid (R10) purified. R10 was conjugated to a d-peptide DP18 and its derivatives. All peptides conjugated with R10 exhibited greater anti-cancer activity compared to the unconjugated peptides. Unconjugated and conjugated peptides were cytocidal for cancer cells. Conjugation of R10 to peptides was essential for enhanced anti-proliferation activity, as unconjugated mixes did not result in enhancement of anti-cancer activity. The conjugation of R10 resulted in more rapid uptake of peptides into HeLa and MiaPaCa cells compared to unconjugated peptide. Both unconjugated and R10 conjugated peptides localized to the mitochondria of HeLa and MiaPaCa cells and induced apoptosis. Peptide conjugated with a terminally hydroxylated decanoic acid (ω-hydroxydecanoic acid) exhibited 3.3 and 6.3 fold higher IC(50) values compared to R10 conjugated peptide indicating a role for the position of the hydroxyl moiety in enhancement of anti-cancer activity. Conjugation of decanoic acid (C10) to peptides resulted in similar or higher IC(50) values compared to R10 conjugates but C10 conjugates did not exhibit any cancer selectivity. Combination studies showed that R10DP18L exhibited synergy with cisplatin, gemcitabine, and taxotere with IC(50) values in the nanomolar range. PMID:23343631

  20. New Peptide-Conjugated Chlorin-Type Photosensitizer Targeting Neuropilin-1 for Anti-Vascular Targeted Photodynamic Therapy

    PubMed Central

    Kamarulzaman, Ezatul Ezleen; Mohd Gazzali, Amirah; Acherar, Samir; Frochot, Céline; Barberi-Heyob, Muriel; Boura, Cédric; Chaimbault, Patrick; Sibille, Estelle; Wahab, Habibah A.; Vanderesse, Régis

    2015-01-01

    Photodynamic therapy (PDT) is a cancer treatment modality that requires three components, namely light, dioxygen and a photosensitizing agent. After light excitation, the photosensitizer (PS) in its excited state transfers its energy to oxygen, which leads to photooxidation reactions. In order to improve the selectivity of the treatment, research has focused on the design of PS covalently attached to a tumor-targeting moiety. In this paper, we describe the synthesis and the physico-chemical and photophysical properties of six new peptide-conjugated photosensitizers designed for targeting the neuropilin-1 (NRP-1) receptor. We chose a TPC (5-(4-carboxyphenyl)-10,15, 20-triphenyl chlorine as photosensitizer, coupled via three different spacers (aminohexanoic acid, 1-amino-3,6-dioxaoctanoic acid, and 1-amino-9-aza-3,6,12,15-tetraoxa-10-on-heptadecanoic acid) to two different peptides (DKPPR and TKPRR). The affinity towards the NRP-1 receptor of the conjugated chlorins was evaluated along with in vitro and in vivo stability levels. The tissue concentration of the TPC-conjugates in animal model shows good distribution, especially for the DKPPR conjugates. The novel peptide–PS conjugates proposed in this study were proven to have potential to be further developed as future NRP-1 targeting photodynamic therapy agent. PMID:26473840

  1. Synergetic Targeted Delivery of Sleeping-Beauty Transposon System to Mesenchymal Stem Cells Using LPD Nanoparticles Modified with a Phage-Displayed Targeting Peptide.

    PubMed

    Ma, Kun; Wang, Dong-Dong; Lin, Yiyang; Wang, Jianglin; Petrenko, Valery; Mao, Chuanbin

    2013-03-01

    An important criterion for effective gene therapy is sufficient chromosomal integration activity. The Sleeping Beauty (SB) transposon system is a plasmid system allowing efficient insertion of transgenes into the host genome. However, such efficient insertion occurs only after the system is delivered to nuclei. Since transposons do not have the transducing abilities of viral vectors, efficient delivery of this system first into cells and then into cell nuclei is still a challenge. Here, a phage display technique using a major coat displayed phage library is employed to identify a peptide (VTAMEPGQ) that can home to rat mesenchymal stem cells (rMSCs). A nanoparticle, called liposome protamine/DNA lipoplex (LPD), is electrostatically assembled from cationic liposomes and an anionic complex of protamine, DNA and targeting peptides. Various peptides are enveloped inside the LPD to improve its targeting capability for rMSCs and nuclei. The rMSC-targeting peptide and nuclear localization signal (NLS) peptide can execute the synergetic effect to promote transfection action of LPD. The homing peptide directs the LPD to target the MSCs, whereas the NLS peptide directs transposon to accumulate into nuclei once LPD is internalized inside the cells, leading to increased gene expression. This suggests that rMSC-targeting peptide and NLS peptide within LPD can target to rMSCs and then guide transposon into nuclei. After entering the nuclei, SB transposon increase the insertion rates into cellular chromosomes. The targeting LPD does not show obvious cell toxicity and influence on the differentiation potential of rMSCs. Therefore, the integration of SB transposon and LPD system is a promising nonviral gene delivery vector in stem cell therapy. PMID:23885226

  2. Construction and application of elastin like polypeptide containing IL-4 receptor targeting peptide.

    PubMed

    Sarangthem, Vijaya; Cho, Eun A; Bae, Sang Mun; Singh, Thoudam Debraj; Kim, Sun-Ji; Kim, Soyoun; Jeon, Won Bae; Lee, Byung-Heon; Park, Rang-Woon

    2013-01-01

    Various human solid tumors highly express IL-4 receptors which amplify the expression of some of anti-apoptotic proteins, preventing drug-induced cancer cell death. Thus, IL-4 receptor targeted drug delivery can possibly increase the therapeutic efficacy in cancer treatment. Macromolecular carriers with multivalent targeting moieties offered great advantages in cancer therapy as they not only increase the plasma half-life of the drug but also allow delivery of therapeutic drugs to the cancer cells with higher specificity, minimizing the deleterious effects of the drug on normal cells. In this study we designed a library of elastin like polypeptide (ELP) polymers containing tumor targeting AP1 peptide using recursive directional ligation method. AP1 was previously discovered as an atherosclerotic plaque and breast tumor tissue homing peptide using phage display screening method, and it can selectively bind to the interleukin 4 receptor (IL-4R). The fluorescently labeled [AP1-V12]6, an ELP polymer containing six AP1 enhanced tumor-specific targeting ability and uptake efficiency in H226 and MDA-MB-231 cancer cell lines in vitro. Surface plasmon resonance analysis showed that multivalent presentation of the targeting ligand in the ELP polymer increased the binding affinity towards IL-4 receptor compared to free peptide. The binding of [AP1-V12]6 to cancer cells was remarkably reduced when IL-4 receptors were blocked by antibody against IL-4 receptor further confirmed its binding. Importantly, the Cy5.5-labeled [AP1-V12]6 demonstrated excellent homing and longer retention in tumor tissues in MDA-MB-231 xenograft mouse model. Immunohistological studies of tumor tissues further validated the targeting efficiency of [AP1-V12]6 to tumor tissue. These results indicate that designed [AP1-V12]6 can serve as a novel carrier for selective delivery of therapeutic drugs to tumors. PMID:24339977

  3. Clinical Efficacy of a Specifically Targeted Antimicrobial Peptide Mouth Rinse: Targeted Elimination of Streptococcus mutans and Prevention of Demineralization

    PubMed Central

    Sullivan, R.; Santarpia, P.; Lavender, S.; Gittins, E.; Liu, Z.; Anderson, M.H.; He, J.; Shi, W.; Eckert, R.

    2011-01-01

    Background/Aims Streptococcus mutans, the major etiological agent of dental caries, has a measurable impact on domestic and global health care costs. Though persistent in the oral cavity despite conventional oral hygiene, S. mutans can be excluded from intact oral biofilms through competitive exclusion by other microorganisms. This suggests that therapies capable of selectively eliminating S. mutans while limiting the damage to the normal oral flora might be effective long-term interventions to fight cariogenesis. To meet this challenge, we designed C16G2, a novel synthetic specifically targeted antimicrobial peptide with specificity for S. mutans. C16G2 consists of a S. mutans-selective ‘targeting region’ comprised of a fragment from S. mutans competence stimulation peptide (CSP) conjoined to a ‘killing region’ consisting of a broad-spectrum antimicrobial peptide (G2). In vitro studies have indicated that C16G2 has robust efficacy and selectivity for S. mutans, and not other oral bacteria, and affects targeted bacteria within seconds of contact. Methods In the present study, we evaluated C16G2 for clinical utility in vitro, followed by a pilot efficacy study to examine the impact of a 0.04% (w/v) C16G2 rinse in an intra-oral remineralization/demineralization model. Results and Conclusions C16G2 rinse usage was associated with reductions in plaque and salivary S. mutans, lactic acid production, and enamel demineralization. The impact on total plaque bacteria was minimal. These results suggest that C16G2 is effective against S. mutans in vivo and should be evaluated further in the clinic. PMID:21860239

  4. MALDI-based identification of stable hazelnut protein derived tryptic marker peptides.

    PubMed

    Cucu, T; De Meulenaer, B; Devreese, B

    2012-01-01

    Food allergy is an important health problem especially in industrialised countries. Tree nuts, among which are hazelnuts (Corylus avellana), are typically causing serious and life-threatening symptoms in sensitive subjects. Hazelnut is used as a food ingredient in pastry, confectionary products, ice cream and meat products, therefore undeclared hazelnut can be often present as a cross-contaminant representing a threat for allergic consumers. Mass spectrometric techniques are used for the detection of food allergens in processed foods, but limited information regarding stable tryptic peptide markers for hazelnut is available. The aim of this study was to detect stable peptide markers from modified hazelnut protein through the Maillard reaction and oxidation in a buffered solution. Peptides ³⁹⁵Gly-Arg⁴⁰³ from Cor a 11 and ²⁰⁹Gln-Arg²¹⁷, ³⁵¹Ile-Arg³⁶³, ⁴⁶⁴Ala-Arg⁴⁷⁸ and ⁴⁰¹Val-Arg⁴¹⁷ from Cor a 9 hazelnut allergens proved to be the most stable and could be detected and confirmed with high scores in most of the modified samples. The identified peptides can be further used as analytical targets for the development of more robust quantitative methods for hazelnut detection in processed foods. PMID:22966848

  5. A PCNA-Derived Cell Permeable Peptide Selectively Inhibits Neuroblastoma Cell Growth

    PubMed Central

    Gu, Long; Smith, Shanna; Li, Caroline; Hickey, Robert J.; Stark, Jeremy M.; Fields, Gregg B.; Lang, Walter H.; Sandoval, John A.; Malkas, Linda H.

    2014-01-01

    Proliferating cell nuclear antigen (PCNA), through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was significantly expressed in a broad range of cancer cells and tumor tissues, but not in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interconnector domain of PCNA that is known to be a major binding site for many of PCNA's interacting proteins. We hypothesized that therapeutic agents targeting protein-protein interactions mediated through this region may confer differential toxicity to normal and malignant cells. To test this hypothesis, we designed a cell permeable peptide containing the PCNA L126-Y133 sequence. Here, we report that this peptide selectively kills human neuroblastoma cells, especially those with MYCN gene amplification, with much less toxicity to non-malignant human cells. Mechanistically, the peptide is able to block PCNA interactions in cancer cells. It interferes with DNA synthesis and homologous recombination-mediated double-stranded DNA break repair, resulting in S-phase arrest, accumulation of DNA damage, and enhanced sensitivity to cisplatin. These results demonstrate conceptually the utility of this peptide for treating neuroblastomas, particularly, the unfavorable MYCN-amplified tumors. PMID:24728180

  6. An alternative bactericidal mechanism of action for lantibiotic peptides that target lipid II.

    PubMed

    Hasper, Hester E; Kramer, Naomi E; Smith, James L; Hillman, J D; Zachariah, Cherian; Kuipers, Oscar P; de Kruijff, Ben; Breukink, Eefjan

    2006-09-15

    Lantibiotics are polycyclic peptides containing unusual amino acids, which have binding specificity for bacterial cells, targeting the bacterial cell wall component lipid II to form pores and thereby lyse the cells. Yet several members of these lipid II-targeted lantibiotics are too short to be able to span the lipid bilayer and cannot form pores, but somehow they maintain their antibacterial efficacy. We describe an alternative mechanism by which members of the lantibiotic family kill Gram-positive bacteria by removing lipid II from the cell division site (or septum) and thus block cell wall synthesis. PMID:16973881

  7. Identification and Characterization of a Small Inhibitory Peptide That Can Target DNA-PKcs Autophosphorylation and Increase Tumor Radiosensitivity

    SciTech Connect

    Sun Xiaonan; Yang Chunying; Liu Hai; Wang Qi; Wu Shixiu; Li Xia; Xie Tian; Brinkman, Kathryn L.; Teh, Bin S.; Butler, E. Brian; Xu Bo; Zheng, Shu

    2012-12-01

    Purpose: The DNA protein kinase catalytic subunit (DNA-PKcs) is one of the critical elements involved in the DNA damage repair process. Inhibition of DNA-PKcs results in hypersensitivity to ionizing radiation (IR); therefore, this approach has been explored to develop molecular targeted radiosensitizers. Here, we aimed to develop small inhibitory peptides that could specifically target DNA-PKcs autophosphorylation, a critical step for the enzymatic activation of the kinase in response to IR. Methods and Materials: We generated several small fusion peptides consisting of 2 functional domains, 1 an internalization domain and the other a DNA-PKcs autophosphorylation inhibitory domain. We characterized the internalization, toxicity, and radiosensitization activities of the fusion peptides. Furthermore, we studied the mechanisms of the inhibitory peptides on DNA-PKcs autophosphorylation and DNA repair. Results: We found that among several peptides, the biotin-labeled peptide 3 (BTW3) peptide, which targets DNA-PKcs threonine 2647 autophosphorylation, can abrogate IR-induced DNA-PKcs activation and cause prolonged {gamma}-H2AX focus formation. We demonstrated that BTW3 exposure led to hypersensitivity to IR in DNA-PKcs-proficient cells but not in DNA-PKcs-deficient cells. Conclusions: The small inhibitory peptide BTW3 can specifically target DNA-PKcs autophosphorylation and enhance radiosensitivity; therefore, it can be further developed as a novel class of radiosensitizer.

  8. Determinants of Human Immunodeficiency Virus Type 1 Resistance to gp41-Derived Inhibitory Peptides

    PubMed Central

    Rimsky, Laurence T.; Shugars, Diane C.; Matthews, Thomas J.

    1998-01-01

    A synthetic peptide, DP178, containing amino acids 127 to 162 of the human immunodeficiency virus type 1 (HIV-1) gp41 Env glycoprotein, is a potent inhibitor of virus infection and virus mediated cell-to-cell fusion (C. Wild, T. Greenwell, and T. Matthews, AIDS Res. Hum. Retroviruses 9:1051–1053, 1993). In an effort to understand the mechanism of action of this peptide, we derived resistant variants of HIV-1IIIB and NL4-3 by serial virus passage in the presence of increasing doses of the peptide. Sequence analysis of the resistant isolates suggested that a contiguous 3-amino-acid sequence within the amino-terminal heptad repeat motif of gp41 was associated with resistance. Site-directed mutagenesis studies confirmed this observation and indicated that changes in two of these three residues were necessary for development of the resistant phenotype. Direct binding of DP178 to recombinant protein and synthetic peptide analogs containing the wild-type and mutant heptad repeat sequences revealed a strong correlation between DP178 binding and the biological sensitivity of the corresponding virus isolates to DP178. The results are discussed from the standpoints of the mechanism of action of DP178 and recent crystallographic information for a core structure of the gp41 ectodomain. PMID:9444991

  9. The Multiplicity of Post-Translational Modifications in Pro-Opiomelanocortin-Derived Peptides

    PubMed Central

    Yasuda, Akikazu; Jones, Leslie Sargent; Shigeri, Yasushi

    2013-01-01

    The precursor protein, pro-opiomelanocortin (POMC) undergoes extensive post-translational processing in a tissue-specific manner to yield various biologically active peptides involved in diverse cellular functions. The recently developed method of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for direct tissue analysis has proved to be a powerful tool for investigating the distribution of peptides and proteins. In particular, topological mass spectrometry analysis using MALDI-MS can selectively provide a mass profile of the hormones included in cell secretory granules. An advantage of this technology is that it is possible to analyze a frozen thin slice section, avoiding an extraction procedure. Subsequently, tandem mass spectrometry (MS/MS) has a profound impact on addressing the modified residues in the hormone molecules. Based on these strategies with mass spectrometry, several interesting molecular forms of POMC-derived peptides have been found in the fish pituitary, such as novel sites of acetylation in α-melanocyte-stimulating hormone (MSH), hydroxylation of a proline residue in β-MSH, and the phosphorylated form of corticotropin-like intermediate lobe peptide. PMID:24348461

  10. Penetration of Milk-Derived Antimicrobial Peptides into Phospholipid Monolayers as Model Biomembranes

    PubMed Central

    Rogalska, Ewa; Więcław-Czapla, Katarzyna

    2013-01-01

    Three antimicrobial peptides derived from bovine milk proteins were examined with regard to penetration into insoluble monolayers formed with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG). Effects on surface pressure (Π) and electric surface potential (ΔV) were measured, Π with a platinum Wilhelmy plate and ΔV with a vibrating plate. The penetration measurements were performed under stationary diffusion conditions and upon the compression of the monolayers. The two type measurements showed greatly different effects of the peptide-lipid interactions. Results of the stationary penetration show that the peptide interactions with DPPC monolayer are weak, repulsive, and nonspecific while the interactions with DPPG monolayer are significant, attractive, and specific. These results are in accord with the fact that antimicrobial peptides disrupt bacteria membranes (negative) while no significant effect on the host membranes (neutral) is observed. No such discrimination was revealed from the compression isotherms. The latter indicate that squeezing the penetrant out of the monolayer upon compression does not allow for establishing the penetration equilibrium, so the monolayer remains supersaturated with the penetrant and shows an under-equilibrium orientation within the entire compression range, practically. PMID:24455264

  11. The TFPI-2 Derived Peptide EDC34 Improves Outcome of Gram-Negative Sepsis

    PubMed Central

    Papareddy, Praveen; Kalle, Martina; Sørensen, Ole E.; Malmsten, Martin; Mörgelin, Matthias; Schmidtchen, Artur

    2013-01-01

    Sepsis is characterized by a dysregulated host-pathogen response, leading to high cytokine levels, excessive coagulation and failure to eradicate invasive bacteria. Novel therapeutic strategies that address crucial pathogenetic steps during infection are urgently needed. Here, we describe novel bioactive roles and therapeutic anti-infective potential of the peptide EDC34, derived from the C-terminus of tissue factor pathway inhibitor-2 (TFPI-2). This peptide exerted direct bactericidal effects and boosted activation of the classical complement pathway including formation of antimicrobial C3a, but inhibited bacteria-induced activation of the contact system. Correspondingly, in mouse models of severe Escherichia coli and Pseudomonas aeruginosa infection, treatment with EDC34 reduced bacterial levels and lung damage. In combination with the antibiotic ceftazidime, the peptide significantly prolonged survival and reduced mortality in mice. The peptide's boosting effect on bacterial clearance paired with its inhibiting effect on excessive coagulation makes it a promising therapeutic candidate for invasive Gram-negative infections. PMID:24339780

  12. Penetration of milk-derived antimicrobial peptides into phospholipid monolayers as model biomembranes.

    PubMed

    Barzyk, Wanda; Rogalska, Ewa; Więcław-Czapla, Katarzyna

    2013-01-01

    Three antimicrobial peptides derived from bovine milk proteins were examined with regard to penetration into insoluble monolayers formed with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG). Effects on surface pressure (Π) and electric surface potential (ΔV) were measured, Π with a platinum Wilhelmy plate and ΔV with a vibrating plate. The penetration measurements were performed under stationary diffusion conditions and upon the compression of the monolayers. The two type measurements showed greatly different effects of the peptide-lipid interactions. Results of the stationary penetration show that the peptide interactions with DPPC monolayer are weak, repulsive, and nonspecific while the interactions with DPPG monolayer are significant, attractive, and specific. These results are in accord with the fact that antimicrobial peptides disrupt bacteria membranes (negative) while no significant effect on the host membranes (neutral) is observed. No such discrimination was revealed from the compression isotherms. The latter indicate that squeezing the penetrant out of the monolayer upon compression does not allow for establishing the penetration equilibrium, so the monolayer remains supersaturated with the penetrant and shows an under-equilibrium orientation within the entire compression range, practically. PMID:24455264

  13. Peptide-Like Molecules (PLMs): A Journey from Peptide Bond Isosteres to Gramicidin S Mimetics and Mitochondrial Targeting Agents

    PubMed Central

    Wipf, Peter; Xiao, Jingbo; Stephenson, Corey R. J.

    2010-01-01

    Peptides are natural ligands and substrates for receptors and enzymes and exhibit broad physiological effects. However, their use as therapeutic agents often suffers from poor bioavailability and insufficient membrane permeability. The success of peptide mimicry hinges on the ability of bioisosteres, in particular peptide bond replacements, to adopt suitable secondary structures relevant to peptide strands and position functional groups in equivalent space. This perspective highlights past and ongoing studies in our group that involve new methods development as well as specific synthetic library preparations and applications in chemical biology, with the goal to enhance the use of alkene and cyclopropane peptide bond isosteres. PMID:20725595

  14. Splicing of distant peptide fragments occurs in the proteasome by transpeptidation and produces the spliced antigenic peptide derived from fibroblast growth factor-5.

    PubMed

    Dalet, Alexandre; Vigneron, Nathalie; Stroobant, Vincent; Hanada, Ken-Ichi; Van den Eynde, Benoît J

    2010-03-15

    Peptide splicing is a newly described mode of production of antigenic peptides presented by MHC class I molecules, whereby two noncontiguous fragments of the parental protein are joined together after excision of the intervening segment. Three spliced peptides have been described. In two cases, splicing involved the excision of a short intervening segment of 4 or 6 aa and was shown to occur in the proteasome by transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by the N terminus of the other peptide fragment. For the third peptide, which is derived from fibroblast growth factor-5 (FGF-5), the splicing mechanism remains unknown. In this case, the intervening segment is 40 aa long. This much greater length made the transpeptidation model more difficult to envision. Therefore, we evaluated the role of the proteasome in the splicing of this peptide. We observed that the spliced FGF-5 peptide was produced in vitro after incubation of proteasomes with a 49-aa-long precursor peptide. We evaluated the catalytic mechanism by incubating proteasomes with various precursor peptides. The results confirmed the transpeptidation model of splicing. By transfecting a series of mutant FGF-5 constructs, we observed that reducing the length of the intervening segment increased the production of the spliced peptide, as predicted by the transpeptidation model. Finally, we observed that trans-splicing (i.e., splicing of fragments from two distinct proteins) can occur in the cell, but with a much lower efficacy than splicing of fragments from the same protein. PMID:20154207

  15. Targeted imaging of esophageal neoplasia with a fluorescently labeled peptide: First in-human results

    PubMed Central

    Sturm, Matthew B.; Joshi, Bishnu P.; Lu, Shaoying; Piraka, Cyrus; Khondee, Supang; Elmunzer, B. Joseph; Kwon, Richard S.; Beer, David G.; Appelman, Henry; Turgeon, D. Kim; Wang, Thomas D.

    2013-01-01

    Esophageal adenocarcinoma is rising rapidly in incidence, and usually develops from Barrett’s esophagus, a precursor condition commonly found in patients with chronic acid reflux. Pre-malignant lesions are challenging to detect on conventional screening endoscopy because of their flat appearance. Molecular changes can be used to improve detection of early neoplasia. We have developed a peptide that binds specifically to high-grade dysplasia and adenocarcinoma. We first applied the peptide ex vivo to esophageal specimens from 17 patients to validate specific binding. Next, we performed confocal endomicroscopy in vivo in 25 human subjects after topical peptide administration and found 3.8-fold greater fluorescence intensity for esophageal neoplasia compared with Barrett’s esophagus and squamous epithelium with 75% sensitivity and 97% specificity. No toxicity was attributed to the peptide in either animal or patient studies. Therefore, our first-in-humans results show that this targeted imaging agent is safe, and may be useful for guiding tissue biopsy and for early detection of esophageal neoplasia and potentially other cancers of epithelial origin, such as bladder, colon, lung, pancreas, and stomach. PMID:23658246

  16. Design of dimeric peptides obtained from a subdominant Epstein-Barr virus LMP2-derived epitope.

    PubMed

    Marastoni, M; Bazzaro, M; Gavioli, R; Micheletti, F; Traniello, S; Tomatis, R

    2000-06-01

    The latent membrane protein 2 (LMP2) is expressed in EBV-associated tumours. LMP2 is a target of HLA-A2 restricted EBV-specific CTL responses and consequently it may represent a good target for specific CTL-based immunotherapies. However, the efficacy of such therapy is limited by the poor immunogenicity of the protein that induces weak cytotoxic T lymphocyte (CTL) responses directed against the CLGGLLTMV (CLG) epitope. Indeed, the CLG peptide presents low affinity for HLA-A2 and does not produce stable complexes. Therefore we synthesized and tested CLG-dimeric analogues with the purpose of characterizing new compounds with the capacity to bind HLA-A2 molecules. By these studies we have identified a few peptides which, compared to the natural epitope, showed higher affinity for HLA-A2 molecules and superior capacity to form a complex. These dimeric peptides may have the potential to induce efficient CTL responses directed to the natural epitope. PMID:10906411

  17. 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.

  18. Structure and activity of human mitochondrial peptide deformylase, a novel cancer target.

    PubMed

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

    2009-04-17

    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. PMID:19236878

  19. Tissue-Factor Targeted Peptide Amphiphile Nanofibers as an Injectable Therapy To Control Hemorrhage.

    PubMed

    Morgan, Courtney E; Dombrowski, Amanda W; Rubert Pérez, Charles M; Bahnson, Edward S M; Tsihlis, Nick D; Jiang, Wulin; Jiang, Qun; Vercammen, Janet M; Prakash, Vivek S; Pritts, Timothy A; Stupp, Samuel I; Kibbe, Melina R

    2016-01-26

    Noncompressible torso hemorrhage is a leading cause of mortality in civilian and battlefield trauma. We sought to develop an i.v.-injectable, tissue factor (TF)-targeted nanotherapy to stop hemorrhage. Tissue factor was chosen as a target because it is only exposed to the intravascular space upon vessel disruption. Peptide amphiphile (PA) monomers that self-assemble into nanofibers were chosen as the delivery vehicle. Three TF-binding sequences were identified (EGR, RLM, and RTL), covalently incorporated into the PA backbone, and shown to self-assemble into nanofibers by cryo-transmission electron microscopy. Both the RLM and RTL peptides bound recombinant TF in vitro. All three TF-targeted nanofibers bound to the site of punch biopsy-induced liver hemorrhage in vivo, but only RTL nanofibers reduced blood loss versus sham (53% reduction, p < 0.05). Increasing the targeting ligand density of RTL nanofibers yielded qualitatively better binding to the site of injury and greater reductions in blood loss in vivo (p < 0.05). In fact, 100% RTL nanofiber reduced overall blood loss by 60% versus sham (p < 0.05). Evaluation of the biocompatibility of the RTL nanofiber revealed that it did not induce RBC hemolysis, did not induce neutrophil or macrophage inflammation at the site of liver injury, and 70% remained intact in plasma after 30 min. In summary, these studies demonstrate successful binding of peptides to TF in vitro and successful homing of a TF-targeted PA nanofiber to the site of hemorrhage with an associated decrease in blood loss in vivo. Thus, this therapeutic may potentially treat noncompressible hemorrhage. PMID:26700464

  20. Differential distribution of VGF-derived peptides in the adrenal medulla and evidence for their selective modulation.

    PubMed

    D'Amato, Filomena; Noli, Barbara; Brancia, Carla; Cocco, Cristina; Flore, Giovanna; Collu, Maria; Nicolussi, Paola; Ferri, Gian-Luca

    2008-05-01

    While vg f gene knockout mice are hyperactive and hypermetabolic, surprisingly the TLQP-21 brain VGF peptide increased energy consumption, suggesting that opposing regulatory effects could be exerted by peptides alternatively cleaved from the VGF precursor. Using antisera to the VGF precursor C-terminus and three cleavage products, we revealed a distinct differential distribution in adrenal, certain peptides (VGF(422-430): PGH peptides) being found throughout bovine and swine medulla, while C-terminus and TLQP peptides were confined to adrenaline cells in the above species and in rat and C-terminally shortened forms (VGF(604-612): HVLL peptides) to nor-adrenaline cells. Random abattoir samples of bovine and swine adrenal contained 520+/-40 and 450+/-60 pmol/g (mean+/-s.e.m. respectively) of C-terminus peptides and similar or lower amounts of others. Upon gel chromatography, bona fide VGF precursor, approximately 7.5 and approximately 3.5 kDa forms were revealed by C-terminus assays, HVLL peptides being limited to small fragments. TLQP peptides included ~7.5 kDa form and peaks accounting for TLQP-21 and predicted TLQP-30 and TLQP-42. Low molecular weight (MW) PGH peptides were revealed, together with a high MW form possibly encompassing the VGF precursor N-terminus. In acutely stressed swine, a striking increase was seen for C-terminus and TLQP peptides, with no significant differences for PGH peptides. A similar response was found in rat TLQP peptides showing a major increase upon an acute swimming stress and 30 min thereafter. A differential processing of the VGF precursor encompassing many areas of its primary sequence and selective modulations of its derived peptides occur in adrenal medullary cells, possibly relevant to adaptive homeostatic responses. PMID:18434366

  1. [Ectopic osteogenesis in vivo using bone morphogenetic protein-2 derived peptide loaded biodegradable hydrogel].

    PubMed

    Zhao, Jingjing; Fang, Zhenhua; Huang, Ruokun; Xiao, Kai; Li, Jing; Xie, Ming; Kan, Wusheng

    2014-08-01

    We investigated the development of an injectable, biodegradable hydrogel composite of poly(trimethylene carbonate)-F127-poly(trimethylene carbonate)(PTMC11-F127-PTMC11 )loaded with bone morphogenetic protein-2 (BMP-2) derived peptide P24 for ectopic bone formation in vivo and evaluated its release kinetics in vitro. Then we evaluated P24 peptide release kinetics from different concentration of PTMC11-F127-PTMC11 hydrogel in vitro using bicinchoninic acid (BCA)assay. P24/ PTMC11-F127-PTMC11 hydrogel was implanted into each rat's erector muscle of spine and ectopic bone formation of the implanted gel in vivo was detected by hematoxylin and eosin stain (HE). PTMC11-F127-PTMC11 hydrogel with concentration more than 20 percent showed sustained slow release for one month after the initial burst release. Bone trabeculae surround the P24/ PTMC11-F127-PTMC11 hydrogel was shown at the end of six weeks by hematoxylin and eosin stain. These results indicated that encapsulated bone morphogenetic protein (BMP-2) derived peptide P24 remained viable in vivo, thus suggesting the potential of PTMC11-F127-PT- MC11 composite hydrogels as part of a novel strategy for localized delivery of bioactive molecules. PMID:25508424

  2. [Ectopic osteogenesis in vivo using bone morphogenetic protein-2 derived peptide loaded biodegradable hydrogel].

    PubMed

    Zhao, Jingjing; Fang, Zhenhua; Huang, Ruokun; Xiao, Kai; Li, Jing; Xie, Ming; Kan, Wusheng

    2014-08-01

    We investigated the development of an injectable, biodegradable hydrogel composite of poly(trimethylene carbonate)-F127-poly(trimethylene carbonate)(PTMC11-F127-PTMC11 )loaded with bone morphogenetic protein-2 (BMP-2) derived peptide P24 for ectopic bone formation in vivo and evaluated its release kinetics in vitro. Then we evaluated P24 peptide release kinetics from different concentration of PTMC11-F127-PTMC11 hydrogel in vitro using bicinchoninic acid (BCA)assay. P24/ PTMC11-F127-PTMC11 hydrogel was implanted into each rat's erector muscle of spine and ectopic bone formation of the implanted gel in vivo was detected by hematoxylin and eosin stain (HE). PTMC11-F127-PTMC11 hydrogel with concentration more than 20 percent showed sustained slow release for one month after the initial burst release. Bone trabeculae surround the P24/ PTMC11-F127-PTMC11 hydrogel was shown at the end of six weeks by hematoxylin and eosin stain. These results indicated that encapsulated bone morphogenetic protein (BMP-2) derived peptide P24 remained viable in vivo, thus suggesting the potential of PTMC11-F127-PT- MC11 composite hydrogels as part of a novel strategy for localized delivery of bioactive molecules. PMID:25464793

  3. Improved tumor-targeting MRI contrast agents: Gd(DOTA) conjugates of a cycloalkane-based RGD peptide

    SciTech Connect

    Park, Ji-Ae; Lee, Yong Jin; Ko, In Ok; Kim, Tae-Jeong; Chang, Yongmin; Lim, Sang Moo; Kim, Kyeong Min; Kim, Jung Young

    2014-12-12

    Highlights: • Development of improved tumor-targeting MRI contrast agents. • To increase the targeting ability of RGD, we developed cycloalkane-based RGD peptides. • Gd(DOTA) conjugates of cycloalkane-based RGD peptide show improved tumor signal enhancement in vivo MR images. - Abstract: Two new MRI contrast agents, Gd-DOTA-c(RGD-ACP-K) (1) and Gd-DOTA-c(RGD-ACH-K) (2), which were designed by incorporating aminocyclopentane (ACP)- or aminocyclohexane (ACH)-carboxylic acid into Gd-DOTA (gadolinium-tetraazacyclo dodecanetetraacetic acid) and cyclic RGDK peptides, were synthesized and evaluated for tumor-targeting ability in vitro and in vivo. Binding affinity studies showed that both 1 and 2 exhibited higher affinity for integrin receptors than cyclic RGDyK peptides, which were used as a reference. These complexes showed high relaxivity and good stability in human serum and have the potential to improve target-specific signal enhancement in vivo MR images.

  4. Human mitochondrial peptide deformylase, a new anticancer target of actinonin-based antibiotics.

    PubMed

    Lee, Mona D; She, Yuhong; Soskis, Michael J; Borella, Christopher P; Gardner, Jeffrey R; Hayes, Paula A; Dy, Benzon M; Heaney, Mark L; Philips, Mark R; Bornmann, William G; Sirotnak, Francis M; Scheinberg, David A

    2004-10-01

    Peptide deformylase activity was thought to be limited to ribosomal protein synthesis in prokaryotes, where new peptides are initiated with an N-formylated methionine. We describe here a new human peptide deformylase (Homo sapiens PDF, or HsPDF) that is localized to the mitochondria. HsPDF is capable of removing formyl groups from N-terminal methionines of newly synthesized mitochondrial proteins, an activity previously not thought to be necessary in mammalian cells. We show that actinonin, a peptidomimetic antibiotic that inhibits HsPDF, also inhibits the proliferation of 16 human cancer cell lines. We designed and synthesized 33 chemical analogs of actinonin; all of the molecules with potent activity against HsPDF also inhibited tumor cell growth, and vice versa, confirming target specificity. Small interfering RNA inhibition of HsPDF protein expression was also antiproliferative. Actinonin treatment of cells led to a tumor-specific mitochondrial membrane depolarization and ATP depletion in a time- and dose-dependent manner; removal of actinonin led to a recovery of the membrane potential consistent with indirect effects on the electron transport chain. In animal models, oral or parenteral actinonin was well tolerated and inhibited human prostate cancer and lung cancer growth. We conclude that HsPDF is a new human mitochondrial enzyme that may provide a novel selective target for anticancer therapy by use of actinonin-based antibiotics. PMID:15489958

  5. Human mitochondrial peptide deformylase, a new anticancer target of actinonin-based antibiotics

    PubMed Central

    Lee, Mona D.; She, Yuhong; Soskis, Michael J.; Borella, Christopher P.; Gardner, Jeffrey R.; Hayes, Paula A.; Dy, Benzon M.; Heaney, Mark L.; Philips, Mark R.; Bornmann, William G.; Sirotnak, Francis M.; Scheinberg, David A.

    2004-01-01

    Peptide deformylase activity was thought to be limited to ribosomal protein synthesis in prokaryotes, where new peptides are initiated with an N-formylated methionine. We describe here a new human peptide deformylase (Homo sapiens PDF, or HsPDF) that is localized to the mitochondria. HsPDF is capable of removing formyl groups from N-terminal methionines of newly synthesized mitochondrial proteins, an activity previously not thought to be necessary in mammalian cells. We show that actinonin, a peptidomimetic antibiotic that inhibits HsPDF, also inhibits the proliferation of 16 human cancer cell lines. We designed and synthesized 33 chemical analogs of actinonin; all of the molecules with potent activity against HsPDF also inhibited tumor cell growth, and vice versa, confirming target specificity. Small interfering RNA inhibition of HsPDF protein expression was also antiproliferative. Actinonin treatment of cells led to a tumor-specific mitochondrial membrane depolarization and ATP depletion in a time- and dose-dependent manner; removal of actinonin led to a recovery of the membrane potential consistent with indirect effects on the electron transport chain. In animal models, oral or parenteral actinonin was well tolerated and inhibited human prostate cancer and lung cancer growth. We conclude that HsPDF is a new human mitochondrial enzyme that may provide a novel selective target for anticancer therapy by use of actinonin-based antibiotics. PMID:15489958

  6. A novel, bipartite transit peptide targets OEP75 to the outer membrane of the chloroplastic envelope.

    PubMed Central

    Tranel, P J; Keegstra, K

    1996-01-01

    OEP75 is an outer envelope membrane component of the chloroplastic protein import apparatus and is synthesized in the cytoplasm as a higher molecular weight precursor (prOEP75). During its own import, prOEP75 is processed first to an intermediate (iOEP75) and subsequently to the mature form (mOEP75). Experiments conducted with stromal extracts indicated that iOEP75 was generated from prOEP75 by the activity of the stromal processing peptidase. The specific processing site was determined and used to divide the prOEP75 transit peptide into N- and C-terminal domains. To determine the targeting functions of the two domains of the transit peptide and of the mature region of prOEP75, we created a deletion mutant construct from prOEP75 and chimeric constructs between domains of prOEP75 and the precursor to a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Analysis of these constructs by in vitro chloroplastic protein import assays revealed that the transit peptide of prOEP75 is bipartite in that the N- and C-terminal portions contain chloroplastic and intraorganellar targeting information, respectively. PMID:8953773

  7. Using PeptideAtlas, SRMAtlas and PASSEL – Comprehensive Resources for discovery and targeted proteomics

    PubMed Central

    Kusebauch, Ulrike; Deutsch, Eric W.; Campbell, David S.; Sun, Zhi; Farrah, Terry; Moritz, Robert L.

    2014-01-01

    PeptideAtlas, SRMAtlas and PASSEL are web-accessible resources to support discovery and targeted proteomics research. PeptideAtlas is a multi-species compendium of shotgun proteomic data provided by the scientific community, SRMAtlas is a resource of high-quality, complete proteome SRM assays generated in a consistent manner for the targeted identification and quantification of proteins, and PASSEL is a repository that compiles and represents selected reaction monitoring data, all in an easy to use interface. The databases are generated from native mass spectrometry data files that are analyzed in a standardized manner including statistical validation of the results. Each resource offers search functionalities and can be queried by user defined constraints; the query results are provided in tables or are graphically displayed. PeptideAtlas, SRMAtlas and PASSEL are publicly available freely via the website http://www.peptideatlas.org. In this protocol, we describe the use of these resources, we highlight how to submit, search, collate and download data. PMID:24939129

  8. An update of radiolabeled bombesin analogs for gastrin-releasing peptide receptor targeting.

    PubMed

    Yu, Zilin; Ananias, Hildo J K; Carlucci, Giuseppe; Hoving, Hilde D; Helfrich, Wijnand; Dierckx, Rudi A J O; Wang, Fan; de Jong, Igle J; Elsinga, Philip H

    2013-01-01

    Prostate cancer is a critical public health problem in USA and Europe. New non-invasive imaging methods are urgently needed, due to the low accuracy and specificity of current screen methods and the desire of localizing primary prostate cancer and bone metastasis. Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) are the non-invasive and sensitive imaging methods which have been widely used for diagnosing diseases in the clinic. Lack of suitable radiotracers is the major issue for nuclear imaging of prostate cancer, although radiolabeled bombesin (BN) peptides targeting the Gastrin-Releasing Peptide Receptor (GRPR) on tumor cells are widely investigated. In this review we discuss the recent trends in the development of GRPR-targeted radiopharmaceuticals based on BN analogs with regard to their potential for imaging and therapy of GRPR-expressing malignancies. Following a brief introduction of GRPR and bombesin peptides, we summarize the properties of prostate cancer specific radiolabeled bombesins. New bombesin tracers published in the last five years are reviewed and compared according to their novelties in biomolecules, radionuclides, labeling methods, bifunctional chelators and linkers. Hot topics such as multimerization, application of agonists and antagonists are highlighted in the review. Lastly, a few clinical trials of cancer nuclear imaging with radiolabeled bombesin have been discussed. PMID:23431995

  9. Non-Peptide Macrocyclic Histone Deacetylase Inhibitors Derived from Tricyclic Ketolide Skeleton

    PubMed Central

    Mwakwari, Sandra C.; Guerrant, William; Patil, Vishal; Khan, Shabana I.; Tekwani, Babu L.; Gurard-Levin, Zachary A.; Mrksich, Milan; Oyelere, Adegboyega K.

    2010-01-01

    Inhibition of histone deacetylase (HDAC) function is a validated therapeutic strategy for cancer treatment. Of the several structurally distinct small molecule histone deacetylase inhibitors (HDACi) reported, macrocyclic depsipeptides possess the most complex cap-groups and have demonstrated excellent HDAC inhibition potency and isoform selectivity. Unfortunately, the development of macrocyclic depsipeptides has been hampered in part due to development problems characteristic of large peptides and the complex reaction schemes required for their synthesis. Herein we report that tricyclic ketolide TE-802 is an excellent mimetic for the peptide backbone of macrocyclic HDACi. Compounds derived from this template are particularly selective against HDAC 1 and 2 with nanomolar inhibitory activity. Interrogation of the association between a subset of these compounds and key HDAC isoforms, using AutoDock, enables a molecular description of the interaction between the HDAC enzyme's outer rim and the inhibitors’ macrocyclic cap group that are responsible for compound affinity and presumably isoform selectivity. PMID:20669972

  10. Identification and Molecular Characterization of Molluskin, a Histone-H2A-Derived Antimicrobial Peptide from Molluscs

    PubMed Central

    Sathyan, Naveen; Philip, Rosamma; Chaithanya, E. R.; Anil Kumar, P. R.

    2012-01-01

    Antimicrobial peptides are humoral innate immune components of molluscs that provide protection against pathogenic microorganisms. Among these, histone-H2A-derived antimicrobial peptides are known to actively participate in host defense responses of molluscs. Present study deals with identification of putative antimicrobial sequences from the histone-H2A of back-water oyster Crassostrea madrasensis, rock oyster Saccostrea cucullata, grey clam Meretrix casta, fig shell Ficus gracilis, and ribbon bullia Bullia vittata. A 75 bp fragment encoding 25 amino acid residues was amplified from cDNA of these five bivalves and was named “Molluskin.” The 25 amino acid peptide exhibited high similarity to previously reported histone-H2A-derived AMPs from invertebrates indicating the presence of an antimicrobial sequence motif. Physicochemical properties of the peptides are in agreement with the characteristic features of antimicrobial peptides, indicating their potential role in innate immunity of molluscs.

  11. Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer

    NASA Astrophysics Data System (ADS)

    McGuire, Michael J.; Gray, Bethany Powell; Li, Shunzi; Cupka, Dorothy; Byers, Lauren Averett; Wu, Lei; Rezaie, Shaghayegh; Liu, Ying-Horng; Pattisapu, Naveen; Issac, James; Oyama, Tsukasa; Diao, Lixia; Heymach, John V.; Xie, Xian-Jin; Minna, John D.; Brown, Kathlynn C.

    2014-03-01

    Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071-40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands.

  12. Amyloid fibril formation of peptides derived from the C-terminus of CETP modulated by lipids

    SciTech Connect

    García-González, Victor; Mas-Oliva, Jaime

    2013-04-26

    Highlights: •The secondary structure of a C-terminal peptide derived from CETP was studied. •Lipids modulate secondary structure changes of a C-terminal peptide derived from CETP. •Lysophosphatidic acid maintains a functional α-helix and prevents fibril formation. •Transfer of lipids by CETP is related to the presence of an α-helix at its C-end. -- Abstract: Cholesteryl-ester transfer protein (CETP) is a plasmatic protein involved in neutral lipid transfer between lipoproteins. Focusing on the last 12 C-terminus residues we have previously shown that mutation D{sub 470}N promotes a conformational change towards a β-secondary structure. In turn, this modification leads to the formation of oligomers and fibrillar structures, which cause cytotoxic effects similar to the ones provoked by amyloid peptides. In this study, we evaluated the role of specific lipid arrangements on the structure of peptide helix-Z (D{sub 470}N) through the use of thioflavin T fluorescence, peptide bond absorbance, circular dichroism and electron microscopy. The results indicate that the use of micelles formed with lysophosphatidylcholine and lysophosphatidic acid (LPA) under neutral pH induce a conformational transition of peptide helix-Z containing a β-sheet conformation to a native α-helix structure, therefore avoiding the formation of amyloid fibrils. In contrast, incubation with phosphatidic acid does not change the profile for the β-sheet conformation. When the electrostatic charge at the surface of micelles or vesicles is regulated through the use of lipids such as phospholipid and LPA, minimal changes and the presence of β-structures were recorded. Mixtures with a positive net charge diminished the percentage of β-structure and the amount of amyloid fibrils. Our results suggest that the degree of solvation determined by the presence of a free hydroxyl group on lipids such as LPA is a key condition that can modulate the secondary structure and the consequent formation of

  13. Spectroscopic and structural elucidation of amino acid derivatives and small peptides: experimental and theoretical tools.

    PubMed

    Kolev, Tsonko; Spiteller, Michael; Koleva, Bojidarka

    2010-01-01

    This mini review deals with the modern aspects of the spectroscopy and structural elucidation of amino acid derivatives and small biologically active compounds. Free peptide bond rotation in these systems yields various conformers, which possess differing biological activities. Another phenomenon is the intermolecular or intramolecular stacking observed in aromatic small peptides. Specifically, the main aim is to illustrate the successful application of the "complex tool", consisting of a combination of the theoretical approximation methods with experimental linear polarized infrared (IR-LD) and/or Raman spectroscopy of oriented colloid suspensions in a nematic host. The possibilities and limitations of the approach for detailed vibrational assignment and structural elucidation of small peptides are discussed. Having in mind that physical and chemical properties of these systems can be precisely calculated by means of ab initio and DFT methods at Hartee-Fock, MP2 and B3LYP level of theory, varying basis sets, the results obtained allow a precise assignment of many vibrational bands to the corresponding normal modes, electronic structures and conformational state. The validity of the conclusions about the structure or vibrational properties of these systems have been supported, compared and/or additionally proved by the results from independent physical methods. In this respect (1)H and (13)C-NMR, single crystal X-ray diffraction, HPLC tandem mass spectrometry as well as thermal methods are all employed. A well ordered crystal must first be grown in order to determine the molecular structure by the absolute method of single crystal X-ray diffraction. Although the 3D structures of peptides have been determined over the past decades, peptide crystallization is still a major obstacle to X-ray diffraction work, the presence of chiral centre/s makes for this difficulty. For this reason the "complex tool" presented can be regarded as an alternative method for obtaining of

  14. Octreotide-Mediated Tumor-Targeted Drug Delivery via a Cleavable Doxorubicin-Peptide Conjugate.

    PubMed

    Lelle, Marco; Kaloyanova, Stefka; Freidel, Christoph; Theodoropoulou, Marily; Musheev, Michael; Niehrs, Christof; Stalla, Günter; Peneva, Kalina

    2015-12-01

    Although recent methods for targeted drug delivery have addressed many of the existing problems of cancer therapy associated with undesirable side effects, significant challenges remain that have to be met before they find significant clinical relevance. One such area is the delicate chemical bond that is applied to connect a cytotoxic drug with targeting moieties like antibodies or peptides. Here we describe a novel platform that can be utilized for the preparation of drug-carrier conjugates in a site-specific manner, which provides excellent versatility and enables triggered release inside cancer cells. Its key feature is a cleavable doxorubicin-octreotide bioconjugate that targets overexpressed somatostatin receptors on tumor cells, where the coupling between the two components was achieved through the first cleavable disulfide-intercalating linker. The tumor targeting ability and suppression of adrenocorticotropic hormone secretion in AtT-20 cells by both octreotide and the doxorubicin hybrid were determined via a specific radioimmunoassay. Both substances reduced the hormone secretion to a similar extent, which demonstrated that the tumor homing peptide is able to interact with the relevant cell surface receptors after the attachment of the drug. Effective drug release was quickly accomplished in the presence of the physiological reducing agent glutathione. We also demonstrate the relevance of this scaffold in biological context in cytotoxicity assays with pituitary, pancreatic, and breast cancer cell lines. PMID:26524088

  15. Targeting dendritic cells in lymph node with an antigen peptide-based nanovaccine for cancer immunotherapy.

    PubMed

    Qian, Yuan; Jin, Honglin; Qiao, Sha; Dai, Yanfeng; Huang, Chuan; Lu, Lisen; Luo, Qingming; Zhang, Zhihong

    2016-08-01

    The design of peptide-based subunit vaccine formulations for the direct delivery of tumor antigen peptides (Aps) to dendritic cells (DCs) localized within draining lymph nodes (DLNs) is challenging. Mature DCs (mDCs) are abundantly distributed within DLNs but have dramatically reduced endocytic uptake and antigen-processing abilities, so their role as potential vaccine targets has been largely overlooked. Here we report an ultra-small biocompatible nanovaccine (α-Ap-FNP) functionalized by avidly targeting delivery of Ap via the scavenger receptor class B1 (SR-B1) pathway to mDCs. The self-assembly, small size (∼30 nm), SR-B1-targeting and optical properties of α-Ap-FNP resulted in its efficient Ap loading, substantial LN accumulation, targeting of mDCs and enhanced Ap presentation, and fluorescence trafficking, respectively. We also demonstrate that the α-Ap-FNP can be either used alone or encapsulated with CpG oligodeoxynucleotide as a prophylactic and therapeutic vaccine. Thus, the excellent properties of α-Ap-FNP provide it potential for clinical applications as a potent nanovaccine for cancer immunotherapy. PMID:27192420

  16. Affinity capture using peptide-functionalized magnetic nanoparticles to target Staphylococcus aureus.

    PubMed

    Kuo, Fang-Yin; Lin, Wei-Lien; Chen, Yu-Chie

    2016-04-28

    Staphylococcus aureus, a commonly found pathogen, can cause food poisoning and infections. Thus, it is necessary to develop analytical methods for the rapid screening of S. aureus in suspicious samples. Magnetic nanoparticles (MNPs) are widely used as affinity probes to selectively enrich target species from complex samples because of their high specific surface area and magnetic properties. The MNP surface should be functionalized to have the capability to target specific species. We herein propose a straightforward method to functionalize aluminum oxide-coated iron oxide (Fe3O4@Al2O3) MNPs with the peptide HHHHHHDEEGLFVD (D). The peptide D was comprised of three domains: polyhistidine (H6) used as the linker, DEE added as the spacer, and GLFVD used for targeting S. aureus. D was immobilized on the surface of Fe3O4@Al2O3 MNPs through H6-Al chelation. Our results showed that the D-functionalized Fe3O4@Al2O3 MNPs (D-Fe3O4 MNPs) possess the capability to target S. aureus. The selective trapping experiments were conducted under microwave-heating for only 60 s, and sufficient bacterial cells were trapped by the MNPs to be identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We demonstrated that the D-Fe3O4@Al2O3 MNPs combined with MALDI-MS can be used to rapidly characterize trace amounts of S. aureus in complex juice and egg samples. PMID:27087258

  17. Peptide array-based screening of human mesenchymal stem cell-adhesive peptides derived from fibronectin type III domain

    SciTech Connect

    Okochi, Mina; Nomura, Shigeyuki; Kaga, Chiaki; Honda, Hiroyuki

    2008-06-20

    Human mesenchymal stem cell-adhesive peptides were screened based on the amino acid sequence of fibronectin type III domain 8-11 (FN-III{sub 8-11}) using a peptide array synthesized by the Fmoc-chemistry. Using hexameric peptide library of FN-III{sub 8-11} scan, we identified the ALNGR (Ala-Leu-Asn-Gly-Arg) peptide that induced cell adhesion as well as RGDS (Arg-Gly-Asp-Ser) peptide. After incubation for 2 h, approximately 68% of inoculated cells adhere to the ALNGR peptide disk. Adhesion inhibition assay with integrin antibodies showed that the ALNGR peptide interacts with integrin {beta}1 but not with {alpha}v{beta}3, indicating that the receptors for ALNGR are different from RGDS. Additionally, the ALNGR peptide expressed cell specificities for adhesion: cell adhesion was promoted for fibroblasts but not for keratinocytes or endotherial cells. The ALNGR peptide induced cell adhesion and promoted cell proliferation without changing its property. It is therefore useful for the construction of functional biomaterials.

  18. Assessment of meat authenticity using bioinformatics, targeted peptide biomarkers and high-resolution mass spectrometry.

    PubMed

    Ruiz Orduna, Alberto; Husby, Erik; Yang, Charles T; Ghosh, Dipankar; Beaudry, Francis

    2015-01-01

    In recent years a significant increase of food fraud has been observed, ranging from false label claims to the use of additives and fillers to increase profitability. Recently in 2013 horse and pig DNAs were detected in beef products sold from several retailers. Mass spectrometry (MS) has become the workhorse in protein research, and the detection of marker proteins could serve for both animal species and tissue authentication. Meat species authenticity is performed in this paper using a well-defined proteogenomic annotation, carefully chosen surrogate tryptic peptides and analysis using a hybrid quadrupole-Orbitrap MS. Selected mammalian meat samples were homogenised and proteins were extracted and digested with trypsin. The samples were analysed using a high-resolution MS. Chromatography was achieved using a 30-min linear gradient along with a BioBasic C8 100 × 1 mm column at a flow rate of 75 µl min(-1). The MS was operated in full-scan high resolution and accurate mass. MS/MS spectra were collected for selected proteotypic peptides. Muscular proteins were methodically analysed in silico in order to generate tryptic peptide mass lists and theoretical MS/MS spectra. Following a comprehensive bottom-up proteomic analysis, we detected and identified a proteotypic myoglobin tryptic peptide (120-134) for each species with observed m/z below 1.3 ppm compared with theoretical values. Moreover, proteotypic peptides from myosin-1, myosin-2 and β-haemoglobin were also identified. This targeted method allowed comprehensive meat speciation down to 1% (w/w) of undesired product. PMID:26241836

  19. Vaccine for human contraception targeting sperm Izumo protein and YLP12 dodecamer peptide

    PubMed Central

    Naz, Rajesh K

    2014-01-01

    There is an urgent need to develop a better method of contraception which is non-steroidal and reversible to control world population explosion and unintended pregnancies. Contraceptive vaccines (CV), especially targeting sperm-specific proteins, can provide an ideal contraceptive modality. Sperm-specific proteins can induce an immune response in women as well as men, thus can be used for CV development in both sexes. In this article, we will review two sperm-specific proteins, namely Izumo protein and YLP12 dodecamer peptide. Gene-knockout studies indicate that Izumo protein is essential for sperm–egg membrane fusion. Vaccination with Izumo protein or its cDNA causes a significant reduction in fertility of female mice. The antibodies to human Izumo inhibit human sperm penetration assay. Recently, our laboratory found that a significant percentage of infertile women have antibodies to Izumo protein. The second sperm-specific protein is YLP12, a peptide mimetic sequence present on human sperm involved in recognition and binding to the human oocyte zona pellucida. Vaccination with YLP12 or its cDNA causes long-term, reversible contraception, without side effects, in female mice. Infertile, but not fertile, men and women have antibodies to YLP12 peptide. Our laboratory has isolated, cloned, and sequenced cDNA encoding human single chain variable fragment (scFv) antibody from infertile men which reacts with YLP12 peptide. The human YLP12 scFv antibody may provide a novel passive immunocontraceptive, the first of its kind. In conclusion, sperm-specific Izumo protein and YLP12 peptide can provide exciting candidates for antisperm CV development. PMID:24723387

  20. Modulation of hydrogel nanoparticle intracellular trafficking by multivalent surface engineering with tumor targeting peptide.

    PubMed

    Karamchand, Leshern; Kim, Gwangseong; Wang, Shouyan; Hah, Hoe Jin; Ray, Aniruddha; Jiddou, Ruba; Koo Lee, Yong-Eun; Philbert, Martin A; Kopelman, Raoul

    2013-11-01

    Surface engineering of a hydrogel nanoparticle (NP) with the tumor-targeting ligand, F3 peptide, enhances both the NP's binding affinity for, and internalization by, nucleolin overexpressing tumor cells. Remarkably, the F3-functionalized NPs consistently exhibited significantly lower trafficking to the degradative lysosomes than the non-functionalized NPs, in the tumor cells, after internalization. This is attributed to the non-functionalized NPs, but not the F3-functionalized NPs, being co-internalized with Lysosome-associated Membrane Protein-1 (LAMP1) from the surface of the tumor cells. Furthermore, it is shown that the intracellular trafficking of the F3-functionalized NPs differs significantly from that of the molecular F3 peptides (untethered to NPs). This has important implications for designing effective, chemically-responsive, controlled-release and multifunctional nanodrugs for multi-drug-resistant cancers. PMID:24056573

  1. A peptide-linked recombinant glucocerebrosidase for targeted neuronal delivery: Design, production, and assessment.

    PubMed

    Gramlich, Paul A; Westbroek, Wendy; Feldman, Ricardo A; Awad, Ola; Mello, Nicholas; Remington, Mary P; Sun, Ying; Zhang, Wujuan; Sidransky, Ellen; Betenbaugh, Michael J; Fishman, Paul S

    2016-03-10

    Although recombinant glucocerebrosidase (GCase) is the standard therapy for the inherited lysosomal storage disease Gaucher's disease (GD), enzyme replacement is not effective when the central nervous system is affected. We created a series of recombinant genes/proteins where GCase was linked to different membrane binding peptides including the Tat peptide, the rabies glycoprotein derived peptide (RDP), the binding domain from tetanus toxin (TTC), and a tetanus like peptide (Tet1). The majority of these proteins were well-expressed in a mammalian producer cell line (HEK 293F). Purified recombinant Tat-GCase and RDP-GCase showed similar GCase protein delivery to a neuronal cell line that genetically lacks the functional enzyme, and greater delivery than control GCase, Cerezyme (Genzyme). This initial result was unexpected based on observations of superior protein delivery to neurons with RDP as a vector. A recombinant protein where a fragment of the flexible hinge region from IgA (IgAh) was introduced between RDP and GCase showed substantially enhanced GCase neuronal delivery (2.5 times over Tat-GCase), suggesting that the original construct resulted in interference with the capacity of RDP to bind neuronal membranes. Extended treatment of these knockout neuronal cells with either Tat-GCase or RDP-IgAh-GCase resulted in an >90% reduction in the lipid substrate glucosylsphingosine, approaching normal levels. Further in vivo studies of RDP-IgAh-GCase as well as Tat-GCase are warranted to assess their potential as treatments for neuronopathic forms of GD. These peptide vectors are especially attractive as they have the potential to carry a protein across the blood-brain barrier, avoiding invasive direct brain delivery. PMID:26795355

  2. A new peptide ligand for colon cancer targeted delivery of micelles.

    PubMed

    Ren, Yachao; Mu, Yu; Song, Yanping; Xie, Jinxin; Yu, Hui; Gao, Sainan; Li, Sen; Peng, Haisheng; Zhou, Yulong; Lu, Weiyue

    2016-06-01

    Ligands are an imperative part of targeted drug delivery systems, and choosing a ligand with high affinity is a subject of considerable interest. In this study, we first synthesized a 12-residue peptide (TK) that interacts with integrin α6β1 overexpressed on colonic cancer cells. The molecular binding affinity assay indicated that TK had a high binding affinity for integrin α6β1. The results of cellular and tumor spheroid uptake suggested that TK peptide not only increases Caco-2 cells uptake, but also effectively increases penetration of the tumor spheroids. TK-conjugated PEG-PLA was synthesized to prepare a novel PEG-PLA micelles loading DOX or coumarin-6 (TK-MS/DOX or TK-MS/C6). The obtained TK-MS/DOX exhibited uniform, spherical shape with a size of 23.80 ± 0.32 nm and zeta potential of 12.21 ± 0.31 mV. The release behavior of DOX from micelles were observed no significant changes after TK modification, however, the release profile exhibited pH-sensitive properties. Compared with MS/DOX, TK-MS/DOX exhibited significantly stronger cytotoxicity for Caco-2. Confocal laser microscopy and flow cytometry data further indicated that the targeting micelles not only had higher uptake by Caco-2 cells, but also more effectively penetrated the tumor spheroids. Therefore, TK peptide appears to be suitable as a targeting ligand with potential applications in colonic targeted therapy. PMID:26289214

  3. Dissociation of Calmodulin-Target Peptide Complexes by the Lipid Mediator Sphingosylphosphorylcholine

    PubMed Central

    Kovacs, Erika; Tóth, Judit; Vértessy, Beáta G.; Liliom, Károly

    2010-01-01

    Previously we have identified the lipid mediator sphingosylphosphorylcholine (SPC) as the first potentially endogenous inhibitor of the ubiquitous Ca2+ sensor calmodulin (CaM) (Kovacs, E., and Liliom, K. (2008) Biochem. J. 410, 427–437). Here we give mechanistic insight into CaM inhibition by SPC, based on fluorescence stopped-flow studies with the model CaM-binding domain melittin. We demonstrate that both the peptide and SPC micelles bind to CaM in a rapid and reversible manner with comparable affinities. Furthermore, we present kinetic evidence that both species compete for the same target site on CaM, and thus SPC can be considered as a competitive inhibitor of CaM-target peptide interactions. We also show that SPC disrupts the complex of CaM and the CaM-binding domain of ryanodine receptor type 1, inositol 1,4,5-trisphosphate receptor type 1, and the plasma membrane Ca2+ pump. By interfering with these interactions, thus inhibiting the negative feedback that CaM has on Ca2+ signaling, we hypothesize that SPC could lead to Ca2+ mobilization in vivo. Hence, we suggest that the action of the sphingolipid on CaM might explain the previously recognized phenomenon that SPC liberates Ca2+ from intracellular stores. Moreover, we demonstrate that unlike traditional synthetic CaM inhibitors, SPC disrupts the complex between not only the Ca2+-saturated but also the apo form of the protein and the target peptide, suggesting a completely novel regulation for target proteins that constitutively bind CaM, such as ryanodine receptors. PMID:19910470

  4. [The Qualitative Analysis of the Amide Derivative of HLDF-6 Peptide and Its Metabolites with the Use of Tritium- and Deuterium-Labeled Derivatives].

    PubMed

    Zolotarev, A; Dadayan, A K; Kost, N V; Voevodina, M E; Sokolov, O Y; Kozik, V S; Shram, S I; Azev, V N; Bocharov, E V; Bogachouk, A P; Lipkin, V M; Myasoedov, N F

    2015-01-01

    The goal of the study was to elaborate the pharmacokinetics methods of the amide derivative of peptide HLDF-6 (TGENHR-NH2) and its range of nootropic and neuroprotective activity is wide. The hexapeptide 41TGENHR46 is a fragment of the HDLF differentiation factor. It forms the basis for the development of preventive and therapeutic preparations for treating cerebrovascular and neurodegenerative conditions. Pharmacokinetic and molecular mechanisms of the action of the HLDF-6 peptide were studied using tritium- and deuterium-labeled derivatives of this peptide, produced with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). This reaction was employed to produce the tritium-labeled peptide [3H]TGENHR-NH2 with a molar radioactivity of 230 Ci/mmol and the deuterium-labeled peptide [2H]TGENHR-NH2 with an average deuterium incorporation equal to 10.5 atoms. It was shown by the NMR spectroscopy that the isotope label distribution over the labeled peptide's molecule was uniform, which allowed qualitative analysis ofboth the peptide itself and its fragments in the organism's tissues to be conducted. The newly developed pharmacokinetics method makes it possible to avoid almost completely losses of the peptides under study due to biodegradation during the analysis of tissues. These labeled peptides were used in mice, rats and rabbits to study the pharmacokinetics of the peptide and to calculate the values of its principal pharmacokinetic parameters. Characteristics of its pharmacokinetic profile in the blood were obtained, the hypothesis of pharmacokinetics linearity tested, its metabolism analyzed and its bioavailability value, 34%, calculated. It has been shown that the studied TGENHR-NH2 peptide shows high resistance to hydrolysis in the blood plasma, with dipeptidyl aminopeptidases making the largest contribution to its hydrolysis. PMID:27125017

  5. Modulation of hydrogel nanoparticle intracellular trafficking by multivalent surface engineering with tumor targeting peptide

    NASA Astrophysics Data System (ADS)

    Karamchand, Leshern; Kim, Gwangseong; Wang, Shouyan; Hah, Hoe Jin; Ray, Aniruddha; Jiddou, Ruba; Koo Lee, Yong-Eun; Philbert, Martin A.; Kopelman, Raoul

    2013-10-01

    Surface engineering of a hydrogel nanoparticle (NP) with the tumor-targeting ligand, F3 peptide, enhances both the NP's binding affinity for, and internalization by, nucleolin overexpressing tumor cells. Remarkably, the F3-functionalized NPs consistently exhibited significantly lower trafficking to the degradative lysosomes than the non-functionalized NPs, in the tumor cells, after internalization. This is attributed to the non-functionalized NPs, but not the F3-functionalized NPs, being co-internalized with Lysosome-associated Membrane Protein-1 (LAMP1) from the surface of the tumor cells. Furthermore, it is shown that the intracellular trafficking of the F3-functionalized NPs differs significantly from that of the molecular F3 peptides (untethered to NPs). This has important implications for designing effective, chemically-responsive, controlled-release and multifunctional nanodrugs for multi-drug-resistant cancers.Surface engineering of a hydrogel nanoparticle (NP) with the tumor-targeting ligand, F3 peptide, enhances both the NP's binding affinity for, and internalization by, nucleolin overexpressing tumor cells. Remarkably, the F3-functionalized NPs consistently exhibited significantly lower trafficking to the degradative lysosomes than the non-functionalized NPs, in the tumor cells, after internalization. This is attributed to the non-functionalized NPs, but not the F3-functionalized NPs, being co-internalized with Lysosome-associated Membrane Protein-1 (LAMP1) from the surface of the tumor cells. Furthermore, it is shown that the intracellular trafficking of the F3-functionalized NPs differs significantly from that of the molecular F3 peptides (untethered to NPs). This has important implications for designing effective, chemically-responsive, controlled-release and multifunctional nanodrugs for multi-drug-resistant cancers. Electronic supplementary information (ESI) available: Effect of Potassium depletion on F3 peptide subcellular localization, MTT

  6. Inhibition of Human Papillomavirus DNA Replication by an E1-Derived p80/UAF1-Binding Peptide

    PubMed Central

    Lehoux, Michaël; Fradet-Turcotte, Amélie; Lussier-Price, Mathieu; Omichinski, James G.

    2012-01-01

    The papillomavirus E1 helicase is recruited by E2 to the viral origin, where it assembles into a double hexamer that orchestrates replication of the viral genome. We previously identified the cellular WD40 repeat-containing protein p80/UAF1 as a novel interaction partner of E1 from anogenital human papillomavirus (HPV) types. p80 was found to interact with the first 40 residues of HPV type 31 (HPV31) E1, and amino acid substitutions within this domain abrogated the maintenance of the viral episome in keratinocytes. In this study, we report that these p80-binding substitutions reduce by 70% the ability of E1 to support transient viral DNA replication without affecting its interaction with E2 and assembly at the origin in vivo. Microscopy studies revealed that p80 is relocalized from the cytoplasm to discrete subnuclear foci by E1 and E2. Chromatin immunoprecipitation assays further revealed that p80 is recruited to the viral origin in an E1- and E2-dependent manner. Interestingly, overexpression of a 40-amino-acid-long p80-binding peptide, derived from HPV31 E1, was found to inhibit viral DNA replication by preventing the recruitment of endogenous p80 to the origin. Mutant peptides defective for p80 interaction were not inhibitory, demonstrating the specificity of this effect. Characterization of this E1 peptide by nuclear magnetic resonance (NMR) showed that it is intrinsically disordered in solution, while mapping studies indicated that the WD repeats of p80 are required for E1 interaction. These results provide additional evidence for the requirement for p80 in anogenital HPV DNA replication and highlight the potential of E1-p80 interaction as a novel antiviral target. PMID:22278251

  7. Inhibition of human papillomavirus DNA replication by an E1-derived p80/UAF1-binding peptide.

    PubMed

    Lehoux, Michaël; Fradet-Turcotte, Amélie; Lussier-Price, Mathieu; Omichinski, James G; Archambault, Jacques

    2012-04-01

    The papillomavirus E1 helicase is recruited by E2 to the viral origin, where it assembles into a double hexamer that orchestrates replication of the viral genome. We previously identified the cellular WD40 repeat-containing protein p80/UAF1 as a novel interaction partner of E1 from anogenital human papillomavirus (HPV) types. p80 was found to interact with the first 40 residues of HPV type 31 (HPV31) E1, and amino acid substitutions within this domain abrogated the maintenance of the viral episome in keratinocytes. In this study, we report that these p80-binding substitutions reduce by 70% the ability of E1 to support transient viral DNA replication without affecting its interaction with E2 and assembly at the origin in vivo. Microscopy studies revealed that p80 is relocalized from the cytoplasm to discrete subnuclear foci by E1 and E2. Chromatin immunoprecipitation assays further revealed that p80 is recruited to the viral origin in an E1- and E2-dependent manner. Interestingly, overexpression of a 40-amino-acid-long p80-binding peptide, derived from HPV31 E1, was found to inhibit viral DNA replication by preventing the recruitment of endogenous p80 to the origin. Mutant peptides defective for p80 interaction were not inhibitory, demonstrating the specificity of this effect. Characterization of this E1 peptide by nuclear magnetic resonance (NMR) showed that it is intrinsically disordered in solution, while mapping studies indicated that the WD repeats of p80 are required for E1 interaction. These results provide additional evidence for the requirement for p80 in anogenital HPV DNA replication and highlight the potential of E1-p80 interaction as a novel antiviral target. PMID:22278251

  8. Monodisperse magnetite nanoparticles coupled with nuclear localization signal peptide for cell-nucleus targeting.

    PubMed

    Xu, Chenjie; Xie, Jin; Kohler, Nathan; Walsh, Edward G; Chin, Y Eugene; Sun, Shouheng

    2008-03-01

    Functionalization of monodisperse superparamagnetic magnetite (Fe(3)O(4)) nanoparticles for cell specific targeting is crucial for cancer diagnostics and therapeutics. Targeted magnetic nanoparticles can be used to enhance the tissue contrast in magnetic resonance imaging (MRI), to improve the efficiency in anticancer drug delivery, and to eliminate tumor cells by magnetic fluid hyperthermia. Herein we report the nucleus-targeting Fe(3)O(4) nanoparticles functionalized with protein and nuclear localization signal (NLS) peptide. These NLS-coated nanoparticles were introduced into the HeLa cell cytoplasm and nucleus, where the particles were monodispersed and non-aggregated. The success of labeling was examined and identified by fluorescence microscopy and MRI. The work demonstrates that monodisperse magnetic nanoparticles can be readily functionalized and stabilized for potential diagnostic and therapeutic applications. PMID:18080259

  9. Innate immunity: Bacterial cell-wall muramyl peptide targets the conserved transcription factor YB-1.

    PubMed

    Laman, A G; Lathe, R; Savinov, G V; Shepelyakovskaya, A O; Boziev, Kh M; Baidakova, L K; Chulin, A N; Brovko, F A; Svirshchevskaya, E V; Kotelevtsev, Y; Eliseeva, I A; Guryanov, S G; Lyabin, D N; Ovchinnikov, L P; Ivanov, V T

    2015-07-01

    The bacterial cell wall muramyl dipeptides MDP and glucosaminyl-MDP (GMDP) are powerful immunostimulators but their binding target remains controversial. We previously reported expression cloning of GMDP-binding polypeptides and identification of Y-box protein 1 (YB-1) as their sole target. Here we show specific binding of GMDP to recombinant YB-1 protein and subcellular colocalization of YB-1 and GMDP. GMDP binding to YB-1 upregulated gene expression levels of NF-κB2, a mediator of innate immunity. Furthermore, YB-1 knockdown abolished GMDP-induced Nfkb2 expression. GMDP/YB-1 stimulation led to NF-κB2 cleavage, transport of activated NF-κB2 p52 to the nucleus, and upregulation of NF-κB2-dependent chemokine Cxcr4 gene expression. Therefore, our findings identify YB-1 as new target for muramyl peptide signaling. PMID:26026270

  10. Dual Functional Peptide-Driven Nanoparticles for Highly Efficient Glioma-Targeting and Drug Codelivery.

    PubMed

    Kuang, Yuyang; Jiang, Xutao; Zhang, Yu; Lu, Yifei; Ma, Haojun; Guo, Yubo; Zhang, Yujie; An, Sai; Li, Jianfeng; Liu, Lisha; Wu, Yinhao; Liang, Jianying; Jiang, Chen

    2016-05-01

    Compared with peripheral tumors, glioma is very difficult to treat, not only because it has general features of tumor but also because the therapy has been restricted by the brain-blood barrier (BBB). The two main features of tumor growth are angiogenesis and proliferation of tumor cells. RNA interference (RNAi) can downregulate VEGF overexpression to inhibit tumor neovascularization. Meanwhile, doxorubicin (DOX) has been used for cytotoxic chemotherapy to kill tumor cells. Thus, combining RNAi and chemotherapy has been regarded as a potential strategy for cancer treatment. However, the BBB limits the shVEGF-DOX codelivery system to direct into glioma. Here, a smart drug delivery system modified with a dual functional peptide was established, which could target to transferrin receptor (TfR) overexpressing on both the BBB and glioma. It showed that the dual-targeting delivery system had high tumor targeting efficiency in vitro and in vivo. PMID:27058780

  11. Cancer Cell Signaling Pathways Targeted by Spice-Derived Nutraceuticals

    PubMed Central

    Sung, Bokyung; Prasad, Sahdeo; Yadav, Vivek R.; Aggarwal, Bharat B.

    2012-01-01

    Extensive research within the last half a century has revealed that cancer is caused by dysregulation of as many as 500 different gene products. Most natural products target multiple gene products and thus are ideally suited for prevention and treatment of various chronic diseases, including cancer. Dietary agents such as spices have been used extensively in the Eastern world for a variety of ailments for millennia, and five centuries ago they took a golden journey to the Western world. Various spice-derived nutraceuticals, including 1′-acetoxychavicol acetate, anethole, capsaicin, car-damonin, curcumin, dibenzoylmethane, diosgenin, eugenol, gambogic acid, gingerol, thymoquinone, ursolic acid, xanthohumol, and zerumbone derived from galangal, anise, red chili, black cardamom, turmeric, licorice, fenugreek, clove, kokum, ginger, black cumin, rosemary, hop, and pinecone ginger, respectively, are the focus of this review. The modulation of various transcription factors, growth factors, protein kinases, and inflammatory mediators by these spice-derived nutraceuticals are described. The anticancer potential through the modulation of various targets is also the subject of this review. Although they have always been used to improve taste and color and as a preservative, they are now also used for prevention and treatment of a wide variety of chronic inflammatory diseases, including cancer. PMID:22149093

  12. Cancer cell signaling pathways targeted by spice-derived nutraceuticals.

    PubMed

    Sung, Bokyung; Prasad, Sahdeo; Yadav, Vivek R; Aggarwal, Bharat B

    2012-01-01

    Extensive research within the last half a century has revealed that cancer is caused by dysregulation of as many as 500 different gene products. Most natural products target multiple gene products and thus are ideally suited for prevention and treatment of various chronic diseases, including cancer. Dietary agents such as spices have been used extensively in the Eastern world for a variety of ailments for millennia, and five centuries ago they took a golden journey to the Western world. Various spice-derived nutraceuticals, including 1'-acetoxychavicol acetate, anethole, capsaicin, cardamonin, curcumin, dibenzoylmethane, diosgenin, eugenol, gambogic acid, gingerol, thymoquinone, ursolic acid, xanthohumol, and zerumbone derived from galangal, anise, red chili, black cardamom, turmeric, licorice, fenugreek, clove, kokum, ginger, black cumin, rosemary, hop, and pinecone ginger, respectively, are the focus of this review. The modulation of various transcription factors, growth factors, protein kinases, and inflammatory mediators by these spice-derived nutraceuticals are described. The anticancer potential through the modulation of various targets is also the subject of this review. Although they have always been used to improve taste and color and as a preservative, they are now also used for prevention and treatment of a wide variety of chronic inflammatory diseases, including cancer. PMID:22149093

  13. Side-chain-to-tail cyclization of ribosomally derived peptides promoted by aryl and alkyl amino-functionalized unnatural amino acids.

    PubMed

    Frost, John R; Wu, Zhijie; Lam, Yick Chong; Owens, Andrew E; Fasan, Rudi

    2016-06-28

    A strategy for the production of side-chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Two genetically encodable unnatural amino acids, bearing either an aryl or alkyl amino group, were investigated for their efficiency toward promoting the formation of medium to large-sized peptide macrocycles via intein-mediated side-chain-to-C-terminus cyclization. While only partial cyclization was observed with precursor proteins containing para-amino-phenylalanine, efficient peptide macrocyclization could be achieved using O-2-aminoethyl-tyrosine as the reactive moiety. Conveniently, the latter was generated upon quantitative, post-translational reduction of the azido-containing counterpart, O-2-azidoethyl-tyrosine, directly in E. coli cells. This methodology could be successfully applied for the production of a 12 mer cyclic peptide with enhanced binding affinity for the model target protein streptavidin as compared to the acyclic counterpart (KD: 5.1 μM vs. 22.4 μM), thus demonstrating its utility toward the creation and investigation of novel, functional macrocyclic peptides. PMID:27064594

  14. Presentation of peptides from Bacillus anthracis protective antigen on Tobacco Mosaic Virus as an epitope targeted anthrax vaccine.

    PubMed

    McComb, Ryan C; Ho, Chi-Lee; Bradley, Kenneth A; Grill, Laurence K; Martchenko, Mikhail

    2015-11-27

    The current anthrax vaccine requires improvements for rapidly invoking longer-lasting neutralizing antibody responses with fewer doses from a well-defined formulation. Designing antigens that target neutralizing antibody epitopes of anthrax protective antigen, a component of anthrax toxin, may offer a solution for achieving a vaccine that can induce strong and long lasting antibody responses with fewer boosters. Here we report implementation of a strategy for developing epitope focused virus nanoparticle vaccines against anthrax by using immunogenic virus particles to present peptides derived from anthrax toxin previously identified in (1) neutralizing antibody epitope mapping studies, (2) toxin crystal structure analyses to identify functional regions, and (3) toxin mutational analyses. We successfully expressed two of three peptide epitopes from anthrax toxin that, in previous reports, bound antibodies that were partially neutralizing against toxin activity, discovered cross-reactivity between vaccine constructs and toxin specific antibodies raised in goats against native toxin and showed that antibodies induced by our vaccine constructs also cross-react with native toxin. While protection against intoxication in cellular and animal studies were not as effective as in previous studies, partial toxin neutralization was observed in animals, demonstrating the feasibility of using plant-virus nanoparticles as a platform for epitope defined anthrax vaccines. PMID:26514421

  15. Purification and characterisation of antibacterial peptide-containing compound derived from palm kernel cake.

    PubMed

    Tan, Yen Nee; Ayob, Mohd Khan; Wan Yaacob, Wan Ahmad

    2013-01-01

    Palm kernel cake (PKC), the most useful by-product resulted from palm kernel oil production. In this study, PKC-derived protein product was found suitable for use as an antimicrobial agent with potent antibacterial activity, particularly against Bacillus species, after enzymatic hydrolysis with alcalase. The hydrolysate was further purified by gel filtration chromatography. The purified fraction was found to have 14.63±0.70% (w/w) protein, a molecular mass of 2.4kDa and low hemolytic activity (<50% hemolysis of human erythrocytes at concentration of 1000μg/ml). The presence of lysine and the major component lauric acid derivative, as indicated by electrospray ionisation-mass spectrometry (ESI-MS) direct infusion and nuclear magnetic resonance (NMR) spectroscopy, may have contributed to the antibacterial effect of purified PKC fraction. This study suggests that the antibacterial PKC compound may be not a pure peptide but instead a peptide-containing compound high in lauric acid derivative. PMID:23017424

  16. Ghrelin-Derived Peptides: A Link between Appetite/Reward, GH Axis, and Psychiatric Disorders?

    PubMed Central

    Labarthe, Alexandra; Fiquet, Oriane; Hassouna, Rim; Zizzari, Philippe; Lanfumey, Laurence; Ramoz, Nicolas; Grouselle, Dominique; Epelbaum, Jacques; Tolle, Virginie

    2014-01-01

    Psychiatric disorders are often associated with metabolic and hormonal alterations, including obesity, diabetes, metabolic syndrome as well as modifications in several biological rhythms including appetite, stress, sleep–wake cycles, and secretion of their corresponding endocrine regulators. Among the gastrointestinal hormones that regulate appetite and adapt the metabolism in response to nutritional, hedonic, and emotional dysfunctions, at the interface between endocrine, metabolic, and psychiatric disorders, ghrelin plays a unique role as the only one increasing appetite. The secretion of ghrelin is altered in several psychiatric disorders (anorexia, schizophrenia) as well as in metabolic disorders (obesity) and in animal models in response to emotional triggers (psychological stress …) but the relationship between these modifications and the physiopathology of psychiatric disorders remains unclear. Recently, a large literature showed that this key metabolic/endocrine regulator is involved in stress and reward-oriented behaviors and regulates anxiety and mood. In addition, preproghrelin is a complex prohormone but the roles of the other ghrelin-derived peptides, thought to act as functional ghrelin antagonists, are largely unknown. Altered ghrelin secretion and/or signaling in psychiatric diseases are thought to participate in altered appetite, hedonic response and reward. Whether this can contribute to the mechanism responsible for the development of the disease or can help to minimize some symptoms associated with these psychiatric disorders is discussed in the present review. We will thus describe (1) the biological actions of ghrelin and ghrelin-derived peptides on food and drugs reward, anxiety and depression, and the physiological consequences of ghrelin invalidation on these parameters, (2) how ghrelin and ghrelin-derived peptides are regulated in animal models of psychiatric diseases and in human psychiatric disorders in relation with the GH axis

  17. Ghrelin-Derived Peptides: A Link between Appetite/Reward, GH Axis, and Psychiatric Disorders?

    PubMed

    Labarthe, Alexandra; Fiquet, Oriane; Hassouna, Rim; Zizzari, Philippe; Lanfumey, Laurence; Ramoz, Nicolas; Grouselle, Dominique; Epelbaum, Jacques; Tolle, Virginie

    2014-01-01

    Psychiatric disorders are often associated with metabolic and hormonal alterations, including obesity, diabetes, metabolic syndrome as well as modifications in several biological rhythms including appetite, stress, sleep-wake cycles, and secretion of their corresponding endocrine regulators. Among the gastrointestinal hormones that regulate appetite and adapt the metabolism in response to nutritional, hedonic, and emotional dysfunctions, at the interface between endocrine, metabolic, and psychiatric disorders, ghrelin plays a unique role as the only one increasing appetite. The secretion of ghrelin is altered in several psychiatric disorders (anorexia, schizophrenia) as well as in metabolic disorders (obesity) and in animal models in response to emotional triggers (psychological stress …) but the relationship between these modifications and the physiopathology of psychiatric disorders remains unclear. Recently, a large literature showed that this key metabolic/endocrine regulator is involved in stress and reward-oriented behaviors and regulates anxiety and mood. In addition, preproghrelin is a complex prohormone but the roles of the other ghrelin-derived peptides, thought to act as functional ghrelin antagonists, are largely unknown. Altered ghrelin secretion and/or signaling in psychiatric diseases are thought to participate in altered appetite, hedonic response and reward. Whether this can contribute to the mechanism responsible for the development of the disease or can help to minimize some symptoms associated with these psychiatric disorders is discussed in the present review. We will thus describe (1) the biological actions of ghrelin and ghrelin-derived peptides on food and drugs reward, anxiety and depression, and the physiological consequences of ghrelin invalidation on these parameters, (2) how ghrelin and ghrelin-derived peptides are regulated in animal models of psychiatric diseases and in human psychiatric disorders in relation with the GH axis

  18. Tetramer-organizing polyproline-rich peptides differ in CHO cell-expressed and plasma-derived human butyrylcholinesterase tetramers.

    PubMed

    Schopfer, Lawrence M; Lockridge, Oksana

    2016-06-01

    Tetrameric butyrylcholinesterase (BChE) in human plasma is the product of multiple genes, namely one BCHE gene on chromosome 3q26.1 and multiple genes that encode polyproline-rich peptides. The function of the polyproline-rich peptides is to assemble BChE into tetramers. CHO cells transfected with human BChE cDNA express BChE monomers and dimers, but only low quantities of tetramers. Our goal was to identify the polyproline-rich peptides in CHO-cell derived human BChE tetramers. CHO cell-produced human BChE tetramers were purified from serum-free culture medium. Peptides embedded in the tetramerization domain were released from BChE tetramers by boiling and identified by liquid chromatography-tandem mass spectrometry. A total of 270 proline-rich peptides were sequenced, ranging in size from 6-41 residues. The peptides originated from 60 different proteins that reside in multiple cell compartments including the nucleus, cytoplasm, and endoplasmic reticulum. No single protein was the source of the polyproline-rich peptides in CHO cell-expressed human BChE tetramers. In contrast, 70% of the tetramer-organizing peptides in plasma-derived BChE tetramers originate from lamellipodin. No protein source was identified for polyproline peptides containing up to 41 consecutive proline residues. In conclusion, the use of polyproline-rich peptides as a tetramerization motif is documented only for the cholinesterases, but is expected to serve other tetrameric proteins as well. The CHO cell data suggest that the BChE tetramer-organizing peptide can arise from a variety of proteins. PMID:26947244

  19. Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics.

    PubMed

    Biniossek, Martin L; Niemer, Melanie; Maksimchuk, Ken; Mayer, Bettina; Fuchs, Julian; Huesgen, Pitter F; McCafferty, Dewey G; Turk, Boris; Fritz, Guenther; Mayer, Jens; Haecker, Georg; Mach, Lukas; Schilling, Oliver

    2016-07-01

    We present protease specificity profiling based on quantitative proteomics in combination with proteome-derived peptide libraries. Peptide libraries are generated by endoproteolytic digestion of proteomes without chemical modification of primary amines before exposure to a protease under investigation. After incubation with a test protease, treated and control libraries are differentially isotope-labeled using cost-effective reductive dimethylation. Upon analysis by liquid chromatography-tandem mass spectrometry, cleavage products of the test protease appear as semi-specific peptides that are enriched for the corresponding isotope label. We validate our workflow with two proteases with well-characterized specificity profiles: trypsin and caspase-3. We provide the first specificity profile of a protease encoded by a human endogenous retrovirus and for chlamydial protease-like activity factor (CPAF). For CPAF, we also highlight the structural basis of negative subsite cooperativity between subsites S1 and S2'. For A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) -4, -5, and -15, we show a canonical preference profile, including glutamate in P1 and glycine in P3'. In total, we report nearly 4000 cleavage sites for seven proteases. Our protocol is fast, avoids enrichment or synthesis steps, and enables probing for lysine selectivity as well as subsite cooperativity. Due to its simplicity, we anticipate usability by most proteomic laboratories. PMID:27122596

  20. Identification of Equine Lactadherin-derived Peptides That Inhibit Rotavirus Infection via Integrin Receptor Competition*

    PubMed Central

    Civra, Andrea; Giuffrida, Maria Gabriella; Donalisio, Manuela; Napolitano, Lorenzo; Takada, Yoshikazu; Coulson, Barbara S.; Conti, Amedeo; Lembo, David

    2015-01-01

    Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the age of 5 years in both developed and developing countries. Human lactadherin, a milk fat globule membrane glycoprotein, inhibits human rotavirus infection in vitro, whereas bovine lactadherin is not active. Moreover, it protects breastfed infants against symptomatic rotavirus infections. To explore the potential antiviral activity of lactadherin sourced by equines, we undertook a proteomic analysis of milk fat globule membrane proteins from donkey milk and elucidated its amino acid sequence. Alignment of the human, bovine, and donkey lactadherin sequences revealed the presence of an Asp-Gly-Glu (DGE) α2β1 integrin-binding motif in the N-terminal domain of donkey sequence only. Because integrin α2β1 plays a critical role during early steps of rotavirus host cell adhesion, we tested a minilibrary of donkey lactadherin-derived peptides containing DGE sequence for anti-rotavirus activity. A 20-amino acid peptide containing both DGE and RGD motifs (named pDGE-RGD) showed the greatest activity, and its mechanism of antiviral action was characterized; pDGE-RGD binds to integrin α2β1 by means of the DGE motif and inhibits rotavirus attachment to the cell surface. These findings suggest the potential anti-rotavirus activity of equine lactadherin and support the feasibility of developing an anti-rotavirus peptide that acts by hindering virus-receptor binding. PMID:25814665

  1. The targeted delivery of the c-Src peptide complexed with schizophyllan to macrophages inhibits polymicrobial sepsis and ulcerative colitis in mice.

    PubMed

    Kim, Ye-Ram; Hwang, Jangsun; Koh, Hyun-Jung; Jang, Kiseok; Lee, Jong-Dae; Choi, Jonghoon; Yang, Chul-Su

    2016-05-01

    Hyper-inflammatory responses triggered by intracellular reactive oxygen species (ROS) can lead to a variety of diseases, including sepsis and colitis. However, the regulators of this process remain poorly defined. In this study, we demonstrate that c-Src is a negative regulator of cellular ROS generation through its binding to p47phox. This molecule also competitively inhibits the NADPH oxidase complex (NOX) assembly. Furthermore, we developed the schizophyllan (SPG)-c-Src SH3 peptide, which is a β-1,3-glucan conjugated c-Src SH3-derived peptide composed of amino acids 91-108 and 121-140 of c-Src. The SPG-SH3 peptide has a significant therapeutic effect on mouse ROS-mediated inflammatory disease models, cecal-ligation-puncture-induced sepsis, and dextran sodium sulfate-induced colitis. It does so by inhibiting the NOX subunit assembly and proinflammatory mediator production. Therefore, the SPG-SH3 peptide is a potential therapeutic agent for ROS-associated lethal inflammatory diseases. Our findings provide clues for the development of new peptide-base drugs that will target p47phox. PMID:26946401

  2. Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens

    PubMed Central

    Abushahba, Mostafa F. N.; Mohammad, Haroon; Thangamani, Shankar; Hussein, Asmaa A. A.; Seleem, Mohamed N.

    2016-01-01

    There is a pressing need for novel and innovative therapeutic strategies to address infections caused by intracellular pathogens. Peptide nucleic acids (PNAs) present a novel method to target intracellular pathogens due to their unique mechanism of action and their ability to be conjugated to cell penetrating peptides (CPP) to overcome challenging delivery barriers. In this study, we targeted the RNA polymerase α subunit (rpoA) using a PNA that was covalently conjugated to five different CPPs. Changing the conjugated CPP resulted in a pronounced improvement in the antibacterial activity observed against Listeria monocytogenes in vitro, in cell culture, and in a Caenorhabditis elegans (C. elegans) infection model. Additionally, a time-kill assay revealed three conjugated CPPs rapidly kill Listeria within 20 minutes without disrupting the bacterial cell membrane. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of other critical virulence genes (Listeriolysin O, and two phospholipases plcA and plcB) in a concentration-dependent manner. Furthermore, PNA-inhibition of bacterial protein synthesis was selective and did not adversely affect mitochondrial protein synthesis. This study provides a foundation for improving and developing PNAs conjugated to CPPs to better target intracellular pathogens. PMID:26860980

  3. Tumor-homing peptides as tools for targeted delivery of payloads to the placenta

    PubMed Central

    King, Anna; Ndifon, Cornelia; Lui, Sylvia; Widdows, Kate; Kotamraju, Venkata R.; Agemy, Lilach; Teesalu, Tambet; Glazier, Jocelyn D.; Cellesi, Francesco; Tirelli, Nicola; Aplin, John D.; Ruoslahti, Erkki; Harris, Lynda K.

    2016-01-01

    The availability of therapeutics to treat pregnancy complications is severely lacking mainly because of the risk of causing harm to the fetus. As enhancement of placental growth and function can alleviate maternal symptoms and improve fetal growth in animal models, we have developed a method for targeted delivery of payloads to the placenta. We show that the tumor-homing peptide sequences CGKRK and iRGD bind selectively to the placental surface of humans and mice and do not interfere with normal development. Peptide-coated nanoparticles intravenously injected into pregnant mice accumulated within the mouse placenta, whereas control nanoparticles exhibited reduced binding and/or fetal transfer. We used targeted liposomes to efficiently deliver cargoes of carboxyfluorescein and insulin-like growth factor 2 to the mouse placenta; the latter significantly increased mean placental weight when administered to healthy animals and significantly improved fetal weight distribution in a well-characterized model of fetal growth restriction. These data provide proof of principle for targeted delivery of drugs to the placenta and provide a novel platform for the development of placenta-specific therapeutics. PMID:27386551

  4. Tumor-homing peptides as tools for targeted delivery of payloads to the placenta.

    PubMed

    King, Anna; Ndifon, Cornelia; Lui, Sylvia; Widdows, Kate; Kotamraju, Venkata R; Agemy, Lilach; Teesalu, Tambet; Glazier, Jocelyn D; Cellesi, Francesco; Tirelli, Nicola; Aplin, John D; Ruoslahti, Erkki; Harris, Lynda K

    2016-05-01

    The availability of therapeutics to treat pregnancy complications is severely lacking mainly because of the risk of causing harm to the fetus. As enhancement of placental growth and function can alleviate maternal symptoms and improve fetal growth in animal models, we have developed a method for targeted delivery of payloads to the placenta. We show that the tumor-homing peptide sequences CGKRK and iRGD bind selectively to the placental surface of humans and mice and do not interfere with normal development. Peptide-coated nanoparticles intravenously injected into pregnant mice accumulated within the mouse placenta, whereas control nanoparticles exhibited reduced binding and/or fetal transfer. We used targeted liposomes to efficiently deliver cargoes of carboxyfluorescein and insulin-like growth factor 2 to the mouse placenta; the latter significantly increased mean placental weight when administered to healthy animals and significantly improved fetal weight distribution in a well-characterized model of fetal growth restriction. These data provide proof of principle for targeted delivery of drugs to the placenta and provide a novel platform for the development of placenta-specific therapeutics. PMID:27386551

  5. Renal targeted delivery of triptolide by conjugation to the fragment peptide of human serum albumin.

    PubMed

    Yuan, Zhi-xiang; Wu, Xiao-juan; Mo, Jingxin; Wang, Yan-li; Xu, Chao-qun; Lim, Lee Yong

    2015-08-01

    We have previously demonstrated that peptide fragments (PFs) of the human serum albumin could be developed as potential renal targeting carriers, in particular, the peptide fragment, PF-A299-585 (A299-585 representing the amino acid sequence of the human serum albumin). In this paper, we conjugated triptolide (TP), the anti-inflammatory Chinese traditional medicine, to PF-A299-585 via a succinic acid spacer to give TPS-PF-A299-585 (TP loading 2.2% w/w). Compared with the free TP, TPS-PF-A299-585 exhibited comparable anti-inflammatory activity in the lipopolysaccharide stimulated MDCK cells, but was significantly less cytotoxic than the free drug. Accumulation of TPS-PF-A299-585 in the MDCK cells in vitro and in rodent kidneys in vivo was demonstrated using FITC-labeled TPS-PF-A299-585. Renal targeting was confirmed in vivo in a membranous nephropathic (MN) rodent model, where optical imaging and analyses of biochemical markers were combined to show that TPS-PF-A299-585 was capable of alleviating the characteristic symptoms of MN. The collective data affirm PF-A299-585 to be a useful carrier for targeting TP to the kidney. PMID:26117184

  6. Kinesin superfamily protein-derived peptides with the ability to induce glioma-reactive cytotoxic T lymphocytes in human leukocyte antigen-A24+ glioma patients.

    PubMed

    Harada, Mamoru; Ishihara, Yuki; Itoh, Kyogo; Yamanaka, Ryuya

    2007-03-01

    One promising modality in the treatment of malignant glioma is specific immunotherapy. However, this modality requires information about target antigens and their epitope peptides that are recognized by T cells. In this study, we searched for new target candidates in specific immunotherapy for malignant glioma by utilizing cDNA microarray technology to compare gene expressions in malignant glioma tissues to those in benign glioma and a panel of normal tissues. The selected genes included three members of the kinesin superfamily proteins (KIFs): KIF1C, KIF3C, and KIF21B. RT-PCR showed that these three genes were expressed in the majority of glioma cell lines. These antigen-derived 25 peptides, which had the ability to bind to human leukocyte antigen (HLA)-A24 molecules, were first screened for their ability to be recognized by the immunoglobulin G of glioma patients, and then tested for their potential to induce peptide-specific and glioma-reactive cytotoxic T lymphocytes (CTLs) from the peripheral blood mononuclear cells of HLA-A24+ glioma patients. The results showed that the KIF1C149-158 and KIF3C512-520 peptides efficiently induced HLA-A24-restricted and glioma-reactive CD8+ T cells. These results suggest the existence of KIF-reactive CTL precursors in glioma patients, and should facilitate the development of specific immunotherapies for malignant glioma. PMID:17273744

  7. Thermodynamic and Kinetics Analysis of Peptides Derived from CapZ, NDR, p53, HDM2, and HDM4 Binding to Human S100B

    PubMed Central

    Wafer, Lucas N.; Streicher, Werner W.; McCallum, Scott A.; Makhatadze, George I.

    2012-01-01

    S100B is a member of the S100 subfamily of EF-hand proteins that has been implicated in malignant melanoma and neurodegenerative conditions such as Alzheimer's and Parkinson's disease. Calcium-induced conformational changes expose a hydrophobic binding cleft, facilitating interactions with a wide variety of nuclear, cytoplasmic, and extracellular target proteins. Previously, peptides derived from CapZ, p53, NDR, HDM2 and HDM4 have been shown to interact with S100B in a calcium-dependent manner. However, the thermodynamic and kinetic basis of these interactions remains largely unknown. To gain further insight, these peptides were screened against the S100B protein using isothermal titration calorimetry and nuclear magnetic resonance. All peptides were found to have binding affinities in the low micromolar to nanomolar range. Binding-induced changes in the line shapes of S100B backbone 1H and 15N were monitored to obtain the dissociation constants and the kinetic binding parameters. The large microscopic Kon rate constants observed in this study, Kon ≥1×107 M-1s-1, suggest that S100B utilizes a “fly casting mechanism” in the recognition of these peptide targets. PMID:22913742

  8. Intervention With an Erythropoietin-Derived Peptide Protects Against Neuroglial and Vascular Degeneration During Diabetic Retinopathy

    PubMed Central

    McVicar, Carmel M.; Hamilton, Ross; Colhoun, Liza M.; Gardiner, Tom A.; Brines, Michael; Cerami, Anthony; Stitt, Alan W.

    2011-01-01

    OBJECTIVE Erythropoietin (EPO) may be protective for early stage diabetic retinopathy, although there are concerns that it could exacerbate retinal angiogenesis and thrombosis. A peptide based on the EPO helix-B domain (helix B-surface peptide [pHBSP]) is nonerythrogenic but retains tissue-protective properties, and this study evaluates its therapeutic potential in diabetic retinopathy. RESEARCH DESIGN AND METHODS After 6 months of streptozotocin-induced diabetes, rats (n = 12) and age-matched nondiabetic controls (n = 12) were evenly split into pHBSP and scrambled peptide groups and injected daily (10 μg/kg per day) for 1 month. The retina was investigated for glial dysfunction, microglial activation, and neuronal DNA damage. The vasculature was dual stained with isolectin and collagen IV. Retinal cytokine expression was quantified using real-time RT-PCR. In parallel, oxygen-induced retinopathy (OIR) was used to evaluate the effects of pHBSP on retinal ischemia and neovascularization (1–30 μg/kg pHBSP or control peptide). RESULTS pHBSP or scrambled peptide treatment did not alter hematocrit. In the diabetic retina, Müller glial expression of glial fibrillary acidic protein was increased when compared with nondiabetic controls, but pHBSP significantly reduced this stress-related response (P < 0.001). CD11b+ microglia and proinflammatory cytokines were elevated in diabetic retina responses, and some of these responses were attenuated by pHBSP (P < 0.01–0.001). pHBSP significantly reduced diabetes-linked DNA damage as determined by 8-hydroxydeoxyguanosine and transferase-mediated dUTP nick-end labeling positivity and also prevented acellular capillary formation (P < 0.05). In OIR, pHBSP had no effect on preretinal neovascularization at any dose. CONCLUSIONS Treatment with an EPO-derived peptide after diabetes is fully established can significantly protect against neuroglial and vascular degenerative pathology without altering hematocrit or exacerbating

  9. Cell adhesion and invasion inhibitory effect of an ovarian cancer targeting peptide selected via phage display in vivo.

    PubMed

    Pu, Ximing; Ma, Chuying; Yin, Guangfu; You, Fei; Wei, Yan

    2014-01-17

    Organ-specific metastasis is of great importance since most of the cancer deaths are caused by spread of the primary cancer to distant sites. Therefore, targeted anti-metastases therapies are needed to prevent cancer cells from metastasizing to different organs. The phage clone pc3-1 displaying peptide WSGPGVWGASVK selected by phage display had been identified which have high binding efficiency and remarkable cell specificity to SK-OV-3 cells. In the present work, the effects of selected cell-binding phage and cognate peptide on the cell adhesion and invasion of targeted cells were investigated. Results showed that the adhesive ability of SK-OV-3 to extracellular matrix was inhibited by pc3-1 and peptide WSGPGVWGASVK, and pc3-1 blocked SK-OV-3 cells attachment more effective than the cognate peptide. The peptide WSGPGVWGASVK suppressed the cell number of SK-OV-3 that attached to HUVECs monolayer up to 24% and could block the spreading of the attaching cells. Forthermore, the cognate peptide could inhibit the invasion of SK-OV-3 significantly. The number of invaded SK-OV-3 cells and invaded SK-OV-3-activated HUVECs pretreated with peptide WSGPGVWGASVK was decreased by 24.3% and 36.6%, respectively. All these results suggested that peptide WSGPGVWGASVK might possess anti-metastasis against SK-OV-3 cells. PMID:24342617

  10. Combretastatin A-4 and Derivatives: Potential Fungicides Targeting Fungal Tubulin.

    PubMed

    Ma, Zhong-lin; Yan, Xiao-jing; Zhao, Lei; Zhou, Jiu-jiu; Pang, Wan; Kai, Zhen-peng; Wu, Fan-hong

    2016-02-01

    Combretastatin A-4, first isolated from the African willow tree Combretum caffrum, is a tubulin polymerization inhibitor in medicine. It was first postulated as a potential fungicide targeting fungal tubulin for plant disease control in this study. Combretastatin A-4 and its derivatives were synthesized and tested against Rhizoctonia solani and Pyricularia oryzae. Several compounds have EC50 values similar to or better than that of isoprothiolane, which is widely used for rice disease control. Structure-activity relationship study indicated the the cis configuration and hydroxyl group in combretastatin A-4 are crucial to the antifungal effect. Molecular modeling indicated the binding sites of combretastatin A-4 and carbendazim on fungal tubulin are totally different. The bioactivity of combretastatin A-4 and its derivatives against carbendazim-resistant strains was demonstrated in this study. The results provide a new approach for fungicide discovery and fungicide resistance management. PMID:26711170

  11. New targets for renal interstitial fibrosis: relaxin family peptide receptor 1-angiotensin type 2 receptor heterodimers.

    PubMed

    Sasser, Jennifer M

    2014-07-01

    The signal transduction mechanisms involved in the renoprotective effects of relaxin are not well understood. Chow et al. demonstrate that relaxin family peptide receptor 1 (RXFP1) forms heterodimer complexes with the angiotensin type 2 receptor (AT2), even in the absence of ligand, and that these heterodimers are required for relaxin's antifibrotic effects. These findings identify a previously unknown link between relaxin and angiotensin II signaling that could be a potential new target for slowing the progression of fibrotic renal diseases. PMID:24978374

  12. High-affinity homologous peptide nucleic acid probes for targeting a quadruplex-forming sequence from a MYC promoter element.

    PubMed

    Roy, Subhadeep; Tanious, Farial A; Wilson, W David; Ly, Danith H; Armitage, Bruce A

    2007-09-18

    Guanine-rich DNA and RNA sequences are known to fold into secondary structures known as G-quadruplexes. Recent biochemical evidence along with the discovery of an increasing number of sequences in functionally important regions of the genome capable of forming G-quadruplexes strongly indicates important biological roles for these structures. Thus, molecular probes that can selectively target quadruplex-forming sequences (QFSs) are envisioned as tools to delineate biological functions of quadruplexes as well as potential therapeutic agents. Guanine-rich peptide nucleic acids have been previously shown to hybridize to homologous DNA or RNA sequences forming PNA-DNA (or RNA) quadruplexes. For this paper we studied the hybridization of an eight-mer G-rich PNA to a quadruplex-forming sequence derived from the promoter region of the MYC proto-oncogene. UV melting analysis, fluorescence assays, and surface plasmon resonance experiments reveal that this PNA binds to the MYC QFS in a 2:1 stoichiometry and with an average binding constant Ka = (2.0 +/- 0.2) x 10(8) M(-1) or Kd = 5.0 nM. In addition, experiments carried out with short DNA targets revealed a dependence of the affinity on the sequence of bases in the loop region of the DNA. A structural model for the hybrid quadruplex is proposed, and implications for gene targeting by G-rich PNAs are discussed. PMID:17718513

  13. Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment.

    PubMed Central

    Yamauchi, K; Tomita, M; Giehl, T J; Ellison, R T

    1993-01-01

    Although the antimicrobial activity of lactoferrin has been well described, its mechanism of action has been poorly characterized. Recent work has indicated that in addition to binding iron, human lactoferrin damages the outer membrane of gram-negative bacteria. In this study, we determined whether bovine lactoferrin and a pepsin-derived bovine lactoferrin peptide (lactoferricin) fragment have similar activities. We found that both 20 microM bovine lactoferrin and 20 microM lactoferricin release intrinsically labeled [3H]lipopolysaccharide ([3H]LPS) from three bacterial strains, Escherichia coli CL99 1-2, Salmonella typhimurium SL696, and Salmonella montevideo SL5222. Under most conditions, more LPS is released by the peptide fragment than by whole bovine lactoferrin. In the presence of either lactoferrin or lactoferricin there is increased killing of E. coli CL99 1-2 by lysozyme. Like human lactoferrin, bovine lactoferrin and lactoferricin have the ability to bind to free intrinsically labeled [3H]LPS molecules. In addition to these effects, whereas bovine lactoferrin was at most bacteriostatic, lactoferricin demonstrated consistent bactericidal activity against gram-negative bacteria. This bactericidal effect is modulated by the cations Ca2+, Mg2+, and Fe3+ but is independent of the osmolarity of the medium. Transmission electron microscopy of bacterial cells exposed to lactoferricin show the immediate development of electron-dense "membrane blisters." These experiments offer evidence that bovine lactoferrin and lactoferricin damage the outer membrane of gram-negative bacteria. Moreover, the peptide fragment lactoferricin has direct bactericidal activity. As lactoferrin is exposed to proteolytic factors in vivo which could cleave the lactoferricin fragment, the effects of this peptide are of both mechanistic and physiologic relevance. Images PMID:8423097

  14. Proteome-derived Peptide Libraries to Study the Substrate Specificity Profiles of Carboxypeptidases*

    PubMed Central

    Tanco, Sebastian; Lorenzo, Julia; Garcia-Pardo, Javier; Degroeve, Sven; Martens, Lennart; Aviles, Francesc Xavier; Gevaert, Kris; Van Damme, Petra

    2013-01-01

    Through processing peptide and protein C termini, carboxypeptidases participate in the regulation of various biological processes. Few tools are however available to study the substrate specificity profiles of these enzymes. We developed a proteome-derived peptide library approach to study the substrate preferences of carboxypeptidases. Our COFRADIC-based approach takes advantage of the distinct chromatographic behavior of intact peptides and the proteolytic products generated by the action of carboxypeptidases, to enrich the latter and facilitate its MS-based identification. Two different peptide libraries, generated either by chymotrypsin or by metalloendopeptidase Lys-N, were used to determine the substrate preferences of human metallocarboxypeptidases A1 (hCPA1), A2 (hCPA2), and A4 (hCPA4). In addition, our approach allowed us to delineate the substrate specificity profile of mouse mast cell carboxypeptidase (MC-CPA or mCPA3), a carboxypeptidase suggested to function in innate immune responses regulation and mast cell granule homeostasis, but which thus far lacked a detailed analysis of its substrate preferences. mCPA3 was here shown to preferentially remove bulky aromatic amino acids, similar to hCPA2. This was also shown by a hierarchical cluster analysis, grouping hCPA1 close to hCPA4 in terms of its P1 primed substrate specificity, whereas hCPA2 and mCPA3 cluster separately. The specificity profile of mCPA3 may further aid to elucidate the function of this mast cell carboxypeptidase and its biological substrate repertoire. Finally, we used this approach to evaluate the substrate preferences of prolylcarboxypeptidase, a serine carboxypeptidase shown to cleave C-terminal amino acids linked to proline and alanine. PMID:23620545

  15. A novel antimicrobial peptide derived from fish goose type lysozyme disrupts the membrane of Salmonella enterica.

    PubMed

    Kumaresan, Venkatesh; Bhatt, Prasanth; Ganesh, Munuswamy-Ramanujam; Harikrishnan, Ramasamy; Arasu, MariadhasValan; Al-Dhabi, Naif Abdullah; Pasupuleti, Mukesh; Marimuthu, Kasi; Arockiaraj, Jesu

    2015-12-01

    In aquaculture, accumulation of antibiotics resulted in development of resistance among bacterial pathogens. Consequently, it became mandatory to find alternative to synthetic antibiotics. Antimicrobial peptides (AMPs) which are described as evolutionary ancient weapons have been considered as promising alternates in recent years. In this study, a novel antimicrobial peptide had been derived from goose type lysozyme (LyzG) which was identified from the cDNA library of freshwater fish Channa striatus (Cs). The identified lysozyme cDNA contains 585 nucleotides which encodes a protein of 194 amino acids. CsLyzG was closely related to Siniperca chuatsi with 92.8% homology. The depicted protein sequence contained a GEWL domain with conserved GLMQ motif, 7 active residues and 2 catalytic residues. Gene expression analysis revealed that CsLyzG was distributed in major immune organs with highest expression in head kidney. Results of temporal expression analysis after bacterial (Aeromonas hydrophila) and fungal (Aphanomyces invadans) challenges indicated a stimulant-dependent expression pattern of CsLyzG. Two antimicrobial peptides IK12 and TS10 were identified from CsLyzG and synthesized. Antibiogram showed that IK12 was active against Salmonella enterica, a major multi-drug resistant (MDR) bacterial pathogen which produces beta lactamase. The IK12 induced loss of cell viability in the bacterial pathogen. Flow cytometry assay revealed that IK12 disrupt the membrane of S. enterica which is confirmed by scanning electron microscope (SEM) analysis that reveals blebs around the bacterial cell membrane. Conclusively, CsLyzG is a potential innate immune component and the identified antimicrobial peptide has great caliber to be used as an ecofriendly antibacterial substance in aquaculture. PMID:26477736

  16. Reaction of phosphorylated and O-glycosylated peptides by chemically targeted identification at ambient temperature.

    PubMed

    Rusnak, Felicia; Zhou, Jie; Hathaway, Gary M

    2004-12-01

    Conditions for carrying out chemically targeted identification of peptides containing phosphorylated or glycosylated serine residues have been investigated. Ba(OH)2 was used at ambient temperature to catalyze the beta-elimination reaction at 25 degrees C. Nucleophilic addition of 2-aminoethanethiol was performed in both parallel and tandem experiments. The method was demonstrated by the reaction of beta-casein tryptic digest phosphopeptides and an O-glycosylated peptide. Contrary to an earlier report by others, the glycopeptide was found to react with essentially the same kinetics as phosphopeptides. Conversion of four phosphoserines in residues 15, 17, 18, and 19 from bovine beta-casein N-terminal tryptic phosphopeptides were followed by monitoring the time course of the addition reaction. The chemistry proceeded rapidly at room temperature with a half-reaction time of 15 min. No side-reaction products were observed; however, care was taken to minimize all counter ions that either precipitate barium or neutralize the base. Digestion of the converted peptides with lysine endopeptidase identified all five phosphoserines in the beta-casein tryptic digest. Alternatively, preincubation with base followed by nucleophilic addition of the thiol was found to work satisfactorily. The use of the water-soluble hydrochloride of 2-aminoethanethiol allowed beta-elimination, nucleophilic addition, and desalting to be carried out on a micro C18 reverse phase pipette tip. PMID:15585826

  17. Black mamba venom peptides target acid-sensing ion channels to abolish pain.

    PubMed

    Diochot, Sylvie; Baron, Anne; Salinas, Miguel; Douguet, Dominique; Scarzello, Sabine; Dabert-Gay, Anne-Sophie; Debayle, Delphine; Friend, Valérie; Alloui, Abdelkrim; Lazdunski, Michel; Lingueglia, Eric

    2012-10-25

    Polypeptide toxins have played a central part in understanding physiological and physiopathological functions of ion channels. In the field of pain, they led to important advances in basic research and even to clinical applications. Acid-sensing ion channels (ASICs) are generally considered principal players in the pain pathway, including in humans. A snake toxin activating peripheral ASICs in nociceptive neurons has been recently shown to evoke pain. Here we show that a new class of three-finger peptides from another snake, the black mamba, is able to abolish pain through inhibition of ASICs expressed either in central or peripheral neurons. These peptides, which we call mambalgins, are not toxic in mice but show a potent analgesic effect upon central and peripheral injection that can be as strong as morphine. This effect is, however, resistant to naloxone, and mambalgins cause much less tolerance than morphine and no respiratory distress. Pharmacological inhibition by mambalgins combined with the use of knockdown and knockout animals indicates that blockade of heteromeric channels made of ASIC1a and ASIC2a subunits in central neurons and of ASIC1b-containing channels in nociceptors is involved in the analgesic effect of mambalgins. These findings identify new potential therapeutic targets for pain and introduce natural peptides that block them to produce a potent analgesia. PMID:23034652

  18. Radiolabeled multimeric cyclic RGD peptides as integrin alphavbeta3 targeted radiotracers for tumor imaging.

    PubMed

    Liu, Shuang

    2006-01-01

    Integrin alphavbeta3 plays a significant role in tumor angiogenesis and is a receptor for the extracellular matrix proteins with the exposed arginine-glycine-aspartic (RGD) tripeptide sequence. These include vitronectin, fibronectin, fibrinogen, lamin, collagen, Von Willibrand's factor, osteoponin, and adenovirus particles. Integrin alphavbeta3 is expressed at low levels on epithelial cells and mature endothelial cells, but it is overexpressed on the activated endothelial cells of tumor neovasculature and some tumor cells. The highly restricted expression of integrin alphavbeta3 during tumor growth, invasion, and metastasis presents an interesting molecular target for both early detection and treatment of rapidly growing solid tumors. In the past decade, many radiolabeled linear and cyclic RGD peptide antagonists have been evaluated as the integrin alphavbeta3 targeted radiotracers. Significant progress has been made on their use for imaging tumors of different origin by single photon emission computed tomography (SPECT) or positron emission tomography (PET) in several tumor-bearing animal models. [18F]Galacto-RGD is under clinical investigation as the first integrin alphavbeta3 targeted radiotracer for noninvasive visualization of the activated integrin alphavbeta3 in cancer patients. This review will focus on the radiolabeled multimeric cyclic RGD peptides (dimers and tetramers) useful as radiotracers to image the tumor integrin alphavbeta3 expression by SPECT and PET, and some fundamental aspects for the development of integrin alphavbeta3 targeted radiotracers. These include the choice of radionuclide and bifunctional chelators, selection of targeting biomolecules, and factors influencing the integrin alphavbeta3 binding affinity and tumor uptake, as well as different approaches for modification of radiotracer pharmacokinetics. PMID:17009846

  19. Dermcidin-Derived Peptides Show a Different Mode of Action than the Cathelicidin LL-37 against Staphylococcus aureus▿

    PubMed Central

    Senyürek, Ilknur; Paulmann, Maren; Sinnberg, Tobias; Kalbacher, Hubert; Deeg, Martin; Gutsmann, Thomas; Hermes, Marina; Kohler, Thomas; Götz, Fritz; Wolz, Christiane; Peschel, Andreas; Schittek, Birgit

    2009-01-01

    Dermcidin (DCD) is an antimicrobial peptide which is constitutively expressed in eccrine sweat glands. By postsecretory proteolytic processing in sweat, the DCD protein gives rise to anionic and cationic DCD peptides with a broad spectrum of antimicrobial activity. Many antimicrobial peptides induce membrane permeabilization as part of their killing mechanism, which is accompanied by a loss of the bacterial membrane potential. In this study we show that there is a time-dependent bactericidal activity of anionic and cationic DCD-derived peptides which is followed by bacterial membrane depolarization. However, DCD-derived peptides do not induce pore formation in the membranes of gram-negative and gram-positive bacteria. This is in contrast to the mode of action of the cathelicidin LL-37. Interestingly, LL-37 as well as DCD-derived peptides inhibit bacterial macromolecular synthesis, especially RNA and protein synthesis, without binding to microbial DNA or RNA. Binding studies with components of the cell envelope of gram-positive and gram-negative bacteria and with model membranes indicated that DCD-derived peptides bind to the bacterial envelope but show only a weak binding to lipopolysaccharide (LPS) from gram-negative bacteria or to peptidoglycan, lipoteichoic acid, and wall teichoic acid, isolated from Staphylococcus aureus. In contrast, LL-37 binds strongly in a dose-dependent fashion to these components. Altogether, these data indicate that the mode of action of DCD-derived peptides is different from that of the cathelicidin LL-37 and that components of the bacterial cell envelope play a role in the antimicrobial activity of DCD. PMID:19364862

  20. Phage display selection of peptides that home to atherosclerotic plaques: IL-4 receptor as a candidate target in atherosclerosis

    PubMed Central

    Hong, Hai-yan; Lee, Hwa Young; Kwak, Wonjung; Yoo, Jeongsoo; Na, Moon-Hee; So, In Seop; Kwon, Tae-Hwan; Park, Heon-Sik; Huh, Seung; Oh, Goo Taeg; Kwon, Ick-Chan; Kim, In-San; Lee, Byung-Heon

    2008-01-01

    Imaging or drug delivery tools for atherosclerosis based on the plaque biology are still insufficient. Here, we attempted to identify peptides that selectively home to atherosclerotic plaques using phage display. A phage library containing random peptides was ex viv screened for binding to human atheroma tissues. After three to four rounds of selection, the DNA inserts of phage clones wer sequenced. A peptide sequence, CRKRLDRNC, was the most frequently occurring one. Intravenously injected phage displaying the CRKRLDRNC peptide was observed to home to atherosclerotic aortic tissues of low-density lipoprotein receptor-deficient (Ldlr−/–) mice at higher levels than to normal aortic tissues of wild-type mice. Moreover, a fluorescein- or radioisotope-conjugated synthetic CRKRLDRNC peptide, but not a control peptide, homed in vivo to atherosclerotic plaques in Ldlr−/– mice, while homing of the peptide to other organs such as brain was minimal. The homing peptide co-localized with endothelial cells, macrophages and smooth muscle cells a mouse and human atherosclerotic plaques. Homology search revealed that the CRKRLDRNC peptide shares a motif of interleukin-receptor (IL-4) that is critical for binding to its receptor. The peptide indeed co-localized with IL-4 receptor (IL-4R) at atherosclerotic plaques. Moreover, the peptide bound to cultured cells expressing IL-4R on the cell surface and the binding was inhibited by the knock-down of IL-4R. These results show that the CRKRLDRNC peptide homes to atherosclerotic plaques through binding to IL-4R as its target and may be a useful tool for selective drug delivery and molecular imaging of atherosclerosis. PMID:19012727

  1. Production of angiotensin-I-converting enzyme inhibitory peptides from β-lactoglobulin- and casein-derived peptides: an integrative approach.

    PubMed

    Welderufael, Fisseha T; Gibson, Trevor; Jauregi, Paula

    2012-01-01

    Angiotensin I-converting enzyme (ACE) inhibition is one of the mechanisms by which reduction in blood pressure is exerted. Whey proteins are a rich source of ACE inhibitory peptides and have shown a blood pressure reduction effect i.e. antihypertensive activity. The aim of this work was to develop a simplified process using a combination of adsorption and microfiltration steps for the production of hydrolysates from whey with high ACE inhibitory activity and potency; the latter was measured as the IC50, which is the peptide concentration required to reduce ACE activity by half. This process integrates the selective separation of β-lactoglobulin- and casein-derived peptides (CDP) from rennet whey and their hydrolysis, which results in partially pure, less complex hydrolysates with high bioactive potency. Hydrolysis was carried out with protease N "Amano" in a thermostatically controlled membrane reactor operated in a batch mode. By applying the integrative approach it was possible to produce from the same feedstock two different hydrolysates that exhibited high ACE inhibition. One hydrolysate was mainly composed of casein-derived peptides with IC50=285 μg/mL. In this hydrolysate we identified the well-known potent ACE-inhibitor and antihypertensive tripeptide Ile-Pro-Pro (IPP) and another novel octapeptide Gln-Asp-Lys-Thr-Glu-Ile-Pro-Thr (QDKTEIPT). The second hydrolysate was mainly composed of β-lactoglobulin derived peptides with IC50=28 μg/mL. This hydrolysate contained a tetrapeptide (Ile-Ile-Ala-Glu) IIAE as one of the two major peptides. A further advantage to this process is that enzyme activity was substantially increased as enzyme product inhibition was reduced. PMID:22467199

  2. Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes.

    PubMed

    Amoutzias, Grigoris D; Chaliotis, Anargyros; Mossialos, Dimitris

    2016-04-01

    Considering that 70% of our planet's surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs) and polyketides (PKs) are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes) and type-I polyketide synthases (PKSes-I), respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS) technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds. PMID:27092515

  3. Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes

    PubMed Central

    Amoutzias, Grigoris D.; Chaliotis, Anargyros; Mossialos, Dimitris

    2016-01-01

    Considering that 70% of our planet’s surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs) and polyketides (PKs) are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes) and type-I polyketide synthases (PKSes-I), respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS) technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds. PMID:27092515

  4. Synthesis and Antiproliferative Activity Evaluation of the Disulfide-Containing Cyclic Peptide Thiochondrilline C and Derivatives.

    PubMed

    Vippila, Mohana Rao; Ly, Phuong Kim; Cuny, Gregory D

    2015-10-23

    Thiochondrilline C (4) was previously isolated from Verrucisispora sp. and reported to have moderate cytotoxicity against human lung adenocarcinoma cells. Herein, we report the synthesis of thiochondrilline C by N-terminal peptide extension, oxidative disulfide bond formation, and heterocycle installation as key steps. Antiproliferative activities for the prepared natural product and several derivatives against the NCI 60 cancer cell line panel are also described. Derivative 22 was identified as a moderately potent antiproliferative agent (50% growth inhibition (GI50) = 0.2-12.2 μM) with leukemia (average GI50 = 1.8 ± 0.1 μM) and colon (average GI50 = 2.4 ± 0.3 μM) cells being most sensitive. PMID:26444379

  5. Fighting microbial infections: A lesson from amphibian skin-derived esculentin-1 peptides.

    PubMed

    Mangoni, Maria Luisa; Luca, Vincenzo; McDermott, Alison M

    2015-09-01

    Due to the growing emergence of resistance to commercially available antibiotics/antimycotics in virtually all clinical microbial pathogens, the discovery of alternative anti-infective agents, is greatly needed. Gene-encoded antimicrobial peptides (AMPs) hold promise as novel therapeutics. In particular, amphibian skin is one of the richest storehouses of AMPs, especially that of the genus Rana, with esculentins-1 being among the longest (46 amino acids) AMPs found in nature to date. Here, we report on the recently discovered in vitro and in vivo activities and mechanism of action of two derivatives of the N-terminal part of esculentin-1a and -1b peptides, primarily against two relevant opportunistic microorganisms causing a large number of life-threatening infections worldwide; i.e. the Gram-negative bacterium Pseudomonas aeruginosa and the yeast Candida albicans. Because of distinct advantages compared to several mammalian AMPs, the two selected frog skin AMP-derivatives represent attractive candidates for the development of new antimicrobial compounds with expanded properties, for both human and veterinary medicine. PMID:25959536

  6. Polycyclic peptide therapeutics.

    PubMed

    Baeriswyl, Vanessa; Heinis, Christian

    2013-03-01

    Owing to their excellent binding properties, high stability, and low off-target toxicity, polycyclic peptides are an attractive molecule format for the development of therapeutics. Currently, only a handful of polycyclic peptides are used in the clinic; examples include the antibiotic vancomycin, the anticancer drugs actinomycin D and romidepsin, and the analgesic agent ziconotide. All clinically used polycyclic peptide drugs are derived from natural sources, such as soil bacteria in the case of vancomycin, actinomycin D and romidepsin, or the venom of a fish-hunting coil snail in the case of ziconotide. Unfortunately, nature provides peptide macrocyclic ligands for only a small fraction of therapeutic targets. For the generation of ligands of targets of choice, researchers have inserted artificial binding sites into natural polycyclic peptide scaffolds, such as cystine knot proteins, using rational design or directed evolution approaches. More recently, large combinatorial libraries of genetically encoded bicyclic peptides have been generated de novo and screened by phage display. In this Minireview, the properties of existing polycyclic peptide drugs are discussed and related to their interesting molecular architectures. Furthermore, technologies that allow the development of unnatural polycyclic peptide ligands are discussed. Recent application of these technologies has generated promising results, suggesting that polycyclic peptide therapeutics could potentially be developed for a broad range of diseases. PMID:23355488

  7. An In-tether Chiral Center Modulates the Helicity, Cell Permeability, and Target Binding Affinity of a Peptide.

    PubMed

    Hu, Kuan; Geng, Hao; Zhang, Qingzhou; Liu, Qisong; Xie, Mingsheng; Sun, Chengjie; Li, Wenjun; Lin, Huacan; Jiang, Fan; Wang, Tao; Wu, Yun-Dong; Li, Zigang

    2016-07-01

    The addition of a precisely positioned chiral center in the tether of a constrained peptide is reported, yielding two separable peptide diastereomers with significantly different helicity, as supported by circular dichroism (CD) and NMR spectroscopy. Single crystal X-ray diffraction analysis suggests that the absolute configuration of the in-tether chiral center in helical form is R, which is in agreement with theoretical simulations. The relationship between the secondary structure of the short peptides and their biochemical/biophysical properties remains elusive, largely because of the lack of proper controls. The present strategy provides the only method for investigating the influence of solely conformational differences upon the biochemical/biophysical properties of peptides. The significant differences in permeability and target binding affinity between the peptide diastereomers demonstrate the importance of helical conformation. PMID:27167181

  8. Poly(NIPAm-AMPS) nanoparticles for targeted delivery of anti-inflammatory cell penetrating peptides

    NASA Astrophysics Data System (ADS)

    Bartlett, Rush Lloyd, II

    Inflammatory diseases such as osteoarthritis and rheumatoid arthritis cause $127.8 billion in US healthcare expenditures each year and are the cause of disability for 27% of disabled persons in the United States. Current treatment options rarely halt disease progression and often result in significant unwanted and debilitating side effects. Our laboratory has previously developed a family of cell penetrating peptides (CPPs) which inhibit the activity of mitogen activated protein kinase activate protein kinase 2 (MK2). MK2 mediates the inflammatory response by activating Tristetraprline (TTP). Once activated, TTP rapidly stabilizes AU rich regions of pro-inflammatory cytokine mRNA which allows translation of pro-inflammatory cytokines to occur. Blocking MK2 with our labs CPPs yields a decrease in inflammatory activity but CPPs by are highly non specific and prone to rapid enzymatic degradation in vivo.. In order to increase the potency of MK2 inhibiting CPPs we have developed a novel nanoparticle drug carrier composed of poly(N-isopropylacrylamide-co-2-acrylamido-2-methyl-1-propanesulfonic acid). This drug carrier has been shown to have preliminary efficacy in vitro and ex vivo for suppressing pro-inflammatory cytokine production when releasing CPPs. This thesis will present progress made on three aims: Specific Aim 1) Create and validate a NIPAm based drug delivery system that mimics the binding and release previously observed between cell penetrating peptides and glycosaminoglycans. Specific Aim 2) Engineer degradability into poly(NIPAm-AMPS) nanoparticles to enable more drug to be released and qualify that system in vitro. Specific Aim 3) Validate poly(NIPAm-AMPS) nanoparticles for targeted drug delivery in an ex vivo inflammatory model. Overall we have developed a novel anionic nanoparticle system that is biocompatible and efficient at loading and releasing cell penetrating peptides to inflamed tissue. Once loaded with a CPP the nanoparticle drug complex is

  9. Affinity capture using peptide-functionalized magnetic nanoparticles to target Staphylococcus aureus

    NASA Astrophysics Data System (ADS)

    Kuo, Fang-Yin; Lin, Wei-Lien; Chen, Yu-Chie

    2016-04-01

    Staphylococcus aureus, a commonly found pathogen, can cause food poisoning and infections. Thus, it is necessary to develop analytical methods for the rapid screening of S. aureus in suspicious samples. Magnetic nanoparticles (MNPs) are widely used as affinity probes to selectively enrich target species from complex samples because of their high specific surface area and magnetic properties. The MNP surface should be functionalized to have the capability to target specific species. We herein propose a straightforward method to functionalize aluminum oxide-coated iron oxide (Fe3O4@Al2O3) MNPs with the peptide HHHHHHDEEGLFVD (D). The peptide D was comprised of three domains: polyhistidine (H6) used as the linker, DEE added as the spacer, and GLFVD used for targeting S. aureus. D was immobilized on the surface of Fe3O4@Al2O3 MNPs through H6-Al chelation. Our results showed that the D-functionalized Fe3O4@Al2O3 MNPs (D-Fe3O4 MNPs) possess the capability to target S. aureus. The selective trapping experiments were conducted under microwave-heating for only 60 s, and sufficient bacterial cells were trapped by the MNPs to be identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We demonstrated that the D-Fe3O4@Al2O3 MNPs combined with MALDI-MS can be used to rapidly characterize trace amounts of S. aureus in complex juice and egg samples.Staphylococcus aureus, a commonly found pathogen, can cause food poisoning and infections. Thus, it is necessary to develop analytical methods for the rapid screening of S. aureus in suspicious samples. Magnetic nanoparticles (MNPs) are widely used as affinity probes to selectively enrich target species from complex samples because of their high specific surface area and magnetic properties. The MNP surface should be functionalized to have the capability to target specific species. We herein propose a straightforward method to functionalize aluminum oxide-coated iron oxide (Fe3O4@Al2O3) MNPs with the

  10. Targeted treatment of liver metastasis from gastric cancer using specific binding peptide

    PubMed Central

    Gong, Jianfeng; Tan, Gewen; Sheng, Nengquan; You, Weiqiang; Wang, Zhigang

    2016-01-01

    Gastric cancer ranks the first in China among all gastrointestinal cancers in terms of incidence, and liver metastasis is the leading cause of death for patients with advanced gastric cancer. Tumor necrosis factor (TNF) is a cytokine commonly chosen as the target for gene therapy against cancers. The specific binding peptide pd20 of gastric cancer cells with a high potential for liver metastasis was fused with human TNF to obtain the pd20-TNF gene using DNA recombinant technique. The expression of the fusion protein was induced and the protein was purified. In vitro activity test showed that the fusion protein greatly improved the membrane permeability of liver cells in nude mice with liver metastasis from gastric cancer. The tumor implantation experiment in nude mice showed that the fusion protein effectively mitigated the cancer lesions. The results provide important clues for developing the drugs for targeted treatment of liver metastasis from gastric cancer. PMID:27347305

  11. Membrane-targeting peptides for nanoparticle-facilitated cellular imaging and analysis

    NASA Astrophysics Data System (ADS)

    Breger, Joyce; Delehanty, James B.; Boeneman Gemmill, Kelly; Field, Lauren D.; Blanco-Canosa, Juan B.; Dawson, Philip E.; Huston, Alan L.; Medintz, Igor L.

    2015-03-01

    The controlled delivery of nanomaterials to the plasma membrane is critical for the development of nanoscale probes that can eventually enable cellular imaging and analysis of membrane processes. Chief among the requisite criteria are delivery/targeting modalities that result in the long-term residence (e.g., days) of the nanoparticles on the plasma membrane while simultaneously not interfering with regular cellular physiology and homeostasis. Our laboratory has developed a suite of peptidyl motifs that target semiconductor nanocrystals (quantum dots (QDs) to the plasma membrane where they remain resident for up to three days. Notably, only small a percentage of the QDs are endocytosed over this time course and cellular viability is maintained. This talk will highlight the utility of these peptide-QD constructs for cellular imaging and analysis.

  12. Cyclic RGD peptide-labeled upconversion nanophosphors for tumor cell-targeted imaging.

    PubMed

    Zako, Tamotsu; Nagata, Hiroyasu; Terada, Naofumi; Utsumi, Arata; Sakono, Masafumi; Yohda, Masafumi; Ueda, Hiroshi; Soga, Kohei; Maeda, Mizuo

    2009-03-27

    One of the great challenges of oncology is to improve methods for early tumor detection. Thus tumor cell-targeted optical imaging has been intensively studied. Bioimaging with upconversion (UC) phosphors (UCPs) is of considerable interest due to a variety of possible applications taking advantage of infrared-to-visible luminescence. Here we report for the first time tumor cell-targeted UC imaging using UCPs modified with cyclic RGD peptide (RGD-Y2O3). Cyclic RGD peptide binds specifically to integrin alphavbeta3 which is highly expressed in a tumor cell surface of certain cancer types but not in normal tissues. Since UC emission from RGD-Y2O3 was observed for U87MG cancer cell (high integrin alphavbeta3 expression), but not for MCF-7 cancer cell (low integrin alphavbeta3 expression), this UC imaging is considered to be integrin alphavbeta3 specific. The non-invasive imaging of integrin alphavbeta3 expression using UCP-based probes will have great potential in cancer imaging in general in living subjects. PMID:19351594

  13. Antibacterial Activity of Synthetic Peptides Derived from Lactoferricin against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212

    PubMed Central

    León-Calvijo, María A.; Leal-Castro, Aura L.; Almanzar-Reina, Giovanni A.; Rosas-Pérez, Jaiver E.; García-Castañeda, Javier E.; Rivera-Monroy, Zuly J.

    2015-01-01

    Peptides derived from human and bovine lactoferricin were designed, synthesized, purified, and characterized using RP-HPLC and MALDI-TOF-MS. Specific changes in the sequences were designed as (i) the incorporation of unnatural amino acids in the sequence, the (ii) reduction or (iii) elongation of the peptide chain length, and (iv) synthesis of molecules with different number of branches containing the same sequence. For each peptide, the antibacterial activity against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 was evaluated. Our results showed that Peptides I.2 (RWQWRWQWR) and I.4 ((RRWQWR)4K2Ahx2C2) exhibit bigger or similar activity against E. coli (MIC 4–33 μM) and E. faecalis (MIC 10–33 μM) when they were compared with lactoferricin protein (LF) and some of its derivate peptides as II.1 (FKCRRWQWRMKKLGA) and IV.1 (FKCRRWQWRMKKLGAPSITCVRRAE). It should be pointed out that Peptides I.2 and I.4, containing the RWQWR motif, are short and easy to synthesize; our results demonstrate that it is possible to design and obtain synthetic peptides that exhibit enhanced antibacterial activity using a methodology that is fast and low-cost and that allows obtaining products with a high degree of purity and high yield. PMID:25815317

  14. Antibacterial activity of synthetic peptides derived from lactoferricin against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212.

    PubMed

    León-Calvijo, María A; Leal-Castro, Aura L; Almanzar-Reina, Giovanni A; Rosas-Pérez, Jaiver E; García-Castañeda, Javier E; Rivera-Monroy, Zuly J

    2015-01-01

    Peptides derived from human and bovine lactoferricin were designed, synthesized, purified, and characterized using RP-HPLC and MALDI-TOF-MS. Specific changes in the sequences were designed as (i) the incorporation of unnatural amino acids in the sequence, the (ii) reduction or (iii) elongation of the peptide chain length, and (iv) synthesis of molecules with different number of branches containing the same sequence. For each peptide, the antibacterial activity against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 was evaluated. Our results showed that Peptides I.2 (RWQWRWQWR) and I.4 ((RRWQWR)4K2Ahx2C2) exhibit bigger or similar activity against E. coli (MIC 4-33 μM) and E. faecalis (MIC 10-33 μM) when they were compared with lactoferricin protein (LF) and some of its derivate peptides as II.1 (FKCRRWQWRMKKLGA) and IV.1 (FKCRRWQWRMKKLGAPSITCVRRAE). It should be pointed out that Peptides I.2 and I.4, containing the RWQWR motif, are short and easy to synthesize; our results demonstrate that it is possible to design and obtain synthetic peptides that exhibit enhanced antibacterial activity using a methodology that is fast and low-cost and that allows obtaining products with a high degree of purity and high yield. PMID:25815317

  15. Osteoblast-Targeting-Peptide Modified Nanoparticle for siRNA/microRNA Delivery.

    PubMed

    Sun, Yao; Ye, Xiongzhen; Cai, Mingxiang; Liu, Xiangning; Xiao, Jia; Zhang, Chenyang; Wang, Yayu; Yang, Li; Liu, Jiafan; Li, Shannai; Kang, Chen; Zhang, Bin; Zhang, Qi; Wang, Zuolin; Hong, An; Wang, Xiaogang

    2016-06-28

    Antiosteoporosis gene-based drug development strategies are presently focused on targeting osteoblasts to either suppress bone loss or increase bone mass. Although siRNA/microRNA-based gene therapy has enormous potential, it is severely limited by the lack of specific cell-targeting delivery systems. We report an osteoblast-targeting peptide (SDSSD) that selectively binds to osteoblasts via periostin. We developed SDSSD-modified polyurethane (PU) nanomicelles encapsulating siRNA/microRNA that delivers drugs to osteoblasts; the data showed that SDSSD-PU could selectively target not only bone-formation surfaces but also osteoblasts without overt toxicity or eliciting an immune response in vivo. We used the SDSSD-PU delivery system to deliver anti-miR-214 to osteoblasts and our results showed increased bone formation, improved bone microarchitecture, and increased bone mass in an ovariectomized osteoporosis mouse model. SDSSD-PU may be a useful osteoblast-targeting small nucleic acid delivery system that could be used as an anabolic strategy to treat osteoblast-induced bone diseases. PMID:27176123

  16. Selection and identification of ligand peptides targeting a model of castrate-resistant osteogenic prostate cancer and their receptors.

    PubMed

    Mandelin, Jami; Cardó-Vila, Marina; Driessen, Wouter H P; Mathew, Paul; Navone, Nora M; Lin, Sue-Hwa; Logothetis, Christopher J; Rietz, Anna Cecilia; Dobroff, Andrey S; Proneth, Bettina; Sidman, Richard L; Pasqualini, Renata; Arap, Wadih

    2015-03-24

    We performed combinatorial peptide library screening in vivo on a novel human prostate cancer xenograft that is androgen-independent and induces a robust osteoblastic reaction in bonelike matrix and soft tissue. We found two peptides, PKRGFQD and SNTRVAP, which were enriched in the tumors, targeted the cell surface of androgen-independent prostate cancer cells in vitro, and homed to androgen receptor-null prostate cancer in vivo. Purification of tumor homogenates by affinity chromatography on these peptides and subsequent mass spectrometry revealed a receptor for the peptide PKRGFQD, α-2-macroglobulin, and for SNTRVAP, 78-kDa glucose-regulated protein (GRP78). These results indicate that GRP78 and α-2-macroglobulin are highly active in osteoblastic, androgen-independent prostate cancer in vivo. These previously unidentified ligand-receptor systems should be considered for targeted drug development against human metastatic androgen-independent prostate cancer. PMID:25762070

  17. Leader Peptide-Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease-Targeted Libraries.

    PubMed

    Reyna-González, Emmanuel; Schmid, Bianca; Petras, Daniel; Süssmuth, Roderich D; Dittmann, Elke

    2016-08-01

    Microviridins are a family of ribosomally synthesized and post-translationally modified peptides with a highly unusual architecture featuring non-canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient in vitro reconstitution approach based on two ATP-grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT-type N-acetyltransferase. The method facilitates the efficient in vitro one-pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo-enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure-activity relationships for respective target proteases. PMID:27336908

  18. Immunological features of T cells induced by human telomerase reverse transcriptase-derived peptides in patients with hepatocellular carcinoma.

    PubMed

    Mizukoshi, Eishiro; Nakagawa, Hidetoshi; Kitahara, Masaaki; Yamashita, Tatsuya; Arai, Kuniaki; Sunagozaka, Hajime; Fushimi, Kazumi; Kobayashi, Eiji; Kishi, Hiroyuki; Muraguchi, Atsushi; Kaneko, Shuichi

    2015-08-10

    Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme required for telomere elongation. In this study, we investigated the safety and immunogenicity of an hTERT-derived peptide (hTERT461) as a vaccine and characterized the hTERT-specific T cell responses induced. Fourteen hepatocellular carcinoma (HCC) patients were enrolled in the study. The hTERT-derived peptide was emulsified in incomplete Freund's adjuvant and administered via subcutaneous immunization three times biweekly. The maximum toxicity observed was grade 2 according to the common terminology criteria and mainly consisted of skin reactions at the site of vaccination. The vaccination induced hTERT-specific immunity in 71.4% of patients and 57.1% of patients administered with hTERT461 peptide-specific T cells could prevent HCC recurrence after vaccination. In phenotypic analysis, the post-vaccinated increase in hTERT-specific T cells was due to an increase in cells with the effector memory phenotype, with the potential to produce multiple cytokines. Seven hTERT-specific T cell receptors were obtained from the vaccinated patients, showing their cytotoxic activities to hTERT-derived peptide-bearing cells. In conclusion, the safety and effects of immune boosting by hTERT461 peptide have shown the potential of the peptide to provide clinical benefits in HCC patients. PMID:25982205

  19. Anti-infectious and anti-inflammatory effects of peptide fragments sequentially derived from the antimicrobial peptide centrocin 1 isolated from the green sea urchin, Strongylocentrotus droebachiensis

    PubMed Central

    2012-01-01

    Bacterial resistance against antibiotic treatment has become a major threat to public health. Antimicrobial peptides (AMPs) have emerged as promising alternative agents for treatment of infectious diseases. This study characterizes novel synthetic peptides sequentially derived from the AMP centrocin 1, isolated from the green sea urchin, for their applicability as anti-infective agents. The microbicidal effect of centrocin 1 heavy chain (CEN1 HC-Br), its debrominated analogue (CEN1 HC), the C-terminal truncated variants of both peptides, i.e. CEN1 HC-Br (1–20) and CEN1 HC (1–20), as well as the cysteine to serine substituted equivalent CEN1 HC (Ser) was evaluated using minimal microbicidal concentration assay. The anti-inflammatory properties were assessed by measuring the inhibition of secretion of pro-inflammatory cytokines. All the peptides tested exhibited marked microbicidal and anti-inflammatory properties. No difference in efficacy was seen comparing CEN1 HC-Br and CEN1 HC, while the brominated variant had higher cytotoxicity. C-terminal truncation of both peptides reduced salt-tolerability of the microbicidal effect as well as anti-inflammatory actions. Also, serine substitution of cysteine residue decreased the microbicidal effect. Thus, from the peptide variants tested, CEN1 HC showed the best efficacy and safety profile. Further, CEN1 HC significantly reduced bacterial counts in two different animal models of infected wounds, while Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) failed to develop resistance against this peptide under continued selection pressure. In summary, CEN1 HC appears a promising new antimicrobial agent, and clinical studies are warranted to evaluate the applicability of this AMP for local treatment of infections in man. PMID:23237525

  20. Discovery of novel peptides targeting pro-atherogenic endothelium in disturbed flow regions -Targeted siRNA delivery to pro-atherogenic endothelium in vivo

    PubMed Central

    Chung, Jihwa; Shim, Hyunbo; Kim, Kwanchang; Lee, Duhwan; Kim, Won Jong; Kang, Dong Hoon; Kang, Sang Won; Jo, Hanjoong; Kwon, Kihwan

    2016-01-01

    Atherosclerosis occurs preferentially in arterial regions exposed to disturbed blood flow. Targeting these pro-atherogenic regions is a potential anti-atherogenic therapeutic approach, but it has been extremely challenging. Here, using in vivo phage display approach and the partial carotid ligation model of flow-induced atherosclerosis in mouse, we identified novel peptides that specifically bind to endothelial cells (ECs) exposed to disturbed flow condition in pro-atherogenic regions. Two peptides, CLIRRTSIC and CPRRSHPIC, selectively bound to arterial ECs exposed to disturbed flow not only in the partially ligated carotids but also in the lesser curvature and branching point of the aortic arch in mice as well as human pulmonary artery branches. Peptides were conjugated to branched polyethylenimine-polyethylene glycol polymer to generate polyplexes carrying siRNA targeting intercellular adhesion molecule-1 (siICAM-1). In mouse model, CLIRRTSIC polyplexes carrying si-ICAM-1 specifically bound to endothelium in disturbed flow regions, reducing endothelial ICAM-1 expression. Mass spectrometry analysis revealed that non-muscle myosin heavy chain II A (NMHC IIA) is a protein targeted by CLIRRTSIC peptide. Further studies showed that shear stress regulates NMHC IIA expression and localization in ECs. The CLIRRTSIC is a novel peptide that could be used for targeted delivery of therapeutics such as siRNAs to pro-atherogenic endothelium. PMID:27173134

  1. A novel role for an RCAN3-derived peptide as a tumor suppressor in breast cancer.

    PubMed

    Martínez-Høyer, Sergio; Solé-Sánchez, Sònia; Aguado, Fernando; Martínez-Martínez, Sara; Serrano-Candelas, Eva; Hernández, José Luis; Iglesias, Mar; Redondo, Juan Miguel; Casanovas, Oriol; Messeguer, Ramon; Pérez-Riba, Mercè

    2015-07-01

    The members of the human regulators of calcineurin (RCAN) protein family are endogenous regulators of the calcineurin (CN)-cytosolic nuclear factor of activated T-cells (NFATc) pathway activation. This function is explained by the presence of a highly conserved calcipressin inhibitor of calcineurin (CIC) motif in RCAN proteins, which has been shown to compete with NFATc for the binding to CN and therefore are able to inhibit NFATc dephosphorylation and activation by CN. Very recently, emerging roles for NFATc proteins in transformation, tumor angiogenesis and metastasis have been described in different cancer cell types. In this work, we report that the overexpression of RCAN3 dramatically inhibits tumor growth and tumor angiogenesis in an orthotopic human breast cancer model. We suggest that RCAN3 exerts these effects in a CN-dependent manner, as mutation of the CIC motif in RCAN3 abolishes the tumor suppressor effect. Moreover, the expression of the EGFP-R3(178-210) peptide, spanning the CIC motif of RCAN3, is able to reproduce all the antitumor effects of RCAN3 full-length protein. Finally, we show that RCAN3 and the EGFP-R3(178-210) peptide inhibit the CN-NFATc signaling pathway and the induction of the NFATc-dependent gene cyclooxygenase-2. Our work suggests that the EGFP-R3(178-210) peptide possess potent tumor suppressor properties and therefore constitutes a novel lead for the development of potent and specific antitumoral agents. Moreover, we propose the targeting of the CN-NFATc pathway in the tumor cells constitutes an effective way to hamper tumor progression by impairing the paracrine network among tumor, endothelial and polymorphonucleated cells. PMID:25916653

  2. Coupling to the surface of liposomes alters the immunogenicity of hepatitis C virus-derived peptides and confers sterile immunity.

    PubMed

    Takagi, Akira; Kobayashi, Nobuharu; Taneichi, Maiko; Uchida, Tetsuya; Akatsuka, Toshitaka

    2013-01-01

    We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen presenting cells to cytotoxic T lymphocytes (CTLs). Liposomal form of immunodominant CTL epitope peptides derived from lymphocytic choriomeningitis virus exhibited highly efficient antiviral CTL responses in immunized mice. In this study, we coupled 15 highly conserved immunodominant CTL epitope peptides derived from hepatitis C virus (HCV) to the surface of liposomes. We also emulsified the peptides in incomplete Freund's adjuvant, and compared the immune responses of the two methods of presenting the peptides by cytotoxicity induction and interferon-gamma (IFN-γ) production by CD8(+) T cells of the immunized mice. We noticed significant variations of the immunogenicity of each peptide between the two antigen delivery systems. In addition, the immunogenicity profiles of the peptides were also different from those observed in the mice infected with recombinant adenoviruses expressing HCV proteins as previously reported. Induction of anti-viral immunity by liposomal peptides was tested by the challenge experiments using recombinant vaccinia viruses expressing corresponding HCV epitopes. One D(b)-restricted and three HLA-A(*)0201-restricted HCV CTL epitope peptides on the surface of liposomes were found to confer complete protection to immunized mice with establishment of long-term memory. Interestingly, their protective efficacy seemed to correlate with the induction of IFN-γ producing cells rather than the cytotoxicity induction suggesting that the immunized mice were protected through non-cytolytic mechanisms. Thus, these liposomal peptides might be useful as HCV vaccines not only for prevention but also for therapeutic use. PMID:23159619

  3. Antiproliferative effect and characterization of a novel antifungal peptide derived from human Chromogranin A

    PubMed Central

    LI, RUI-FANG; LU, YA-LI; LU, YAN-BO; ZHANG, HUI-RU; HUANG, LIANG; YIN, YANLI; ZHANG, LIN; LIU, SHUAI; LU, ZHIFANG; SUN, YANAN

    2015-01-01

    CGA-N46 is a novel antifungal peptide derived from the N-terminus of human Chromogranin A, corresponding to the 31st to 76th amino acids. Further research on its activities and characteristics may be helpful for the application of CGA-N46 in medical or other situations. In the present study, the antifungal spectrum and physicochemical characteristics of CGA-N46 were investigated using an antifungal assay, its antiproliferative effects on cancer and normal cells were assessed using MTT assay and its combinatorial effect with other antibiotics was analyzed using checkerboard analysis. The results showed that CGA-N46 exhibited antifungal activity against the tested Candidas (C. glabrata, C. parapsilosis, C. krusei, C. tropicalis and C. albicans) at a concentration of <0.8 mM, but had no effect on the growth of filamentous fungi or other types of fungi (Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Fusarium moniliforme, Microsporum canis, Microsporum gypseum, Trichophyton rubrum and Trichophyton mentagrophytes), even at a concentration of 3.2 mM. CGA-N46 had an inhibitory effect on the proliferation of lung cancer A549 cells and a reversible effect on the growth of normal primary chicken embryo fibroblast cells, but no hemolytic activity on human erythrocytes at the minimum inhibitory concentration of CGA-N46 against yeasts. The antifungal activity of CGA-N46 was stable at a temperature <40°C or within a broad pH range (pH 5.0–7.0). Its antifungal activity was enhanced when the peptide was used in combination with fluconazole and terbinafine. The present results indicate that CGA-N46 is a safe, physicochemically stable, antifungal peptide with anticancer cell activity that exhibits an additive effect with conventional antibiotics. PMID:26668630

  4. Photosensitizer and peptide-conjugated PAMAM dendrimer for targeted in vivo photodynamic therapy

    PubMed Central

    Narsireddy, Amreddy; Vijayashree, Kurra; Adimoolam, Mahesh G; Manorama, Sunkara V; Rao, Nalam M

    2015-01-01

    Challenges in photodynamic therapy (PDT) include development of efficient near infrared-sensitive photosensitizers (5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphine [PS]) and targeted delivery of PS to the tumor tissue. In this study, a dual functional dendrimer was synthesized for targeted PDT. For targeting, a poly(amidoamine) dendrimer (G4) was conjugated with a PS and a nitrilotriacetic acid (NTA) group. A peptide specific to human epidermal growth factor 2 was expressed in Escherichia coli with a His-tag and was specifically bound to the NTA group on the dendrimer. Reaction conditions were optimized to result in dendrimers with PS and the NTA at a fractional occupancy of 50% and 15%, respectively. The dendrimers were characterized by nuclear magnetic resonance, matrix-assisted laser desorption/ionization, absorbance, and fluorescence spectroscopy. Using PS fluorescence, cell uptake of these particles was confirmed by confocal microscopy and fluorescence-activated cell sorting. PS-dendrimers are more efficient than free PS in PDT-mediated cell death assays in HER2 positive cells, SK-OV-3. Similar effects were absent in HER2 negative cell line, MCF-7. Compared to free PS, the PS-dendrimers have shown significant tumor suppression in a xenograft animal tumor model. Conjugation of a PS with dendrimers and with a targeting agent has enhanced photodynamic therapeutic effects of the PS. PMID:26604753

  5. Photosensitizer and peptide-conjugated PAMAM dendrimer for targeted in vivo photodynamic therapy.

    PubMed

    Narsireddy, Amreddy; Vijayashree, Kurra; Adimoolam, Mahesh G; Manorama, Sunkara V; Rao, Nalam M

    2015-01-01

    Challenges in photodynamic therapy (PDT) include development of efficient near infrared-sensitive photosensitizers (5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphine [PS]) and targeted delivery of PS to the tumor tissue. In this study, a dual functional dendrimer was synthesized for targeted PDT. For targeting, a poly(amidoamine) dendrimer (G4) was conjugated with a PS and a nitrilotriacetic acid (NTA) group. A peptide specific to human epidermal growth factor 2 was expressed in Escherichia coli with a His-tag and was specifically bound to the NTA group on the dendrimer. Reaction conditions were optimized to result in dendrimers with PS and the NTA at a fractional occupancy of 50% and 15%, respectively. The dendrimers were characterized by nuclear magnetic resonance, matrix-assisted laser desorption/ionization, absorbance, and fluorescence spectroscopy. Using PS fluorescence, cell uptake of these particles was confirmed by confocal microscopy and fluorescence-activated cell sorting. PS-dendrimers are more efficient than free PS in PDT-mediated cell death assays in HER2 positive cells, SK-OV-3. Similar effects were absent in HER2 negative cell line, MCF-7. Compared to free PS, the PS-dendrimers have shown significant tumor suppression in a xenograft animal tumor model. Conjugation of a PS with dendrimers and with a targeting agent has enhanced photodynamic therapeutic effects of the PS. PMID:26604753

  6. Peptide-Targeted Gold Nanoparticles for Photodynamic Therapy of Brain Cancer

    PubMed Central

    Meyers, Joseph D.; Cheng, Yu; Agnes, Richard S.; Schluchter, Mark D.; Margevicius, Seunghee; Wang, Xinning; Kenney, Malcolm E.

    2015-01-01

    Targeted drug delivery using epidermal growth factor peptide-targeted gold nanoparticles (EGFpep-Au NPs) is investigated as a novel approach for delivery of photodynamic therapy (PDT) agents, specifically Pc 4, to cancer. In vitro studies of PDT show that EGFpep-Au NP-Pc 4 is twofold better at killing tumor cells than free Pc 4 after increasing localization in early endosomes. In vivo studies show that targeting with EGFpep-Au NP-Pc 4 improves accumulation of fluorescence of Pc 4 in subcutaneous tumors by greater than threefold compared with untargeted Au NPs. Targeted drug delivery and treatment success can be imaged via the intrinsic fluorescence of the PDT drug Pc 4. Using Pc 4 fluorescence, it is demonstrated in vivo that EGFpep-Au NP-Pc 4 impacts biodistribution of the NPs by decreasing the initial uptake by the reticuloendothelial system (RES) and by increasing the amount of Au NPs circulating in the blood 4 h after IV injection. Interestingly, in vivo PDT with EGFpep-Au NP-Pc 4 results in interrupted tumor growth when compared with EGFpep-Au NP control mice when selectively activated with light. These data demonstrate that EGFpep-Au NP-Pc 4 utilizes cancer-specific biomarkers to improve drug delivery and therapeutic efficacy over untargeted drug delivery. PMID:25999665

  7. Role of Proopiomelanocortin-Derived Peptides and Their Receptors in the Osteoarticular System: From Basic to Translational Research

    PubMed Central

    Grässel, Susanne

    2012-01-01

    Proopiomelanocortin (POMC)-derived peptides such as melanocortins and β-endorphin (β-ED) exert their pleiotropic effects via binding to melanocortin receptors (MCR) and opioid receptors (OR). There is now compelling evidence for the existence of a functional POMC system within the osteoarticular system. Accordingly, distinct cell types of the synovial tissue and bone have been identified to generate POMC-derived peptides like β-ED, ACTH, or α-MSH. MCR subtypes, especially MC1R, MC2R (the ACTH receptor), MC3R, and MC4R, but also the μ-OR and δ-OR, have been detected in various cells of the synovium, cartilage, and bone. The respective ligands of these POMC-derived peptide receptors mediate an increasing number of newly recognized biological effects in the osteoarticular system. These include bone mineralization and longitudinal growth, cell proliferation and differentiation, extracellular matrix synthesis, osteoprotection, and immunomodulation. Importantly, bone formation is also regulated by the central melanocortin system via a complex hormonal interplay with other organs and tissues involved in energy metabolism. Among the POMC-derived peptides examined in cell culture systems from osteoarticular tissue and in animal models of experimentally induced arthritis, α-MSH, ACTH, and MC3R-specific agonists appear to have the most promising antiinflammatory actions. The effects of these melanocortin peptides may be exploited in future for the treatment of patients with inflammatory and degenerative joint diseases. PMID:22736674

  8. Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

    SciTech Connect

    Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

    2008-08-01

    Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs.

  9. Application of Collagen-Model Triple-Helical Peptide-Amphiphiles for CD44-Targeted Drug Delivery Systems

    PubMed Central

    Ndinguri, Margaret W.; Zheleznyak, Alexander; Lauer, Janelle L.; Anderson, Carolyn J.; Fields, Gregg B.

    2012-01-01

    Cancer treatment by chemotherapy is typically accompanied by deleterious side effects, attributed to the toxic action of chemotherapeutics on proliferating cells from nontumor tissues. The cell surface proteoglycan CD44 has been recognized as a cancer stem cell marker. The present study has examined CD44 targeting as a way to selectively deliver therapeutic agents encapsulated inside colloidal delivery systems. CD44/chondroitin sulfate proteoglycan binds to a triple-helical sequence derived from type IV collagen, α1(IV)1263–1277. We have assembled a peptide-amphiphile (PA) in which α1(IV)1263–1277 was sandwiched between 4 repeats of Gly-Pro-4-hydroxyproline and conjugated to palmitic acid. The PA was incorporated into liposomes composed of DSPG, DSPC, cholesterol, and DSPE-PEG-2000 (1 : 4 : 5 : 0.5). Doxorubicin-(DOX-)loaded liposomes with and without 10% α1(IV)1263–1277 PA were found to exhibit similar stability profiles. Incubation of DOX-loaded targeted liposomes with metastatic melanoma M14#5 and M15#11 cells and BJ fibroblasts resulted in IC50 values of 9.8, 9.3, and >100 μM, respectively. Nontargeted liposomes were considerably less efficacious for M14#5 cells. In the CD44+ B16F10 mouse melanoma model, CD44-targeted liposomes reduced the tumor size to 60% of that of the untreated control, whereas nontargeted liposomes were ineffective. These results suggest that PA targeted liposomes may represent a new class of nanotechnology-based drug delivery systems. PMID:23213537

  10. Targeting Multidrug-resistant Staphylococci with an anti-rpoA Peptide Nucleic Acid Conjugated to the HIV-1 TAT Cell Penetrating Peptide.

    PubMed

    Abushahba, Mostafa Fn; Mohammad, Haroon; Seleem, Mohamed N

    2016-01-01

    Staphylococcus aureus infections present a serious challenge to healthcare practitioners due to the emergence of resistance to numerous conventional antibiotics. Due to their unique mode of action, peptide nucleic acids are novel alternatives to traditional antibiotics to tackle the issue of bacterial multidrug resistance. In this study, we designed a peptide nucleic acid covalently conjugated to the HIV-TAT cell penetrating peptide (GRKKKRRQRRRYK) in order to target the RNA polymerase α subunit gene (rpoA) required for bacterial genes transcription. We explored the antimicrobial activity of the anti-rpoA construct (peptide nucleic acid-TAT) against methicillin-resistant S. aureus, vancomycin-intermediate S. aureus, vancomycin-resistant S. aureus, linezolid-resistant S. aureus, and methicillin-resistant S. epidermidis in pure culture, infected mammalian cell culture, and in an in vivo Caenorhabditis elegans infection model. The anti-rpoA construct led to a concentration-dependent inhibition of bacterial growth (at micromolar concentrations) in vitro and in both infected cell culture and in vivo in C. elegans. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of two important methicillin-resistant S. aureus USA300 toxins (α-hemolysin and Panton-Valentine leukocidin). This study confirms that rpoA gene is a potential target for development of novel antisense therapeutics to treat infections caused by methicillin-resistant S. aureus. PMID:27434684

  11. Peptide-functionalized polymeric nanoparticles for active targeting of damaged tissue in animals with experimental autoimmune encephalomyelitis.

    PubMed

    Führmann, Tobias; Ghosh, Mousumi; Otero, Anthony; Goss, Ben; Dargaville, Tim R; Pearse, Damien D; Dalton, Paul D

    2015-08-18

    Increased permeability of blood vessels is an indicator for various injuries and diseases, including multiple sclerosis (MS), of the central nervous system. Nanoparticles have the potential to deliver drugs locally to sites of tissue damage, reducing the drug administered and limiting associated side effects, but efficient accumulation still remains a challenge. We developed peptide-functionalized polymeric nanoparticles to target blood clots and the extracellular matrix molecule nidogen, which are associated with areas of tissue damage. Using the induction of experimental autoimmune encephalomyelitis in rats to provide a model of MS associated with tissue damage and blood vessel lesions, all targeted nanoparticles were delivered systemically. In vivo data demonstrates enhanced accumulation of peptide functionalized nanoparticles at the injury site compared to scrambled and naive controls, particularly for nanoparticles functionalized to target fibrin clots. This suggests that further investigations with drug laden, peptide functionalized nanoparticles might be of particular interest in the development of treatment strategies for MS. PMID:26141613

  12. Interaction between tachyplesin I, an antimicrobial peptide derived from horseshoe crab, and lipopolysaccharide.

    PubMed

    Kushibiki, Takahiro; Kamiya, Masakatsu; Aizawa, Tomoyasu; Kumaki, Yasuhiro; Kikukawa, Takashi; Mizuguchi, Mineyuki; Demura, Makoto; Kawabata, Shun-ichiro; Kawano, Keiichi

    2014-03-01

    Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria and is the very first site of interactions with antimicrobial peptides (AMPs). In order to gain better insight into the interaction between LPS and AMPs, we determined the structure of tachyplesin I (TP I), an antimicrobial peptide derived from horseshoe crab, in its bound state with LPS and proposed the complex structure of TP I and LPS using a docking program. CD and NMR measurements revealed that binding to LPS slightly extends the two β-strands of TP I and stabilizes the whole structure of TP I. The fluorescence wavelength of an intrinsic tryptophan of TP I and fluorescence quenching in the presence or absence of LPS indicated that a tryptophan residue is incorporated into the hydrophobic environment of LPS. Finally, we succeeded in proposing a structural model for the complex of TP I and LPS by using a docking program. The calculated model structure suggested that the cationic residues of TP I interact with phosphate groups and saccharides of LPS, whereas hydrophobic residues interact with the acyl chains of LPS. PMID:24389234

  13. Anti-biofilm activity of ultrashort cinnamic acid peptide derivatives against medical device-related pathogens.

    PubMed

    Laverty, Garry; McCloskey, Alice P; Gorman, Sean P; Gilmore, Brendan F

    2015-10-01

    The threat of antimicrobial resistance has placed increasing emphasis on the development of innovative approaches to eradicate multidrug-resistant pathogens. Biofilm-forming microorganisms, for example, Staphylococcus epidermidis and Staphylococcus aureus, are responsible for increased incidence of biomaterial infection, extended hospital stays and patient morbidity and mortality. This paper highlights the potential of ultrashort tetra-peptide conjugated to hydrophobic cinnamic acid derivatives. These peptidomimetic molecules demonstrate selective and highly potent activity against resistant biofilm forms of Gram-positive medical device-related pathogens. 3-(4-Hydroxyphenyl)propionic)-Orn-Orn-Trp-Trp-NH2 displays particular promise with minimum biofilm eradication concentration (MBEC) values of 125 µg/ml against methicillin sensitive (ATCC 29213) and resistant (ATCC 43300) S. aureus and activity shown against biofilm forms of Escherichia coli (MBEC: 1000 µg/ml). Kill kinetics confirms complete eradication of established 24-h biofilms at MBEC with 6-h exposure. Reduced cell cytotoxicity, relative to Gram-positive pathogens, was proven via tissue culture (HaCaT) and haemolysis assays (equine erythrocytes). Existing in nature as part of the immune response, antimicrobial peptides display great promise for exploitation by the pharmaceutical industry in order to increase the library of available therapeutic molecules. Ultrashort variants are particularly promising for translation as clinical therapeutics as they are more cost-effective, easier to synthesise and can be tailored to specific functional requirements based on the primary sequence allowing factors such as spectrum of activity to be varied. PMID:26310860

  14. Targeting of follicle stimulating hormone peptide-conjugated dendrimers to ovarian cancer cells

    NASA Astrophysics Data System (ADS)

    Modi, Dimple A.; Sunoqrot, Suhair; Bugno, Jason; Lantvit, Daniel D.; Hong, Seungpyo; Burdette, Joanna E.

    2014-02-01

    Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side effects. To address these issues, we have designed poly(amidoamine) (PAMAM) dendrimers to selectively target the follicle stimulating hormone receptor (FSHR), which is overexpressed by tumorigenic ovarian cancer cells but not by immature primordial follicles and other non-tumorigenic cells. Fluorescein-labeled generation 5 (G5) PAMAM dendrimers were conjugated with the binding peptide domain of FSH (FSH33) that has a high affinity to FSHR. The targeted dendrimers exhibited high receptor selectivity to FSHR-expressing OVCAR-3 cells, resulting in significant uptake and downregulation of an anti-apoptotic protein survivin, while showing minimal interactions with SKOV-3 cells that do not express FSHR. The selectivity of the FSH33-targeted dendrimers was further validated in 3D organ cultures of normal mouse ovaries. Immunostaining of the conjugates revealed their selective binding and uptake by ovarian surface epithelium (OSE) cells that express FSHR, while sparing the immature primordial follicles. In addition, an in vivo study monitoring tissue accumulation following a single intraperitoneal (i.p.) injection of the conjugates showed significantly higher accumulation of FSH33-targeted dendrimers in the ovary and oviduct compared to the non-targeted conjugates. These proof-of-concept findings highlight the potential of these FSH33-targeted dendrimers to serve as a delivery platform for anti-ovarian cancer drugs, while reducing their systemic side effects by preventing nonspecific uptake by the primordial follicles.Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side

  15. Selection of Novel Peptides Homing the 4T1 CELL Line: Exploring Alternative Targets for Triple Negative Breast Cancer.

    PubMed

    Silva, Vera L; Ferreira, Debora; Nobrega, Franklin L; Martins, Ivone M; Kluskens, Leon D; Rodrigues, Ligia R

    2016-01-01

    The use of bacteriophages to select novel ligands has been widely explored for cancer therapy. Their application is most warranted in cancer subtypes lacking knowledge on how to target the cancer cells in question, such as the triple negative breast cancer, eventually leading to the development of alternative nanomedicines for cancer therapeutics. Therefore, the following study aimed to select and characterize novel peptides for a triple negative breast cancer murine mammary carcinoma cell line- 4T1. Using phage display, 7 and 12 amino acid random peptide libraries were screened against the 4T1 cell line. A total of four rounds, plus a counter-selection round using the 3T3 murine fibroblast cell line, was performed. The enriched selective peptides were characterized and their binding capacity towards 4T1 tissue samples was confirmed by immunofluorescence and flow cytometry analysis. The selected peptides (4T1pep1 -CPTASNTSC and 4T1pep2-EVQSSKFPAHVS) were enriched over few rounds of selection and exhibited specific binding to the 4T1 cell line. Interestingly, affinity to the human MDA-MB-231 cell line was also observed for both peptides, promoting the translational application of these novel ligands between species. Additionally, bioinformatics analysis suggested that both peptides target human Mucin-16. This protein has been implicated in different types of cancer, as it is involved in many important cellular functions. This study strongly supports the need of finding alternative targeting systems for TNBC and the peptides herein selected exhibit promising future application as novel homing peptides for breast cancer therapy. PMID:27548261

  16. Selection of Novel Peptides Homing the 4T1 CELL Line: Exploring Alternative Targets for Triple Negative Breast Cancer

    PubMed Central

    Nobrega, Franklin L.; Martins, Ivone M.

    2016-01-01

    The use of bacteriophages to select novel ligands has been widely explored for cancer therapy. Their application is most warranted in cancer subtypes lacking knowledge on how to target the cancer cells in question, such as the triple negative breast cancer, eventually leading to the development of alternative nanomedicines for cancer therapeutics. Therefore, the following study aimed to select and characterize novel peptides for a triple negative breast cancer murine mammary carcinoma cell line– 4T1. Using phage display, 7 and 12 amino acid random peptide libraries were screened against the 4T1 cell line. A total of four rounds, plus a counter-selection round using the 3T3 murine fibroblast cell line, was performed. The enriched selective peptides were characterized and their binding capacity towards 4T1 tissue samples was confirmed by immunofluorescence and flow cytometry analysis. The selected peptides (4T1pep1 –CPTASNTSC and 4T1pep2—EVQSSKFPAHVS) were enriched over few rounds of selection and exhibited specific binding to the 4T1 cell line. Interestingly, affinity to the human MDA-MB-231 cell line was also observed for both peptides, promoting the translational application of these novel ligands between species. Additionally, bioinformatics analysis suggested that both peptides target human Mucin-16. This protein has been implicated in different types of cancer, as it is involved in many important cellular functions. This study strongly supports the need of finding alternative targeting systems for TNBC and the peptides herein selected exhibit promising future application as novel homing peptides for breast cancer therapy. PMID:27548261

  17. Synthesis, antibacterial activity, and biological evaluation of formyl hydroxyamino derivatives as novel potent peptide deformylase inhibitors against drug-resistant bacteria.

    PubMed

    Yang, Shouning; Shi, Wei; Xing, Dong; Zhao, Zheng; Lv, Fengping; Yang, Liping; Yang, Yushe; Hu, Wenhao

    2014-10-30

    Peptide deformylase (PDF) has been identified as a promising target for novel antibacterial agents. In this study, a series of novel formyl hydroxyamino derivatives were designed and synthesized as PDF inhibitors and their antibacterial activities were evaluated. Among the potent PDF inhibitors (1o, 1q, 1o', 1q', and 1x), in vivo studies showed that compound 1q possesses mild toxicity, a good pharmacokinetic profile and protective effects. The good in vivo efficacy and low toxicity suggest that this class of compounds has potential for development and use in future antibacterial drugs. PMID:25151577

  18. Enhanced In Vivo Tumor Detection by Active Tumor Cell Targeting Using Multiple Tumor Receptor-Binding Peptides Presented on Genetically Engineered Human Ferritin Nanoparticles.

    PubMed

    Kwon, Koo Chul; Ko, Ho Kyung; Lee, Jiyun; Lee, Eun Jung; Kim, Kwangmeyung; Lee, Jeewon

    2016-08-01

    Human ferritin heavy-chain nanoparticle (hFTH) is genetically engineered to present tumor receptor-binding peptides (affibody and/or RGD-derived cyclic peptides, named 4CRGD here) on its surface. The affibody and 4CRGD specifically and strongly binds to human epidermal growth factor receptor I (EGFR) and human integrin αvβ3, respectively, which are overexpressed on various tumor cells. Through in vitro culture of EGFR-overexpressing adenocarcinoma (MDA-MB-468) and integrin-overexpressing glioblastoma cells (U87MG), it is clarified that specific interactions between receptors on tumor cells and receptor-binding peptides on engineered hFTH is critical in active tumor cell targeting. After labeling with the near-infrared fluorescence dye (Cy5.5) and intravenouse injection into MDA-MB-468 or U87MG tumor-bearing mice, the recombinant hFTHs presenting either peptide or both of affibody and 4CRGD are successfully delivered to and retained in the tumor for a prolonged period of time. In particular, the recombinant hFTH presenting both affibody and 4CRGD notably enhances in vivo detection of U87MG tumors that express heterogeneous receptors, integrin and EGFR, compared to the other recombinant hFTHs presenting either affibody or 4CRGD only. Like affibody and 4CRGD used in this study, other multiple tumor receptor-binding peptides can be also genetically introduced to the hFTH surface for actively targeting of in vivo tumors with heterogenous receptors. PMID:27356892

  19. Conserved Peptide Fragmentation as a Benchmarking Tool for Mass Spectrometers and a Discriminating Feature for Targeted Proteomics*

    PubMed Central

    Toprak, Umut H.; Gillet, Ludovic C.; Maiolica, Alessio; Navarro, Pedro; Leitner, Alexander; Aebersold, Ruedi

    2014-01-01

    Quantifying the similarity of spectra is an important task in various areas of spectroscopy, for example, to identify a compound by comparing sample spectra to those of reference standards. In mass spectrometry based discovery proteomics, spectral comparisons are used to infer the amino acid sequence of peptides. In targeted proteomics by selected reaction monitoring (SRM) or SWATH MS, predetermined sets of fragment ion signals integrated over chromatographic time are used to identify target peptides in complex samples. In both cases, confidence in peptide identification is directly related to the quality of spectral matches. In this study, we used sets of simulated spectra of well-controlled dissimilarity to benchmark different spectral comparison measures and to develop a robust scoring scheme that quantifies the similarity of fragment ion spectra. We applied the normalized spectral contrast angle score to quantify the similarity of spectra to objectively assess fragment ion variability of tandem mass spectrometric datasets, to evaluate portability of peptide fragment ion spectra for targeted mass spectrometry across different types of mass spectrometers and to discriminate target assays from decoys in targeted proteomics. Altogether, this study validates the use of the normalized spectral contrast angle as a sensitive spectral similarity measure for targeted proteomics, and more generally provides a methodology to assess the performance of spectral comparisons and to support the rational selection of the most appropriate similarity measure. The algorithms used in this study are made publicly available as an open source toolset with a graphical user interface. PMID:24623587

  20. Factors affecting antimicrobial activity of MUC7 12-mer, a human salivary mucin-derived peptide

    PubMed Central

    Wei, Guo-Xian; Campagna, Alexander N; Bobek, Libuse A

    2007-01-01

    Background MUC7 12-mer (RKSYKCLHKRCR), a cationic antimicrobial peptide derived from the human low-molecular-weight salivary mucin MUC7, possesses potent antimicrobial activity in vitro. In order to evaluate the potential therapeutic application of the MUC7 12-mer, we examined the effects of mono- and divalent cations, EDTA, pH, and temperature on its antimicrobial activity. Methods Minimal Inhibitory Concentrations (MICs) were determined using a liquid growth inhibition assay in 96-well microtiter plates. MUC7 12-mer was added at concentrations of 1.56–50 μM. MICs were determined at three endpoints: MIC-0, MIC-1, and MIC-2 (the lowest drug concentration showing 10%, 25% and 50% of growth, respectively). To examine the effect of salts or EDTA, a checkerboard microdilution technique was used. Fractional inhibitory concentration index (FICi) was calculated on the basis of MIC-0. The viability of microbial cells treated with MUC7 12-mer in the presence of sodium or potassium was also determined by killing assay or flow cytometry. Results The MICs of MUC7 12-mer against organisms tested ranged from 6.25–50 μM. For C. albicans, antagonism (FICi 4.5) was observed for the combination of MUC7 12-mer and calcium; however, there was synergism (FICi 0.22) between MUC7 12-mer and EDTA, and the synergism was retained in the presence of calcium at its physiological concentration (1–2 mM). No antagonism but additivity or indifference (FICi 0.55–2.5) was observed for the combination of MUC7 12-mer and each K+, Na+, Mg2+, or Zn2+. MUC7 12-mer peptide (at 25 μM) also exerted killing activity in the presence of NaCl, (up to 25 mM for C. albicans and up to 150 mM for E. coli, a physiological concentration of sodium in the oral cavity and serum, respectively) and retained candidacidal activity in the presence of KCl (up to 40 mM). The peptide exhibited higher inhibitory activity against C. albicans at pH 7, 8, and 9 than at pH 5 and 6, and temperature up to 60°C did not

  1. In vitro immunostimulatory properties of Abrus lectins derived peptides in tumor bearing mice.

    PubMed

    Bhutia, Sujit K; Mallick, Sanjaya K; Maiti, Tapas K

    2009-08-01

    In vitro immunostimulatory effect of Abrus lectins derived peptide fractions (AGP and ABP) was investigated in DL bearing mice. Both AGP and ABP were found to activate splenocytes and induced production of cytokines like IL-2, IFN-gamma and TNF-alpha indicating a Th1 type of immune response. Analysis of in vitro treated splenocytes by flow cytometry revealed an increase in percentage of T and B cell with high expression of activation markers (CD25(+) and CD71(+)). At the same time, expression of co-stimulatory markers was significantly high compared to tumor control. The tumor associated macrophages were able to stimulate NO production, IL-1 secretion, increased phagocytosis and decreased expression of mannose receptor. It was also observed that NK cell was activated by AGP and ABP. These results suggest that both AGP and ABP act as immunostimulants in vitro in DL bearing mice. PMID:19303750

  2. A Nonribosomal Peptide Synthetase-derived Iron(III) Complex from the Pathogenic Fungus Aspergillus fumigatus

    PubMed Central

    Yin, Wen-Bing; Baccile, Joshua A.; Bok, Jin Woo; Chen, Yiming; Keller, Nancy P.; Schroeder, Frank C.

    2013-01-01

    Small molecules (SMs) play central roles as virulence factors of pathogenic fungi and bacteria; however, genomic analyses suggest that the majority of microbial SMs have remained uncharacterized. Based on microarray analysis followed by comparative metabolomics of overexpression/knockout mutants we identified a tryptophan-derived iron(III)-complex, hexadehydroastechrome (HAS), as the major product of the cryptic has non-ribosomal peptide synthetase (NRPS) gene cluster in the human pathogen Aspergillus fumigatus. Activation of the has cluster created a highly virulent A. fumigatus strain that increased mortality of infected mice. Comparative metabolomics of different mutant strains allowed to propose a pathway for HAS biosynthesis and further revealed cross-talk with another NRPS pathway producing the anti-cancer fumitremorgins. PMID:23360537

  3. Food-derived sensory cues modulate longevity via distinct neuroendocrine insulin-like peptides.

    PubMed

    Artan, Murat; Jeong, Dae-Eun; Lee, Dongyeop; Kim, Young-Il; Son, Heehwa G; Husain, Zahabiya; Kim, Jinmahn; Altintas, Ozlem; Kim, Kyuhyung; Alcedo, Joy; Lee, Seung-Jae V

    2016-05-01

    Environmental fluctuations influence organismal aging by affecting various regulatory systems. One such system involves sensory neurons, which affect life span in many species. However, how sensory neurons coordinate organismal aging in response to changes in environmental signals remains elusive. Here, we found that a subset of sensory neurons shortens Caenorhabditis elegans' life span by differentially regulating the expression of a specific insulin-like peptide (ILP), INS-6. Notably, treatment with food-derived cues or optogenetic activation of sensory neurons significantly increases ins-6 expression and decreases life span. INS-6 in turn relays the longevity signals to nonneuronal tissues by decreasing the activity of the transcription factor DAF-16/FOXO. Together, our study delineates a mechanism through which environmental sensory cues regulate aging rates by modulating the activities of specific sensory neurons and ILPs. PMID:27125673

  4. Metallamacrocycle formation through dimerization of metal bioconjugates derived from amino acids and peptides.

    PubMed

    Álvarez, Celedonio M; García-Rodríguez, Raúl; Miguel, Daniel

    2016-01-21

    Metallamacrocycles of 12, 16, and 22 members are obtained by deprotonation of the carboxylic group of the side chain of iminopyridine complexes derived from the amino acid β-alanine, and the peptides Gly-Gly and Gly-Gly-Gly. Instead of the expected intramolecular attack to give tridentate (N,N,O) ligands, the deprotonated carboxylate attacks in an inter-molecular manner to give dimers in which the ligand acts as a bridge bonded in a κ(2)(N,N') chelating fashion to one metal and as κ(O) to the other metal. The formation of the dimers is supported by NMR spectroscopy, mass spectrometry and X-ray crystallography. PMID:26645303

  5. Histatin 5-Derived Peptide with Improved Fungicidal Properties Enhances Human Immunodeficiency Virus Type 1 Replication by Promoting Viral Entry

    PubMed Central

    Groot, Fedde; Sanders, Rogier W.; ter Brake, Olivier; Nazmi, Kamran; Veerman, Enno C. I.; Bolscher, Jan G. M.; Berkhout, Ben

    2006-01-01

    Antimicrobial peptides are found in a number of body compartments and are secreted at mucosal surfaces, where they form part of the innate immune system. Many of these small peptides have a broad spectrum of inhibitory activity against bacteria, fungi, parasites, and viruses. Generally, the peptide's mode of action is binding and disruption of membranes due to its amphipathic properties. Histatin 5 is a salivary peptide that inhibits Candida albicans, an opportunistic fungus that causes oropharyngeal candidiasis in a majority of human immunodeficiency virus type 1 (HIV-1)-infected patients progressing towards AIDS. Previously, we increased the fungicidal properties of histatin 5 by replacing amino acids in the active domain of histatin 5 (Dh-5) (A. L. Ruissen, J. Groenink, E. J. Helmerhorst, E. Walgreen-Weterings, W. van’t Hof, E. C. Veerman, and A. V. Nieuw Amerongen, Biochem. J. 356:361-368, 2001). In the current study, we tested the anti-HIV-1 activity of Dh-5 and its derivatives. Although Dh-5 inhibited HIV-1 replication, none of the peptide variants were more effective in this respect. In contrast, one of the derivatives, Dhvar2, significantly increased HIV-1 replication by promoting the envelope-mediated cell entry process. Most likely, Dhvar2 affects membranes, thereby facilitating fusion of viral and cellular membranes. This study shows that modification of antimicrobial peptides in order to improve their activity against a pathogen may have unpredictable and unwanted side effects on other pathogens. PMID:16940535

  6. Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic and intranuclear translocation of macromolecules.

    PubMed

    Avrameas, A; Ternynck, T; Nato, F; Buttin, G; Avrameas, S

    1998-05-12

    Naturally occurring polyreactive anti-DNA mAbs derived from a nonimmunized (NZB x NZW)F1 mouse with spontaneous lupus erythematosus penetrated and accumulated in the nuclei of a variety of cultured cells. These mAbs and their F(ab')2 and Fab' fragments, covalently coupled to fluorescein, peroxidase, or a 15-mer polynucleotide, also translocated to the cell nuclei. A 30-amino acid peptide corresponding to the combined sequences of the complementary-determining regions 2 and 3 of the heavy chain variable region of one mAb was able to penetrate into the cytoplasm and nucleus of cells of several lines. This peptide recognized DNA and was strongly polyreactive. Streptavidin-peroxidase conjugates complexed with the N-biotinylated peptide were rapidly translocated into cells. Similarly, peroxidase or anti-peroxidase polyclonal antibodies covalently coupled to the N-cysteinylated peptide through an heterobifunctional maleimide cross-linker were also rapidly internalized and frequently accumulated in nuclei. The peptide carrying 19 lysine residues at its N-terminal was highly effective in transfecting 3T3 cells with a plasmid containing the luciferase gene. Thus, penetrating mAbs and derived peptides are versatile vectors for the intracellular delivery of proteins and genes. PMID:9576929

  7. Recombinant vascular basement-membrane-derived multifunctional peptide inhibits angiogenesis and growth of hepatocellular carcinoma

    PubMed Central

    Wu, You-Hua; Cao, Jian-Guo; Xiang, Hong-Lin; Xia, Hong; Qin, Yong; Huang, A-Ji; Xiao, Di; Xu, Fang

    2009-01-01

    AIM: To investigate the anti-angiogenic and anti-tumor activities of recombinant vascular basement membrane-derived multifunctional peptide (rVBMDMP) in hepatocellular carcinoma (HCC). METHODS: HepG2, Bel-7402, Hep-3B, HUVE-12 and L-02 cell lines were cultured in vitro and the inhibitory effect of rVBMDMP on proliferation of cells was detected by MTT assay. The in vivo antitumor efficacy of rVBMDMP on HCC was assessed by HepG2 xenografts in nude mice. Distribution of rVBMDMP, mechanism by which the growth of HepG2 xenografts is inhibited, and microvessel area were observed by proliferating cell nuclear antigen (PCNA) and CD31 immunohistochemistry. RESULTS: MTT assay showed that rVBMDMP markedly inhibited the proliferation of human HCC (HepG2, Bel-7402, Hep-3B) cells and human umbilical vein endothelial (HUVE-12) cells in a dose-dependent manner, with little effect on the growth of L-02 cells. When the IC50 was 4.68, 7.65, 8.96, 11.65 and 64.82 μmol/L, respectively, the potency of rVBMDMP to HepG2 cells was similar to 5-fluorouracil (5-FU) with an IC50 of 4.59 μmol/L. The selective index of cytotoxicity to HepG2 cells of rVBMDMP was 13.8 (64.82/4.68), which was higher than that of 5-FU [SI was 1.9 (8.94/4.59)]. The VEGF-targeted recombinant humanized monoclonal antibody bevacizumab (100 mg/L) did not affect the proliferation of HepG2, Bel-7402, Hep-3B and L-02 cells, but the growth inhibitory rate of bevacizumab (100 mg/L) to HUVE-12 cells was 87.6% ± 8.2%. Alternis diebus intraperitoneal injection of rVBMDMP suppressed the growth of HepG2 xenografts in a dose-dependent manner. rVBMDMP (1, 3, 10 mg/kg) decreased the tumor weight by 12.6%, 55.9% and 79.7%, respectively, compared with the vehicle control. Immunohistochemical staining of rVBMDMP showed that the positive area rates (2.2% ± 0.73%, 4.5% ± 1.3% and 11.5% ± 3.8%) in rVBMDMP treated group (1, 3, 10 mg/kg) were significantly higher than that (0.13% ± 0.04%) in the control group (P < 0.01). The positive

  8. Experimental and Computational Investigation of the Effect of Hydrophobicity on Aggregation and Osteoinductive Potential of BMP-2-Derived Peptide in a Hydrogel Matrix

    PubMed Central

    Moeinzadeh, Seyedsina; Barati, Danial; Sarvestani, Samaneh K.; Karimi, Tahereh

    2015-01-01

    An attractive approach to reduce the undesired side effects of bone morphogenetic proteins (BMPs) in regenerative medicine is to use osteoinductive peptide sequences derived from BMPs. Although the structure and function of BMPs have been studied extensively, there is limited data on structure and activity of BMP-derived peptides immobilized in hydrogels. The objective of this work was to investigate the effect of concentration and hydrophobicity of the BMP-2 peptide, corresponding to residues 73–92 of the knuckle epitope of BMP-2 protein, on peptide aggregation and osteogenic differentiation of human mesenchymal stem cells encapsulated in a polyethylene glycol (PEG) hydrogel. The peptide hydrophobicity was varied by capping PEG chain ends with short lactide segments. The BMP-2 peptide with a positive index of hydrophobicity had a critical micelle concentration (CMC) and formed aggregates in aqueous solution. Based on simulation results, there was a slight increase in the concentration of free peptide in solution with 1000-fold increase in peptide concentration. The dose-osteogenic response curve of the BMP-2 peptide was in the 0.0005–0.005 mM range, and osteoinductive potential of the BMP-2 peptide was significantly less than that of BMP-2 protein even at 1000-fold higher concentrations, which was attributed to peptide aggregation. Further, the peptide or PEG-peptide aggregates had significantly higher interaction energy with the cell membrane compared with the free peptide, which led to a higher nonspecific interaction with the cell membrane and loss of osteoinductive potential. Conjugation of the BMP-2 peptide to PEG increased CMC and osteoinductive potential of the peptide whereas conjugation to lactide-capped PEG reduced CMC and osteoinductive potential of the peptide. Experimental and simulation results revealed that osteoinductive potential of the BMP-2 peptide is correlated with its CMC and the free peptide concentration in aqueous medium and not the

  9. Peptides targeting chemokine receptor CXCR4: structural behavior and biological binding studies.

    PubMed

    Costantini, Susan; Raucci, Raffaele; Colonna, Giovanni; Mercurio, Flavia Anna; Trotta, Anna Maria; Paola, Ringhieri; Leone, Marilisa; Rossi, Filomena; Pellegrino, Carmela; Castello, Giuseppe; Scala, Stefania

    2014-04-01

    CXCR4 is a G-protein-coupled receptor involved in a number of physiological processes in the hematopoietic and immune systems. CXCL12/CXCR4 axis plays a central role in diseases, such as HIV, cancer, WHIM syndrome, rheumatoid arthritis, pulmonary fibrosis, and lupus and, hence, indicated as putative therapeutic target. Although multiple CXCR4 antagonists have been developed, there is only one marketed drug, plerixafor, indicated for stem cell mobilization in poor mobilizer patients. In this work, we have designed and synthesized two peptides, six and seven residues long, using as template the N-terminal region of CXCL12; analyzed their conformations by CD, NMR, and molecular dynamics simulations; simulated their complexes with CXCR4 by docking methods; and validated these data by in vitro studies. The results showed that the two peptides are rather flexible in aqueous solution lacking ordered secondary structure elements and present a promising affinity for CXCR4. This affinity is not revealed for CXCR7, indicating a specificity for CXCR4. PMID:24474664

  10. Formyl Peptide Receptor as a Novel Therapeutic Target for Anxiety-Related Disorders

    PubMed Central

    Piras, Giuseppa; Gobbetti, Thomas; Panza, Elisabetta; Perretti, Mauro; Dalley, Jeffrey W.; D'Acquisto, Fulvio

    2014-01-01

    Formyl peptide receptors (FPR) belong to a family of sensors of the immune system that detect microbe-associated molecules and inform various cellular and sensorial mechanisms to the presence of pathogens in the host. Here we demonstrate that Fpr2/3-deficient mice show a distinct profile of behaviour characterised by reduced anxiety in the marble burying and light-dark box paradigms, increased exploratory behaviour in an open-field, together with superior performance on a novel object recognition test. Pharmacological blockade with a formyl peptide receptor antagonist, Boc2, in wild type mice reproduced most of the behavioural changes observed in the Fpr2/3-/- mice, including a significant improvement in novel object discrimination and reduced anxiety in a light/dark shuttle test. These effects were associated with reduced FPR signalling in the gut as shown by the significant reduction in the levels of p-p38. Collectively, these findings suggest that homeostatic FPR signalling exerts a modulatory effect on anxiety-like behaviours. These findings thus suggest that therapies targeting FPRs may be a novel approach to ameliorate behavioural abnormalities present in neuropsychiatric disorders at the cognitive-emotional interface. PMID:25517119

  11. Formyl peptide receptor as a novel therapeutic target for anxiety-related disorders.

    PubMed

    Gallo, Irene; Rattazzi, Lorenza; Piras, Giuseppa; Gobbetti, Thomas; Panza, Elisabetta; Perretti, Mauro; Dalley, Jeffrey W; D'Acquisto, Fulvio

    2014-01-01

    Formyl peptide receptors (FPR) belong to a family of sensors of the immune system that detect microbe-associated molecules and inform various cellular and sensorial mechanisms to the presence of pathogens in the host. Here we demonstrate that Fpr2/3-deficient mice show a distinct profile of behaviour characterised by reduced anxiety in the marble burying and light-dark box paradigms, increased exploratory behaviour in an open-field, together with superior performance on a novel object recognition test. Pharmacological blockade with a formyl peptide receptor antagonist, Boc2, in wild type mice reproduced most of the behavioural changes observed in the Fpr2/3(-/-) mice, including a significant improvement in novel object discrimination and reduced anxiety in a light/dark shuttle test. These effects were associated with reduced FPR signalling in the gut as shown by the significant reduction in the levels of p-p38. Collectively, these findings suggest that homeostatic FPR signalling exerts a modulatory effect on anxiety-like behaviours. These findings thus suggest that therapies targeting FPRs may be a novel approach to ameliorate behavioural abnormalities present in neuropsychiatric disorders at the cognitive-emotional interface. PMID:25517119

  12. A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines

    PubMed Central

    Solarte, Víctor A.; Rosas, Jaiver E.; Rivera, Zuly J.; Arango-Rodríguez, Martha L.; García, Javier E.; Vernot, Jean-Paul

    2015-01-01

    Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC) cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20–25)4, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90%) in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC. PMID:26609531

  13. Rescuing neuronal cell death by RAIDD- and PIDD- derived peptides and its implications for therapeutic intervention in neurodegenerative diseases

    PubMed Central

    Jang, Tae-Ho; Lim, In-Hye; Kim, Chang Min; Choi, Jae Young; Kim, Eun-Ae; Lee, Tae-Jin; Park, Hyun Ho

    2016-01-01

    Caspase-2 is known to be involved in oxidative-stress mediated neuronal cell death. In this study, we demonstrated that rotenone-induced neuronal cell death is mediated by caspase-2 activation via PIDDosome formation. Our newly designed TAT-fused peptides, which contains wild-type helix number3 (H3) from RAIDD and PIDD, blocked the PIDDosome formation in vitro. Furthermore, peptides inhibited rotenone-induced caspase-2-dependent apoptosis in neuronal cells. These results suggest that PIDD- or RAIDD-targeted peptides might be effective at protecting against rotenone-induced neurotoxicity. Our peptides are novel neuronal cell apoptosis inhibitors that might serve as a prototype for development of drugs for the treatment of neurodegenerative diseases. PMID:27502430

  14. Rescuing neuronal cell death by RAIDD- and PIDD- derived peptides and its implications for therapeutic intervention in neurodegenerative diseases.

    PubMed

    Jang, Tae-Ho; Lim, In-Hye; Kim, Chang Min; Choi, Jae Young; Kim, Eun-Ae; Lee, Tae-Jin; Park, Hyun Ho

    2016-01-01

    Caspase-2 is known to be involved in oxidative-stress mediated neuronal cell death. In this study, we demonstrated that rotenone-induced neuronal cell death is mediated by caspase-2 activation via PIDDosome formation. Our newly designed TAT-fused peptides, which contains wild-type helix number3 (H3) from RAIDD and PIDD, blocked the PIDDosome formation in vitro. Furthermore, peptides inhibited rotenone-induced caspase-2-dependent apoptosis in neuronal cells. These results suggest that PIDD- or RAIDD-targeted peptides might be effective at protecting against rotenone-induced neurotoxicity. Our peptides are novel neuronal cell apoptosis inhibitors that might serve as a prototype for development of drugs for the treatment of neurodegenerative diseases. PMID:27502430

  15. Development of a recombinant antibody to target peptides and proteins to sialoadhesin-expressing macrophages

    PubMed Central

    2013-01-01

    Background Sialoadhesin (Sn)-expressing monocytes/macrophages have been associated with several diseases like inflammatory and autoimmune disorders as well as viral infections, and they also appear to play a role in the initiation of an adaptive immune response. This makes Sn-expressing cells not only attractive targets for cell-directed therapies, but also an appealing target for vaccination. Furthermore, since Sn was shown to be an endocytic receptor, the conjugation of effector molecules to an Sn-specific ligand should allow intracellular delivery of these conjugates. Previously, we developed functional Sn-specific immunoconjugates that were generated via chemical coupling. Although successful, the system requires significant optimization for each immunoconjugate to be made. To generate a more flexible and controlled system, we developed a recombinant antibody vector allowing the creation of genetic antibody fusion constructs. This paper reports on the characterization of the recombinant antibody and the evaluation of its use for Sn-directed targeting. Results The variable domains of the porcine Sn-specific monoclonal antibody 41D3 were sequenced and cloned in frame with a mouse IgG1 backbone. Transfection of HEK293T cells with the resulting plasmid led to the secretion of fully assembled IgG into the culture medium. This recombinant antibody rec41D3 was shown to specifically bind to porcine Sn with a comparable affinity as the native monoclonal antibody. In addition, rec41D3 also induced Sn endocytosis in primary macrophages and resided for prolonged times in early/late endosomes. To allow the generation of antibody fusion constructs, a multiple cloning site was introduced at the C-terminus of the heavy chain. Two fusion constructs were generated, one containing a V5 peptide tag and one containing an eGFP molecule. Both constructs were shown to be efficiently produced in HEK293T cells and easily purified using standard protein G chromatography. In addition

  16. Single amino acid variation underlies species-specific sensitivity to amphibian skin-derived opioid-like peptides

    PubMed Central

    Vardy, Eyal; Sassano, Maria F.; Rennekamp, Andrew J.; Kroeze, Wesley K.; Mosier, Philip D.; Westkaemper, Richard B.; Stevens, Craig W.; Katritch, Vsevolod; Stevens, Raymond C.; Peterson, Randel T.; Roth, Bryan L.

    2015-01-01

    It has been suggested that the evolution of vertebrate opioid receptors (ORs) follow a vector of increased functionality. Here we test this idea comparing human and frog ORs. Interestingly, some of the most potent opioid peptides known have been isolated from amphibian skin secretions. Here we show that such peptides (dermorphin and deltorphin) are highly potent in the human receptors and inactive in frog ORs. The molecular basis for the insensitivity of the frog ORs to these peptides was studied using chimeras and molecular modeling. Interestingly, the insensitivity of the delta opioid receptor (DOR) to deltorphin was due to variation of a single amino acid– Trp7.35—which is a leucine in mammalian DORs. Notably, Trp7.35 is completely conserved in all known DOR sequences from lamprey, fish and amphibians. The deltorphin-insensitive phenotype was verified in fish. Our results provide a molecular explanation for the species selectivity of skin-derived opioid peptides. PMID:26091169

  17. Target tissue distribution of the proenkephalin peptides F, E, and B

    SciTech Connect

    Katzenstein, G.E.; Lund, D.; Schultz, P.; Lewis, R.V.

    1987-08-14

    The adrenal medulla is a rich source of endogenous opioid peptides. These peptides exist predominantly in the form of larger (25-34 amino acid long) enkephalin containing peptides, whose biological roles have yet to be elucidated. We report here the tissue binding distribution of three iodinated enkephalin containing peptides, Peptides F, E, and B, in rats, rabbits, and guinea pigs following intravenous injection. Each (/sup 125/I)peptide has a unique distribution profile but all three are found to bind to the pituitary in each species of animal examined. A number of lines of evidence point toward the enkephalin containing peptides, Peptides F, E, and B, having physiological roles distinct from the enkephalins. The distribution profile of these (/sup 125/I)peptides reported here gives insight into their potential effector sites.

  18. Effect of peptide assay library size and composition in targeted data-independent acquisition-MS analyses.

    PubMed

    Parker, Sarah J; Venkatraman, Vidya; Van Eyk, Jennifer E

    2016-08-01

    The quantification of peptides using targeted analysis of data-independent acquisition MS (DIA-MS) is dependent on the size and characteristics of the assay library. We addressed several important questions on how library composition influences: (1) the number of peptides extracted from DIA-MS datasets, (2) the quality of these peptides and proteins, and (3) the biological conclusions inferred. To answer these questions we constructed five libraries from mouse vascular smooth muscle cell (VSMC) lysate, each unique in depth, input sample complexity, data acquisition mode (DDA-MS or DIA-MS), and precursor fragmentation mode (TOF-CID or Orbitrap HCD) and extracted them against the same eight DIA-MS files of VSMCs treated with vehicle or transforming growth factor β-1 (TGF-β1). We found that along with differences in peptide and protein composition, the fragments representing a given peptide differed between the libraries. Collectively these differences impacted both peak group score profile and protein abundance estimates. Surprisingly, there was little overlap in the TGF-β1 response proteome between libraries. We conclude that additional work is needed to optimize peptide assay library building for DIA-MS applications, particularly in terms of selecting optimal peptides and their respective fragments for protein quantification. PMID:27432805

  19. Targeted Delivery of Ubiquitin-Conjugated BH3 Peptide-Based Mcl-1 Inhibitors into Cancer Cells

    PubMed Central

    2015-01-01

    BH3 peptides are key mediators of apoptosis and have served as the lead structures for the development of anticancer therapeutics. Previously, we reported the application of a simple cysteine-based side chain cross-linking chemistry to NoxaBH3 peptides that led to the generation of the cross-linked NoxaBH3 peptides with increased cell permeability and higher inhibitory activity against Mcl-1 (Muppidi, A., Doi, K., Edwardraja, S., Drake, E. J., Gulick, A. M., Wang, H.-G., Lin, Q. (2012) J. Am. Chem. Soc.134, 1473422920569). To deliver cross-linked NoxaBH3 peptides selectively into cancer cells for enhanced efficacy and reduced systemic toxicity, here we report the conjugation of the NoxaBH3 peptides with the extracellular ubiquitin, a recently identified endogenous ligand for CXCR4, a chemokine receptor overexpressed in cancer cells. The resulting ubiquitin-NoxaBH3 peptide conjugates showed increased inhibitory activity against Mcl-1 and selective killing of the CXCR4-expressing cancer cells. The successful delivery of the NoxaBH3 peptides by ubiquitin into cancer cells suggests that the ubiquitin/CXCR4 axis may serve as a general route for the targeted delivery of anticancer agents. PMID:24410055

  20. Antibacterial activity of peptides derived from the C-terminal region of a hemolytic lectin, CEL-III, from the marine invertebrate Cucumaria echinata.

    PubMed

    Hatakeyama, Tomomitsu; Suenaga, Tomoko; Eto, Seiichiro; Niidome, Takuro; Aoyagi, Haruhiko

    2004-01-01

    Several synthetic peptides derived from the C-terminal domain sequence of a hemolytic lectin, CEL-III, were examined as to their action on bacteria and artificial lipid membranes. Peptide P332 (KGVIFAKASVSVKVTASLSK-NH(2)), corresponding to the sequence from residue 332, exhibited strong antibacterial activity toward Gram-positive bacteria. Replacement of each Lys in P332 by Ala markedly decreased the activity. However, when all Lys were replaced by Arg, the antibacterial activity increased, indicating the importance of positively charged residues at these positions. Replacement of Val by Leu also led to higher antibacterial activity, especially toward Gram-negative bacteria. The antibacterial activity of these peptides was correlated with their membrane-permeabilizing activity toward the bacterial inner membrane and artificial lipid vesicles, indicating that the antibacterial action is due to perturbation of bacterial cell membranes, leading to enhancement of their permeability. These results also suggest that the hydrophobic region of CEL-III, from which P332 and its analogs were derived, may play some role in the interaction with target cell membranes to trigger hemolysis. PMID:14999010

  1. Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

    PubMed Central

    Birkedal, Henrik

    2011-01-01

    Gene correction activation effects of a small series of triplex forming peptide nucleic acid (PNA) covalently conjugated to the DNA interacting ligands psoralen, chlorambucil and camptothecin targeted proximal to a stop codon mutation in an EGFP reporter gene were studied. A 15-mer homopyrimidine PNA conjugated to the topoisomerase I inhibitor camptothecin was found to increase the frequency of repair domain mediated gene correctional events of the EGFP reporter in an in vitro HeLa cell nuclear extract assay, whereas PNA psoralen or chlorambucil conjugates both of which form covalent and also interstrand crosslinked adducts with dsDNA dramatically decreased the frequency of targeted repair/correction. The PNA conjugates were also studied in mammalian cell lines upon transfection of PNA bound EGFP reporter vector and scoring repair of the EGFP gene by FACS analysis of functional EGFP expression. Consistent with the extract experiments, treatment with adduct forming PNA conjugates (psoralen and chlorambucil) resulted in a decrease in background correction frequencies in transiently transfected cells, whereas unmodified PNA or the PNA-camptothecin conjugate had little or no effect. These results suggest that simple triplex forming PNAs have little effect on proximal gene correctional events whereas PNA conjugates capable of forming DNA adducts and interstrand crosslinks are strong inhibitors. Most interestingly the PNA conjugated to the topoisomerase inhibitor, camptothecin enhanced repair in nuclear extract. Thus the effects and use of camptothecin conjugates in gene targeted repair merit further studies. PMID:21686249

  2. Development of synthetic of peptide-functionalized liposome for enhanced targeted ovarian carcinoma therapy

    PubMed Central

    Wu, Hong; Yao, Li; Mei, Jiazhuan; Li, Feng

    2015-01-01

    In this study, we report an active targeting liposomal formulation directed by a novel peptide (T7) that specifically binds to the transferrin receptor (TfR) overexpressed on ovarian carcinoma cells. The objectives of this study were to evaluate the in vitro and in vivo tumor drug targeting delivery of T7-anchored liposomes on A2780 cells. T7 conjugated to the distal end of DSPE-PEG2000-maleimide was incorporated into the liposomes via a post-insertion method, the liposome could keep stability in 50% FBS for more than 24 h. The uptake efficiency of T7-LP was 3.7 times higher than that of LP on A2780 cells. The anti-proliferative activity of T7-LP-PTX against A2780 cells was much stronger compared to that of LP-PTX and free PTX, respectively. The homing specificity and anticancer efficacy of T7-LP-PTX were also evaluated on the tumor spheroids, which revealed that T7-LP-PTX was more efficaciously internalized into tumor cells than LP. Compared to LP, T7-LP-PTX showed the highest accumulation capability into tumor spheroids, and the greatest tumor growth inhibitory effect in vitro. In the in vivo study, the T7-LP-PTX showed the best inhibition effect of the tumor growth for the A2780-bearing mice and tumor accumulation. In brief, the T7-LP may be an efficient targeting drug delivery system for ovarian carcinoma. PMID:25755707

  3. Development of synthetic of peptide-functionalized liposome for enhanced targeted ovarian carcinoma therapy

    PubMed Central

    Wu, Hong; Yao, Li; Mei, Jiazhuan; Li, Feng

    2014-01-01

    In this study, we report an active targeting liposomal formulation directed by a novel peptide (T7) that specifically binds to the transferrin receptor (TfR) overexpressed on ovarian carcinoma cells. The objectives of this study were to evaluate the in vitro and in vivo tumor drug targeting delivery of T7-anchored liposomes on A2780 cells. T7 conjugated to the distal end of DSPE-PEG2000-maleimide was incorporated into the liposomes via a post-insertion method, the liposome could keep stability in 50% FBS for more than 24 h. The uptake efficiency of T7-LP was 3.7 times higher than that of LP on A2780 cells. The anti-proliferative activity of T7-LP-PTX against A2780 cells was much stronger compared to that of LP-PTX and free PTX, respectively. The homing specificity and anticancer efficacy of T7-LP-PTX were also evaluated on the tumor spheroids, which revealed that T7-LP-PTX was more efficaciously internalized into tumor cells than LP. Compared to LP, T7-LP-PTX showed the highest accumulation capability into tumor spheroids, and the greatest tumor growth inhibitory effect in vitro. In the in vivo study, the T7-LP-PTX showed the best inhibition effect of the tumor growth for the A2780-bearing mice and tumor accumulation. In brief, the T7-LP may be an efficient targeting drug delivery system for ovarian carcinoma. PMID:25663977

  4. Peptide deformylase: a new target in antibacterial, antimalarial and anticancer drug discovery.

    PubMed

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

    2015-01-01

    Peptide deformylase (PDF) is a class of metalloenzyme responsible for catalyzing the removal of the N-formyl group from N-terminal methionine following translation. PDF inhibitors are moving into new phase of drug development. Initially, PDF was considered as an important target in antibacterial drug discovery; however genome database searches have revealed PDF-like sequences in parasites (P. falciparum) and human, widening the utility of this target in antimalarial and anticancer drug discovery along with antibacterial. Using structural and mechanistic information together with high throughput screening, several types of chemical classes of PDF inhibitors with improved efficacy and specificity have been identified. Various drugs like, GSK-1322322 (Phase II), BB-83698 (Phase I), and LBM-415 (Phase I) have entered into clinical developments. Developments in the field have prompted us to review the current aspects of PDFs, especially their structures, different classes of PDF inhibitors, and molecular modeling studies. In nut shell, this review enlightens PDF as a versatile target along with its inhibitors and future perspectives of different PDF inhibitors. PMID:25174923

  5. Microbiome changes in healthy volunteers treated with GSK1322322, a novel antibiotic targeting bacterial peptide deformylase.

    PubMed

    Arat, Seda; Spivak, Aaron; Van Horn, Stephanie; Thomas, Elizabeth; Traini, Christopher; Sathe, Ganesh; Livi, George P; Ingraham, Karen; Jones, Lori; Aubart, Kelly; Holmes, David J; Naderer, Odin; Brown, James R

    2015-02-01

    GSK1322322 is a novel antibacterial agent under development, and it has known antibacterial activities against multidrug-resistant respiratory and skin pathogens through its inhibition of the bacterial peptide deformylase. Here, we used next-generation sequencing (NGS) of the bacterial 16S rRNA genes from stool samples collected from 61 healthy volunteers at the predosing and end-of-study time points to determine the effects of GSK1322322 on the gastrointestinal (GI) microbiota in a phase I, randomized, double-blind, and placebo-controlled study. GSK1322322 was administered either intravenously (i.v.) only or in an oral-i.v. combination in single- and repeat-dose-escalation infusions. Analysis of the 16S rRNA sequence data found no significant changes in the relative abundances of GI operational taxonomic units (OTUs) between the prestudy and end-of-study samples for either the placebo- or i.v.-only-treated subjects. However, oral-i.v. treatment resulted in significant decreases in some bacterial taxa, the Firmicutes and Bacteroidales, and increases in others, the Betaproteobacteria, Gammaproteobacteria, and Bifidobacteriaceae. Microbiome diversity plots clearly differentiated the end-of-study oral-i.v.-dosed samples from all others collected. The changes in genome function as inferred from species composition suggest an increase in bacterial transporter and xenobiotic metabolism pathways in these samples. A phylogenetic analysis of the peptide deformylase protein sequences collected from the published genomes of clinical isolates previously tested for GSK1322322 in vitro susceptibility and GI bacterial reference genomes suggests that antibiotic target homology is one of several factors that influences the response of GI microbiota to this antibiotic. Our study shows that dosing regimen and target class are important factors when considering the impact of antibiotic usage on GI microbiota. (This clinical trial was registered at the GlaxoSmithKline Clinical Study

  6. Microbiome Changes in Healthy Volunteers Treated with GSK1322322, a Novel Antibiotic Targeting Bacterial Peptide Deformylase

    PubMed Central

    Arat, Seda; Spivak, Aaron; Van Horn, Stephanie; Thomas, Elizabeth; Traini, Christopher; Sathe, Ganesh; Livi, George P.; Ingraham, Karen; Jones, Lori; Aubart, Kelly; Holmes, David J.; Naderer, Odin

    2014-01-01

    GSK1322322 is a novel antibacterial agent under development, and it has known antibacterial activities against multidrug-resistant respiratory and skin pathogens through its inhibition of the bacterial peptide deformylase. Here, we used next-generation sequencing (NGS) of the bacterial 16S rRNA genes from stool samples collected from 61 healthy volunteers at the predosing and end-of-study time points to determine the effects of GSK1322322 on the gastrointestinal (GI) microbiota in a phase I, randomized, double-blind, and placebo-controlled study. GSK1322322 was administered either intravenously (i.v.) only or in an oral-i.v. combination in single- and repeat-dose-escalation infusions. Analysis of the 16S rRNA sequence data found no significant changes in the relative abundances of GI operational taxonomic units (OTUs) between the prestudy and end-of-study samples for either the placebo- or i.v.-only-treated subjects. However, oral-i.v. treatment resulted in significant decreases in some bacterial taxa, the Firmicutes and Bacteroidales, and increases in others, the Betaproteobacteria, Gammaproteobacteria, and Bifidobacteriaceae. Microbiome diversity plots clearly differentiated the end-of-study oral-i.v.-dosed samples from all others collected. The changes in genome function as inferred from species composition suggest an increase in bacterial transporter and xenobiotic metabolism pathways in these samples. A phylogenetic analysis of the peptide deformylase protein sequences collected from the published genomes of clinical isolates previously tested for GSK1322322 in vitro susceptibility and GI bacterial reference genomes suggests that antibiotic target homology is one of several factors that influences the response of GI microbiota to this antibiotic. Our study shows that dosing regimen and target class are important factors when considering the impact of antibiotic usage on GI microbiota. (This clinical trial was registered at the GlaxoSmithKline Clinical Study

  7. Peptide-Decorated Gold Nanoparticles as Functional Nano-Capping Agent of Mesoporous Silica Container for Targeting Drug Delivery.

    PubMed

    Chen, Ganchao; Xie, Yusheng; Peltier, Raoul; Lei, Haipeng; Wang, Ping; Chen, Jun; Hu, Yi; Wang, Feng; Yao, Xi; Sun, Hongyan

    2016-05-11

    A stimuli-responsive drug delivery system (DDS) with bioactive surface is constructed by end-capping mesoporous silica nanoparticles (MSNs) with functional peptide-coated gold nanoparticles (GNPs). MSNs are first functionalized with acid-labile α-amide-β-carboxyl groups to carry negative charges, and then capped with positively charged GNPs that are decorated with oligo-lysine-containing peptide. The resulting hybrid delivery system exhibits endo/lysosomal pH triggered drug release, and the incorporation of RGD peptide facilitates targeting delivery to αvβ3 integrin overexpressing cancer cells. The system can serve as a platform for preparing diversified multifunctional nanocomposites using various functional inorganic nanoparticles and bioactive peptides. PMID:27102225

  8. Proenkephalin system in human polymorphonuclear cells. Production and release of a novel 1.0-kD peptide derived from synenkephalin.

    PubMed Central

    Vindrola, O; Padrós, M R; Sterin-Prync, A; Ase, A; Finkielman, S; Nahmod, V

    1990-01-01

    In the hematopoietic system a pluripotent stem cell generates precursors for lymphoid and myeloid lineages. Proenkephalin-derived peptides were previously detected in differentiated lymphoid cells. We have studied whether the proenkephalin system is expressed in a typical differentiated cell of the myeloid lineage, the neutrophil. Human peripheral polymorphonuclear cells contain and release proenkephalin-derived peptides. The opioid portion of proenkephalin (met-enkephalin-containing peptides) was incompletely processed, resulting in the absence of low molecular weight products. The nonopioid synenkephalin (proenkephalin 1-70) molecule was completely processed to a 1.0-kD peptide derived from the COOH-terminal. This molecule was characterized in neutrophils by biochemical and immunocytochemical methods. The chemotactic peptide FMLP and the calcium ionophore A23187 induced the release of the proenkephalin-derived peptides, and this effect was potentiated by cytochalasin B. The materials secreted were similar to those present in the cell, although in the supernatant a higher proportion corresponded to more processed products. The 1.0-kD peptide was detected in human, bovine, and rat neutrophils, but the chromatographic pattern of synenkephalin-derived peptides suggests a differential posttranslational processing among species. These findings demonstrate the existence of the proenkephalin system in human neutrophils and the production and release of a novel 1.0-kD peptide derived from the synenkephalin molecule. The presence of opioid peptides in neutrophils suggests their participation in the inflammatory process, including a local analgesic effect. Images PMID:2117023

  9. Identification of a novel peptide ligand targeting visceral adipose tissue via transdermal route by in vivo phage display.

    PubMed

    Lee, Nam Kyung; Kim, Hong Shin; Kim, Kyung Hyun; Kim, Eun-Bae; Cho, Chong Su; Kang, Sang Kee; Choi, Yun Jaie

    2011-11-01

    To find novel peptide ligands targeting visceral adipose tissue (visceral fat) via transdermal route, in vivo phage display screening was conducted by dermal administration of a phage-peptide library to rats and a peptide sequence, CGLHPAFQC (designated as TDA1), was identified as a targeting ligand to visceral adipose tissue through the consecutive transdermal biopannings. Adipocyte-specific affinity and transdermal activity of the TDA1 were validated in vitro and targeting ability of the dermally administered TDA1 to visceral adipose tissue was also confirmed in vivo. TDA1 was effectively translocated into systemic circulation after dermal administration and selectively targeted visceral adipose tissue without any preference to other organs tested. Fluorescent microscopic analysis revealed that the TDA1 could be specifically localized in the hair follicles of the skin, as well as in the visceral adipose tissue. Thus, we inferred that dermally administered TDA1 would first access systemic circulation via hair follicles as its transdermal route and then could target visceral fat effectively. The overall results suggest that the TDA1 peptide could be potentially applied as a homing moiety for delivery of anti-obesity therapeutics to visceral fat through the convenient transdermal pathway. PMID:21999821

  10. A salt-regulated peptide derived from the CAP superfamily protein negatively regulates salt-stress tolerance in Arabidopsis

    PubMed Central

    Chien, Pei-Shan; Nam, Hong Gil; Chen, Yet-Ran

    2015-01-01

    High salinity has negative impacts on plant growth through altered water uptake and ion-specific toxicities. Plants have therefore evolved an intricate regulatory network in which plant hormones play significant roles in modulating physiological responses to salinity. However, current understanding of the plant peptides involved in this regulatory network remains limited. Here, we identified a salt-regulated peptide in Arabidopsis. The peptide was 11 aa and was derived from the C terminus of a cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily. This peptide was found by searching homologues in Arabidopsis using the precursor of a tomato CAP-derived peptide (CAPE) that was initially identified as an immune signal. In searching for a CAPE involved in salt responses, we screened CAPE precursor genes that showed salt-responsive expression and found that the PROAtCAPE1 (AT4G33730) gene was regulated by salinity. We confirmed the endogenous Arabidopsis CAP-derived peptide 1 (AtCAPE1) by mass spectrometry and found that a key amino acid residue in PROAtCAPE1 is critical for AtCAPE1 production. Moreover, although PROAtCAPE1 was expressed mainly in the roots, AtCAPE1 was discovered to be upregulated systemically upon salt treatment. The salt-induced AtCAPE1 negatively regulated salt tolerance by suppressing several salt-tolerance genes functioning in the production of osmolytes, detoxification, stomatal closure control, and cell membrane protection. This discovery demonstrates that AtCAPE1, a homologue of tomato immune regulator CAPE1, plays an important role in the regulation of salt stress responses. Our discovery thus suggests that the peptide may function in a trade-off between pathogen defence and salt tolerance. PMID:26093145

  11. [Prospects for use of peptides and their derivatives, structurally corresponding to the G protein-coupled receptors, in medicine].

    PubMed

    Shpakov, A O; Shpakova, E A

    2015-01-01

    The regulation of signaling pathways involved in the control of many physiological functions is carried out via the heterotrimeric G protein-coupled receptors (GPCR). The search of effective and selective regulators of GPCR and intracellular signaling cascades coupled with them is one of the important problems of modern fundamental and clinical medicine. Recently data suggest that synthetic peptides and their derivatives, structurally corresponding to the intracellular and transmembrane regions of GPCR, can interact with high efficiency and selectivity with homologous receptors and influence, thus, the functional activity of intracellular signaling cascades and fundamental cellular processes controlled by them. GPCR-peptides are active in both in vitro and in vivo. They regulate hematopoiesis, angiogenesis and cell proliferation, inhibit tumor growth and metastasis, and prevent the inflammatory diseases and septic shock. These data show greatest prospects in the development of the new generations of drugs based on GPCR-derived peptides, capable of regulating the important functions of the organism. PMID:25762596

  12. Synthesis of linear and cyclic peptide-PEG-lipids for stabilization and targeting of cationic liposome-DNA complexes.

    PubMed

    Ewert, Kai K; Kotamraju, Venkata Ramana; Majzoub, Ramsey N; Steffes, Victoria M; Wonder, Emily A; Teesalu, Tambet; Ruoslahti, Erkki; Safinya, Cyrus R

    2016-03-15

    Because nucleic acids (NAs) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for cationic liposome-DNA (CL-DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL-DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL-DNA nanoparticles. PMID:26874401

  13. Synthesis of a Cross-Bridged Cyclam Derivative for Peptide Conjugation and 64Cu Radiolabeling

    PubMed Central

    Boswell, C. Andrew; Regino, Celeste A. S.; Baidoo, Kwamena E.; Wong, Karen J.; Bumb, Ambika; Xu, Heng; Milenic, Diane E.; Kelley, James A.; Lai, Christopher C.; Brechbiel, Martin W.

    2008-01-01

    The increased use of copper radioisotopes in radiopharmaceutical applications has created a need for bifunctional chelators (BFCs) that form stable radiocopper complexes and allow covalent attachment to biological molecules. Previous studies have established that 4,11-bis-(carbo-tert-butoxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (H2CB-TE2A), a member of the ethylene “cross-bridged” cyclam (CB-cyclam) class of bicyclic tetraaza macrocycles, forms highly kinetically stable complexes with Cu(II) and is less susceptible to in vivo transchelation than its non-bridged analog, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). Herein, we report a convenient synthesis of a novel cross-bridged BFC that is structurally analogous to CB-TE2A in that it possesses two coordinating acetate arms, but in addition possesses a third orthogonally-protected arm for conjugation to peptides and other targeting agents. Application of this strategy to cross-bridged chelators may also enable the development of even further improved agents for 64Cu-mediated diagnostic positron emission tomography (PET) imaging as well as for targeted radiotherapeutic applications. PMID:18597510

  14. Distinguishing of tumor cell-targeting peptide ligands through a color-encoding microarray.

    PubMed

    Wang, Zihua; Wang, Weizhi; Geng, Lingling; Hu, Zhiyuan

    2015-12-21

    A silicon-based microarray system was constructed to discover the affinity peptides and to distinguish the specific peptides from a high throughput library. Using a color-encoding strategy, in situ peptide distinguishing between HER1 ligands and HER2 ligands was achieved. Novel affinity peptide sequences H1P (HER1 ligand) and H2P (HER2 ligand) were determined with nmol affinity. PMID:26530232

  15. Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness

    PubMed Central

    2012-01-01

    Background The Biopeptide BP100 is a synthetic and strongly cationic α-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. It was selected from a library of synthetic peptides, along with other peptides with activities against relevant bacterial and fungal species. Expression of the BP100 series of peptides in plants is of major interest to establish disease-resistant plants and facilitate molecular farming. Specific challenges were the small length, peptide degradation by plant proteases and toxicity to the host plant. Here we approached the expression of the BP100 peptide series in plants using BP100 as a proof-of-concept. Results Our design considered up to three tandemly arranged BP100 units and peptide accumulation in the endoplasmic reticulum (ER), analyzing five BP100 derivatives. The ER retention sequence did not reduce the antimicrobial activity of chemically synthesized BP100 derivatives, making this strategy possible. Transformation with sequences encoding BP100 derivatives (bp100der) was over ten-fold less efficient than that of the hygromycin phosphotransferase (hptII) transgene. The BP100 direct tandems did not show higher antimicrobial activity than BP100, and genetically modified (GM) plants constitutively expressing them were not viable. In contrast, inverted repeats of BP100, whether or not elongated with a portion of a natural antimicrobial peptide (AMP), had higher antimicrobial activity, and fertile GM rice lines constitutively expressing bp100der were produced. These GM lines had increased resistance to the pathogens Dickeya chrysanthemi and Fusarium verticillioides, and tolerance to oxidative stress, with agronomic performance comparable to untransformed lines. Conclusions Constitutive expression of transgenes encoding short cationic α-helical synthetic peptides can have a strong negative impact on rice fitness. However, GM plants expressing, for

  16. Calcitonin Gene-Related Peptide Enhances Release of Native Brain-Derived Neurotrophic Factor from Trigeminal Ganglion Neurons

    PubMed Central

    Buldyrev, Ilya; Tanner, Nathan M.; Hsieh, Hui-ya; Dodd, Emily G.; Nguyen, Loi T.; Balkowiec, Agnieszka

    2008-01-01

    Activity-dependent plasticity in nociceptive pathways has been implicated in pathomechanisms of chronic pain syndromes. Calcitonin gene-related peptide (CGRP), which is expressed by trigeminal nociceptors, has recently been identified as a key player in the mechanism of migraine headaches. Here we show that CGRP is co-expressed with brain-derived neurotrophic factor (BDNF) in a large subset of adult rat trigeminal ganglion neurons in vivo. Using ELISA in situ, we show that CGRP (1–1000 nM) potently enhances BDNF release from cultured trigeminal neurons. The effect of CGRP is dose–dependent and abolished by pretreatment with CGRP receptor antagonist, CGRP(8–37). Intriguingly, CGRP-mediated BDNF release, unlike BDNF release evoked by physiological patterns of electrical stimulation, is independent of extracellular calcium. Depletion of intracellular calcium stores with thapsigargin blocks the CGRP-mediated BDNF release. Using transmission electron microscopy, our study also shows that BDNF-immunoreactivity is present in dense core vesicles of unmyelinated axons and axon terminals in the subnucleus caudalis of the spinal trigeminal nucleus, the primary central target of trigeminal nociceptors. Together, these results reveal a previously unknown role for CGRP in regulating BDNF availability, and point to BDNF as a candidate mediator of trigeminal nociceptive plasticity. PMID:17064360

  17. Calcitonin gene-related peptide enhances release of native brain-derived neurotrophic factor from trigeminal ganglion neurons.

    PubMed

    Buldyrev, Ilya; Tanner, Nathan M; Hsieh, Hui-ya; Dodd, Emily G; Nguyen, Loi T; Balkowiec, Agnieszka

    2006-12-01

    Activity-dependent plasticity in nociceptive pathways has been implicated in pathomechanisms of chronic pain syndromes. Calcitonin gene-related peptide (CGRP), which is expressed by trigeminal nociceptors, has recently been identified as a key player in the mechanism of migraine headaches. Here we show that CGRP is coexpressed with brain-derived neurotrophic factor (BDNF) in a large subset of adult rat trigeminal ganglion neurons in vivo. Using ELISA in situ, we show that CGRP (1-1000 nM) potently enhances BDNF release from cultured trigeminal neurons. The effect of CGRP is dose-dependent and abolished by pretreatment with CGRP receptor antagonist, CGRP(8-37). Intriguingly, CGRP-mediated BDNF release, unlike BDNF release evoked by physiological patterns of electrical stimulation, is independent of extracellular calcium. Depletion of intracellular calcium stores with thapsigargin blocks the CGRP-mediated BDNF release. Using transmission electron microscopy, our study also shows that BDNF-immunoreactivity is present in dense core vesicles of unmyelinated axons and axon terminals in the subnucleus caudalis of the spinal trigeminal nucleus, the primary central target of trigeminal nociceptors. Together, these results reveal a previously unknown role for CGRP in regulating BDNF availability, and point to BDNF as a candidate mediator of trigeminal nociceptive plasticity. PMID:17064360

  18. Anti-infective efficacy of the lactoferrin-derived antimicrobial peptide HLR1r.

    PubMed

    Björn, Camilla; Mahlapuu, Margit; Mattsby-Baltzer, Inger; Håkansson, Joakim

    2016-07-01

    Antimicrobial peptides (AMPs) have emerged as a new class of drug candidates for the treatment of infectious diseases. Here we describe a novel AMP, HLR1r, which is structurally derived from the human milk protein lactoferrin and demonstrates a broad spectrum microbicidal action in vitro. The minimum concentration of HLR1r needed for killing ≥99% of microorganisms in vitro, was in the range of 3-50μg/ml for common Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and for the yeast Candida albicans, when assessed in diluted brain-heart infusion medium. We found that HLR1r also possesses anti-inflammatory properties as evidenced by inhibition of tumor necrosis factor alpha (TNF-α) secretion from human monocyte-derived macrophages and by repression of interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1) secretion from human mesothelial cells, without any cytotoxic effect observed at the concentration range tested (up to 400μg/ml). HLR1r demonstrated pronounced anti-infectious effect in in vivo experimental models of cutaneous candidiasis in mice and of excision wounds infected with MRSA in rats as well as in an ex vivo model of pig skin infected with S. aureus. In conclusion, HLR1r may constitute a new therapeutic alternative for local treatment of skin infections. PMID:27155369

  19. The FGF-2-Derived Peptide FREG Inhibits Melanoma Growth In Vitro and In Vivo

    PubMed Central

    Aguzzi, Maria S; Faraone, Debora; D'Arcangelo, Daniela; De Marchis, Francesco; Toietta, Gabriele; Ribatti, Domenico; Parazzoli, Alberto; Colombo, Paolo; Capogrossi, Maurizio C; Facchiano, Antonio

    2011-01-01

    Previous data report that fibroblast growth factor-2 (FGF-2)-derived peptide FREG potently inhibits FGF-2-dependent angiogenesis in vitro and in vivo. Here, we show that FREG inhibits up to 70% in vitro growth and invasion/migration of smooth muscle and melanoma cells. Such inhibition is mediated by platelet-derived growth factor-receptor-α (PDGF-Rα); in fact, proliferation and migration were restored upon PDGF-Rα neutralization. Further experiments demonstrated that FREG interacts with PDGF-Rα both in vitro and in vivo and stimulates its phosphorylation. We have previously shown that overexpressing PDGF-Rα strongly inhibits melanoma growth in vivo; we, therefore, hypothesized that PDGF-Rα agonists may represent a novel tool to inhibit melanoma growth in vivo. To support this hypothesis, FREG was inoculated intravenously (i.v.) in a mouse melanoma model and markedly inhibited pulmonary metastases formation. Immunohistochemical analyses showed less proliferation, less angiogenesis, and more apoptosis in metastasized lungs upon FREG treatment, as compared to untreated controls. Finally, in preliminary acute toxicity studies, FREG showed no toxicity signs in healthy animals, and neither microscopic nor macroscopic toxicity at the liver, kidney, and lungs level. Altogether, these data indicate that FREG systemic treatment strongly inhibits melanoma metastases development and indicate for the first time that agonists of PDGF-Rα may control melanoma both in vitro and in vivo. PMID:20924364

  20. Synthetic Amphipathic Helical Peptides Targeting CD36 Attenuate Lipopolysaccharide-Induced Inflammation and Acute Lung Injury.

    PubMed

    Bocharov, Alexander V; Wu, Tinghuai; Baranova, Irina N; Birukova, Anna A; Sviridov, Denis; Vishnyakova, Tatyana G; Remaley, Alan T; Eggerman, Thomas L; Patterson, Amy P; Birukov, Konstantin G

    2016-07-15

    Synthetic amphipathic helical peptides (SAHPs) designed as apolipoprotein A-I mimetics are known to bind to class B scavenger receptors (SR-Bs), SR-BI, SR-BII, and CD36, receptors that mediate lipid transport and facilitate pathogen recognition. In this study, we evaluated SAHPs, selected for targeting human CD36, by their ability to attenuate LPS-induced inflammation, endothelial barrier dysfunction, and acute lung injury (ALI). L37pA, which targets CD36 and SR-BI equally, inhibited LPS-induced IL-8 secretion and barrier dysfunction in cultured endothelial cells while reducing lung neutrophil infiltration by 40% in a mouse model of LPS-induced ALI. A panel of 20 SAHPs was tested in HEK293 cell lines stably transfected with various SR-Bs to identify SAHPs with preferential selectivity toward CD36. Among several SAHPs targeting both SR-BI/BII and CD36 receptors, ELK-B acted predominantly through CD36. Compared with L37pA, 5A, and ELK SAHPs, ELK-B was most effective in reducing the pulmonary barrier dysfunction, neutrophil migration into the lung, and lung inflammation induced by LPS. We conclude that SAHPs with relative selectivity toward CD36 are more potent at inhibiting acute pulmonary inflammation and dysfunction. These data indicate that therapeutic strategies using SAHPs targeting CD36, but not necessarily mimicking all apolipoprotein A-I functions, may be considered a possible new treatment approach for inflammation-induced ALI and pulmonary edema. PMID:27316682

  1. The Synthetic Parasite-Derived Peptide GK1 Increases Survival in a Preclinical Mouse Melanoma Model

    PubMed Central

    Vera-Aguilera, Jesús; Hernaiz-Leonardo, Juan Carlos; Moreno-Aguilera, Eduardo; Monteverde-Suarez, Diego; Vera-Aguilera, Carlos; Estrada-Bárcenas, Daniel

    2013-01-01

    Abstract Purpose The therapeutic efficacy of a synthetic parasite-derived peptide GK1, an immune response booster, was evaluated in a mouse melanoma model. This melanoma model correlates with human stage IIb melanoma, which is treated with wide surgical excision; a parallel study employing a surgical treatment was carried out as an instructive goal. Experimental Design C57BL/6 mice were injected subcutaneously in the flank with 2×105 B16-F10 murine melanoma cells. When the tumors reached 20 mm3, mice were separated into two different groups; the GK1 group, treated weekly with peritumoral injections of GK1 (10 μg/100 μL of sterile saline solution) and the control group, treated weekly with an antiseptic peritumoral injection of 100 μL of sterile saline solution without further intervention. All mice were monitored daily for clinical appearance, tumor size, and survival. Surgical treatment was performed in parallel when the tumor size was 20 mm3 (group A), 500 mm3 (group B), and >500 mm3 (group C). Results The GK1 peptide effectively increased the mean survival time by 9.05 days, corresponding to an increase of 42.58%, and significantly delayed tumor growth from day 3 to 12 of treatment. In addition, tumor necrosis was significantly increased (p<0.05) in the treated mice. The overall survival rates obtained with surgical treatment at 6 months were 83.33% for group A, 40% for group B, and 0% for group C, with significant differences (p<0.05) among the groups. Conclusions The GK1 peptide demonstrated therapeutic properties in a mouse melanoma model, as treatment resulted in a significant increase in the mean survival time of the treated animals (42.58%). The potential for GK1 to be used as a primary or adjuvant component of chemotherapeutic cocktails for the treatment of experimental and human cancers remains to be determined, and surgical removal remains a challenge for any new experimental treatment of melanoma in mouse models. PMID:23841709

  2. Prodynorphin-derived peptides are critical modulators of anxiety and regulate neurochemistry and corticosterone.

    PubMed

    Wittmann, Walter; Schunk, Eduard; Rosskothen, Iris; Gaburro, Stefano; Singewald, Nicolas; Herzog, Herbert; Schwarzer, Christoph

    2009-02-01

    Stress and anxiety are mainly regulated by amygdala and hypothalamic circuitries involving several neurotransmitter systems and providing physiological responses to peripheral organs via the hypothalamic-pituitary-adrenal axis and other pathways. The role of endogenous opioid peptides in this process is largely unknown. Here we show for the first time that anxiolytic parameters of explorative behavior in mice lacking prodynorphin were increased 2-4-fold in the open field, the elevated plus maze and the light-dark test. Consistent with this, treatment of wild-type mice with selective kappa-opioid receptor antagonists GNTI or norbinaltorphimine showed the same effects. Furthermore, treatment of prodynorphin knockout animals with U-50488H, a selective kappa-opioid receptor agonist, fully reversed their anxiolytic phenotype. These behavioral data are supported by an approximal 30% reduction in corticotropin-releasing hormone (CRH) mRNA expression in the hypothalamic paraventricular nucleus and central amygdala and an accompanying 30-40% decrease in corticosterone serum levels in prodynorphin knockout mice. Although stress-induced increases in corticosterone levels were attenuated in prodynorphin knockout mice, they were associated with minor increases in depression-like behavior in the tail suspension and forced swim tests. Taken together, our data suggest a pronounced impact of endogenous prodynorphin-derived peptides on anxiety, but not stress coping ability and that these effects are mediated via kappa-opioid receptors. The delay in the behavioral response to kappa-opioid receptor agonists and antagonist treatment suggests an indirect control level for the action of dynorphin, probably by modulating the expression of CRH or neuropeptide Y, and subsequently influencing behavior. PMID:18800067

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

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

    PubMed

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

    2016-01-01

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

  5. SARS Coronavirus Fusion Peptide-Derived Sequence Suppresses Collagen-Induced Arthritis in DBA/1J Mice.

    PubMed

    Shen, Zu T; Sigalov, Alexander B

    2016-01-01

    During the co-evolution of viruses and their hosts, the viruses have evolved numerous strategies to counter and evade host antiviral immune responses in order to establish a successful infection, replicate and persist in the host. Recently, based on our model of immune signaling, the Signaling Chain HOmoOLigomerization (SCHOOL) model, we suggested specific molecular mechanisms used by different viruses such as severe acute respiratory syndrome coronavirus (SARS-CoV) to modulate the host immune response mediated by members of the family of multichain immune recognition receptors (MIRRs). This family includes T cell receptor (TCR) that is critically involved in immune diseases such as autoimmune arthritis. In the present study, we provide compelling experimental in vivo evidence in support of our hypothesis. Using the SCHOOL approach and the SARS-CoV fusion peptide sequence, we rationally designed a novel immunomodulatory peptide that targets TCR. We showed that this peptide ameliorates collagen-induced arthritis in DBA/1J mice and protects against bone and cartilage damage. Incorporation of the peptide into self-assembling lipopeptide nanoparticles that mimic native human high density lipoproteins significantly increases peptide dosage efficacy. Together, our data further confirm that viral immune evasion strategies that target MIRRs can be transferred to therapeutic strategies that require similar functionalities. PMID:27349522

  6. SARS Coronavirus Fusion Peptide-Derived Sequence Suppresses Collagen-Induced Arthritis in DBA/1J Mice

    PubMed Central

    Shen, Zu T.; Sigalov, Alexander B.

    2016-01-01

    During the co-evolution of viruses and their hosts, the viruses have evolved numerous strategies to counter and evade host antiviral immune responses in order to establish a successful infection, replicate and persist in the host. Recently, based on our model of immune signaling, the Signaling Chain HOmoOLigomerization (SCHOOL) model, we suggested specific molecular mechanisms used by different viruses such as severe acute respiratory syndrome coronavirus (SARS-CoV) to modulate the host immune response mediated by members of the family of multichain immune recognition receptors (MIRRs). This family includes T cell receptor (TCR) that is critically involved in immune diseases such as autoimmune arthritis. In the present study, we provide compelling experimental in vivo evidence in support of our hypothesis. Using the SCHOOL approach and the SARS-CoV fusion peptide sequence, we rationally designed a novel immunomodulatory peptide that targets TCR. We showed that this peptide ameliorates collagen-induced arthritis in DBA/1J mice and protects against bone and cartilage damage. Incorporation of the peptide into self-assembling lipopeptide nanoparticles that mimic native human high density lipoproteins significantly increases peptide dosage efficacy. Together, our data further confirm that viral immune evasion strategies that target MIRRs can be transferred to therapeutic strategies that require similar functionalities. PMID:27349522

  7. Actively-targeted LTVSPWY peptide-modified magnetic nanoparticles for tumor imaging

    PubMed Central

    Jie, Li-Yong; Cai, Li-Li; Wang, Le-Jian; Ying, Xiao-Ying; Yu, Ri-Sheng; Zhang, Min-Ming; Du, Yong-Zhong

    2012-01-01

    Background Magnetic resonance imaging (MRI) is widely used in modern clinical medicine as a diagnostic tool, and provides noninvasive and three-dimensional visualization of biological phenomena in living organisms with high spatial and temporal resolution. Therefore, considerable attention has been paid to magnetic nanoparticles as MRI contrast agents with efficient targeting ability and cellular internalization ability, which make it possible to offer higher contrast and information-rich images for detection of disease. Methods LTVSPWY peptide-modified PEGylated chitosan (LTVSPWY-PEG-CS) was synthesized by chemical reaction, and the chemical structure was confirmed by 1H-NMR. LTVSPWY-PEG-CS-modified magnetic nanoparticles were prepared successfully using the solvent diffusion method. Their particle size, size distribution, and zeta potential were measured by dynamic light scattering and electrophoretic mobility, and their surface morphology was investigated by transmission electron microscopy. To investigate their selective targeting ability, the cellular uptake of the LTVSPWY-PEG-CS-modified magnetic nanoparticles was observed in a cocultured system of SKOV-3 cells which overexpress HER2 and A549 cells which are HER2-negative. The in vitro cytotoxicity of these nanoparticles in SKOV-3 and A549 cells was measured using the MTT method. The SKOV-3-bearing nude mouse model was used to investigate the tumor targeting ability of the magnetic nanoparticles in vivo. Results The average diameter and zeta potential of the LTVSPWY-PEG-CS-modified magnetic nanoparticles was 267.3 ± 23.4 nm and 30.5 ± 7.0 mV, respectively, with a narrow size distribution and spherical morphology. In vitro cytotoxicity tests demonstrated that these magnetic nanoparticles were carriers suitable for use in cancer diagnostics with low toxicity. With modification of the LTVSPWY homing peptide, magnetic nanoparticles could be selectively taken up by SKOV-3 cells overexpressing HER2 when

  8. Topical and Targeted Delivery of siRNAs to Melanoma Cells Using a Fusion Peptide Carrier

    PubMed Central

    Ruan, Renquan; Chen, Ming; Sun, Sijie; Wei, Pengfei; Zou, Lili; Liu, Jing; Gao, Dayong; Wen, Longping; Ding, Weiping

    2016-01-01

    Topical application of siRNAs through the skin is a potentially effective strategy for the treatment of melanoma tumors. In this study, we designed a new and safe fusion peptide carrier SPACE-EGF to improve the skin and cell penetration function of the siRNAs and their targeting ability to B16 cells, such that the apoptosis of B16 cells can be induced. The results show that the carrier is stable and less toxic. The EGF motif does not affect the skin and cell penetration function of the SPACE. Because EGF can strongly bind EGFR, which is overexpressed in cancer cells, the targeting ability of the SPACE-EGF-siRNA complex is increased. In vitro experiments indicate that GAPDH siRNAs conjugated with SPACE-EGF can significantly reduce the GAPDH concentration in B16 cells, and c-Myc siRNAs can cause the gene silencing of c-Myc and thus the apoptosis of cells. In vivo experiments show that the topical application of c-Myc siRNAs delivered by SPACE-EGF through the skin can significantly inhibit the growth of melanoma tumors. This work may provide insight into the development of new transdermal drug carriers to treat a variety of skin disorders. PMID:27374619

  9. Efficient Microscale Basic Reverse Phase Peptide Fractionation for Global and Targeted Proteomics.

    PubMed

    Lee, Hyoung-Joo; Kim, Hye-Jung; Liebler, Daniel C

    2016-07-01

    Analysis of small biological samples would benefit from an efficient microscale fractionation strategy that minimizes sample handling, transfer steps, and accompanying losses. Here we describe a microscale basic reverse phase liquid chromatographic (bRPLC) fractionation method that offers high reproducibility and efficiency for peptide mixtures from small (5-20 μg) samples. We applied our platform to detect differentially expressed proteins from lung tumor cell lines that are sensitive (11-18) and resistant (11-18R) to the tyrosine kinase inhibitor erlotinib. Label-free analyses of 5-20 μg samples yielded identifications of approximately 3,200 to 4,000 proteins with coefficients of variation of 1.9-8.9% in replicate analyses. iTRAQ analyses produced similar protein inventories. Label-free and iTRAQ analyses displayed high concordance in identifications of proteins differentially expressed in 11-18 and 11-18R cells. Micro-bRPLC fractionation of cell proteomes increased sensitivity by an average of 4.5-fold in targeted quantitation using parallel reaction monitoring for three representative receptor tyrosine kinases (EGFR, PDGFRA, and BMX), which are present at low abundance in 11-18 and 11-18R cells. These data illustrate the broad utility of micro-bRPLC fractionation for global and targeted proteomic analyses. Data are available through Proteome eXchange Accession PXD003604. PMID:27255222

  10. Isolation and identification of renal cell carcinoma-derived peptides associated with GP96.

    PubMed

    Li, H-Z; Li, C-W; Li, C-Y; Zhang, B-F; Li, L-T; Li, J-M; Zheng, J-N; Chang, J-W

    2013-08-01

    We determined the possible associated determinants and analyzed whether gp96-_associated antigenic peptides can be found in renal cell carcinoma (RCC). The gp96-peptide complexes were chromatographically purified from resected tumor tissue of RCC patients. SDS-PAGE and Western blot analysis confirmed gp96 using the gp96 monoclonal antibody, and its concentration was measured using BCA. Approximately 20 to 50 μg gp96-peptide complexes was obtained from