WorldWideScience

Sample records for promiscuous peptide substrates

  1. Catalytic promiscuity and heme-dependent redox regulation of H2S synthesis.

    Science.gov (United States)

    Banerjee, Ruma

    2017-04-01

    The view of enzymes as punctilious catalysts has been shifting as examples of their promiscuous behavior increase. However, unlike a number of cases where the physiological relevance of breached substrate specificity is questionable, the very synthesis of H 2 S relies on substrate and reaction promiscuity, which presents the enzymes with a multitude of substrate and reaction choices. The transsulfuration pathway, a major source of H 2 S, is inherently substrate-ambiguous. A heme-regulated switch embedded in the first enzyme in the pathway can help avert the stochastic production of cysteine versus H 2 S and control switching between metabolic tracks to meet cellular needs. This review discusses the dominant role of enzyme promiscuity in pathways that double as sulfur catabolic and H 2 S synthetic tracks. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. A Cell-Signaling Network Temporally Resolves Specific versus Promiscuous Phosphorylation

    Directory of Open Access Journals (Sweden)

    Evgeny Kanshin

    2015-02-01

    Full Text Available If specific and functional kinase- or phosphatase-substrate interactions are optimized for binding compared to promiscuous interactions, then changes in phosphorylation should occur faster on functional versus promiscuous substrates. To test this hypothesis, we designed a high temporal resolution global phosphoproteomics protocol to study the high-osmolarity glycerol (HOG response in the budding yeast Saccharomyces cerevisiae. The method provides accurate, stimulus-specific measurement of phosphoproteome changes, quantitative analysis of phosphodynamics at sub-minute temporal resolution, and detection of more phosphosites. Rates of evolution of dynamic phosphosites were comparable to those of known functional phosphosites and significantly lower than static or longer-time-frame dynamic phosphosites. Kinetic profile analyses indicated that putatively functional kinase- or phosphatase-substrate interactions occur more rapidly, within 60 s, than promiscuous interactions. Finally, we report many changes in phosphorylation of proteins implicated in cytoskeletal and mitotic spindle dynamics that may underlie regulation of cell cycle and morphogenesis.

  3. Computer-assisted prediction of HLA-DR binding and experimental analysis for human promiscuous Th1-cell peptides in the 24 kDa secreted lipoprotein (LppX) of Mycobacterium tuberculosis.

    Science.gov (United States)

    Al-Attiyah, R; Mustafa, A S

    2004-01-01

    The secreted 24 kDa lipoprotein (LppX) is an antigen that is specific for Mycobacterium tuberculosis complex and M. leprae. The present study was carried out to identify the promiscuous T helper 1 (Th1)-cell epitopes of the M. tuberculosis LppX (MT24, Rv2945c) antigen by using 15 overlapping synthetic peptides (25 mers overlapping by 10 residues) covering the sequence of the complete protein. The analysis of Rv2945c sequence for binding to 51 alleles of nine serologically defined HLA-DR molecules, by using a virtual matrix-based prediction program (propred), showed that eight of the 15 peptides of Rv2945c were predicted to bind promiscuously to >/=10 alleles from more than or equal to three serologically defined HLA-DR molecules. The Th1-cell reactivity of all the peptides was assessed in antigen-induced proliferation and interferon-gamma (IFN-gamma)-secretion assays with peripheral blood mononuclear cells (PBMCs) from 37 bacille Calmette-Guérin (BCG)-vaccinated healthy subjects. The results showed that 17 of the 37 donors, which represented an HLA-DR-heterogeneous group, responded to one or more peptides of Rv2945c in the Th1-cell assays. Although each peptide stimulated PBMCs from one or more donors in the above assays, the best positive responses (12/17 (71%) responders) were observed with the peptide p14 (aa 196-220). This suggested a highly promiscuous presentation of p14 to Th1 cells. In addition, the sequence of p14 is completely identical among the LppX of M. tuberculosis, M. bovis and M. leprae, which further supports the usefulness of Rv2945c and p14 in the subunit vaccine design against both tuberculosis and leprosy.

  4. Harnessing natural product assembly lines: structure, promiscuity, and engineering.

    Science.gov (United States)

    Ladner, Christopher C; Williams, Gavin J

    2016-03-01

    Many therapeutically relevant natural products are biosynthesized by the action of giant mega-enzyme assembly lines. By leveraging the specificity, promiscuity, and modularity of assembly lines, a variety of strategies has been developed that enables the biosynthesis of modified natural products. This review briefly summarizes recent structural advances related to natural product assembly lines, discusses chemical approaches to probing assembly line structures in the absence of traditional biophysical data, and surveys efforts that harness the inherent or engineered promiscuity of assembly lines for the synthesis of non-natural polyketides and non-ribosomal peptide analogues.

  5. A Novel Semi-biosynthetic Route for Artemisinin Production Using Engineered Substrate-Promiscuous P450BM3

    Energy Technology Data Exchange (ETDEWEB)

    Dietrich, Jeffrey; Yoshikuni, Yasuo; Fisher, Karl; Woolard, Frank; Ockey, Denise; McPhee, Derek; Renninger, Neil; Chang, Michelle; Baker, David; Keasling, Jay

    2009-11-30

    Production of fine heterologus pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved and enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450BM3 from Bacillus megaterium. Using a computer model, we illustrate how key P450BM3 activ site mutations enable binding of non-native substrate amorphadiene, incorporating these mutations into P450BM3 enabled the selective oxidation of amorphadiene arteminsinic-11s,12-epoxide, at titers of 250 mg L"1 in E. coli. We also demonstrate high-yeilding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high value anti-malarial drug artemisinin.

  6. Substrate interactions and promiscuity in a viral DNA packaging motor.

    Science.gov (United States)

    Aathavan, K; Politzer, Adam T; Kaplan, Ariel; Moffitt, Jeffrey R; Chemla, Yann R; Grimes, Shelley; Jardine, Paul J; Anderson, Dwight L; Bustamante, Carlos

    2009-10-01

    The ASCE (additional strand, conserved E) superfamily of proteins consists of structurally similar ATPases associated with diverse cellular activities involving metabolism and transport of proteins and nucleic acids in all forms of life. A subset of these enzymes consists of multimeric ringed pumps responsible for DNA transport in processes including genome packaging in adenoviruses, herpesviruses, poxviruses and tailed bacteriophages. Although their mechanism of mechanochemical conversion is beginning to be understood, little is known about how these motors engage their nucleic acid substrates. Questions remain as to whether the motors contact a single DNA element, such as a phosphate or a base, or whether contacts are distributed over several parts of the DNA. Furthermore, the role of these contacts in the mechanochemical cycle is unknown. Here we use the genome packaging motor of the Bacillus subtilis bacteriophage varphi29 (ref. 4) to address these questions. The full mechanochemical cycle of the motor, in which the ATPase is a pentameric-ring of gene product 16 (gp16), involves two phases-an ATP-loading dwell followed by a translocation burst of four 2.5-base-pair (bp) steps triggered by hydrolysis product release. By challenging the motor with a variety of modified DNA substrates, we show that during the dwell phase important contacts are made with adjacent phosphates every 10-bp on the 5'-3' strand in the direction of packaging. As well as providing stable, long-lived contacts, these phosphate interactions also regulate the chemical cycle. In contrast, during the burst phase, we find that DNA translocation is driven against large forces by extensive contacts, some of which are not specific to the chemical moieties of DNA. Such promiscuous, nonspecific contacts may reflect common translocase-substrate interactions for both the nucleic acid and protein translocases of the ASCE superfamily.

  7. Promiscuous survivin peptide induces robust CD4+ T-cell responses in the majority of vaccinated cancer patients.

    Science.gov (United States)

    Widenmeyer, Melanie; Griesemann, Heinrich; Stevanović, Stefan; Feyerabend, Susan; Klein, Reinhild; Attig, Sebastian; Hennenlotter, Jörg; Wernet, Dorothee; Kuprash, Dmitri V; Sazykin, Alexei Y; Pascolo, Steve; Stenzl, Arnulf; Gouttefangeas, Cécile; Rammensee, Hans-Georg

    2012-07-01

    CD4(+) T cells have been shown to be crucial for the induction and maintenance of cytotoxic T cell responses and to be also capable of mediating direct tumor rejection. Therefore, the anticancer therapeutic efficacy of peptide-based vaccines may be improved by addition of HLA class II epitopes to stimulate T helper cells. Survivin is an apoptosis inhibiting protein frequently overexpressed in tumors. Here we describe the first immunological evaluation of a survivin-derived CD4(+) T cell epitope in a multipeptide immunotherapy trial for prostate carcinoma patients. The survivin peptide is promiscuously presented by several human HLA-DRB1 molecules and, most importantly, is naturally processed by dendritic cells. In vaccinated patients, it was able to induce frequent, robust and multifunctional CD4(+) T cell responses, as monitored by IFN-γ ELISPOT and intracellular cytokine staining. Thus, this HLA-DR restricted epitope is broadly immunogenic and should be valuable for stimulating T helper cells in patients suffering from a wide range of tumors. Copyright © 2011 UICC.

  8. Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.

    Directory of Open Access Journals (Sweden)

    Yu Zhang

    Full Text Available Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL from Geobacillus kaustophilus HTA426 (GkaP exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a "hot spot" in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

  9. Improved surface bioactivity of stainless steel substrates using osteocalcin mimetic peptide

    International Nuclear Information System (INIS)

    Hosseini, Samaneh; Naderi-Manesh, Hossein; Vali, Hojatollah; Faghihi, Shahab

    2014-01-01

    Although stainless steel has a good biocompatibility for most clinical cases, the higher tissue response (bone bonding property) is required in orthopedic field. In this study, to improve bone-bonding ability of stainless steel substrates, a specific sequence of osteocalcin mimetic peptide is used as bioactive coating material to biochemically modify the surface of metallic samples. This sequence consists of thirteen amino acids present in the first helix of osteocalcin is synthesized in amidic form and physically adsorbed on the surface of 316LS (316 low carbon surgical grade) stainless steel substrates. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used to characterize the surface of peptide coated and uncoated substrates. The bioactivity and bone bonding ability of coated and uncoated substrates are assessed by level of hydroxyapatite formation, using transmission electron microscopy (TEM), energy-dispersive x-ray (EDS), and scanning electron microscopy (SEM). The pre-osteoblast cell attachment and proliferation are also evaluated by MTT assay. The results show that the surface of coated sample is homogenously covered by the peptide and display a rougher surface relative to uncoated sample. TEM images reveal the formation of plate-like hydroxyapatite crystals in the presence of the peptide and an amorphous calcium phosphate phase without the peptide. Pre-osteoblast cells proliferation is significantly higher on the surface of peptide coated substrate, while cell attachment remains unaffected by the peptide coatings. Pre-osteoblast cells also demonstrate a higher degree of spreading on the surface of coated sample. It is believed that osteocalcin mimetic peptide improve surface bioactivity and promote hydroxyapatite crystal formation may lead to increased mineralization and bone formation on the surface of metallic biomedical devices. - Graphical abstract: A peptide sequence located in the first helix of OC is selected based on its

  10. Improved surface bioactivity of stainless steel substrates using osteocalcin mimetic peptide

    Energy Technology Data Exchange (ETDEWEB)

    Hosseini, Samaneh [Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Tissue Engineering and Biomaterials Division, National Institute of Genetic Engineering and Biotechnology, Tehran 14965/161 (Iran, Islamic Republic of); Naderi-Manesh, Hossein, E-mail: naderman@modares.ac.ir [Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Vali, Hojatollah [Department of Anatomy and Cell Biology, McGill University, 3640 University Street, Montréal, QC H3A 0C7 (Canada); Faghihi, Shahab, E-mail: sfaghihi@nigeb.ac.ir [Tissue Engineering and Biomaterials Division, National Institute of Genetic Engineering and Biotechnology, Tehran 14965/161 (Iran, Islamic Republic of)

    2014-02-14

    Although stainless steel has a good biocompatibility for most clinical cases, the higher tissue response (bone bonding property) is required in orthopedic field. In this study, to improve bone-bonding ability of stainless steel substrates, a specific sequence of osteocalcin mimetic peptide is used as bioactive coating material to biochemically modify the surface of metallic samples. This sequence consists of thirteen amino acids present in the first helix of osteocalcin is synthesized in amidic form and physically adsorbed on the surface of 316LS (316 low carbon surgical grade) stainless steel substrates. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used to characterize the surface of peptide coated and uncoated substrates. The bioactivity and bone bonding ability of coated and uncoated substrates are assessed by level of hydroxyapatite formation, using transmission electron microscopy (TEM), energy-dispersive x-ray (EDS), and scanning electron microscopy (SEM). The pre-osteoblast cell attachment and proliferation are also evaluated by MTT assay. The results show that the surface of coated sample is homogenously covered by the peptide and display a rougher surface relative to uncoated sample. TEM images reveal the formation of plate-like hydroxyapatite crystals in the presence of the peptide and an amorphous calcium phosphate phase without the peptide. Pre-osteoblast cells proliferation is significantly higher on the surface of peptide coated substrate, while cell attachment remains unaffected by the peptide coatings. Pre-osteoblast cells also demonstrate a higher degree of spreading on the surface of coated sample. It is believed that osteocalcin mimetic peptide improve surface bioactivity and promote hydroxyapatite crystal formation may lead to increased mineralization and bone formation on the surface of metallic biomedical devices. - Graphical abstract: A peptide sequence located in the first helix of OC is selected based on its

  11. Peptide substrates for Rho-associated kinase 2 (Rho-kinase 2/ROCK2.

    Directory of Open Access Journals (Sweden)

    Jeong-Hun Kang

    Full Text Available Peptide substrates sensitive for a certain protein kinase could be important for new-drug development and to understand the mechanism of diseases. Rho-associated kinase (Rho-kinase/ROCK is a serine/threonine kinase, and plays an important part in cardiovascular disease, migration and invasion of tumor cells, and in neurological disorders. The purpose of this study was to find substrates with high affinity and sensitivity for ROCK2. We synthesized 136 peptide substrates from protein substrates for ROCK2 with different lengths and charged peptides. Incorporation of (32P [counts per minute (CPM] for each peptide substrate was determined by the radiolabel assay using [γ-(32P]ATP. When the top five peptide substrates showing high CPMs (R4, R22, R133, R134, and R135 were phosphorylated by other enzymes (PKA, PKCα, and ERK1, R22, R133, and R135 displayed the highest CPM level for ROCK2 compared with other enzymes, whereas R4 and R134 showed similar CPM levels for ROCK2 and PKCα. We hypothesize that R22, R133, and R135 can be useful peptide substrates for ROCK2.

  12. Predicted MHC peptide binding promiscuity explains MHC class I 'hotspots' of antigen presentation defined by mass spectrometry eluted ligand data.

    Science.gov (United States)

    Jappe, Emma Christine; Kringelum, Jens; Trolle, Thomas; Nielsen, Morten

    2018-02-15

    Peptides that bind to and are presented by MHC class I and class II molecules collectively make up the immunopeptidome. In the context of vaccine development, an understanding of the immunopeptidome is essential, and much effort has been dedicated to its accurate and cost-effective identification. Current state-of-the-art methods mainly comprise in silico tools for predicting MHC binding, which is strongly correlated with peptide immunogenicity. However, only a small proportion of the peptides that bind to MHC molecules are, in fact, immunogenic, and substantial work has been dedicated to uncovering additional determinants of peptide immunogenicity. In this context, and in light of recent advancements in mass spectrometry (MS), the existence of immunological hotspots has been given new life, inciting the hypothesis that hotspots are associated with MHC class I peptide immunogenicity. We here introduce a precise terminology for defining these hotspots and carry out a systematic analysis of MS and in silico predicted hotspots. We find that hotspots defined from MS data are largely captured by peptide binding predictions, enabling their replication in silico. This leads us to conclude that hotspots, to a great degree, are simply a result of promiscuous HLA binding, which disproves the hypothesis that the identification of hotspots provides novel information in the context of immunogenic peptide prediction. Furthermore, our analyses demonstrate that the signal of ligand processing, although present in the MS data, has very low predictive power to discriminate between MS and in silico defined hotspots. © 2018 John Wiley & Sons Ltd.

  13. Formation and electrochemical investigation of ordered cobalt coordinated peptide monolayers on gold substrates

    International Nuclear Information System (INIS)

    Wang Xinxin; Nagata, Kenji; Higuchi, Masahiro

    2012-01-01

    The monolayers composed of cobalt coordinated peptides were prepared on gold substrates by two different approaches. One was the self-assembly method, which was used to prepare a peptide monolayer on the gold substrate via the spontaneous attachment of peptides owing to the interaction between gold and sulfur at the N-terminal of the peptide. The other one was the stepwise polymerization method that was utilized to fabricate the unidirectionally arranged peptide monolayer by the stepwise condensation of amino acids from the initiator fixed on the gold substrate. Leu 2 Ala(4-Pyri)Leu 6 Ala(4-Pyri)Leu 6 sequence was chosen as the cobalt coordinated peptide. The 4-pyridyl alanines, Ala(4-Pyri)s, were introduced as ligands for cobalt to the leucine-rich sequential peptide. The complexation between cobalt and pyridyl groups of the peptide induced the formation of a stable α-helical bundle, which oriented perpendicularly to the substrate surface. In the case of the monolayer fabricated by the stepwise polymerization method, the direction of the peptide macro-dipole moment aligned unidirectionally, and the cobalt complexes were fixed in the monolayer to form the ordered arrangement. On the other hand, the peptides prepared by the self-assembly method formed the mixture of parallel and antiparallel packing owing to the dipole-dipole interaction. The spatial location of the cobalt complexes in the monolayer prepared by the self-assembly method was distorted, compared with that in the monolayer fabricated by the stepwise polymerization method. The vectorial electron flow through the peptide monolayer was achieved by the regular alignment of the peptide macro-dipole moment and the cobalt complexes in the monolayer fabricated by the stepwise polymerization method. - Highlights: ► We fabricated ordered Co coordinated peptide monolayers on the gold substrates. ► The Co complexes in peptide monolayer formed an ordered arrangement of the peptide. ► The peptide macro

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

    Directory of Open Access Journals (Sweden)

    Linde A Miles

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

  15. Differential active site loop conformations mediate promiscuous activities in the lactonase SsoPox.

    Directory of Open Access Journals (Sweden)

    Julien Hiblot

    Full Text Available Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263 that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability.

  16. A distributive peptide cyclase processes multiple microviridin core peptides within a single polypeptide substrate.

    Science.gov (United States)

    Zhang, Yi; Li, Kunhua; Yang, Guang; McBride, Joshua L; Bruner, Steven D; Ding, Yousong

    2018-05-03

    Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an important family of natural products. Their biosynthesis follows a common scheme in which the leader peptide of a precursor peptide guides the modifications of a single core peptide. Here we describe biochemical studies of the processing of multiple core peptides within a precursor peptide, rare in RiPP biosynthesis. In a cyanobacterial microviridin pathway, an ATP-grasp ligase, AMdnC, installs up to two macrolactones on each of the three core peptides within AMdnA. The enzyme catalysis occurs in a distributive fashion and follows an unstrict N-to-C overall directionality, but a strict order in macrolactonizing each core peptide. Furthermore, AMdnC is catalytically versatile to process unnatural substrates carrying one to four core peptides, and kinetic studies provide insights into its catalytic properties. Collectively, our results reveal a distinct biosynthetic logic of RiPPs, opening up the possibility of modular production via synthetic biology approaches.

  17. Development of a peptide substrate for detection of Sunn pest damage in wheat flour.

    Science.gov (United States)

    Hançerlioğulları, Begüm Zeynep; Köksel, Hamit; Dudak, Fahriye Ceyda

    2018-05-07

    Since the common protease substrates did not give satisfactory results for the determination of Sunn pest protease activity in damaged wheat, different peptide substrates derived from the repeat sequences of high molecular weight glutenin subunits were synthesized. Hydrolysis of peptides by pest protease was determined by HPLC. Among three peptides having the same consensus motifs, peptide1 (PGQGQQGYYPTSPQQ) showed the best catalytic efficiency. A novel assay was described for monitoring the enzymatic activity of protease extracted from damaged wheat flour. The selected peptide was labeled with a fluorophore (EDANS) and quencher (Dabcyl) to display fluorescence resonance energy transfer (FRET). The proteolytic activity was measured by the change in fluorescence intensity that occurred when the protease cleaved the peptide substrate. Furthermore, the developed assay was modified for rapid and easy detection of bug damage in flour. Flour samples were suspended in water and mixed with fluorescence peptide substrate. After centrifugation, the fluorescence intensities of the supernatants were determined which is proportional with the protease content of the flour. The total analysis time for the developed assay is estimated as 15 minutes. The developed assay permits a significant decrease in time and labor, offering sensitive detection of Sunn pest damage in wheat flour. This article is protected by copyright. All rights reserved.

  18. Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen

    KAUST Repository

    Hohl, Adrian

    2017-12-06

    The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl are used to facilitate the incorporation of non-canonical amino acids (ncAAs) into the genetic code of bacterial and eukaryotic cells by orthogonally reassigning the amber codon. Currently, the incorporation of new ncAAs requires a cumbersome engineering process composed of several positive and negative selection rounds to select the appropriate PylRS/tRNAPyl pair. Our fast and sensitive engineering approach required only a single FACS selection round to identify 110 orthogonal PylRS variants for the aminoacylation of 20 ncAAs. Pocket-substrate relationship from these variants led to the design of a highly promiscuous PylRS (HpRS), which catalyzed the aminoacylation of 31 structurally diverse lysine derivatives bearing clickable, fluorinated, fluorescent, and biotinylated entities. The high speed and sensitivity of our approach provides a competitive alternative to existing screening methodologies, and delivers insights into the complex PylRS-substrate interactions to facilitate the generation of additional promiscuous variants.

  19. Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen

    KAUST Repository

    Hohl, Adrian; Karan, Ram; Akal, Anstassja; Renn, Dominik; Liu, Xuechao; Dharamarajnadar, Alaguraj; Ghoprade, Seema Arun; Groll, Michael; Rueping, Magnus; Eppinger, Jö rg

    2017-01-01

    The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl are used to facilitate the incorporation of non-canonical amino acids (ncAAs) into the genetic code of bacterial and eukaryotic cells by orthogonally reassigning the amber codon. Currently, the incorporation of new ncAAs requires a cumbersome engineering process composed of several positive and negative selection rounds to select the appropriate PylRS/tRNAPyl pair. Our fast and sensitive engineering approach required only a single FACS selection round to identify 110 orthogonal PylRS variants for the aminoacylation of 20 ncAAs. Pocket-substrate relationship from these variants led to the design of a highly promiscuous PylRS (HpRS), which catalyzed the aminoacylation of 31 structurally diverse lysine derivatives bearing clickable, fluorinated, fluorescent, and biotinylated entities. The high speed and sensitivity of our approach provides a competitive alternative to existing screening methodologies, and delivers insights into the complex PylRS-substrate interactions to facilitate the generation of additional promiscuous variants.

  20. Female economic dependence and the morality of promiscuity.

    Science.gov (United States)

    Price, Michael E; Pound, Nicholas; Scott, Isabel M

    2014-10-01

    In environments in which female economic dependence on a male mate is higher, male parental investment is more essential. In such environments, therefore, both sexes should value paternity certainty more and thus object more to promiscuity (because promiscuity undermines paternity certainty). We tested this theory of anti-promiscuity morality in two studies (N = 656 and N = 4,626) using U.S. samples. In both, we examined whether opposition to promiscuity was higher among people who perceived greater female economic dependence in their social network. In Study 2, we also tested whether economic indicators of female economic dependence (e.g., female income, welfare availability) predicted anti-promiscuity morality at the state level. Results from both studies supported the proposed theory. At the individual level, perceived female economic dependence explained significant variance in anti-promiscuity morality, even after controlling for variance explained by age, sex, religiosity, political conservatism, and the anti-promiscuity views of geographical neighbors. At the state level, median female income was strongly negatively related to anti-promiscuity morality and this relationship was fully mediated by perceived female economic dependence. These results were consistent with the view that anti-promiscuity beliefs may function to promote paternity certainty in circumstances where male parental investment is particularly important.

  1. Engineered P450 biocatalysts show improved activity and regio-promiscuity in aromatic nitration.

    Science.gov (United States)

    Zuo, Ran; Zhang, Yi; Jiang, Chao; Hackett, John C; Loria, Rosemary; Bruner, Steven D; Ding, Yousong

    2017-04-12

    Nitroaromatics are among the most important and commonly used chemicals but their production often suffers from multiple unsolved challenges. We have previously described the development of biocatalytic nitration processes driven by an engineered P450 TxtE fusion construct. Herein we report the creation of improved nitration biocatalysts through constructing and characterizing fusion proteins of TxtE with the reductase domain of CYP102A1 (P450BM3, BM3R). The majority of constructs contained variable linker length while one was rationally designed for optimizing protein-protein interactions. Detailed biochemical characterization identified multiple active chimeras that showed improved nitration activity, increased coupling efficiency and higher total turnover numbers compared with TxtE. Substrate promiscuity of the most active chimera was further assessed with a substrate library. Finally, a biocatalytic nitration process was developed to nitrate 4-Me-DL-Trp. The production of both 4-Me-5-NO 2 -L-Trp and 4-Me-7-NO 2 -L-Trp uncovered remarkable regio-promiscuity of nitration biocatalysts.

  2. Synthetic peptides and ribosomal proteins as substrate for 60S ribosomal protein kinase from yeast cells

    DEFF Research Database (Denmark)

    Grankowski, N; Gasior, E; Issinger, O G

    1993-01-01

    Kinetic studies on the 60S protein kinase were conducted with synthetic peptides and ribosomal proteins as substrate. Peptide RRREEESDDD proved to be the best synthetic substrate for this enzyme. The peptide has a sequence of amino acids which most closely resembles the structure of potential...... phosphorylation sites in natural substrates, i.e., acidic ribosomal proteins. The superiority of certain kinetic parameters for 60S kinase obtained with the native whole 80S ribosomes over those of the isolated fraction of acidic ribosomal proteins indicates that the affinity of 60S kinase to the specific protein...

  3. Insights into the evolution of enzyme substrate promiscuity after the discovery of (βα)₈ isomerase evolutionary intermediates from a diverse metagenome.

    Science.gov (United States)

    Noda-García, Lianet; Juárez-Vázquez, Ana L; Ávila-Arcos, María C; Verduzco-Castro, Ernesto A; Montero-Morán, Gabriela; Gaytán, Paul; Carrillo-Tripp, Mauricio; Barona-Gómez, Francisco

    2015-06-10

    Current sequence-based approaches to identify enzyme functional shifts, such as enzyme promiscuity, have proven to be highly dependent on a priori functional knowledge, hampering our ability to reconstruct evolutionary history behind these mechanisms. Hidden Markov Model (HMM) profiles, broadly used to classify enzyme families, can be useful to distinguish between closely related enzyme families with different specificities. The (βα)8-isomerase HisA/PriA enzyme family, involved in L-histidine (HisA, mono-substrate) biosynthesis in most bacteria and plants, but also in L-tryptophan (HisA/TrpF or PriA, dual-substrate) biosynthesis in most Actinobacteria, has been used as model system to explore evolutionary hypotheses and therefore has a considerable amount of evolutionary, functional and structural knowledge available. We searched for functional evolutionary intermediates between the HisA and PriA enzyme families in order to understand the functional divergence between these families. We constructed a HMM profile that correctly classifies sequences of unknown function into the HisA and PriA enzyme sub-families. Using this HMM profile, we mined a large metagenome to identify plausible evolutionary intermediate sequences between HisA and PriA. These sequences were used to perform phylogenetic reconstructions and to identify functionally conserved amino acids. Biochemical characterization of one selected enzyme (CAM1) with a mutation within the functionally essential N-terminus phosphate-binding site, namely, an alanine instead of a glycine in HisA or a serine in PriA, showed that this evolutionary intermediate has dual-substrate specificity. Moreover, site-directed mutagenesis of this alanine residue, either backwards into a glycine or forward into a serine, revealed the robustness of this enzyme. None of these mutations, presumably upon functionally essential amino acids, significantly abolished its enzyme activities. A truncated version of this enzyme (CAM2

  4. Proteolytic Activity of Prostate-Specific Antigen (PSA) towards Protein Substrates and Effect of Peptides Stimulating PSA Activity

    Science.gov (United States)

    Mattsson, Johanna M.; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu

    2014-01-01

    Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA. PMID:25237904

  5. Proteolytic activity of prostate-specific antigen (PSA towards protein substrates and effect of peptides stimulating PSA activity.

    Directory of Open Access Journals (Sweden)

    Johanna M Mattsson

    Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  6. Efficient, crosswise catalytic promiscuity among enzymes that catalyze phosphoryl transfer.

    Science.gov (United States)

    Mohamed, Mark F; Hollfelder, Florian

    2013-01-01

    The observation that one enzyme can accelerate several chemically distinct reactions was at one time surprising because the enormous efficiency of catalysis was often seen as inextricably linked to specialization for one reaction. Originally underreported, and considered a quirk rather than a fundamental property, enzyme promiscuity is now understood to be important as a springboard for adaptive evolution. Owing to the large number of promiscuous enzymes that have been identified over the last decade, and the increased appreciation for promiscuity's evolutionary importance, the focus of research has shifted to developing a better understanding of the mechanistic basis for promiscuity and the origins of tolerant or restrictive specificity. We review the evidence for widespread crosswise promiscuity amongst enzymes that catalyze phosphoryl transfer, including several members of the alkaline phosphatase superfamily, where large rate accelerations between 10(6) and 10(17) are observed for both native and multiple promiscuous reactions. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Sexual promiscuity: knowledge of dangers in institutions of higher learning.

    Science.gov (United States)

    Ebong, R D

    1994-06-01

    Knowledge of dangers of sexual promiscuity was assessed in 2 institutions of higher learning. The objectives were to find out the knowledge of medical and social consequences as well as the factors responsible for sexual promiscuity among Nigerian youths. The study also assessed the discrepancies in societal concept of sex norms for males and females. The result was used as an index to determine the need for sex education for Nigerian youths. A total of 200 students (100 from each school) was assessed by random selection and use of a questionnaire. The result showed that students had a fair knowledge of sexual promiscuity, although in terms of medical consequences the knowledge was low for both groups. On social consequences, the knowledge was fair for both groups. Students agreed that lack of financial support and of supervision from parents and teachers were among the causes of sexual promiscuity. Recommendations were made for Health Education in these areas in institutions of higher learning. Also, recommendations were made for parental education on how to bring up, and care for, their adolescents to reduce the problems of sexual promiscuity. It was also recommended that a compulsory course on sexual promiscuity should be included in the syllabus in institutions of higher learning.

  8. Prediction of binding modes between protein L-isoaspartyl (D-aspartyl) O-methyltransferase and peptide substrates including isomerized aspartic acid residues using in silico analytic methods for the substrate screening.

    Science.gov (United States)

    Oda, Akifumi; Noji, Ikuhiko; Fukuyoshi, Shuichi; Takahashi, Ohgi

    2015-12-10

    Because the aspartic acid (Asp) residues in proteins are occasionally isomerized in the human body, not only l-α-Asp but also l-β-Asp, D-α-Asp and D-β-Asp are found in human proteins. In these isomerized aspartic acids, the proportion of D-β-Asp is the largest and the proportions of l-β-Asp and D-α-Asp found in human proteins are comparatively small. To explain the proportions of aspartic acid isomers, the possibility of an enzyme able to repair l-β-Asp and D-α-Asp is frequently considered. The protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT) is considered one of the possible repair enzymes for l-β-Asp and D-α-Asp. Human PIMT is an enzyme that recognizes both l-β-Asp and D-α-Asp, and catalyzes the methylation of their side chains. In this study, the binding modes between PIMT and peptide substrates containing l-β-Asp or D-α-Asp residues were investigated using computational protein-ligand docking and molecular dynamics simulations. The results indicate that carboxyl groups of both l-β-Asp and D-α-Asp were recognized in similar modes by PIMT and that the C-terminal regions of substrate peptides were located in similar positions on PIMT for both the l-β-Asp and D-α-Asp peptides. In contrast, for peptides containing l-α-Asp or D-β-Asp residues, which are not substrates of PIMT, the computationally constructed binding modes between PIMT and peptides greatly differed from those between PIMT and substrates. In the nonsubstrate peptides, not inter- but intra-molecular hydrogen bonds were observed, and the conformations of peptides were more rigid than those of substrates. Thus, the in silico analytical methods were able to distinguish substrates from nonsubstrates and the computational methods are expected to complement experimental analytical methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. The preferred substrates for transglutaminase 2 in a complex wheat gluten digest are Peptide fragments harboring celiac disease T-cell epitopes.

    Directory of Open Access Journals (Sweden)

    Siri Dørum

    Full Text Available BACKGROUND: Celiac disease is a T-cell mediated chronic inflammatory disorder of the gut that is induced by dietary exposure to gluten proteins. CD4+ T cells of the intestinal lesion recognize gluten peptides in the context of HLA-DQ2.5 or HLA-DQ8 and the gluten derived peptides become better T-cell antigens after deamidation catalyzed by the enzyme transglutaminase 2 (TG2. In this study we aimed to identify the preferred peptide substrates of TG2 in a heterogeneous proteolytic digest of whole wheat gluten. METHODS: A method was established to enrich for preferred TG2 substrates in a complex gluten peptide mixture by tagging with 5-biotinamido-pentylamine. Tagged peptides were isolated and then identified by nano-liquid chromatography online-coupled to tandem mass spectrometry, database searching and final manual data validation. RESULTS: We identified 31 different peptides as preferred substrates of TG2. Strikingly, the majority of these peptides were harboring known gluten T-cell epitopes. Five TG2 peptide substrates that were predicted to bind to HLA-DQ2.5 did not contain previously characterized sequences of T-cell epitopes. Two of these peptides elicited T-cell responses when tested for recognition by intestinal T-cell lines of celiac disease patients, and thus they contain novel candidate T-cell epitopes. We also found that the intact 9mer core sequences of the respective epitopes were not present in all peptide substrates. Interestingly, those epitopes that were represented by intact forms were frequently recognized by T cells in celiac disease patients, whereas those that were present in truncated versions were infrequently recognized. CONCLUSION: TG2 as well as gastrointestinal proteolysis play important roles in the selection of gluten T-cell epitopes in celiac disease.

  10. The Preferred Substrates for Transglutaminase 2 in a Complex Wheat Gluten Digest Are Peptide Fragments Harboring Celiac Disease T-Cell Epitopes

    Science.gov (United States)

    Dørum, Siri; Arntzen, Magnus Ø.; Qiao, Shuo-Wang; Holm, Anders; Koehler, Christian J.; Thiede, Bernd; Sollid, Ludvig M.; Fleckenstein, Burkhard

    2010-01-01

    Background Celiac disease is a T-cell mediated chronic inflammatory disorder of the gut that is induced by dietary exposure to gluten proteins. CD4+ T cells of the intestinal lesion recognize gluten peptides in the context of HLA-DQ2.5 or HLA-DQ8 and the gluten derived peptides become better T-cell antigens after deamidation catalyzed by the enzyme transglutaminase 2 (TG2). In this study we aimed to identify the preferred peptide substrates of TG2 in a heterogeneous proteolytic digest of whole wheat gluten. Methods A method was established to enrich for preferred TG2 substrates in a complex gluten peptide mixture by tagging with 5-biotinamido-pentylamine. Tagged peptides were isolated and then identified by nano-liquid chromatography online-coupled to tandem mass spectrometry, database searching and final manual data validation. Results We identified 31 different peptides as preferred substrates of TG2. Strikingly, the majority of these peptides were harboring known gluten T-cell epitopes. Five TG2 peptide substrates that were predicted to bind to HLA-DQ2.5 did not contain previously characterized sequences of T-cell epitopes. Two of these peptides elicited T-cell responses when tested for recognition by intestinal T-cell lines of celiac disease patients, and thus they contain novel candidate T-cell epitopes. We also found that the intact 9mer core sequences of the respective epitopes were not present in all peptide substrates. Interestingly, those epitopes that were represented by intact forms were frequently recognized by T cells in celiac disease patients, whereas those that were present in truncated versions were infrequently recognized. Conclusion TG2 as well as gastrointestinal proteolysis play important roles in the selection of gluten T-cell epitopes in celiac disease. PMID:21124911

  11. Pyranose dehydrogenase ligand promiscuity: a generalized approach to simulate monosaccharide solvation, binding, and product formation.

    Directory of Open Access Journals (Sweden)

    Michael M H Graf

    2014-12-01

    Full Text Available The flavoenzyme pyranose dehydrogenase (PDH from the litter decomposing fungus Agaricus meleagris oxidizes many different carbohydrates occurring during lignin degradation. This promiscuous substrate specificity makes PDH a promising catalyst for bioelectrochemical applications. A generalized approach to simulate all 32 possible aldohexopyranoses in the course of one or a few molecular dynamics (MD simulations is reported. Free energy calculations according to the one-step perturbation (OSP method revealed the solvation free energies (ΔGsolv of all 32 aldohexopyranoses in water, which have not yet been reported in the literature. The free energy difference between β- and α-anomers (ΔGβ-α of all d-stereoisomers in water were compared to experimental values with a good agreement. Moreover, the free-energy differences (ΔG of the 32 stereoisomers bound to PDH in two different poses were calculated from MD simulations. The relative binding free energies (ΔΔGbind were calculated and, where available, compared to experimental values, approximated from Km values. The agreement was very good for one of the poses, in which the sugars are positioned in the active site for oxidation at C1 or C2. Distance analysis between hydrogens of the monosaccharide and the reactive N5-atom of the flavin adenine dinucleotide (FAD revealed that oxidation is possible at HC1 or HC2 for pose A, and at HC3 or HC4 for pose B. Experimentally detected oxidation products could be rationalized for the majority of monosaccharides by combining ΔΔGbind and a reweighted distance analysis. Furthermore, several oxidation products were predicted for sugars that have not yet been tested experimentally, directing further analyses. This study rationalizes the relationship between binding free energies and substrate promiscuity in PDH, providing novel insights for its applicability in bioelectrochemistry. The results suggest that a similar approach could be applied to study

  12. Promiscuity and selectivity of bitter molecules and their receptors.

    Science.gov (United States)

    Di Pizio, Antonella; Niv, Masha Y

    2015-07-15

    Bitter taste is essential for survival, as it protects against consuming poisonous compounds, which are often bitter. Bitter taste perception is mediated by bitter taste receptors (TAS2Rs), a subfamily of G-protein coupled receptors (GPCRs). The number of TAS2R subtypes is species-dependent, and varies from 3 in chicken to 50 in frog. TAS2Rs present an intriguing case for studying promiscuity: some of the receptors are still orphan, or have few known agonists, while others can be activated by numerous, structurally dissimilar compounds. The ligands also vary in the repertoire of TAS2Rs that they activate: some bitter compounds are selective toward a single TAS2R, while others activate multiple TAS2Rs. Selectivity/promiscuity profile of bitter taste receptors and their compounds was explored by a chemoinformatic approach. TAS2R-promiscuous and TAS2R-selective bitter molecules were found to differ in chemical features, such as AlogP, E-state, total charge, number of rings, globularity, and heavy atom count. This allowed the prediction of bitter ligand selectivity toward TAS2Rs. Interestingly, while promiscuous TAS2Rs are activated by both TAS2R-promiscuous and TAS2R-selective compounds, almost all selective TAS2Rs in human are activated by promiscuous compounds, which are recognized by other TAS2Rs anyway. Thus, unique ligands, that may have been the evolutionary driving force for development of selective TAS2Rs, still need to be unraveled. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Limitations to the use of radioactively labelled substrates for studying peptide transport in microorganisms

    International Nuclear Information System (INIS)

    Payne, J.W.; Nisbet, T.M.

    1980-01-01

    The authors wished to investigate the stoicheiometry of energy coupling to peptide transport in whole cells of several organisms, a study that requires accurate measurements of the rate and amount of peptide translocation. They show that using radioactively-labelled substrates can lead to severe miscalculation of these parameters and produce misleading data on the kinetics of uptake. These conclusions are based on comparative studies using the fluorescamine and dansyl procedures. (Auth.)

  14. Expanding the Bioactive Chemical Space of Anthrabenzoxocinones through Engineering the Highly Promiscuous Biosynthetic Modification Steps.

    Science.gov (United States)

    Mei, Xianyi; Yan, Xiaoli; Zhang, Hui; Yu, Mingjia; Shen, Guangqing; Zhou, Linjun; Deng, Zixin; Lei, Chun; Qu, Xudong

    2018-01-19

    Anthrabenzoxocinones (ABXs) including (-)-ABXs and (+)-ABXs are a group of bacterial FabF-specific inhibitors with potent antimicrobial activity of resistant strains. Optimization of their chemical structures is a promising method to develop potent antibiotics. Through biosynthetic investigation, we herein identified and characterized two highly promiscuous enzymes involved in the (-)-ABX structural modification. The promiscuous halogenase and methyltransferase can respectively introduce halogen-modifications into various positions of the ABX scaffolds and methylation to highly diverse substrates. Manipulation of their activity in both of the (-)-ABXs and (+)-ABXs biosyntheses led to the generation of 14 novel ABX analogues of both enantiomers. Bioactivity assessment revealed that a few of the analogues showed significantly improved antimicrobial activity, with the C3-hydroxyl and chlorine substitutions critical for their activity. This study enormously expands the bioactive chemical space of the ABX family and FabF-specific inhibitors. The disclosed broad-selective biosynthetic machineries and structure-activity relationship provide a solid basis for further generation of potent antimicrobial agents.

  15. Evaluation of a FRET-peptide substrate to predict virulence in Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Wendy E Kaman

    Full Text Available Pseudomonas aeruginosa produces a number of proteases that are associated with virulence and disease progression. A substrate able to detect P. aeruginosa-specific proteolytic activity could help to rapidly alert clinicians to the virulence potential of individual P. aeruginosa strains. For this purpose we designed a set of P. aeruginosa-specific fluorogenic substrates, comprising fluorescence resonance energy transfer (FRET-labeled peptides, and evaluated their applicability to P. aeruginosa virulence in a range of clinical isolates. A FRET-peptide comprising three glycines (3xGly was found to be specific for the detection of P. aeruginosa proteases. Further screening of 97 P. aeruginosa clinical isolates showed a wide variation in 3xGly cleavage activity. The absence of 3xGly degradation by a lasI knock out strain indicated that 3xGly cleavage by P. aeruginosa could be quorum sensing (QS-related, a hypothesis strengthened by the observation of a strong correlation between 3xGly cleavage, LasA staphylolytic activity and pyocyanin production. Additionally, isolates able to cleave 3xGly were more susceptible to the QS inhibiting antibiotic azithromycin (AZM. In conclusion, we designed and evaluated a 3xGly substrate possibly useful as a simple tool to predict virulence and AZM susceptibility.

  16. Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase 2.

    Science.gov (United States)

    Keresztessy, Zsolt; Csosz, Eva; Hársfalvi, Jolán; Csomós, Krisztián; Gray, Joe; Lightowlers, Robert N; Lakey, Jeremy H; Balajthy, Zoltán; Fésüs, László

    2006-11-01

    Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross-linking enzyme, which forms isopeptide bonds between protein-linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine-donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine-donor substrate. Twenty-six Gln-containing sequences from the second and third biopanning rounds were susceptible for TG2-mediated incorporation of 5-(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage-selected sequences, and the N-terminal glutamine-rich domain of SWI1/SNF1-related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry-based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q(6), Q(8), and Q(22) are modified by TG2. Kinetic parameters of SnQ1 transamidation (K(M)(app) = 250 microM, k(cat) = 18.3 sec(-1), and k(cat)/K(M)(app) = 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full-length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research.

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

    Science.gov (United States)

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

    2014-09-26

    Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. A hybrid approach for predicting promiscuous MHC class I restricted ...

    Indian Academy of Sciences (India)

    Prakash

    2006-09-15

    Sep 15, 2006 ... with existing MHC binder prediction methods for alleles studied by both ... in locating the promiscuous MHC binding regions from antigen sequence. ... Artificial neural network; MHC class I alleles; promiscuous binders; ... this problem by developing methods for prediction for ... In case equal number of.

  19. A library of fluorescent peptides for exploring the substrate specificities of prolyl isomerases

    NARCIS (Netherlands)

    Zoldak, G.; Aumuller, T.; Lucke, C.; Hritz, J.; Oostenbrink, C.; Fischer, G.; Schmid, F.X.

    2009-01-01

    To fully explore the substrate specificities of prolyl isomerases, we synthesized a library of 20 tetrapeptides that are labeled with a 2-aminobenzoyl (Abz) group at the amino terminus and a p-nitroanilide (pNA) group at the carboxy terminus. In this peptide library of the general formula

  20. Predicting novel substrates for enzymes with minimal experimental effort with active learning.

    Science.gov (United States)

    Pertusi, Dante A; Moura, Matthew E; Jeffryes, James G; Prabhu, Siddhant; Walters Biggs, Bradley; Tyo, Keith E J

    2017-11-01

    Enzymatic substrate promiscuity is more ubiquitous than previously thought, with significant consequences for understanding metabolism and its application to biocatalysis. This realization has given rise to the need for efficient characterization of enzyme promiscuity. Enzyme promiscuity is currently characterized with a limited number of human-selected compounds that may not be representative of the enzyme's versatility. While testing large numbers of compounds may be impractical, computational approaches can exploit existing data to determine the most informative substrates to test next, thereby more thoroughly exploring an enzyme's versatility. To demonstrate this, we used existing studies and tested compounds for four different enzymes, developed support vector machine (SVM) models using these datasets, and selected additional compounds for experiments using an active learning approach. SVMs trained on a chemically diverse set of compounds were discovered to achieve maximum accuracies of ~80% using ~33% fewer compounds than datasets based on all compounds tested in existing studies. Active learning-selected compounds for testing resolved apparent conflicts in the existing training data, while adding diversity to the dataset. The application of these algorithms to wide arrays of metabolic enzymes would result in a library of SVMs that can predict high-probability promiscuous enzymatic reactions and could prove a valuable resource for the design of novel metabolic pathways. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  1. Predicting novel substrates for enzymes with minimal experimental effort with active learning

    Energy Technology Data Exchange (ETDEWEB)

    Pertusi, Dante A.; Moura, Matthew E.; Jeffryes, James G.; Prabhu, Siddhant; Walters Biggs, Bradley; Tyo, Keith E. J.

    2017-11-01

    Enzymatic substrate promiscuity is more ubiquitous than previously thought, with significant consequences for understanding metabolism and its application to biocatalysis. This realization has given rise to the need for efficient characterization of enzyme promiscuity. Enzyme promiscuity is currently characterized with a limited number of human-selected compounds that may not be representative of the enzyme's versatility. While testing large numbers of compounds may be impractical, computational approaches can exploit existing data to determine the most informative substrates to test next, thereby more thoroughly exploring an enzyme's versatility. To demonstrate this, we used existing studies and tested compounds for four different enzymes, developed support vector machine (SVM) models using these datasets, and selected additional compounds for experiments using an active learning approach. SVMs trained on a chemically diverse set of compounds were discovered to achieve maximum accuracies of similar to 80% using similar to 33% fewer compounds than datasets based on all compounds tested in existing studies. Active learning-selected compounds for testing resolved apparent conflicts in the existing training data, while adding diversity to the dataset. The application of these algorithms to wide arrays of metabolic enzymes would result in a library of SVMs that can predict high-probability promiscuous enzymatic reactions and could prove a valuable resource for the design of novel metabolic pathways.

  2. Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures

    Czech Academy of Sciences Publication Activity Database

    Semashko, T. A.; Vorotnikova, E. A.; Sharikova, V. F.; Vinokurov, Konstantin; Smirnova, Y. A.; Dunaevsky, Y. E.; Belozersky, M. A.; Oppert, B.; Elpidina, E. N.; Filippova, I. Y.

    2014-01-01

    Roč. 449, č. 1 (2014), s. 179-187 ISSN 0003-2697 Grant - others:Russian Foundation for Basic Research(RU) 12-04-01562-a; Russian Foundation for Basic Research(RU) 12-03-01057-a; ISTC (RU) 3455 Institutional support: RVO:60077344 Keywords : cysteine peptidases * substrates of peptidases * selective peptide substrates Subject RIV: CE - Biochemistry Impact factor: 2.219, year: 2014 http://www.sciencedirect.com/science/article/pii/S0003269713006180#

  3. Investigation of the substrate specificity of the proton coupled peptide transporter PepTSo from Shewanella oneidensis

    DEFF Research Database (Denmark)

    Prabhala, Bala Krishna; Aduri, Nanda Gowtham; Hald, Helle

    2015-01-01

    a strikingly high sequence identity, can be used to rationalize its mechanism and substrate preference. However, very little is known about the substrate specificity of PepTSo. To elaborate on this, the natural peptide specificity of PepTSo was investigated. Di and tri-peptides were found to be substrates...... for PepTSo in contrast to mono- and tetrapeptides as was indicated by previous competition studies. Interestingly, a negatively charged side chain was better accommodated on the dipeptide N- than the C-terminus position. Inversely, a positive charged side chain appeared to be tolerated better...

  4. Promiscuous behaviour of the bacterial metallohydrolase DapE : an evolutionary and mechanistic perspective

    OpenAIRE

    Uda, Narasimha Rao

    2015-01-01

    Enzyme promiscuity, defined as functional properties other than those for which they are evolved, is considered a key factor in the evolution of new enzyme functions. Many metalloproteins can be alternatively metallated, which may lead to metal-dependent promiscuity. The mechanisms and evolutionary implications of metal-mediated promiscuity appear to be underexplored, especially considering that approximately one-third of structurally characterized proteins are thought to be metalloproteins. ...

  5. Promiscuous mating in the harem-roosting fruit bat, Cynopterus sphinx.

    Science.gov (United States)

    Garg, Kritika M; Chattopadhyay, Balaji; Doss D, Paramanatha Swami; A K, Vinoth Kumar; Kandula, Sripathi; Ramakrishnan, Uma

    2012-08-01

    Observations on mating behaviours and strategies guide our understanding of mating systems and variance in reproductive success. However, the presence of cryptic strategies often results in situations where social mating system is not reflective of genetic mating system. We present such a study of the genetic mating system of a harem-forming bat Cynopterus sphinx where harems may not be true indicators of male reproductive success. This temporal study using data from six seasons on paternity reveals that social harem assemblages do not play a role in the mating system, and variance in male reproductive success is lower than expected assuming polygynous mating. Further, simulations reveal that the genetic mating system is statistically indistinguishable from promiscuity. Our results are in contrast to an earlier study that demonstrated high variance in male reproductive success. Although an outcome of behavioural mating patterns, standardized variance in male reproductive success (I(m)) affects the opportunity for sexual selection. To gain a better understanding of the evolutionary implications of promiscuity for mammals in general, we compared our estimates of I(m) and total opportunity for sexual selection (I(m) /I(f), where I(f) is standardized variance in female reproductive success) with those of other known promiscuous species. We observed a broad range of I(m) /I(f) values across known promiscuous species, indicating our poor understanding of the evolutionary implications of promiscuous mating. © 2012 Blackwell Publishing Ltd.

  6. Endoprotease profiling with double-tagged peptide substrates: a new diagnostic approach in oncology.

    Science.gov (United States)

    Peccerella, Teresa; Lukan, Nadine; Hofheinz, Ralf; Schadendorf, Dirk; Kostrezewa, Markus; Neumaier, Michael; Findeisen, Peter

    2010-02-01

    The measurement of disease-related proteolytic activity in complex biological matrices like serum is of emerging interest to improve the diagnosis of malignant diseases. We developed a mass spectrometry (MS)-based functional proteomic profiling approach that tracks degradation of artificial endoprotease substrates in serum specimens. The synthetic reporter peptides that are cleaved by tumor-associated endopeptidases were systematically optimized with regard to flanking affinity tags, linkers, and stabilizing elements. Serum specimens were incubated with reporter peptides under standardized conditions and the peptides subsequently extracted with affinity chromatography before MS. In a pilot study an optimized reporter peptide with the cleavage motif WKPYDAADL was added to serum specimens from colorectal tumor patients (n = 50) and healthy controls (n = 50). This reporter peptide comprised a known cleavage site for the cysteine-endopeptidase "cancer procoagulant." Serial affinity chromatography using biotin- and 6xHis tags was superior to the single affinity enrichment using only 6xHis tags. Furthermore, protease-resistant stop elements ensured signal accumulation after prolonged incubation. In contrast, signals from reporter peptides without stop elements vanished completely after prolonged incubation owing to their total degradation. Reporter-peptide spiking showed good reproducibility, and the difference in proteolytic activity between serum specimens from cancer patients and controls was highly significant (P < 0.001). The introduction of a few structural key elements (affinity tags, linkers, d-amino acids) into synthetic reporter peptides increases the diagnostic sensitivity for MS-based protease profiling of serum specimens. This new approach might lead to functional MS-based protease profiling for improved disease classification.

  7. Protein interaction networks by proteome peptide scanning.

    Directory of Open Access Journals (Sweden)

    Christiane Landgraf

    2004-01-01

    Full Text Available A substantial proportion of protein interactions relies on small domains binding to short peptides in the partner proteins. Many of these interactions are relatively low affinity and transient, and they impact on signal transduction. However, neither the number of potential interactions mediated by each domain nor the degree of promiscuity at a whole proteome level has been investigated. We have used a combination of phage display and SPOT synthesis to discover all the peptides in the yeast proteome that have the potential to bind to eight SH3 domains. We first identified the peptides that match a relaxed consensus, as deduced from peptides selected by phage display experiments. Next, we synthesized all the matching peptides at high density on a cellulose membrane, and we probed them directly with the SH3 domains. The domains that we have studied were grouped by this approach into five classes with partially overlapping specificity. Within the classes, however, the domains display a high promiscuity and bind to a large number of common targets with comparable affinity. We estimate that the yeast proteome contains as few as six peptides that bind to the Abp1 SH3 domain with a dissociation constant lower than 100 microM, while it contains as many as 50-80 peptides with corresponding affinity for the SH3 domain of Yfr024c. All the targets of the Abp1 SH3 domain, identified by this approach, bind to the native protein in vivo, as shown by coimmunoprecipitation experiments. Finally, we demonstrate that this strategy can be extended to the analysis of the entire human proteome. We have developed an approach, named WISE (whole interactome scanning experiment, that permits rapid and reliable identification of the partners of any peptide recognition module by peptide scanning of a proteome. Since the SPOT synthesis approach is semiquantitative and provides an approximation of the dissociation constants of the several thousands of interactions that are

  8. The effect of polylysine on casein-kinase-2 activity is influenced by both the structure of the protein/peptide substrates and the subunit composition of the enzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Marin, O

    1992-01-01

    , moreover, is variably accounted for by changes in Vmax and/or Km, depending on the structure of the peptide substrate. Maximum stimulation with all protein/peptide substrates tested requires the presence of the beta subunit, since the recombinant alpha subunit is much less responsive than CK2 holoenzyme......The mechanism by which polybasic peptides stimulate the activity of casein kinase 2 (CK2) has been studied by comparing the effect of polylysine on the phosphorylation of a variety of protein and peptide substrates by the native CK2 holoenzyme and by its recombinant catalytic alpha subunit, either...

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

    Science.gov (United States)

    Swedberg, Joakim E; Harris, Jonathan M

    2011-10-04

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

  10. Coding Military Command as a Promiscuous Practice

    DEFF Research Database (Denmark)

    Ashcraft, Karen Lee; Muhr, Sara Louise

    2018-01-01

    by translating the vague promise of queering leadership into a tangible method distinguished by specific habits. The article formulates this analytical practice out of empirical provocations encountered by the authors: namely, a striking mismatch between their experiences in military fields and the dominant......Despite abundant scholarship addressed to gender equity in leadership, much leadership literature remains invested in gender binaries. Metaphors of leadership are especially dependent on gender oppositions, and this article treats the scholarly practice of coding leadership through gendered...... metaphor as a consequential practice of leadership unto itself. Drawing on queer theory, the article develops a mode of analysis, called ‘promiscuous coding’, conducive to disrupting the gender divisions that currently anchor most leadership metaphors. Promiscuous coding can assist leadership scholars...

  11. Identification of a preferred substrate peptide for transglutaminase 3 and detection of in situ activity in skin and hair follicles.

    Science.gov (United States)

    Yamane, Asaka; Fukui, Mina; Sugimura, Yoshiaki; Itoh, Miho; Alea, Mileidys Perez; Thomas, Vincent; El Alaoui, Said; Akiyama, Masashi; Hitomi, Kiyotaka

    2010-09-01

    Transglutaminases (TGases) are a family of enzymes that catalyze cross-linking reactions between proteins. During epidermal differentiation, these enzymatic reactions are essential for formation of the cornified envelope, which consists of cross-linked structural proteins. Two main transglutaminases isoforms, epidermal-type (TGase 3) and keratinocyte-type (TGase 1), are cooperatively involved in this process of differentiating keratinocytes. Information regarding their substrate preference is of great importance to determine the functional role of these isozymes and clarify their possible co-operative action. Thus far, we have identified highly reactive peptide sequences specifically recognized by TGases isozymes such as TGase 1, TGase 2 (tissue-type isozyme) and the blood coagulation isozyme, Factor XIII. In this study, several substrate peptide sequences for human TGase 3 were screened from a phage-displayed peptide library. The preferred substrate sequences for TGase 3 were selected and evaluated as fusion proteins with mutated glutathione S-transferase. From these studies, a highly reactive and isozyme-specific sequence (E51) was identified. Furthermore, this sequence was found to be a prominent substrate in the peptide form and was suitable for detection of in situ TGase 3 activity in the mouse epidermis. TGase 3 enzymatic activity was detected in the layers of differentiating keratinocytes and hair follicles with patterns distinct from those of TGase 1. Our findings provide new information on the specific distribution of TGase 3 and constitute a useful tool to clarify its functional role in the epidermis.

  12. Probing the mutational interplay between primary and promiscuous protein functions: a computational-experimental approach.

    Science.gov (United States)

    Garcia-Seisdedos, Hector; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

    2012-01-01

    Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous

  13. Development of Novel Promiscuous Anti-Chemokine Peptibodies for Treating Autoimmunity and Inflammation

    Directory of Open Access Journals (Sweden)

    Michal Abraham

    2017-11-01

    Full Text Available Chemokines and their receptors play critical roles in the progression of autoimmunity and inflammation. Typically, multiple chemokines are involved in the development of these pathologies. Indeed, targeting single chemokines or chemokine receptors has failed to achieve significant clinical benefits in treating autoimmunity and inflammation. Moreover, the binding of host atypical chemokine receptors to multiple chemokines as well as the binding of chemokine-binding proteins secreted by various pathogens can serve as a strategy for controlling inflammation. In this work, promiscuous chemokine-binding peptides that could bind and inhibit multiple inflammatory chemokines, such as CCL2, CCL5, and CXCL9/10/11, were selected from phage display libraries. These peptides were cloned into human mutated immunoglobulin Fc-protein fusions (peptibodies. The peptibodies BKT120Fc and BKT130Fc inhibited the ability of inflammatory chemokines to induce the adhesion and migration of immune cells. Furthermore, BKT120Fc and BKT130Fc also showed a significant inhibition of disease progression in a variety of animal models for autoimmunity and inflammation. Developing a novel class of antagonists that can control the courses of diseases by selectively blocking multiple chemokines could be a novel way of generating effective therapeutics.

  14. Turning a Substrate Peptide into a Potent Inhibitor for the Histone Methyltransferase SETD8

    Energy Technology Data Exchange (ETDEWEB)

    Judge, Russell A.; Zhu, Haizhong; Upadhyay, Anup K.; Bodelle, Pierre M.; Hutchins, Charles W.; Torrent, Maricel; Marin, Violeta L.; Yu, Wenyu; Vedadi, Masoud; Li, Fengling; Brown, Peter J.; Pappano, William N.; Sun, Chaohong; Petros, Andrew M.

    2016-12-08

    SETD8 is a histone H4–K20 methyltransferase that plays an essential role in the maintenance of genomic integrity during mitosis and in DNA damage repair, making it an intriguing target for cancer research. While some small molecule inhibitors for SETD8 have been reported, the structural binding modes for these inhibitors have not been revealed. Using the complex structure of the substrate peptide bound to SETD8 as a starting point, different natural and unnatural amino acid substitutions were tested, and a potent (Ki 50 nM, IC50 0.33 μM) and selective norleucine containing peptide inhibitor has been obtained.

  15. Construction of a multifunctional coating consisting of phospholipids and endothelial progenitor cell-specific peptides on titanium substrates

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Huiqing; Li, Xiaojing [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Zhao, Yuancong, E-mail: zhaoyc7320@163.com [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Li, Jingan; Chen, Jiang [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Yang, Ping, E-mail: yangping8@263.net [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Maitz, Manfred F. [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Max Bergmann Center of Biomaterials Dresden, Leibniz of Polymer Research Dresden, 01069 Dresden (Germany); Huang, Nan [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China)

    2015-08-30

    Graphical abstract: The phospholipid groups of PMMDP can inhibit platele adhesion, and the EPCs-specific peptide of the PMMDP showed special recognition and capture for EPCs. The catechol groups of PMMDP play a critical role as molecular anchor for balancing the binding between the coating and the substrate. - Highlights: • The uniform coating of PMMDP can be constructed on titanium surface successfully through the catechol groups. • The phospholipid groups of PMMDP can inhibit platele adhesion, fibrinogen denaturation and improve the hydrophilicity of substrate. • The EPCs-specific peptide of the PMMDP showed special recognition and capture for EPCs. - Abstract: A phospholipid/peptide polymer (PMMDP) with phosphorylcholine groups, endothelial progenitor cell (EPC)-specific peptides and catechol groups was anchored onto a titanium (Ti) surface to fabricate a biomimetic multifunctional surface. The PMMDP coating was characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements and atomic force microscopy (AFM), respectively. The amount of PMMDP coating on the Ti surface was quantified by using the quartz crystal microbalance with dissipation (QCM-D). Interactions between blood components and the coated and bare Ti substrates were evaluated by platelet adhesion and activation assays and fibrinogen denaturation test using platelet rich plasma (PRP). The results revealed that the PMMDP-modified surface inhibited fibrinogen denaturation and reduced platelet adhesion and activation. EPC cell culture on the PMMDP-modified surface showed increased adhesion and proliferation of EPCs when compared to the cells cultured on untreated Ti surface. The inhibition of fibrinogen denaturation and platelet adhesion and support of EPCs attachment and proliferation indicated that this coating might be beneficial for future applications in blood-contacting implants, such as vascular stents.

  16. Statistical Profiling of One Promiscuous Protein Binding Site: Illustrated by Urokinase Catalytic Domain.

    Science.gov (United States)

    Cerisier, Natacha; Regad, Leslie; Triki, Dhoha; Petitjean, Michel; Flatters, Delphine; Camproux, Anne-Claude

    2017-10-01

    While recent literature focuses on drug promiscuity, the characterization of promiscuous binding sites (ability to bind several ligands) remains to be explored. Here, we present a proteochemometric modeling approach to analyze diverse ligands and corresponding multiple binding sub-pockets associated with one promiscuous binding site to characterize protein-ligand recognition. We analyze both geometrical and physicochemical profile correspondences. This approach was applied to examine the well-studied druggable urokinase catalytic domain inhibitor binding site, which results in a large number of complex structures bound to various ligands. This approach emphasizes the importance of jointly characterizing pocket and ligand spaces to explore the impact of ligand diversity on sub-pocket properties and to establish their main profile correspondences. This work supports an interest in mining available 3D holo structures associated with a promiscuous binding site to explore its main protein-ligand recognition tendency. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Z linkage of female promiscuity genes in the moth Utetheisa ornatrix: support for the sexy-sperm hypothesis?

    Science.gov (United States)

    Iyengar, Vikram K; Reeve, Hudson K

    2010-05-01

    Female preference genes for large males in the highly promiscuous moth Utetheisa ornatrix (Lepidoptera: Arctiidae) have previously been shown to be mostly Z-linked, in accordance with the hypothesis that ZZ-ZW sex chromosome systems should facilitate Fisherian sexual selection. We determined the heritability of both female and male promiscuity in the highly promiscuous moth U. ornatrix (Lepidoptera: Arctiidae) through parent-offspring and grandparent-offspring regression analyses. Our data show that male promiscuity is not sex-limited and either autosomal or sex-linked whereas female promiscuity is primarily determined by sex-limited, Z-linked genes. These data are consistent with the "sexy-sperm hypothesis," which posits that multiple-mating and sperm competitiveness coevolve through a Fisherian-like process in which female promiscuity is a kind of mate choice in which sperm-competitiveness is the trait favored in males. Such a Fisherian process should also be more potent when female preferences are Z-linked and sex-limited than when autosomal or not limited.

  18. Exploiting Catalytic Promiscuity for Biocatalysis : Carbon-Carbon Bond Formation by a Proline-Based Tautomerase

    NARCIS (Netherlands)

    Miao, Yufeng

    2015-01-01

    Een belangrijk thema bij het ontwikkelen van nieuwe biokatalysatoren is katalytische promiscuïteit, waarbij een enzym alternatieve reacties katalyseert naast de reactie die biologisch relevant is. Promiscue enzymactiviteiten zijn een veelbelovende bron van synthetisch bruikbare katalytische

  19. Experimental evolution under hyper-promiscuity in Drosophila melanogaster.

    Science.gov (United States)

    Perry, Jennifer C; Joag, Richa; Hosken, David J; Wedell, Nina; Radwan, Jacek; Wigby, Stuart

    2016-06-16

    The number of partners that individuals mate with over their lifetime is a defining feature of mating systems, and variation in mate number is thought to be a major driver of sexual evolution. Although previous research has investigated the evolutionary consequences of reductions in the number of mates, we know little about the costs and benefits of increased numbers of mates. Here, we use a genetic manipulation of mating frequency in Drosophila melanogaster to create a novel, highly promiscuous mating system. We generated D. melanogaster populations in which flies were deficient for the sex peptide receptor (SPR) gene - resulting in SPR- females that mated more frequently - and genetically-matched control populations, and allowed them to evolve for 55 generations. At several time-points during this experimental evolution, we assayed behavioural, morphological and transcriptional reproductive phenotypes expected to evolve in response to increased population mating frequencies. We found that males from the high mating frequency SPR- populations evolved decreased ability to inhibit the receptivity of their mates and decreased copulation duration, in line with predictions of decreased per-mating investment with increased sperm competition. Unexpectedly, SPR- population males also evolved weakly increased sex peptide (SP) gene expression. Males from SPR- populations initially (i.e., before experimental evolution) exhibited more frequent courtship and faster time until mating relative to controls, but over evolutionary time these differences diminished or reversed. In response to experimentally increased mating frequency, SPR- males evolved behavioural responses consistent with decreased male post-copulatory investment at each mating and decreased overall pre-copulatory performance. The trend towards increased SP gene expression might plausibly relate to functional differences in the two domains of the SP protein. Our study highlights the utility of genetic

  20. Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.

    Science.gov (United States)

    Wang, Hong; Brautigan, David L

    2006-11-01

    Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

  1. Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases: 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore.

    Science.gov (United States)

    Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

    2007-01-15

    Fluorescence protease assays were investigated with peptide substrates containing a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as a fluorescent amino acid. The special characteristic of the fluorophore Dbo is its exceedingly long fluorescence lifetime (ca. 300 ns in water under air), which allows the use of nanosecond time-resolved fluorescence (Nano-TRF) detection to efficiently suppress shorter-lived background emission. In addition, the natural amino acids tryptophan and tyrosine can be employed as intramolecular fluorescence quenchers, which facilitates substrate design. Fourteen synthetic peptide substrates (composed of 2-19 amino acids) and five enzymes (trypsin, pepsin, carboxypeptidase A, leucine aminopeptidase, and chymotrypsin) were investigated and, in all 28 examined combinations, enzymatic activity was detected by monitoring the increase in steady state fluorescence with time and determining the reaction rates as kcat/Km values, which ranged from 0.2 to 80x10(6) M-1 min-1. The results suggest an excellent compatibility of the very small and hydrophilic fluorescent probe Dbo with solid-phase peptide synthesis and the investigated proteases. For all 14 peptides the fluorescence lifetimes before and after enzymatic cleavage were measured and Nano-TRF measurements were performed in 384-well microplates. The fluorescence lifetimes of the different peptides provide the basis for the rational design of Dbo-based fluorescent substrates for protease assays. Measurements in Nano-TRF mode revealed, in addition to efficient suppression of background fluorescence, an increased differentiation between cleaved and uncleaved substrate. The Dbo-based assays can be adapted for high-throughput screening.

  2. Causes of Sexual Promiscuity Among Female Undergraduate ...

    African Journals Online (AJOL)

    This study investigated causes of sexual promiscuity among female undergraduate students in university of Lagos. The sample comprised 150 female undergraduate students randomly selected from all the five female hostels in University of Lagos. A researcher-constructed questionnaire was administered to test the three ...

  3. Promiscuity, sexual selection, and genetic diversity: a reply to Spurgin.

    Science.gov (United States)

    Lifjeld, Jan T; Gohli, Jostein; Johnsen, Arild

    2013-10-01

    We recently reported a positive association between female promiscuity and genetic diversity across passerine birds, and launched the hypothesis that female promiscuity acts as a balancing selection, pressure maintaining genetic diversity in populations (Gohli et al.2013). Spurgin (2013) questions both our analyses and interpretations. While we agree that the hypothesis needs more comprehensive empirical testing, we find his specific points of criticism unjustified. In a more general perspective, we call for a more explicit recognition of female mating preferences as mechanisms of selection in population genetics theory. © 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.

  4. Highly Promiscuous Oxidases Discovered in the Bovine Rumen Microbiome

    Directory of Open Access Journals (Sweden)

    Lisa Ufarté

    2018-05-01

    Full Text Available The bovine rumen hosts a diverse microbiota, which is highly specialized in the degradation of lignocellulose. Ruminal bacteria, in particular, are well equipped to deconstruct plant cell wall polysaccharides. Nevertheless, their potential role in the breakdown of the lignin network has never been investigated. In this study, we used functional metagenomics to identify bacterial redox enzymes acting on polyaromatic compounds. A new methodology was developed to explore the potential of uncultured microbes to degrade lignin derivatives, namely kraft lignin and lignosulfonate. From a fosmid library covering 0.7 Gb of metagenomic DNA, three hit clones were identified, producing enzymes able to oxidize a wide variety of polyaromatic compounds without the need for the addition of copper, manganese, or mediators. These promiscuous redox enzymes could thus be of potential interest both in plant biomass refining and dye remediation. The enzymes were derived from uncultured Clostridia, and belong to complex gene clusters involving proteins of different functional types, including hemicellulases, which likely work in synergy to produce substrate degradation.

  5. The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules.

    Science.gov (United States)

    Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

    2006-05-01

    Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including adherence and immunomodulation, thus contributing to S. aureus pathogenesis. Eap binding to host macromolecules is unusually promiscuous and includes matrix or matricellular proteins as well as plasma proteins. The structural basis of this promiscuity is poorly understood. Here, we show that in spite of the preferential location of the binding epitopes within triple helical regions in some collagens there is a striking specificity of Eap binding to different collagen types. Collagen I, but not collagen II, is a binding substrate in monomolecular form. However, collagen I is virtually unrecognized by Eap when incorporated into banded fibrils. By contrast, microfibrils containing collagen VI as well as basement membrane-associated networks containing collagen IV, or aggregates containing fibronectin bound Eap as effectively as the monomeric proteins. Therefore, Eap-binding to extracellular matrix ligands is promiscuous at the molecular level but not indiscriminate with respect to supramolecular structures containing the same macromolecules. In addition, Eap bound to banded fibrils after their partial disintegration by matrix-degrading proteinases, including matrix metalloproteinase 1. Therefore, adherence to matrix suprastructures by S. aureus can be supported by inflammatory reactions.

  6. Computational Identification and Characterization of a Promiscuous T-Cell Epitope on the Extracellular Protein 85B of Mycobacterium spp. for Peptide-Based Subunit Vaccine Design

    Directory of Open Access Journals (Sweden)

    Md. Saddam Hossain

    2017-01-01

    Full Text Available Tuberculosis (TB is a reemerging disease that remains as a leading cause of morbidity and mortality in humans. To identify and characterize a T-cell epitope suitable for vaccine design, we have utilized the Vaxign server to assess all antigenic proteins of Mycobacterium spp. recorded to date in the Protegen database. We found that the extracellular protein 85B displayed the most robust antigenicity among the proteins identified. Computational tools for identifying T-cell epitopes predicted an epitope, 181-QQFIYAGSLSALLDP-195, that could bind to at least 13 major histocompatibility complexes, revealing the promiscuous nature of the epitope. Molecular docking simulation demonstrated that the epitope could bind to the binding groove of MHC II and MHC I molecules by several hydrogen bonds. Molecular docking analysis further revealed that the epitope had a distinctive binding pattern to all DRB1 and A and B series of MHC molecules and presented almost no polymorphism in its binding site. Moreover, using “Allele Frequency Database,” we checked the frequency of HLA alleles in the worldwide population and found a higher frequency of both class I and II HLA alleles in individuals living in TB-endemic regions. Our results indicate that the identified peptide might be a universal candidate to produce an efficient epitope-based vaccine for TB.

  7. sexual promiscuity among female undergraduates in tertiary

    African Journals Online (AJOL)

    Elizabeth Egbochuku

    female undergraduates, the causes and effects of such unhealthy behaviour on healthy living. Hence ... larger scale what they started in secondary schools. Again ... 1, 2010. 103 mostly under the influence of “psychotropic drugs” that put them in a ... the respondents on issues relating to sexual promiscuity and its attendant.

  8. Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function

    NARCIS (Netherlands)

    Ingólfsson, Helgi I; Thakur, Pratima; Herold, Karl F; Hobart, E Ashley; Ramsey, Nicole B; Periole, Xavier; de Jong, Djurre H; Zwama, Martijn; Yilmaz, Duygu; Hall, Katherine; Maretzky, Thorsten; Hemmings, Hugh C; Blobel, Carl; Marrink, Siewert J; Kocer, Armagan; Sack, Jon T; Andersen, Olaf S

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous

  9. Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function

    NARCIS (Netherlands)

    Ingólfsson, Helgi I; Thakur, Pratima; Herold, Karl F; Hobart, E Ashley; Ramsey, Nicole B; Periole, Xavier; de Jong, Djurre H; Zwama, Martijn; Yilmaz, Duygu; Hall, Katherine; Maretzky, Thorsten; Hemmings, Hugh C; Blobel, Carl; Marrink, Siewert J; Kocer, Armagan; Sack, Jon T; Andersen, Olaf S

    2014-01-01

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous

  10. Predicting peptides binding to MHC class II molecules using multi-objective evolutionary algorithms

    Directory of Open Access Journals (Sweden)

    Feng Lin

    2007-11-01

    Full Text Available Abstract Background Peptides binding to Major Histocompatibility Complex (MHC class II molecules are crucial for initiation and regulation of immune responses. Predicting peptides that bind to a specific MHC molecule plays an important role in determining potential candidates for vaccines. The binding groove in class II MHC is open at both ends, allowing peptides longer than 9-mer to bind. Finding the consensus motif facilitating the binding of peptides to a MHC class II molecule is difficult because of different lengths of binding peptides and varying location of 9-mer binding core. The level of difficulty increases when the molecule is promiscuous and binds to a large number of low affinity peptides. In this paper, we propose two approaches using multi-objective evolutionary algorithms (MOEA for predicting peptides binding to MHC class II molecules. One uses the information from both binders and non-binders for self-discovery of motifs. The other, in addition, uses information from experimentally determined motifs for guided-discovery of motifs. Results The proposed methods are intended for finding peptides binding to MHC class II I-Ag7 molecule – a promiscuous binder to a large number of low affinity peptides. Cross-validation results across experiments on two motifs derived for I-Ag7 datasets demonstrate better generalization abilities and accuracies of the present method over earlier approaches. Further, the proposed method was validated and compared on two publicly available benchmark datasets: (1 an ensemble of qualitative HLA-DRB1*0401 peptide data obtained from five different sources, and (2 quantitative peptide data obtained for sixteen different alleles comprising of three mouse alleles and thirteen HLA alleles. The proposed method outperformed earlier methods on most datasets, indicating that it is well suited for finding peptides binding to MHC class II molecules. Conclusion We present two MOEA-based algorithms for finding motifs

  11. Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis.

    Science.gov (United States)

    Shin, Byung-Sik; Katoh, Takayuki; Gutierrez, Erik; Kim, Joo-Ran; Suga, Hiroaki; Dever, Thomas E

    2017-08-21

    Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid proline is a poor substrate for protein synthesis. Previous studies have shown that the translation factor eIF5A and its bacterial ortholog EF-P bind in the E site of the ribosome where they contact the peptidyl-tRNA in the P site and play a critical role in promoting the synthesis of polyproline peptides. Using misacylated Pro-tRNAPhe and Phe-tRNAPro, we show that the imino acid proline and not tRNAPro imposes the primary eIF5A requirement for polyproline synthesis. Though most proline analogs require eIF5A for efficient peptide synthesis, azetidine-2-caboxylic acid, a more flexible four-membered ring derivative of proline, shows relaxed eIF5A dependency, indicating that the structural rigidity of proline might contribute to the requirement for eIF5A. Finally, we examine the interplay between eIF5A and polyamines in promoting translation elongation. We show that eIF5A can obviate the polyamine requirement for general translation elongation, and that this activity is independent of the conserved hypusine modification on eIF5A. Thus, we propose that the body of eIF5A functionally substitutes for polyamines to promote general protein synthesis and that the hypusine modification on eIF5A is critically important for poor substrates like proline. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

  12. Catalytic promiscuity of 4-oxalocrotonate tautomerase : discovery and characterization of C-C bond-forming activities

    NARCIS (Netherlands)

    Zandvoort, Ellen

    2012-01-01

    Het verschijnsel katalytische promiscuïteit, waarin een enzym naast zijn natuurlijke activiteit een alternatieve reactie (of reacties) katalyseert, is een krachtig, recent opgekomen concept, dat erg relevant is voor het ontwerpen van nieuwe enzymen. Enzym promiscuïteit is een veelbelovende bron van

  13. Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures.

    Science.gov (United States)

    Semashko, Tatiana A; Vorotnikova, Elena A; Sharikova, Valeriya F; Vinokurov, Konstantin S; Smirnova, Yulia A; Dunaevsky, Yakov E; Belozersky, Mikhail A; Oppert, Brenda; Elpidina, Elena N; Filippova, Irina Y

    2014-03-15

    This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp=pyroglutamyl; Xaa=Phe or Val; and Y=pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes. Published by Elsevier Inc.

  14. Targeting ligand-gated ion channels in neurology and psychiatry: is pharmacological promiscuity an obstacle or an opportunity?

    Science.gov (United States)

    Bianchi, Matt T; Botzolakis, Emmanuel J

    2010-03-02

    The traditional emphasis on developing high specificity pharmaceuticals ("magic bullets") for the treatment of Neurological and Psychiatric disorders is being challenged by emerging pathophysiology concepts that view disease states as abnormal interactions within complex networks of molecular and cellular components. So-called network pharmacology focuses on modifying the behavior of entire systems rather than individual components, a therapeutic strategy that would ideally employ single pharmacological agents capable of interacting with multiple targets ("magic shotguns"). For this approach to be successful, however, a framework for understanding pharmacological "promiscuity"--the ability of individual agents to modulate multiple molecular targets--is needed. Pharmacological promiscuity is more often the rule than the exception for drugs that target the central nervous system (CNS). We hypothesize that promiscuity is an important contributor to clinical efficacy. Modulation patterns of existing therapeutic agents may provide critical templates for future drug discovery in Neurology and Psychiatry. To demonstrate the extent of pharmacological promiscuity and develop a framework for guiding drug screening, we reviewed the ability of 170 therapeutic agents and endogenous molecules to directly modulate neurotransmitter receptors, a class of historically attractive therapeutic targets in Neurology and Psychiatry. The results are summarized in the form of 1) receptor-centric maps that illustrate the degree of promiscuity for GABA-, glycine-, serotonin-, and acetylcholine-gated ion channels, and 2) drug-centric maps that illustrated how characterization of promiscuity can guide drug development. Developing promiscuity maps of approved neuro-pharmaceuticals will provide therapeutic class-based templates against which candidate compounds can be screened. Importantly, compounds previously rejected in traditional screens due to poor specificity could be reconsidered in this

  15. Substrate promiscuity of a rosmarinic acid synthase from lavender (Lavandula angustifolia L.).

    Science.gov (United States)

    Landmann, Christian; Hücherig, Stefanie; Fink, Barbara; Hoffmann, Thomas; Dittlein, Daniela; Coiner, Heather A; Schwab, Wilfried

    2011-08-01

    One of the most common types of modification of secondary metabolites is the acylation of oxygen- and nitrogen-containing substrates to produce esters and amides, respectively. Among the known acyltransferases, the members of the plant BAHD family are capable of acylating a wide variety of substrates. Two full-length acyltransferase cDNAs (LaAT1 and 2) were isolated from lavender flowers (Lavandula angustifolia L.) by reverse transcriptase-PCR using degenerate primers based on BAHD sequences. Recombinant LaAT1 exhibited a broad substrate tolerance accepting (hydroxy)cinnamoyl-CoAs as acyl donors and not only tyramine, tryptamine, phenylethylamine and anthranilic acid but also shikimic acid and 4-hydroxyphenyllactic acid as acceptors. Thus, LaLT1 forms esters and amides like its phylogenetic neighbors. In planta LaAT1 might be involved in the biosynthesis of rosmarinic acid, the ester of caffeic acid and 3,4-dihydroxyphenyllactic acid, a major constituent of lavender flowers. LaAT2 is one of three members of clade VI with unknown function.

  16. Nosology, ontology and promiscuous realism.

    Science.gov (United States)

    Binney, Nicholas

    2015-06-01

    Medics may consider worrying about their metaphysics and ontology to be a waste of time. I will argue here that this is not the case. Promiscuous realism is a metaphysical position which holds that multiple, equally valid, classification schemes should be applied to objects (such as patients) to capture different aspects of their complex and heterogeneous nature. As medics at the bedside may need to capture different aspects of their patients' problems, they may need to use multiple classification schemes (multiple nosologies), and thus consider adopting a different metaphysics to the one commonly in use. © 2014 John Wiley & Sons, Ltd.

  17. Molecular pharmacology of promiscuous seven transmembrane receptors sensing organic nutrients

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Johansen, Lars Dan; Bräuner-Osborne, Hans

    2009-01-01

    drug targets, to treat, for example, type II diabetes by mimicking food intake by potent agonists or positive allosteric modulators. The ligand-receptor interactions of the promiscuous receptors of organic nutrients thus remain an interesting subject of emerging functional importance....... in taste tissue, the gastrointestinal tract, endocrine glands, adipose tissue, and/or kidney. These receptors thus hold the potential to act as sensors of food intake, regulating, for example, release of incretin hormones from the gut, insulin/glucagon from the pancreas, and leptin from adipose tissue....... The promiscuous tendency in ligand recognition of these receptors is in contrast to the typical specific interaction with one physiological agonist seen for most receptors, which challenges the classic "lock-and-key" concept. We here review the molecular mechanisms of nutrient sensing of the calcium...

  18. Directed divergent evolution of a thermostable D-tagatose epimerase towards improved activity for two hexose substrates.

    Science.gov (United States)

    Bosshart, Andreas; Hee, Chee Seng; Bechtold, Matthias; Schirmer, Tilman; Panke, Sven

    2015-03-02

    Functional promiscuity of enzymes can often be harnessed as the starting point for the directed evolution of novel biocatalysts. Here we describe the divergent morphing of an engineered thermostable variant (Var8) of a promiscuous D-tagatose epimerase (DTE) into two efficient catalysts for the C3 epimerization of D-fructose to D-psicose and of L-sorbose to L-tagatose. Iterative single-site randomization and screening of 48 residues in the first and second shells around the substrate-binding site of Var8 yielded the eight-site mutant IDF8 (ninefold improved kcat for the epimerization of D-fructose) and the six-site mutant ILS6 (14-fold improved epimerization of L-sorbose), compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site; this presumably facilitates entry of the polar substrate. The improvement in catalytic activity of variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures can now be used to select additional sites for further directed evolution of the ketohexose epimerase. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Promiscuous 2-aminothiazoles (PrATs): a frequent hitting scaffold.

    Science.gov (United States)

    Devine, Shane M; Mulcair, Mark D; Debono, Cael O; Leung, Eleanor W W; Nissink, J Willem M; Lim, San Sui; Chandrashekaran, Indu R; Vazirani, Mansha; Mohanty, Biswaranjan; Simpson, Jamie S; Baell, Jonathan B; Scammells, Peter J; Norton, Raymond S; Scanlon, Martin J

    2015-02-12

    We have identified a class of molecules, known as 2-aminothiazoles (2-ATs), as frequent-hitting fragments in biophysical binding assays. This was exemplified by 4-phenylthiazol-2-amine being identified as a hit in 14/14 screens against a diverse range of protein targets, suggesting that this scaffold is a poor starting point for fragment-based drug discovery. This prompted us to analyze this scaffold in the context of an academic fragment library used for fragment-based drug discovery (FBDD) and two larger compound libraries used for high-throughput screening (HTS). This analysis revealed that such "promiscuous 2-aminothiazoles" (PrATs) behaved as frequent hitters under both FBDD and HTS settings, although the problem was more pronounced in the fragment-based studies. As 2-ATs are present in known drugs, they cannot necessarily be deemed undesirable, but the combination of their promiscuity and difficulties associated with optimizing them into a lead compound makes them, in our opinion, poor scaffolds for fragment libraries.

  20. Bladder cancer detection using a peptide substrate of the 20S proteasome.

    Science.gov (United States)

    Gruba, Natalia; Wysocka, Magdalena; Brzezińska, Magdalena; Dębowski, Dawid; Sieńczyk, Marcin; Gorodkiewicz, Ewa; Guszcz, Tomasz; Czaplewski, Cezary; Rolka, Krzysztof; Lesner, Adam

    2016-08-01

    The 20S catalytic core of the human 26S proteasome can be secreted from cells, and high levels of extracellular 20S proteasome have been linked to many types of cancers and autoimmune diseases. Several diagnostic approaches have been developed that detect 20S proteasome activity in plasma, but these suffer from problems with efficiency and sensitivity. In this report, we describe the optimization and synthesis of an internally quenched fluorescent substrate of the 20S proteasome, and investigate its use as a potential diagnostic test in bladder cancer. This peptide, 2-aminobenzoic acid (ABZ)-Val-Val-Ser-Tyr-Ala-Met-Gly-Tyr(3-NO2 )-NH2 , is cleaved by the chymotrypsin 20S proteasome subunit and displays an excellent specificity constant value (9.7 × 10(5)  m(-1) ·s(-1) ) and a high kcat (8 s(-1) ). Using this peptide, we identified chymotrypsin-like proteasome activity in the majority of urine samples obtained from patients with bladder cancer, whereas the proteasome activity in urine samples from healthy volunteers was below the detection limit (0.5 pm). These findings were confirmed by an inhibitory study and immunochemistry methods. © 2016 Federation of European Biochemical Societies.

  1. Unattractive, promiscuous and heavy drinkers: perceptions of women with tattoos.

    Science.gov (United States)

    Swami, Viren; Furnham, Adrian

    2007-12-01

    This study examined social and physical perceptions of blonde and brunette women with different degrees of tattooing. Eighty-four female and 76 male undergraduates rated a series of 16 female line drawings that varied in 2 levels of hair colour and 8 levels of tattooing. Ratings were made for physical attractiveness and sexual promiscuity, as well as estimates of the number of alcohol units consumed on a typical night out. Results showed that tattooed women were rated as less physically attractive, more sexually promiscuous and heavier drinkers than untattooed women, with more negative ratings with increasing number of tattoos. There were also weak interactions between body art and hair colour, with blonde women in general rated more negatively than brunettes. Results are discussed in terms of stereotypes about women who have tattoos and the effects of such stereotypes on well-being.

  2. Mechanism of papain-catalyzed synthesis of oligo-tyrosine peptides.

    Science.gov (United States)

    Mitsuhashi, Jun; Nakayama, Tsutomu; Narai-Kanayama, Asako

    2015-01-01

    Di-, tri-, and tetra-tyrosine peptides with angiotensin I-converting enzyme inhibitory activity were synthesized by papain-catalyzed polymerization of L-tyrosine ethyl ester in aqueous media at 30 °C. Varying the reaction pH from 6.0 to 7.5 and the initial concentration of the ester substrate from 25 to 100 mM, the highest yield of oligo-tyrosine peptides (79% on a substrate basis) was produced at pH 6.5 and 75 mM, respectively. In the reaction initiated with 100 mM of the substrate, approx. 50% yield of insoluble, highly polymerized peptides accumulated. At less than 15 mM, the reaction proceeded poorly; however, from 30 mM to 120 mM a dose-dependent increase in the consumption rate of the substrate was observed with a sigmoidal curve. Meanwhile, each of the tri- and tetra-tyrosine peptides, even at approx. 5mM, was consumed effectively by papain but was not elongated to insoluble polymers. For deacylation of the acyl-papain intermediate through which a new peptide bond is made, L-tyrosine ethyl ester, even at 5mM, showed higher nucleophilic activity than di- and tri-tyrosine. These results indicate that the mechanism through which papain polymerizes L-tyrosine ethyl ester is as follows: the first interaction between papain and the ester substrate is a rate-limiting step; oligo-tyrosine peptides produced early in the reaction period are preferentially used as acyl donors, while the initial ester substrate strongly contributes as a nucleophile to the elongation of the peptide product; and the balance between hydrolytic fragmentation and further elongation of oligo-tyrosine peptides is dependent on the surrounding concentration of the ester substrate. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. A highly scalable peptide-based assay system for proteomics.

    Directory of Open Access Journals (Sweden)

    Igor A Kozlov

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

  4. TM0416, a Hyperthermophilic Promiscuous Nonphosphorylated Sugar Isomerase, Catalyzes Various C5 and C6 Epimerization Reactions.

    Science.gov (United States)

    Shin, Sun-Mi; Cao, Thinh-Phat; Choi, Jin Myung; Kim, Seong-Bo; Lee, Sang-Jae; Lee, Sung Haeng; Lee, Dong-Woo

    2017-05-15

    There is currently little information on nonphosphorylated sugar epimerases, which are of potential interest for producing rare sugars. We found a gene (the TM0416 gene) encoding a putative d-tagatose-3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima We overexpressed the TM0416 gene in Escherichia coli and purified the resulting recombinant protein for detailed characterization. Amino acid sequence alignment and a structural similarity search revealed that TM0416 is a putative nonphosphorylated sugar epimerase. The recombinant enzyme exhibited maximal C-3 epimerization of l-ribulose to l-xylulose at ∼80°C and pH 7 in the presence of 1 mM Mn 2+ In addition, this enzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose, with a conversion yield of 20% after 6 h at 80°C. Remarkably, the enzyme catalyzed the isomerization of d-erythrose or d-threose to d-erythrulose significantly, with conversion yields of 71% and 54.5%, respectively, after 6 h at 80°C at pH 7. To further investigate the substrate specificity of TM0416, we determined its crystal structures in complex with divalent metal ions and l-erythrulose at resolutions of 1.5 and 1.6 Å. Detailed inspection of the structural features and biochemical data clearly demonstrated that this metalloenzyme, with a freely accessible substrate-binding site and neighboring hydrophobic residues, exhibits different and promiscuous substrate preferences, compared with its mesophilic counterparts. Therefore, this study suggests that TM0416 can be functionally classified as a novel type of l-ribulose 3-epimerase (R3E) with d-erythrose isomerase activity. IMPORTANCE Rare sugars, which occur naturally in small amounts, have attracted considerable attention in the food and drug industries. However, there is little information on nonphosphorylated sugar epimerases, which might potentially be applied for the production of rare sugars. This study describes the

  5. Tailoring Agility: Promiscuous Pair Story Authoring and Value Calculation

    Science.gov (United States)

    Tendon, Steve

    This chapter describes how a multi-national software organization created a business plan involving business units from eight countries that followed an agile way, after two previously failed attempts with traditional approaches. The case is told by the consultant who initiated implementation of agility into requirements gathering, estimation and planning processes in an international setting. The agile approach was inspired by XP, but then tailored to meet the peculiar requirements. Two innovations were critical. The first innovation was promiscuous pair story authoring, where user stories were written by two people (similarly to pair programming), and the pairing changed very often (as frequently as every 15-20 minutes) to achieve promiscuity and cater for diverse point of views. The second innovation was an economic value evaluation (and not the cost) which was attributed to stories. Continuous recalculation of the financial value of the stories allowed to assess the projects financial return. In this case implementation of agility in the international context allowed the involved team members to reach consensus and unanimity of decisions, vision and purpose.

  6. Kinetics in Signal Transduction Pathways Involving Promiscuous Oligomerizing Receptors Can Be Determined by Receptor Specificity : Apoptosis Induction by TRAIL

    NARCIS (Netherlands)

    Szegezdi, Eva; van der Sloot, Almer M.; Mahalingam, Devalingam; O'Leary, Lynda; Cool, Robbert H.; Munoz, Ines G.; Montoya, Guillermo; Quax, Wim J.; de Jong, Steven; Samali, Afshin; Serrano, Luis

    Here we show by computer modeling that kinetics and outcome of signal transduction in case of hetero-oligomerizing receptors of a promiscuous ligand largely depend on the relative amounts of its receptors. Promiscuous ligands can trigger the formation of nonproductive receptor complexes, which slows

  7. Entering the 'big data' era in medicinal chemistry: molecular promiscuity analysis revisited.

    Science.gov (United States)

    Hu, Ye; Bajorath, Jürgen

    2017-06-01

    The 'big data' concept plays an increasingly important role in many scientific fields. Big data involves more than unprecedentedly large volumes of data that become available. Different criteria characterizing big data must be carefully considered in computational data mining, as we discuss herein focusing on medicinal chemistry. This is a scientific discipline where big data is beginning to emerge and provide new opportunities. For example, the ability of many drugs to specifically interact with multiple targets, termed promiscuity, forms the molecular basis of polypharmacology, a hot topic in drug discovery. Compound promiscuity analysis is an area that is much influenced by big data phenomena. Different results are obtained depending on chosen data selection and confidence criteria, as we also demonstrate.

  8. Much More than Power: The Pedagogy of Promiscuous Black Feminism

    Science.gov (United States)

    Huckaby, M. Francyne

    2013-01-01

    This paper explores promiscuous black feminism by juxtaposing black feminism, Foucualt's poststructuralism, and my grandmother. The tensions created by these juxtapositions illuminate the ways black feminism and poststructuralism are resources and challenges to each other, and how both offer understandings of the relations at play that shape…

  9. High-Throughput Quantification of SH2 Domain-Phosphopeptide Interactions with Cellulose-Peptide Conjugate Microarrays.

    Science.gov (United States)

    Engelmann, Brett W

    2017-01-01

    The Src Homology 2 (SH2) domain family primarily recognizes phosphorylated tyrosine (pY) containing peptide motifs. The relative affinity preferences among competing SH2 domains for phosphopeptide ligands define "specificity space," and underpins many functional pY mediated interactions within signaling networks. The degree of promiscuity exhibited and the dynamic range of affinities supported by individual domains or phosphopeptides is best resolved by a carefully executed and controlled quantitative high-throughput experiment. Here, I describe the fabrication and application of a cellulose-peptide conjugate microarray (CPCMA) platform to the quantitative analysis of SH2 domain specificity space. Included herein are instructions for optimal experimental design with special attention paid to common sources of systematic error, phosphopeptide SPOT synthesis, microarray fabrication, analyte titrations, data capture, and analysis.

  10. A parasitic selfish gene that affects host promiscuity.

    Science.gov (United States)

    Giraldo-Perez, Paulina; Goddard, Matthew R

    2013-11-07

    Selfish genes demonstrate transmission bias and invade sexual populations despite conferring no benefit to their hosts. While the molecular genetics and evolutionary dynamics of selfish genes are reasonably well characterized, their effects on hosts are not. Homing endonuclease genes (HEGs) are one well-studied family of selfish genes that are assumed to be benign. However, we show that carrying HEGs is costly for Saccharomyces cerevisiae, demonstrating that these genetic elements are not necessarily benign but maybe parasitic. We estimate a selective load of approximately 1-2% in 'natural' niches. The second aspect we examine is the ability of HEGs to affect hosts' sexual behaviour. As all selfish genes critically rely on sex for spread, then any selfish gene correlated with increased host sexuality will enjoy a transmission advantage. While classic parasites are known to manipulate host behaviour, we are not aware of any evidence showing a selfish gene is capable of affecting host promiscuity. The data presented here show a selfish element may increase the propensity of its eukaryote host to undergo sex and along with increased rates of non-Mendelian inheritance, this may counterbalance mitotic selective load and promote spread. Demonstration that selfish genes are correlated with increased promiscuity in eukaryotes connects with ideas suggesting that selfish genes promoted the evolution of sex initially.

  11. Genetic monogamy despite social promiscuity in the pot-bellied seahorse (Hippocampus abdominalis).

    Science.gov (United States)

    Wilson, A B; Martin-Smith, K M

    2007-06-01

    Sexual selection theory predicts a positive correlation between relative parental investment and mate choice. In syngnathid fishes (seahorses and pipefish), males brood offspring in specialized brooding structures. While female-female mating competition has been demonstrated in some pipefishes, all seahorses (genus Hippocampus) studied to date have been found to have conventional sex roles with greater male-male competition for access to mates despite possessing the most complex brood structures in the family. Although multiple mating is common in pipefish, seahorses are again exceptional, exhibiting strict genetic monogamy. Both demographic and behavioural explanations have been offered to explain the lack of multiple mating in seahorse species, but these hypotheses have not yet been explicitly addressed. We investigated mating systems and brood parentage of the pot-bellied seahorse, Hippocampus abdominalis, a temperate-water species that is socially promiscuous with conventional sex roles in laboratory populations. We observed promiscuous courtship behaviour and sex-role reversal in high density, female-biased field populations of H. abdominalis. We hypothesize that sex roles are plastic in H. abdominalis, depending on local population density and sex ratio. Despite promiscuous courtship behaviour, all assayed male seahorses were genetically monogamous in both laboratory and wild populations. Physiological limitations associated with embryo incubation may explain the absence of multiple mating in seahorses and may have played an important role in the development of the unique reproductive behaviour typical in these species.

  12. Promiscuity is related to masculine and feminine body traits in both men and women: evidence from Brazilian and Czech samples.

    Science.gov (United States)

    Varella, Marco Antonio Correa; Valentova, Jaroslava Varella; Pereira, Kamila Janaina; Bussab, Vera Silvia Raad

    2014-11-01

    One of the possible explanations for human within-sex variation in promiscuity stems from conditional strategies dependent on the level of body sex-dimorphism. There is some evidence that masculine men and feminine women are more promiscuous than their sex-atypical counterparts, although mixed results persist. Moreover, another line of evidence shows that more promiscuous women are rather sex-atypical. We tested whether diverse sex-dimorphic body measures (2D:4D, WHR/WSR, handgrip strength, and height and weight) influence sociosexual desires, attitudes, promiscuous behavior, and age of first intercourse in a sex-typical or sex-atypical direction. Participants were 185 young adults, 51 men and 54 women from Brazil, and 40 men and 40 women from the Czech Republic. In men stronger handgrip and more feminine 2D:4D predicted higher sociosexual behaviors, desires, and lower age of the first sexual intercourse. While in women, sociosexual desires were predicted by lower handgrip strength and more feminine 2D:4D. It thus seems that it is rather a mixture of masculine and feminine traits in men, and feminine traits in women that increase their sociosexuality. Masculine traits (height) predicting female promiscuous behavior were specific for only one population. In conclusion, a mosaic combination of sex-typical but also sex-atypical independent body traits can lead to higher promiscuity, particularly in men. Limitations, implications, and future directions for research are considered. This article is part of a Special Issue entitled: Neotropical Behaviour. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

    Science.gov (United States)

    Nalbone, Joseph M; Lahankar, Neelam; Buissereth, Lyssa; Raj, Monika

    2016-03-04

    Site-specific hydrolysis of peptide bonds at glutamic acid under neutral aqueous conditions is reported. The method relies on the activation of the backbone amide chain at glutamic acid by the formation of a pyroglutamyl (pGlu) imide moiety. This activation increases the susceptibility of a peptide bond toward hydrolysis. The method is highly specific and demonstrates broad substrate scope including cleavage of various bioactive peptides with unnatural amino acid residues, which are unsuitable substrates for enzymatic hydrolysis.

  14. Carotenoid β-Ring Hydroxylase and Ketolase from Marine Bacteria—Promiscuous Enzymes for Synthesizing Functional Xanthophylls

    Science.gov (United States)

    Misawa, Norihiko

    2011-01-01

    Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C40-type dicyclic carotenoids containing two β-end groups (β rings) that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4′)-ketolase (4(4′)-oxygenase; CrtW) and hydroxylated by carotenoid β-ring 3(3′)-hydroxylase (CrtZ). In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2′)-hydroxylase (CrtG). This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids) in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s)-2(2′)-hydroxylated carotenoids). PMID:21673887

  15. Carotenoid β-Ring Hydroxylase and Ketolase from Marine Bacteria—Promiscuous Enzymes for Synthesizing Functional Xanthophylls

    Directory of Open Access Journals (Sweden)

    Norihiko Misawa

    2011-05-01

    Full Text Available Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C40-type dicyclic carotenoids containing two β-end groups (β rings that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4′-ketolase (4(4′-oxygenase; CrtW and hydroxylated by carotenoid β-ring 3(3′-hydroxylase (CrtZ. In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2′-hydroxylase (CrtG. This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s-2(2′-hydroxylated carotenoids.

  16. Carotenoid β-ring hydroxylase and ketolase from marine bacteria-promiscuous enzymes for synthesizing functional xanthophylls.

    Science.gov (United States)

    Misawa, Norihiko

    2011-01-01

    Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C₄₀-type dicyclic carotenoids containing two β-end groups (β rings) that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4')-ketolase (4(4')-oxygenase; CrtW) and hydroxylated by carotenoid β-ring 3(3')-hydroxylase (CrtZ). In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2')-hydroxylase (CrtG). This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids) in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s)-2(2')-hydroxylated carotenoids).

  17. The v-Src and c-Src tyrosine kinases immunoprecipitated from Rous sarcoma virus-transformed cells display different peptide substrate specificities

    Czech Academy of Sciences Publication Activity Database

    Vojtěchová, Martina; Tuháčková, Zdena; Hlaváček, Jan; Velek, Jiří; Sovová, Vlasta

    2004-01-01

    Roč. 421, č. 2 (2004), s. 277-282 ISSN 0003-9861 R&D Projects: GA ČR GV312/96/K205; GA ČR GA301/00/0269 Institutional research plan: CEZ:AV0Z4055905; CEZ:AV0Z1003909 Keywords : Src kinase, in vitro phosphorylation, peptide substrate specificity Subject RIV: CE - Biochemistry Impact factor: 2.657, year: 2004

  18. Molecular pharmacology of promiscuous seven transmembrane receptors sensing organic nutrients.

    Science.gov (United States)

    Wellendorph, Petrine; Johansen, Lars Dan; Bräuner-Osborne, Hans

    2009-09-01

    A number of highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized within the last few years. It is noteworthy that many of these receptors are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids and are expressed in taste tissue, the gastrointestinal tract, endocrine glands, adipose tissue, and/or kidney. These receptors thus hold the potential to act as sensors of food intake, regulating, for example, release of incretin hormones from the gut, insulin/glucagon from the pancreas, and leptin from adipose tissue. The promiscuous tendency in ligand recognition of these receptors is in contrast to the typical specific interaction with one physiological agonist seen for most receptors, which challenges the classic "lock-and-key" concept. We here review the molecular mechanisms of nutrient sensing of the calcium-sensing receptor, the G protein-coupled receptor family C, group 6, subtype A (GPRC6A), and the taste1 receptor T1R1/T1R3, which are sensing L-alpha-amino acids, the carbohydrate-sensing T1R2/T1R3 receptor, the proteolytic degradation product sensor GPR93 (also termed GPR92), and the free fatty acid (FFA) sensing receptors FFA1, FFA2, FFA3, GPR84, and GPR120. The involvement of the individual receptors in sensing of food intake has been validated to different degrees because of limited availability of specific pharmacological tools and/or receptor knockout mice. However, as a group, the receptors represent potential drug targets, to treat, for example, type II diabetes by mimicking food intake by potent agonists or positive allosteric modulators. The ligand-receptor interactions of the promiscuous receptors of organic nutrients thus remain an interesting subject of emerging functional importance.

  19. Entering the ‘big data’ era in medicinal chemistry: molecular promiscuity analysis revisited

    Science.gov (United States)

    Hu, Ye; Bajorath, Jürgen

    2017-01-01

    The ‘big data’ concept plays an increasingly important role in many scientific fields. Big data involves more than unprecedentedly large volumes of data that become available. Different criteria characterizing big data must be carefully considered in computational data mining, as we discuss herein focusing on medicinal chemistry. This is a scientific discipline where big data is beginning to emerge and provide new opportunities. For example, the ability of many drugs to specifically interact with multiple targets, termed promiscuity, forms the molecular basis of polypharmacology, a hot topic in drug discovery. Compound promiscuity analysis is an area that is much influenced by big data phenomena. Different results are obtained depending on chosen data selection and confidence criteria, as we also demonstrate. PMID:28670471

  20. Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites.

    Directory of Open Access Journals (Sweden)

    Jose L S Lopes

    Full Text Available Diacylglycerol acyltransferase 1 (DGAT1 is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.

  1. Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity.

    Directory of Open Access Journals (Sweden)

    Miriam Kaltenbach

    2016-10-01

    Full Text Available The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with "evolvability" was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1 from phosphotriesterase to arylesterase, and (2 from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak

  2. Engineering substrate promiscuity in halophilic alcohol dehydrogenase (HvADH2 by in silico design.

    Directory of Open Access Journals (Sweden)

    Jennifer Cassidy

    Full Text Available An alcohol dehydrogenase from the halophilic archaeon Haloferax volcanii (HvADH2 has been engineered by rational design to broaden its substrate scope towards the conversion of a range of aromatic substrates, including flurbiprofenol, that is an intermediate of the non-steroidal anti-inflammatory drug, flurbiprofen. Wild-type HvADH2 showed minimal activity with flurbiprofenol (11.1 mU/mg. A homology model of HvADH2 was built and docking experiments with this substrate revealed that the biphenyl rings of flurbiprofenol formed strong interactions with residues F85 and F108, preventing its optimal binding in the active site. Mutations at position 85 however did not increase activity. Site directed mutagenesis at position F108 allowed the identification of three variants showing a significant (up to 2.3-fold enhancement of activity towards flurbiprofenol, when compared to wild-type HvADH2. Interestingly, F108G variant did not show the classic inhibition in the presence of (R-enantiomer when tested with rac-1-phenylethanol, underling its potential in racemic resolution of secondary alcohols.

  3. PNA Peptide chimerae

    DEFF Research Database (Denmark)

    Koch, T.; Næsby, M.; Wittung, P.

    1995-01-01

    Radioactive labelling of PNA has been performed try linking a peptide segment to the PNA which is substrate for protein kinase A. The enzymatic phosphorylation proceeds in almost quantitative yields....

  4. Predictors of Drug/Alcohol Abuse and Sexual Promiscuity of College Students.

    Science.gov (United States)

    Nam, Jeong Sook; And Others

    This study examined the relationship between the individual's purpose in life, existential anxiety, powerlessness and use of alcohol/drugs and the tendency to be sexually promiscuous. The study is rooted in the work of Viktor E. Frankl, which suggested that a lack of meaning and purpose can cause socially deviant behavior and psychological…

  5. Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

    Science.gov (United States)

    Castro-Fernandez, Víctor; Herrera-Morande, Alejandra; Zamora, Ricardo; Merino, Felipe; Gonzalez-Ordenes, Felipe; Padilla-Salinas, Felipe; Pereira, Humberto M; Brandão-Neto, Jose; Garratt, Richard C; Guixe, Victoria

    2017-09-22

    One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales , Methanosarcinales , and Methanococcales , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. High-throughput functional screening of steroid substrates with wild-type and chimeric P450 enzymes.

    Science.gov (United States)

    Urban, Philippe; Truan, Gilles; Pompon, Denis

    2014-01-01

    The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.

  7. Daphnia HR96 is a promiscuous xenobiotic and endobiotic nuclear receptor

    International Nuclear Information System (INIS)

    Karimullina, Elina; Li Yangchun; Ginjupalli, Gautam K.; Baldwin, William S.

    2012-01-01

    Daphnia pulex is the first crustacean to have its genome sequenced. The genome project provides new insight and data into how an aquatic crustacean may respond to environmental stressors, including toxicants. We cloned Daphnia pulex HR96 (DappuHR96), a nuclear receptor orthologous to the CAR/PXR/VDR group of nuclear receptors. In Drosophila melanogaster, (hormone receptor 96) HR96 responds to phenobarbital exposure and has been hypothesized as a toxicant receptor. Therefore, we set up a transactivation assay to test whether DappuHR96 is a promiscuous receptor activated by xenobiotics and endobiotics similar to the constitutive androstane receptor (CAR) and the pregnane X-receptor (PXR). Transactivation assays performed with a GAL4-HR96 chimera demonstrate that HR96 is a promiscuous toxicant receptor activated by a diverse set of chemicals such as pesticides, hormones, and fatty acids. Several environmental toxicants activate HR96 including estradiol, pyriproxyfen, chlorpyrifos, atrazine, and methane arsonate. We also observed repression of HR96 activity by chemicals such as triclosan, androstanol, and fluoxetine. Nearly 50% of the chemicals tested activated or inhibited HR96. Interestingly, unsaturated fatty acids were common activators or inhibitors of HR96 activity, indicating a link between diet and toxicant response. The omega-6 and omega-9 unsaturated fatty acids linoleic and oleic acid activated HR96, but the omega-3 unsaturated fatty acids alpha-linolenic acid and docosahexaenoic acid inhibited HR96, suggesting that these two distinct sets of lipids perform opposing roles in Daphnia physiology. This also provides a putative mechanism by which the ratio of dietary unsaturated fats may affect the ability of an organism to respond to a toxic insult. In summary, HR96 is a promiscuous nuclear receptor activated by numerous endo- and xenobiotics.

  8. Daphnia HR96 is a promiscuous xenobiotic and endobiotic nuclear receptor

    Energy Technology Data Exchange (ETDEWEB)

    Karimullina, Elina [Environmental Toxicology Program, Clemson University, Clemson, SC 29634 (United States); Institute of Plant and Animal Ecology, Russian Academy of Sciences, Ural Branch, Yekaterinburg 620144 (Russian Federation); Li Yangchun; Ginjupalli, Gautam K. [Environmental Toxicology Program, Clemson University, Clemson, SC 29634 (United States); Baldwin, William S., E-mail: baldwin@clemson.edu [Environmental Toxicology Program, Clemson University, Clemson, SC 29634 (United States); Biological Sciences, Clemson University, Clemson, SC (United States)

    2012-07-15

    Daphnia pulex is the first crustacean to have its genome sequenced. The genome project provides new insight and data into how an aquatic crustacean may respond to environmental stressors, including toxicants. We cloned Daphnia pulex HR96 (DappuHR96), a nuclear receptor orthologous to the CAR/PXR/VDR group of nuclear receptors. In Drosophila melanogaster, (hormone receptor 96) HR96 responds to phenobarbital exposure and has been hypothesized as a toxicant receptor. Therefore, we set up a transactivation assay to test whether DappuHR96 is a promiscuous receptor activated by xenobiotics and endobiotics similar to the constitutive androstane receptor (CAR) and the pregnane X-receptor (PXR). Transactivation assays performed with a GAL4-HR96 chimera demonstrate that HR96 is a promiscuous toxicant receptor activated by a diverse set of chemicals such as pesticides, hormones, and fatty acids. Several environmental toxicants activate HR96 including estradiol, pyriproxyfen, chlorpyrifos, atrazine, and methane arsonate. We also observed repression of HR96 activity by chemicals such as triclosan, androstanol, and fluoxetine. Nearly 50% of the chemicals tested activated or inhibited HR96. Interestingly, unsaturated fatty acids were common activators or inhibitors of HR96 activity, indicating a link between diet and toxicant response. The omega-6 and omega-9 unsaturated fatty acids linoleic and oleic acid activated HR96, but the omega-3 unsaturated fatty acids alpha-linolenic acid and docosahexaenoic acid inhibited HR96, suggesting that these two distinct sets of lipids perform opposing roles in Daphnia physiology. This also provides a putative mechanism by which the ratio of dietary unsaturated fats may affect the ability of an organism to respond to a toxic insult. In summary, HR96 is a promiscuous nuclear receptor activated by numerous endo- and xenobiotics.

  9. Effect of Ca2+ on the promiscuous target-protein binding of calmodulin.

    Directory of Open Access Journals (Sweden)

    Annie M Westerlund

    2018-04-01

    Full Text Available Calmodulin (CaM is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity.

  10. Promiscuous activities of heterologous enzymes lead to unintended metabolic rerouting in Saccharomyces cerevisiae engineered to assimilate various sugars from renewable biomass.

    Science.gov (United States)

    Yun, Eun Ju; Oh, Eun Joong; Liu, Jing-Jing; Yu, Sora; Kim, Dong Hyun; Kwak, Suryang; Kim, Kyoung Heon; Jin, Yong-Su

    2018-01-01

    Understanding the global metabolic network, significantly perturbed upon promiscuous activities of foreign enzymes and different carbon sources, is crucial for systematic optimization of metabolic engineering of yeast Saccharomyces cerevisiae . Here, we studied the effects of promiscuous activities of overexpressed enzymes encoded by foreign genes on rerouting of metabolic fluxes of an engineered yeast capable of assimilating sugars from renewable biomass by profiling intracellular and extracellular metabolites. Unbiased metabolite profiling of the engineered S. cerevisiae strain EJ4 revealed promiscuous enzymatic activities of xylose reductase and xylitol dehydrogenase on galactose and galactitol, respectively, resulting in accumulation of galactitol and tagatose during galactose fermentation. Moreover, during glucose fermentation, a trisaccharide consisting of glucose accumulated outside of the cells probably owing to the promiscuous and transglycosylation activity of β-glucosidase expressed for hydrolyzing cellobiose. Meanwhile, higher accumulation of fatty acids and secondary metabolites was observed during xylose and cellobiose fermentations, respectively. The heterologous enzymes functionally expressed in S. cerevisiae showed promiscuous activities that led to unintended metabolic rerouting in strain EJ4. Such metabolic rerouting could result in a low yield and productivity of a final product due to the formation of unexpected metabolites. Furthermore, the global metabolic network can be significantly regulated by carbon sources, thus yielding different patterns of metabolite production. This metabolomic study can provide useful information for yeast strain improvement and systematic optimization of yeast metabolism to manufacture bio-based products.

  11. High-Throughput Functional Screening of Steroid Substrates with Wild-Type and Chimeric P450 Enzymes

    Directory of Open Access Journals (Sweden)

    Philippe Urban

    2014-01-01

    Full Text Available The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.

  12. Photoinduced electron transfer through peptide-based self-assembled monolayers chemisorbed on gold electrodes: directing the flow-in and flow-out of electrons through peptide helices.

    Science.gov (United States)

    Venanzi, Mariano; Gatto, Emanuela; Caruso, Mario; Porchetta, Alessandro; Formaggio, Fernando; Toniolo, Claudio

    2014-08-21

    Photoinduced electron transfer (PET) experiments have been carried out on peptide self-assembled monolayers (SAM) chemisorbed on a gold substrate. The oligopeptide building block was exclusively formed by C(α)-tetrasubstituted α-aminoisobutyric residues to attain a helical conformation despite the shortness of the peptide chain. Furthermore, it was functionalized at the C-terminus by a pyrene choromophore to enhance the UV photon capture cross-section of the compound and by a lipoic group at the N-terminus for linking to gold substrates. Electron transfer across the peptide SAM has been studied by photocurrent generation experiments in an electrochemical cell employing a gold substrate modified by chemisorption of a peptide SAM as a working electrode and by steady-state and time-resolved fluorescence experiments in solution and on a gold-coated glass. The results show that the electronic flow through the peptide bridge is strongly asymmetric; i.e., PET from the C-terminus to gold is highly favored with respect to PET in the opposite direction. This effect arises from the polarity of the Au-S linkage (Au(δ+)-S(δ-), junction effect) and from the electrostatic field generated by the peptide helix.

  13. Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates

    Directory of Open Access Journals (Sweden)

    Amy eCurran

    2011-08-01

    Full Text Available The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs. While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16 and 34. Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ~70 µM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites. Of these, 74 (27% were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.

  14. In vitro evaluation of N-methyl amide tripeptidomimetics as substrates for the human intestinal di-/tri-peptide transporter hPEPT1

    DEFF Research Database (Denmark)

    Andersen, Rikke; Nielsen, Carsten Uhd; Begtrup, Mikael

    2006-01-01

    application of N-methyl amide bioisosteres as peptide bond replacements in tripeptides in order to decrease degradation by peptidases and yet retain affinity for and transport via hPEPT1. Seven structurally diverse N-methyl amide tripeptidomimetics were selected based on a principal component analysis...... of structural properties of 6859 N-methyl amide tripeptidomimetics. In vitro extracellular degradation of the selected tripeptidomimetics as well as affinity for and transepithelial transport via hPEPT1 were investigated in Caco-2 cells. Decreased apparent degradation was observed for all tripeptidomimetics...... to be substrates for hPEPT1 than tripeptidomimetics with charged side chains. The results of the present study indicate that the N-methyl amide peptide bond replacement approach for increasing bioavailability of tripeptidomimetic drug candidates is not generally applicable to all tripeptides. Nevertheless...

  15. Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity

    Directory of Open Access Journals (Sweden)

    Charlotte Kürten

    2016-06-01

    Full Text Available The discovery and generation of biocatalysts with extended catalytic versatilities are of immense relevance in both chemistry and biotechnology. An enhanced atomistic understanding of enzyme promiscuity, a mechanism through which living systems acquire novel catalytic functions and specificities by evolution, would thus be of central interest. Using esterase-catalyzed amide bond hydrolysis as a model system, we pursued a simplistic in silico discovery program aiming for the identification of enzymes with an internal backbone hydrogen bond acceptor that could act as a reaction specificity shifter in hydrolytic enzymes. Focusing on stabilization of the rate limiting transition state of nitrogen inversion, our mechanism-guided approach predicted that the acyl hydrolase patatin of the α/β phospholipase fold would display reaction promiscuity. Experimental analysis confirmed previously unknown high amidase over esterase activity displayed by the first described esterase machinery with a protein backbone hydrogen bond acceptor to the reacting NH-group of amides. The present work highlights the importance of a fundamental understanding of enzymatic reactions and its potential for predicting enzyme scaffolds displaying alternative chemistries amenable to further evolution by enzyme engineering.

  16. The effect of the promiscuity stereotype on opposition to gay rights.

    Science.gov (United States)

    Pinsof, David; Haselton, Martie G

    2017-01-01

    Opposition to gay rights is prevalent in countries around the world. Recent correlational research suggests that opposition to gay rights may be driven by an interaction between one's own short-term mating orientation (i.e. willingness to engage in casual sex) and representations of gay people as sexually promiscuous. Here, we experimentally manipulated representations of gay men by randomly assigning participants to read one of two versions of a fictitious newspaper article, one of which contained faux scientific evidence confirming the stereotype that gay men are promiscuous, and the other containing faux scientific evidence refuting the stereotype. We found that the manipulation interacted with short-term mating orientation (STMO) to predict opposition to gay rights, such that low-STMO individuals (i.e. more averse to casual sex) exhibited more support for gay rights when assigned to read the stereotype-refuting article compared to the stereotype-confirming article, whereas high-STMO individuals (i.e. less averse to casual sex) were not significantly influenced by the manipulation. We discuss the implications of these findings for the study of antigay attitudes, as well as for recent societal changes in acceptance of homosexuality.

  17. Testing whether Metazoan Tyrosine Loss Was Driven by Selection against Promiscuous Phosphorylation

    Science.gov (United States)

    Pandya, Siddharth; Struck, Travis J.; Mannakee, Brian K.; Paniscus, Mary; Gutenkunst, Ryan N.

    2015-01-01

    Protein tyrosine phosphorylation is a key regulatory modification in metazoans, and the corresponding kinase enzymes have diversified dramatically. This diversification is correlated with a genome-wide reduction in protein tyrosine content, and it was recently suggested that this reduction was driven by selection to avoid promiscuous phosphorylation that might be deleterious. We tested three predictions of this intriguing hypothesis. 1) Selection should be stronger on residues that are more likely to be phosphorylated due to local solvent accessibility or structural disorder. 2) Selection should be stronger on proteins that are more likely to be promiscuously phosphorylated because they are abundant. We tested these predictions by comparing distributions of tyrosine within and among human and yeast orthologous proteins. 3) Selection should be stronger against mutations that create tyrosine versus remove tyrosine. We tested this prediction using human population genomic variation data. We found that all three predicted effects are modest for tyrosine when compared with the other amino acids, suggesting that selection against deleterious phosphorylation was not dominant in driving metazoan tyrosine loss. PMID:25312910

  18. Promiscuous activity of ER glucosidase II discovered through donor specificity analysis of UGGT

    Energy Technology Data Exchange (ETDEWEB)

    Miyagawa, Atsushi, E-mail: miyagawa.atsushi@nitech.ac.jp [RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Department of Materials Science and Engineering, Nagoya Institute of Technology, Showa-Ku, Nagoya 466-8555 (Japan); Totani, Kiichiro [Department of Materials and Life Science, Seikei University, Musashino, Tokyo 180-8633 (Japan); Matsuo, Ichiro [Department of Chemistry and Chemical Biology, Gunma University, Kiryu, Gunma 376-8515 (Japan); Ito, Yukishige, E-mail: yukito@riken.jp [RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); ERATO Japan Science and Technology Agency, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)

    2010-12-17

    Research highlights: {yields} UGGT has a narrow donor specificity. {yields} UGGT gave several non-natural high-mannose-type glycans. {yields} G-II has a promiscuous activity as broad specificity hexosidase. -- Abstract: In glycoprotein quality control system in the endoplasmic reticulum (ER), UGGT (UDP-glucose:glycoprotein glucosyltransferase) and glucosidase II (G-II) play key roles. UGGT serves as a glycoprotein folding sensor by virtue of its unique specificity to glucosylate glycoproteins at incompletely folded stage. By using various UDP-Glc analogues, we first analyzed donor specificity of UGGT, which was proven to be rather narrow. However, marginal activity was observed with UDP-galactose and UDP-glucuronic acid as well as with 3-, 4- and 6-deoxy glucose analogues to give corresponding transfer products. Intriguingly, G-II smoothly converted all of them back to Man{sub 9}GlcNAc{sub 2}, providing an indication that G-II has a promiscuous activity as a broad specificity hexosidase.

  19. Promiscuous Seven Transmembrane Receptors Sensing L-α-amino Acids

    DEFF Research Database (Denmark)

    Smajilovic, Sanela; Wellendorph, Petrine; Bräuner-Osborne, Hans

    2014-01-01

    A number of nutrient sensing seven trans-membrane (7TM) receptors have been identified and characterized over the past few years. While the sensing mechanisms to carbohydrates and free fatty acids are well understood, the molecular basis of amino acid sensing has recently come to the limelight....... The present review describes the current status of promiscuous L-α-amino acid sensors, the calcium sensing receptor (CaSR), the GPRC6A receptor, the T1R1/T1R3 receptor and also their molecular pharmacology, expression pattern and physiological significance....

  20. Insights into the substrate specificity of plant peptide deformylase, an essential enzyme with potential for the development of novel biotechnology applications in agriculture.

    Science.gov (United States)

    Dirk, Lynnette M A; Schmidt, Jack J; Cai, Yiying; Barnes, Jonathan C; Hanger, Katherine M; Nayak, Nihar R; Williams, Mark A; Grossman, Robert B; Houtz, Robert L; Rodgers, David W

    2008-08-01

    The crystal structure of AtPDF1B [Arabidopsis thaliana PDF (peptide deformylase) 1B; EC 3.5.1.88], a plant specific deformylase, has been determined at a resolution of 2.4 A (1 A=0.1 nm). The overall fold of AtPDF1B is similar to other peptide deformylases that have been reported. Evidence from the crystal structure and gel filtration chromatography indicates that AtPDF1B exists as a symmetric dimer. PDF1B is essential in plants and has a preferred substrate specificity towards the PS II (photosystem II) D1 polypeptide. Comparative analysis of AtPDF1B, AtPDF1A, and the type 1B deformylase from Escherichia coli, identifies a number of differences in substrate binding subsites that might account for variations in sequence preference. A model of the N-terminal five amino acids from the D1 polypeptide bound in the active site of AtPDF1B suggests an influence of Tyr(178) as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr(2) in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-terminus was performed on AtPDF1B mutated at Tyr(178) to alanine, phenylalanine or arginine (equivalent residue in AtPDF1A). The results suggest that, whereas Tyr(178) can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.

  1. Studies on peptide amidase-catalysed C-terminal peptide amidation in organic media with respect to its substrate specificity

    Czech Academy of Sciences Publication Activity Database

    Čeřovský, Václav; Kula, M. R.

    2001-01-01

    Roč. 33, - (2001), s. 183-187 ISSN 0885-4513 R&D Projects: GA ČR GA203/99/1458 Keywords : enzymic amidation * peptide amides * peptide synthesis Subject RIV: CC - Organic Chemistry Impact factor: 1.408, year: 2001

  2. Superior Antifouling Performance of a Zwitterionic Peptide Compared to an Amphiphilic, Non-Ionic Peptide.

    Science.gov (United States)

    Ye, Huijun; Wang, Libing; Huang, Renliang; Su, Rongxin; Liu, Boshi; Qi, Wei; He, Zhimin

    2015-10-14

    The aim of this study was to explore the influence of amphiphilic and zwitterionic structures on the resistance of protein adsorption to peptide self-assembled monolayers (SAMs) and gain insight into the associated antifouling mechanism. Two kinds of cysteine-terminated heptapeptides were studied. One peptide had alternating hydrophobic and hydrophilic residues with an amphiphilic sequence of CYSYSYS. The other peptide (CRERERE) was zwitterionic. Both peptides were covalently attached onto gold substrates via gold-thiol bond formation. Surface plasmon resonance analysis results showed that both peptide SAMs had ultralow or low protein adsorption amounts of 1.97-11.78 ng/cm2 in the presence of single proteins. The zwitterionic peptide showed relatively higher antifouling ability with single proteins and natural complex protein media. We performed molecular dynamics simulations to understand their respective antifouling behaviors. The results indicated that strong surface hydration of peptide SAMs contributes to fouling resistance by impeding interactions with proteins. Compared to the CYSYSYS peptide, more water molecules were predicted to form hydrogen-bonding interactions with the zwitterionic CRERERE peptide, which is in agreement with the antifouling test results. These findings reveal a clear relation between peptide structures and resistance to protein adsorption, facilitating the development of novel peptide-containing antifouling materials.

  3. Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2.

    Science.gov (United States)

    Mpakali, Anastasia; Giastas, Petros; Mathioudakis, Nikolas; Mavridis, Irene M; Saridakis, Emmanuel; Stratikos, Efstratios

    2015-10-23

    Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Identification of the key determinant of the transport promiscuity in Na+-translocating rhodopsins.

    Science.gov (United States)

    Mamedov, Adalyat M; Bertsova, Yulia V; Anashkin, Viktor A; Mamedov, Mahir D; Baykov, Alexander A; Bogachev, Alexander V

    2018-05-15

    Bacterial Na + -transporting rhodopsins convert solar energy into transmembrane ion potential difference. Typically, they are strictly specific for Na + , but some can additionally transport H + . To determine the structural basis of cation promiscuity in Na + -rhodopsins, we compared their primary structures and found a single position that harbors a cysteine in strictly specific Na + -rhodopsins and a serine in the promiscuous Krokinobacter eikastus Na + -rhodopsin (Kr2). A Cys253Ser variant of the strictly specific Dokdonia sp. PRO95 Na + -rhodopsin (NaR) was indeed found to transport both Na + and H + in a light-dependent manner when expressed in retinal-producing Escherichia coli cells. The dual specificity of the NaR variant was confirmed by analysis of its photocycle, which revealed an acceleration of the cation-capture step by comparison with the wild-type NaR in a Na + -deficient medium. The structural basis for the dependence of the Na + /H + specificity in Na + -rhodopsin on residue 253 remains to be determined. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Specificity and Application of the Lantibiotic Protease NisP

    Directory of Open Access Journals (Sweden)

    Manuel Montalbán-López

    2018-02-01

    Full Text Available Lantibiotics are ribosomally produced and posttranslationally modified peptides containing several lanthionine residues. They exhibit substantial antimicrobial activity against Gram-positive bacteria, including relevant pathogens. The production of the model lantibiotic nisin minimally requires the expression of the modification and export machinery. The last step during nisin maturation is the cleavage of the leader peptide. This liberates the active compound and is catalyzed by the cell wall-anchored protease NisP. Here, we report the production and purification of a soluble variant of NisP. This has enabled us to study its specificity and test its suitability for biotechnological applications. The ability of soluble NisP to cleave leaders from various substrates was tested with two sets of nisin variants. The first set was designed to investigate the influence of amino acid variations in the leader peptide or variations around the cleavage site. The second set was designed to study the influence of the lanthionine ring topology on the proteolytic efficiency. We show that the substrate promiscuity is higher than has previously been suggested. Our results demonstrate the importance of the arginine residue at the end of the leader peptide and the importance of lanthionine rings in the substrate for specific cleavage. Collectively, these data indicate that NisP is a suitable protease for the activation of diverse heterologously expressed lantibiotics, which is required to release active antimicrobial compounds.

  6. Hypersensitive detection and quantitation of BoNT/A by IgY antibody against substrate linear-peptide.

    Directory of Open Access Journals (Sweden)

    Tao Li

    Full Text Available Botulinum neurotoxin A (BoNT/A, the most acutely poisonous substance to humans known, cleave its SNAP-25 substrate with high specificity. Based on the endopeptidase activity, different methods have been developed to detect BoNT/A, but most lack ideal reproducibility or sensitivity, or suffer from long-term or unwanted interferences. In this study, we developed a simple method to detect and quantitate trace amounts of botulinum neurotoxin A using the IgY antibody against a linear-peptide substrate. The effects of reaction buffer, time, and temperature were analyzed and optimized. When the optimized assay was used to detect BoNT/A, the limit of detection of the assay was 0.01 mouse LD50 (0.04 pg, and the limit of quantitation was 0.12 mouse LD50/ml (0.48 pg. The findings also showed favorable specificity of detecting BoNT/A. When used to detect BoNT/A in milk or human serum, the proposed assay exhibited good quantitative accuracy (88% < recovery < 111%; inter- and intra-assay CVs < 18%. This method of detection took less than 3 h to complete, indicating that it can be a valuable method of detecting BoNT/A in food or clinical diagnosis.

  7. Activity-Based Profiling of a Physiologic Aglycone Library Reveals Sugar Acceptor Promiscuity of Family 1 UDP-Glucosyltransferases from Grape1[W

    Science.gov (United States)

    Bönisch, Friedericke; Frotscher, Johanna; Stanitzek, Sarah; Rühl, Ernst; Wüst, Matthias; Bitz, Oliver; Schwab, Wilfried

    2014-01-01

    Monoterpenols serve various biological functions and accumulate in grape (Vitis vinifera), where a major fraction occurs as nonvolatile glycosides. We have screened the grape genome for sequences with similarity to terpene URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASES (UGTs) from Arabidopsis (Arabidopsis thaliana). A ripening-related expression pattern was shown for three candidates by spatial and temporal expression analyses in five grape cultivars. Transcript accumulation correlated with the production of monoterpenyl β-d-glucosides in grape exocarp during ripening and was low in vegetative tissue. Targeted functional screening of the recombinant UGTs for their biological substrates was performed by activity-based metabolite profiling (ABMP) employing a physiologic library of aglycones built from glycosides isolated from grape. This approach led to the identification of two UDP-glucose:monoterpenol β-d-glucosyltransferases. Whereas VvGT14a glucosylated geraniol, R,S-citronellol, and nerol with similar efficiency, the three allelic forms VvGT15a, VvGT15b, and VvGT15c preferred geraniol over nerol. Kinetic resolution of R,S-citronellol and R,S-linalool was shown for VvGT15a and VvGT14a, respectively. ABMP revealed geraniol as the major biological substrate but also disclosed that these UGTs may add to the production of further glycoconjugates in planta. ABMP of aglycone libraries provides a versatile tool to uncover novel biologically relevant substrates of small-molecule glycosyltransferases that often show broad sugar acceptor promiscuity. PMID:25073706

  8. Ervaringen met een solid phase enzyme immunoassay voor het aantonen van gonorroe bij promiscue vrouwen

    NARCIS (Netherlands)

    Ulsen; J.van*; Michel; M.F.*; Strik; R.van*; Joost; T.H.van*; Stolz; E.*; Eijk; R.V.W.van

    1985-01-01

    De Gonozyme test (Abbott Laboratories), een nieuwe enzyme immunoassay (EIA) voor het aantonen van Neisseria gonorrhoeae werd geevalueerd in een grote groep promiscue vrouwen. Als de EIA werd uitgevoerd met materiaal afkomstig van de cervix, bedroeg de prevalentie van gonorroe 8,2%. Vergeleken

  9. Lactoferricin-related peptides with inhibitory effects on ACE-dependent vasoconstriction.

    Science.gov (United States)

    Centeno, José M; Burguete, María C; Castelló-Ruiz, María; Enrique, María; Vallés, Salvador; Salom, Juan B; Torregrosa, Germán; Marcos, José F; Alborch, Enrique; Manzanares, Paloma

    2006-07-26

    A selection of lactoferricin B (LfcinB)-related peptides with an angiotensin I-converting enzyme (ACE) inhibitory effect have been examined using in vitro and ex vivo functional assays. Peptides that were analyzed included a set of sequence-related antimicrobial hexapeptides previously reported and two representative LfcinB-derived peptides. In vitro assays using hippuryl-L-histidyl-L-leucine (HHL) and angiotensin I as substrates allowed us to select two hexapeptides, PACEI32 (Ac-RKWHFW-NH2) and PACEI34 (Ac-RKWLFW-NH2), and also a LfcinB-derived peptide, LfcinB17-31 (Ac-FKCRRWQWRMKKLGA-NH2). Ex vivo functional assays using rabbit carotid arterial segments showed PACEI32 (both D- and L-enantiomers) and LfcinB17-31 have inhibitory effects on ACE-dependent angiotensin I-induced contraction. None of the peptides exhibited in vitro ACE inhibitory activity using bradykinin as the substrate. In conclusion, three bioactive lactoferricin-related peptides exhibit inhibitory effects on both ACE activity and ACE-dependent vasoconstriction with potential to modulate hypertension that deserves further investigation.

  10. Lactoferricin B-derived peptides with inhibitory effects on ECE-dependent vasoconstriction.

    Science.gov (United States)

    Fernández-Musoles, Ricardo; López-Díez, José Javier; Torregrosa, Germán; Vallés, Salvador; Alborch, Enrique; Manzanares, Paloma; Salom, Juan B

    2010-10-01

    Endothelin-converting enzyme (ECE), a key peptidase in the endothelin (ET) system, cleaves inactive big ET-1 to produce active ET-1, which binds to ET(A) receptors to exert its vasoconstrictor and pressor effects. ECE inhibition could be beneficial in the treatment of hypertension. In this study, a set of eight lactoferricin B (LfcinB)-derived peptides, previously characterized in our laboratory as angiotensin-converting enzyme (ACE) inhibitory peptides, was examined for their inhibitory effects on ECE. In vitro inhibitory effects on ECE activity were assessed using both the synthetic fluorogenic peptide substrate V (FPS V) and the natural substrate big ET-1. To study vasoactive effects, an ex vivo functional assay was developed using isolated rabbit carotid artery segments. With FPS V, only four LfcinB-derived peptides induced inhibition of ECE activity, whereas the eight peptides showed ECE inhibitory effects with big ET-1 as substrate. Regarding the ex vivo assays, six LfcinB-derived peptides showed inhibition of big ET-1-induced, ECE-dependent vasoconstriction. A positive correlation between the inhibitory effects of LfcinB-derived peptides on ECE activity when using big ET-1 and the inhibitory effects on ECE-dependent vasoconstriction was shown. ECE-independent vasoconstriction induced by ET-1 was not affected, thus discarding effects of LfcinB-derived peptides on ET(A) receptors or intracellular signal transduction mechanisms. In conclusion, a combined in vitro and ex vivo method to assess the effects of potentially antihypertensive peptides on the ET system has been developed and applied to show the inhibitory effects on ECE-dependent vasoconstriction of six LfcinB-derived peptides, five of which were dual vasopeptidase (ACE/ECE) inhibitors. Copyright © 2010 Elsevier Inc. All rights reserved.

  11. Bacillus subtilis RapA phosphatase domain interaction with its substrate, phosphorylated Spo0F, and its inhibitor, the PhrA peptide.

    Science.gov (United States)

    Diaz, Alejandra R; Core, Leighton J; Jiang, Min; Morelli, Michela; Chiang, Christina H; Szurmant, Hendrik; Perego, Marta

    2012-03-01

    Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0F∼P), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNA-binding domain of ComA, rather than Spo0F∼P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the amino-terminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.

  12. ClbM is a versatile, cation-promiscuous MATE transporter found in the colibactin biosynthetic gene cluster

    International Nuclear Information System (INIS)

    Mousa, Jarrod J.; Newsome, Rachel C.; Yang, Ye; Jobin, Christian; Bruner, Steven D.

    2017-01-01

    Multidrug transporters play key roles in cellular drug resistance to toxic molecules, yet these transporters are also involved in natural product transport as part of biosynthetic clusters in bacteria and fungi. The genotoxic molecule colibactin is produced by strains of virulent and pathobiont Escherichia coli and Klebsiella pneumoniae. In the biosynthetic cluster is a multidrug and toxic compound extrusion protein (MATE) proposed to transport the prodrug molecule precolibactin across the cytoplasmic membrane, for subsequent cleavage by the peptidase ClbP and cellular export. We recently determined the X-ray structure of ClbM, and showed preliminary data suggesting its specific role in precolibactin transport. Here, we define a functional role of ClbM by examining transport capabilities under various biochemical conditions. Our data indicate ClbM responds to sodium, potassium, and rubidium ion gradients, while also having substantial transport activity in the absence of alkali cations. - Highlights: • ClbM is a cation promiscuous MATE multidrug transporter. • The role of key residues were identified in both the cation and proton binding. • The biologically relevant substrate for ClbM is the natural product precolibactin.

  13. Mycobacterium leprae virulence-associated peptides are indicators of exposure to M. leprae in Brazil, Ethiopia and Nepal.

    Science.gov (United States)

    Bobosha, Kidist; Tang, Sheila Tuyet; van der Ploeg-van Schip, Jolien J; Bekele, Yonas; Martins, Marcia V S B; Lund, Ole; Franken, Kees L M C; Khadge, Saraswoti; Pontes, Maria Araci de Andrade; Gonçalves, Heitor de Sá; Hussien, Jemal; Thapa, Pratibha; Kunwar, Chhatra B; Hagge, Deanna A; Aseffa, Abraham; Pessolani, Maria Cristina Vidal; Pereira, Geraldo M B; Ottenhoff, Tom H M; Geluk, Annemieke

    2012-12-01

    Silent transmission of Mycobacterium leprae, as evidenced by stable leprosy incidence rates in various countries, remains a health challenge despite the implementation of multidrug therapy worldwide. Therefore, the development of tools for the early diagnosis of M. leprae infection should be emphasised in leprosy research. As part of the continuing effort to identify antigens that have diagnostic potential, unique M. leprae peptides derived from predicted virulence-associated proteins (group IV.A) were identified using advanced genome pattern programs and bioinformatics. Based on human leukocyte antigen (HLA)-binding motifs, we selected 21 peptides that were predicted to be promiscuous HLA-class I T-cell epitopes and eight peptides that were predicted to be HLA-class II restricted T-cell epitopes for field-testing in Brazil, Ethiopia and Nepal. High levels of interferon (IFN)-γ were induced when peripheral blood mononuclear cells (PBMCs) from tuberculoid/borderline tuberculoid leprosy patients located in Brazil and Ethiopia were stimulated with the ML2055 p35 peptide. PBMCs that were isolated from healthy endemic controls living in areas with high leprosy prevalence (EChigh) in Ethiopia also responded to the ML2055 p35 peptide. The Brazilian EChigh group recognised the ML1358 p20 and ML1358 p24 peptides. None of the peptides were recognised by PBMCs from healthy controls living in non-endemic region. In Nepal, mixtures of these peptides induced the production of IFN-γ by the PBMCs of leprosy patients and EChigh. Therefore, the M. leprae virulence-associated peptides identified in this study may be useful for identifying exposure to M. leprae in population with differing HLA polymorphisms.

  14. Activity artifacts in drug discovery and different facets of compound promiscuity [v1; ref status: indexed, http://f1000r.es/4gz

    Directory of Open Access Journals (Sweden)

    Jürgen Bajorath

    2014-10-01

    Full Text Available Compounds with apparent activity in a variety of assays might disable target proteins or produce false assay signals in the absence of specific interactions. In some instances, such effects are easy to detect, in others they are not. Observed promiscuity of compounds might be due to such non-specific assay artifacts. By contrast, promiscuity might also result from specific interactions with multiple targets. In the latter case, promiscuous compounds can be attractive candidates for certain therapeutic applications. However, compounds with artificial activity readouts are often not recognized and are further progressed, which presents a substantial problem for drug discovery. In this context, the concept of PAINS (pan-assay interference compounds should be seriously considered, which makes it possible to eliminate flawed compounds from the discovery pipeline, even if their activities appear to be sound at a first glance.

  15. Coexistence of unlimited bipartite and genuine multipartite entanglement: Promiscuous quantum correlations arising from discrete to continuous-variable systems

    International Nuclear Information System (INIS)

    Adesso, Gerardo; Ericsson, Marie; Illuminati, Fabrizio

    2007-01-01

    Quantum mechanics imposes 'monogamy' constraints on the sharing of entanglement. We show that, despite these limitations, entanglement can be fully 'promiscuous', i.e., simultaneously present in unlimited two-body and many-body forms in states living in an infinite-dimensional Hilbert space. Monogamy just bounds the divergence rate of the various entanglement contributions. This is demonstrated in simple families of N-mode (N≥4) Gaussian states of light fields or atomic ensembles, which therefore enable infinitely more freedom in the distribution of information, as opposed to systems of individual qubits. Such a finding is of importance for the quantification, understanding, and potential exploitation of shared quantum correlations in continuous variable systems. We discuss how promiscuity gradually arises when considering simple families of discrete variable states, with increasing Hilbert space dimension towards the continuous variable limit. Such models are somehow analogous to Gaussian states with asymptotically diverging, but finite, squeezing. In this respect, we find that non-Gaussian states (which in general are more entangled than Gaussian states) exhibit also the interesting feature that their entanglement is more shareable: in the non-Gaussian multipartite arena, unlimited promiscuity can be already achieved among three entangled parties, while this is impossible for Gaussian, even infinitely squeezed states

  16. The Suzuki–Miyaura Cross-Coupling as a Versatile Tool for Peptide Diversification and Cyclization

    Directory of Open Access Journals (Sweden)

    Tom Willemse

    2017-02-01

    Full Text Available The (site-selective derivatization of amino acids and peptides represents an attractive field with potential applications in the establishment of structure–activity relationships and labeling of bioactive compounds. In this respect, bioorthogonal cross-coupling reactions provide valuable means for ready access to peptide analogues with diversified structure and function. Due to the complex and chiral nature of peptides, mild reaction conditions are preferred; hence, a suitable cross-coupling reaction is required for the chemical modification of these challenging substrates. The Suzuki reaction, involving organoboron species, is appropriate given the stability and environmentally benign nature of these reactants and their amenability to be applied in (partial aqueous reaction conditions, an expected requirement upon the derivatization of peptides. Concerning the halogenated reaction partner, residues bearing halogen moieties can either be introduced directly as halogenated amino acids during solid-phase peptide synthesis (SPPS or genetically encoded into larger proteins. A reversed approach building in boron in the peptidic backbone is also possible. Furthermore, based on this complementarity, cyclic peptides can be prepared by halogenation, and borylation of two amino acid side chains present within the same peptidic substrate. Here, the Suzuki–Miyaura reaction is a tool to induce the desired cyclization. In this review, we discuss diverse amino acid and peptide-based applications explored by means of this extremely versatile cross-coupling reaction. With the advent of peptide-based drugs, versatile bioorthogonal conversions on these substrates have become highly valuable.

  17. Probing ADAMTS13 substrate specificity using phage display.

    Directory of Open Access Journals (Sweden)

    Karl C Desch

    Full Text Available Von Willebrand factor (VWF is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2' and P11', for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13-VWF exosite interactions outside of VWF73.

  18. Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors.

    Science.gov (United States)

    Thorsell, Ann-Gerd; Ekblad, Torun; Karlberg, Tobias; Löw, Mirjam; Pinto, Ana Filipa; Trésaugues, Lionel; Moche, Martin; Cohen, Michael S; Schüler, Herwig

    2017-02-23

    Selective inhibitors could help unveil the mechanisms by which inhibition of poly(ADP-ribose) polymerases (PARPs) elicits clinical benefits in cancer therapy. We profiled 10 clinical PARP inhibitors and commonly used research tools for their inhibition of multiple PARP enzymes. We also determined crystal structures of these compounds bound to PARP1 or PARP2. Veliparib and niraparib are selective inhibitors of PARP1 and PARP2; olaparib, rucaparib, and talazoparib are more potent inhibitors of PARP1 but are less selective. PJ34 and UPF1069 are broad PARP inhibitors; PJ34 inserts a flexible moiety into hydrophobic subpockets in various ADP-ribosyltransferases. XAV939 is a promiscuous tankyrase inhibitor and a potent inhibitor of PARP1 in vitro and in cells, whereas IWR1 and AZ-6102 are tankyrase selective. Our biochemical and structural analysis of PARP inhibitor potencies establishes a molecular basis for either selectivity or promiscuity and provides a benchmark for experimental design in assessment of PARP inhibitor effects.

  19. Substrate- and Cofactor-independent Inhibition of Histone Demethylase KDM4C

    DEFF Research Database (Denmark)

    Leurs, Ulrike; Lohse, Brian; Rand, Kasper Dyrberg

    2014-01-01

    Inhibition of histone demethylases has within recent years advanced into a new strategy for treating cancer and other diseases. Targeting specific histone demethylases can be challenging as the active sites of KDM1A-B and KDM-4A-D histone demethylases, respectively, are highly conserved. Most...... inhibitors developed up-to-date target either the cofactor- or substrate-binding sites of these enzymes, resulting in a lack of selectivity and off-target effects. This study describes the discovery of the first peptide-based inhibitors of KDM4 histone demethylases that do not share the histone peptide...... sequence, or inhibit through substrate competition. Through screening of DNA-encoded peptide libraries against KDM1 and -4 histone demethylases by phage display, two cyclic peptides targeting the histone demethylase KDM4C were identified and developed as inhibitors by amino acid replacement, truncation...

  20. Site-selective modification of peptides: From "customizable units" to novel α-aryl and α-alkyl glycine derivatives, and components of branched peptides.

    Science.gov (United States)

    Romero-Estudillo, Iván; Saavedra, Carlos; Boto, Alicia; Álvarez, Eleuterio

    2015-09-01

    The creation of peptide libraries by site-selective modification of a few peptide substrates would increase the efficiency of discovery processes, but still is a real synthetic challenge. The site-selective modification of small peptides at serine or threonine residues, by using a short scission-addition procedure, allows the preparation of peptides with unnatural α-aryl glycines. In a similar way, the scission of hydroxyproline residues is the key step in the production of optically pure α-alkyl glycines which are precursors or components of branched peptides. With these versatile processes, a single peptide can be transformed into a variety of peptide derivatives. The process takes place under mild conditions, and good global yields are obtained. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 104: 650-662, 2015. © 2015 Wiley Periodicals, Inc.

  1. Dual-affinity peptides to generate dense surface coverages of nanoparticles

    International Nuclear Information System (INIS)

    Del Re, Julia; Blum, Amy Szuchmacher

    2014-01-01

    Graphical abstract: - Highlights: • Stable nanoparticles were created with the Flg-A3 fusion peptide as a ligand. • Interactions of transition metal ions with Flg control aggregation of the nanoparticles in solution. • The QBP1-A3 fusion peptide improves surface attachment of gold nanoparticles. • Solution pre-aggregation of nanoparticles results in dense surface coverage. - Abstract: Depositing gold nanoparticles is of great interest because of the many potential applications of nanoparticle films; however, generating dense surface nanoparticle coverage remains a difficult challenge. Using dual-affinity peptides we have synthesized gold nanoparticles and then pre-aggregated the particles in solution via interactions with metal ions. These nanoparticle aggregates were then deposited onto silicon dioxide surfaces using another dual-affinity peptide to control binding to the substrate. The results demonstrate that when divalent ions like Zn 2+ or Ni 2+ are used, densely packed gold nanoparticle monolayers are formed on the silicon dioxide substrate, which may have applications in fields like molecular electronics

  2. Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12

    Energy Technology Data Exchange (ETDEWEB)

    Chan,K.; Fedorov, A.; Almo, S.; Gerlt, J.

    2008-01-01

    Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies of d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the

  3. Probing ADAMTS13 Substrate Specificity using Phage Display

    Science.gov (United States)

    Desch, Karl C.; Kretz, Colin; Yee, Andrew; Gildersleeve, Robert; Metzger, Kristin; Agrawal, Nidhi; Cheng, Jane; Ginsburg, David

    2015-01-01

    Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2’ and P11’, for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13–VWF exosite interactions outside of VWF73. PMID:25849793

  4. Targeting Biological Sensing with Commercial SERS Substrates

    Science.gov (United States)

    2012-09-01

    substrate substrate. Sl x 4 mm waf urement to re ossible contam substrates, fiv resented as an e being used f tigated, and nam eived in an ac cations...targeted nanotechnology-based inhalation co-delivery of anticancer drugs and siRNA," Journal of Drug Targeting, 900-914 (2011). [35] Jong , H.J., Na...J.H., Jin, B.S., Lee, W.K., Lee, W.H., Jung, H.J., Kim , S.C., Lim, S.H., Yu, Y.G., "Identification of Dinitrotoluene Selective Peptides by Phage

  5. SANDPUMA: ensemble predictions of nonribosomal peptide chemistry reveal biosynthetic diversity across Actinobacteria

    NARCIS (Netherlands)

    Chevrette, Marc G.; Aicheler, Fabian; Kohlbacher, Oliver; Currie, Cameron R.; Medema, M.H.

    2017-01-01

    Nonribosomally synthesized peptides (NRPs) are natural products with widespread applications in medicine and biotechnology. Many algorithms have been developed to predict the substrate specificities of nonribosomal peptide synthetase adenylation (A) domains from DNA sequences, which enables

  6. Competition between bound and free peptides in an ELISA-based procedure that assays peptides derived from protein digests

    Directory of Open Access Journals (Sweden)

    Pace Umberto

    2006-05-01

    Full Text Available Abstract Background We describe an ELISA-based method that can be used to identify and quantitate proteins in biological samples. In this method, peptides in solution, derived from proteolytic digests of the sample, compete with substrate-attached synthetic peptides for antibodies, also in solution, generated against the chosen peptides. The peptides used for the ELISA are chosen on the basis of their being (i products of the proteolytic (e.g. tryptic digestion of the protein to be identified and (ii unique to the target protein, as far as one can know from the published sequences. Results In this paper we describe the competition assay and we define the optimal conditions for the most effective assay. We have performed an analysis of the kinetics of interaction between the four components of the assay: the plastic substratum to which the peptide is bound, the bound peptide itself, the competing added peptide, and the antibody that is specific for the peptide and we compare the results of theoretical simulations to the actual data in some model systems. Conclusion The data suggest that the peptides bind to the plastic substratum in more than one conformation and that, once bound, the peptide displays different affinities for the antibody, depending on how it has bound to the plate

  7. Peptides for functionalization of InP semiconductors.

    Science.gov (United States)

    Estephan, Elias; Saab, Marie-belle; Larroque, Christian; Martin, Marta; Olsson, Fredrik; Lourdudoss, Sebastian; Gergely, Csilla

    2009-09-15

    The challenge is to achieve high specificity in molecular sensing by proper functionalization of micro/nano-structured semiconductors by peptides that reveal specific recognition for these structures. Here we report on surface modification of the InP semiconductors by adhesion peptides produced by the phage display technique. An M13 bacteriophage library has been used to screen 10(10) different peptides against the InP(001) and the InP(111) surfaces to finally isolate specific peptides for each orientation of the InP. MALDI-TOF/TOF mass spectrometry has been employed to study real affinity of the peptide towards the InP surfaces. The peptides serve for controlled placement of biotin onto InP to bind then streptavidin. Our Atomic Force Microscopy study revealed a total surface coverage of molecules when the InP surface was functionalized by its specific biotinylated peptide (YAIKGPSHFRPS). Finally, fluorescence microscopy has been employed to demonstrate the preferential attachment of the peptide onto a micro-patterned InP surface. Use of substrate specific peptides could present an alternative solution for the problems encountered in the actually existing sensing methods and molecular self-assembly due to the unwanted unspecific interactions.

  8. Enzymatic Detoxication, Conformational Selection, and the Role of Molten Globule Active Sites*

    Science.gov (United States)

    Honaker, Matthew T.; Acchione, Mauro; Zhang, Wei; Mannervik, Bengt; Atkins, William M.

    2013-01-01

    The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning “induced fit” versus “conformational selection” has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1–1 (GSTA1–1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1–1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that “local” molten globule behavior optimizes detoxication enzymes. PMID:23649628

  9. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang

    2014-01-01

    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...... pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending......, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity...

  10. The Post-polyketide Synthase Steps in iso-Migrastatin Biosynthesis Featuring Tailoring Enzymes with Broad Substrate Specificity

    Science.gov (United States)

    Ma, Ming; Kwong, Thomas; Lim, Si-Kyu; Ju, Jianhua; Lohman, Jeremy R.; Shen, Ben

    2013-01-01

    The iso-migrastatin (iso-MGS) biosynthetic gene cluster from Streptomyces platensis NRRL 18993 consists of 11 genes, featuring an acyltransferase (AT)-less type I polyketide synthase (PKS) and three tailoring enzymes MgsIJK. Systematic inactivation of mgsIJK in S. platensis enabled us to (i) identify two nascent products (10 and 13) of the iso-MGS AT-less type I PKS, establishing an unprecedented novel feature for AT-less type I PKSs, and (ii) account for the formation of all known post-PKS biosynthetic intermediates (10-17) generated by the three tailoring enzymes MgsIJK, which possessed significant substrate promiscuities. PMID:23394593

  11. Rapid Analysis of Protein Farnesyltransferase Substrate Specificity Using Peptide Libraries and Isoprenoid Diphosphate Analogues

    OpenAIRE

    Wang, Yen-Chih; Dozier, Jonathan K.; Beese, Lorena S.; Distefano, Mark D.

    2014-01-01

    Protein farnesytransferase (PFTase) catalyzes the farnesylation of proteins with a carboxy-terminal tetrapeptide sequence denoted as a Ca1a2X box. To explore the specificity of this enzyme, an important therapeutic target, solid-phase peptide synthesis in concert with a peptide inversion strategy was used to prepare two libraries, each containing 380 peptides. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequen...

  12. Convenience polyandry or convenience polygyny? Costly sex under female control in a promiscuous primate

    OpenAIRE

    Huchard, Elise; Canale, Cindy I.; Le Gros, Chloé; Perret, Martine; Henry, Pierre-Yves; Kappeler, Peter M.

    2011-01-01

    Classic sex roles depict females as choosy, but polyandry is widespread. Empirical attempts to understand the evolution of polyandry have often focused on its adaptive value to females, whereas ‘convenience polyandry’ might simply decrease the costs of sexual harassment. We tested whether constraint-free female strategies favour promiscuity over mating selectivity through an original experimental design. We investigated variation in mating behaviour in response to a reversible alteration of s...

  13. Structural basis for polyspecificity in the POT family of proton-coupled oligopeptide transporters

    DEFF Research Database (Denmark)

    Lyons, Joseph A.; Parker, Joanne L.; Solcan, Nicolae

    2014-01-01

    An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as exemplified by PepT1, the mammalian plasma membrane peptide transporter. Here, we present crystal structures of di‐ and tripeptide‐bound complexes of a bacterial homologue of PepT1, which reveal at least...... two mechanisms for peptide recognition that operate within a single, centrally located binding site. The dipeptide was orientated laterally in the binding site, whereas the tripeptide revealed an alternative vertical binding mode. The co‐crystal structures combined with functional studies reveal...... that biochemically distinct peptide‐binding sites likely operate within the POT/PTR family of proton‐coupled symporters and suggest that transport promiscuity has arisen in part through the ability of the binding site to accommodate peptides in multiple orientations for transport...

  14. Structural Simulation of MHC-peptide Interactions using T-cell Epitope in Iron-acquisition Protein of N. meningitides for Vaccine Design

    Directory of Open Access Journals (Sweden)

    Namrata Mishra

    2010-12-01

    Full Text Available The present work uses a structural simulation approach to identify the potential target vaccine candidates or T cell epitopes (antigenic region that can activate T cell response in two iron acquisition proteins from Neisseria. An iron regulated outer membrane protein frpB: extracellular, [NMB1988], and a Major ferric Iron-binding protein fbpA: periplasmic, [NMB0634] critical for the survival of the pathogen in the host were used. Ten novel promiscuous epitopes from the two iron acquisition proteins were identified using bioinformatics interface. Of these epitopes, 630VQKAVGSIL638 present on frpB with high binding affinity for allele HLA*DR1 was identified with an anchor position at P2, an aliphatic residue at P4 and glycine at P6 making it thereby a potential quality choice for linking peptide-loaded MHC dynamics to T-cell activation and vaccine constructs. The feasibility and structural binding of predicted peptide to the respective HLA allele was investigated by molecular modeling and template-based structural simulation. The conformational properties of the linear peptide were investigated by molecular dynamics using GROMOS96 package and Swiss PDB viewer.

  15. Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli.

    Science.gov (United States)

    Wu, Meizhi; Zhao, Lin; Zhu, Lei; Chen, Zhange; Li, Huangjin

    2013-03-01

    Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. QM/MM and classical molecular dynamics simulation of histidine-tagged peptide immobilization on nickel surface

    Energy Technology Data Exchange (ETDEWEB)

    Yang Zhenyu [State Key Laboratory of Nonlinear Mechanics (LNM), Institute of Mechanics, Chinese Academy of Sciences, Beijing 100080(China); Zhao Yapu [State Key Laboratory of Nonlinear Mechanics (LNM), Institute of Mechanics, Chinese Academy of Sciences, Beijing 100080 (China)]. E-mail: yzhao@lnm.imech.ac.cn

    2006-05-15

    The hybrid quantum mechanics (QM) and molecular mechanics (MM) method is employed to simulate the His-tagged peptide adsorption to ionized region of nickel surface. Based on the previous experiments, the peptide interaction with one Ni ion is considered. In the QM/MM calculation, the imidazoles on the side chain of the peptide and the metal ion with several neighboring water molecules are treated as QM part calculated by 'GAMESS', and the rest atoms are treated as MM part calculated by 'TINKER'. The integrated molecular orbital/molecular mechanics (IMOMM) method is used to deal with the QM part with the transitional metal. By using the QM/MM method, we optimize the structure of the synthetic peptide chelating with a Ni ion. Different chelate structures are considered. The geometry parameters of the QM subsystem we obtained by QM/MM calculation are consistent with the available experimental results. We also perform a classical molecular dynamics (MD) simulation with the experimental parameters for the synthetic peptide adsorption on a neutral Ni(1 0 0) surface. We find that half of the His-tags are almost parallel with the substrate, which enhance the binding strength. Peeling of the peptide from the Ni substrate is simulated in the aqueous solvent and in vacuum, respectively. The critical peeling forces in the two environments are obtained. The results show that the imidazole rings are attached to the substrate more tightly than other bases in this peptide.

  17. Identification of Cyclin A Binders with a Fluorescent Peptide Sensor.

    Science.gov (United States)

    Pazos, Elena; Mascareñas, José L; Vázquez, M Eugenio

    2016-01-01

    A peptide sensor that integrates the 4-dimethylaminophthalimide (4-DMAP) fluorophore in a short cyclin A binding sequence displays a large fluorescence emission increase upon interacting with the cyclin A Binding Groove (CBG). Competitive displacement assays of this probe allow the straightforward identification of peptides that interact with the CBG, which could potentially block the recognition of CDK/cyclin A kinase substrates.

  18. Galactokinase promiscuity: a question of flexibility?

    Science.gov (United States)

    McAuley, Megan; Kristiansson, Helena; Huang, Meilan; Pey, Angel L; Timson, David J

    2016-02-01

    Galactokinase catalyses the first committed step of the Leloir pathway, i.e. the ATP-dependent phosphorylation of α-D-galactose at C1-OH. Reduced galactokinase activity results in the inherited metabolic disease type II galactosaemia. However, inhibition of galactokinase is considered a viable approach to treating more severe forms of galactosaemia (types I and III). Considerable progress has been made in the identification of high affinity, selective inhibitors. Although the structure of galactokinase from a variety of species is known, its catalytic mechanism remains uncertain. Although the bulk of evidence suggests that the reaction proceeds via an active site base mechanism, some experimental and theoretical studies contradict this. The enzyme has potential as a biocatalyst in the production of sugar 1-phosphates. This potential is limited by its high specificity. A variety of approaches have been taken to identify galactokinase variants which are more promiscuous. These have broadened galactokinase's specificity to include a wide range of D- and L-sugars. Initial studies suggest that some of these alterations result in increased flexibility at the active site. It is suggested that modulation of protein flexibility is at least as important as structural modifications in determining the success or failure of enzyme engineering. © 2016 Authors; published by Portland Press Limited.

  19. Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates.

    Science.gov (United States)

    Adediran, S A; Kumar, Ish; Nagarajan, Rajesh; Sauvage, Eric; Pratt, R F

    2011-01-25

    The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.

  20. Nanoparticles and peptides: a fruitful liaison for biomimetic catalysis.

    Science.gov (United States)

    Stodulski, Maciej; Gulder, Tanja

    2012-11-05

    Inspired by nature: self-assembled peptide nanoparticles have been designed that accelerate ester hydrolysis (see picture; Cbz=carbobenzyloxy, NP=p-NO(2)-C(6) H(4)). The concerted interplay of the multivalent surface with the catalytically active peptide and the substrate at the same time combines several aspects decisive for the catalyst's efficiency, a property characteristic of enzymes. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Developing a capillary electrophoresis based method for dynamically monitoring enzyme cleavage activity using quantum dots-peptide assembly.

    Science.gov (United States)

    Wang, Jianhao; Fan, Jie; Liu, Li; Ding, Shumin; Liu, Xiaoqian; Wang, Jianpeng; Gao, Liqian; Chattopadhaya, Souvik; Miao, Peng; Xia, Jiang; Qiu, Lin; Jiang, Pengju

    2017-10-01

    Herein, a novel assay has been developed for monitoring PreScission protease (His-PSP) mediated enzyme cleavage of ATTO 590 labeled peptide substrate (ATTO-LEV). This novel method is based on combining the use of capillary electrophoresis and fluorescence detection (CE-FL) to dynamically monitor the enzyme cleavage activity. A multivalent peptide substrate was first constructed by immobilizing His-tagged ATTO 590 labeled peptide substrate (ATTO-LEVH6) onto the surface of CdSe/ZnS quantum dots (QDs). Once successfully immobilized, the novel multivalent peptide substrate resulted in the Förster resonance energy transfer (FRET) from QDs to ATTO 590. The ATTO-LEVH6-QD assembly was then incubated with His-PSP to study the proteolytic cleavage of surface bound ATTO-LEVH6 by CE-FL. Our data suggests that PreScission-mediated proteolytic cleavage is enzyme concentration- and incubation time-dependent. By combining capillary electrophoresis, QDs and FRET, our study herein not only provides a new method for the detection and dynamically monitoring of PSP enzyme cleavage activity, but also can be extended to the detection of many other enzymes and proteases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Substrate-driven mapping of the degradome by comparison of sequence logos.

    Directory of Open Access Journals (Sweden)

    Julian E Fuchs

    Full Text Available Sequence logos are frequently used to illustrate substrate preferences and specificity of proteases. Here, we employed the compiled substrates of the MEROPS database to introduce a novel metric for comparison of protease substrate preferences. The constructed similarity matrix of 62 proteases can be used to intuitively visualize similarities in protease substrate readout via principal component analysis and construction of protease specificity trees. Since our new metric is solely based on substrate data, we can engraft the protease tree including proteolytic enzymes of different evolutionary origin. Thereby, our analyses confirm pronounced overlaps in substrate recognition not only between proteases closely related on sequence basis but also between proteolytic enzymes of different evolutionary origin and catalytic type. To illustrate the applicability of our approach we analyze the distribution of targets of small molecules from the ChEMBL database in our substrate-based protease specificity trees. We observe a striking clustering of annotated targets in tree branches even though these grouped targets do not necessarily share similarity on protein sequence level. This highlights the value and applicability of knowledge acquired from peptide substrates in drug design of small molecules, e.g., for the prediction of off-target effects or drug repurposing. Consequently, our similarity metric allows to map the degradome and its associated drug target network via comparison of known substrate peptides. The substrate-driven view of protein-protein interfaces is not limited to the field of proteases but can be applied to any target class where a sufficient amount of known substrate data is available.

  3. MFPred: Rapid and accurate prediction of protein-peptide recognition multispecificity using self-consistent mean field theory.

    Directory of Open Access Journals (Sweden)

    Aliza B Rubenstein

    2017-06-01

    Full Text Available Multispecificity-the ability of a single receptor protein molecule to interact with multiple substrates-is a hallmark of molecular recognition at protein-protein and protein-peptide interfaces, including enzyme-substrate complexes. The ability to perform structure-based prediction of multispecificity would aid in the identification of novel enzyme substrates, protein interaction partners, and enable design of novel enzymes targeted towards alternative substrates. The relatively slow speed of current biophysical, structure-based methods limits their use for prediction and, especially, design of multispecificity. Here, we develop a rapid, flexible-backbone self-consistent mean field theory-based technique, MFPred, for multispecificity modeling at protein-peptide interfaces. We benchmark our method by predicting experimentally determined peptide specificity profiles for a range of receptors: protease and kinase enzymes, and protein recognition modules including SH2, SH3, MHC Class I and PDZ domains. We observe robust recapitulation of known specificities for all receptor-peptide complexes, and comparison with other methods shows that MFPred results in equivalent or better prediction accuracy with a ~10-1000-fold decrease in computational expense. We find that modeling bound peptide backbone flexibility is key to the observed accuracy of the method. We used MFPred for predicting with high accuracy the impact of receptor-side mutations on experimentally determined multispecificity of a protease enzyme. Our approach should enable the design of a wide range of altered receptor proteins with programmed multispecificities.

  4. Developing a Dissociative Nanocontainer for Peptide Drug Delivery

    Directory of Open Access Journals (Sweden)

    Patrick Kelly

    2015-10-01

    Full Text Available The potency, selectivity, and decreased side effects of bioactive peptides have propelled these agents to the forefront of pharmacological research. Peptides are especially promising for the treatment of neurological disorders and pain. However, delivery of peptide therapeutics often requires invasive techniques, which is a major obstacle to their widespread application. We have developed a tailored peptide drug delivery system in which the viral capsid of P22 bacteriophage is modified to serve as a tunable nanocontainer for the packaging and controlled release of bioactive peptides. Recent efforts have demonstrated that P22 nanocontainers can effectively encapsulate analgesic peptides and translocate them across blood-brain-barrier (BBB models. However, release of encapsulated peptides at their target site remains a challenge. Here a Ring Opening Metathesis Polymerization (ROMP reaction is applied to trigger P22 nanocontainer disassembly under physiological conditions. Specifically, the ROMP substrate norbornene (5-Norbornene-2-carboxylic acid is conjugated to the exterior of a loaded P22 nanocontainer and Grubbs II Catalyst is used to trigger the polymerization reaction leading to nanocontainer disassembly. Our results demonstrate initial attempts to characterize the ROMP-triggered release of cargo peptides from P22 nanocontainers. This work provides proof-of-concept for the construction of a triggerable peptide drug delivery system using viral nanocontainers.

  5. Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase.

    Science.gov (United States)

    Costa, Sara R; Marek, Magdalena; Axelsen, Kristian B; Theorin, Lisa; Pomorski, Thomas G; López-Marqués, Rosa L

    2016-06-01

    P-type ATPases of subfamily IV (P4-ATPases) constitute a major group of phospholipid flippases that form heteromeric complexes with members of the Cdc50 (cell division control 50) protein family. Some P4-ATPases interact specifically with only one β-subunit isoform, whereas others are promiscuous and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues, Asn(181) and Asn(231) Whereas mutation of Asn(231) seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn(181) disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys(86) and Cys(107) compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys(158) and Cys(172) has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications in the β-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  6. Nodulation and nitrogen uptake characteristics as selection indices for promiscuous soybean breeding in Africa

    International Nuclear Information System (INIS)

    Ababio, R.C.; Sanginga, K.E.; Dashiell, K.E.

    2001-01-01

    Selection for atmospheric dinitrogen fixation in promiscuous soybeans by indigenous rhizobia populations is usually determined indirectly based on visual assessment of nodulation. As the method has been criticized extensively, we have examined this assay in relation to other N 2 fixation indices by evaluating growth of promiscuous soybeans in four ecological zones of Nigeria. Nodulation was affected considerably by rainfall indigenous, rhizobia populations and soil P. The N 2 fixation indices measured, nodule development, apparent effectiveness, total N accumulation and N derived from fixation, varied among soybean genotypes. The visual nodule assessment index correlated with nodule weight (r = 0.74; P = 0.01), but had no significant correlation (r = 0.13) with proportion of apparent effective nodules. All nodule parameters, however, correlated significantly (r = 0.16-0.71; P=0.05) with total N accumulation and seed yield. The percent apparent effectiveness of nodules was a superior index to nodule score and nodule number but inferior to nodule mass. Total N accumulation patterns in the genotypes indicated that N 2 fixation in some genotypes could be substantial during the reproductive growth phase and may serve as suitable criteria for selection. Therefore, in low N soils, percent nodule effectiveness and total N accumulation may be used to augment the visual scores particularly for genotypes, which seem to nodulate profusely with indigenous rhizohia populations (author)

  7. Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides

    Directory of Open Access Journals (Sweden)

    Protti Maria

    2005-12-01

    Full Text Available Abstract Background MHC class I-peptide tetramers are currently utilised to characterize CD8+ T cell responses at single cell level. The generation and use of MHC class II tetramers to study antigen-specific CD4+ T cells appears less straightforward. Most MHC class II tetramers are produced with a homogeneously built-in peptide, reducing greatly their flexibility of use. We attempted the generation of "empty" functional HLA-DR*1101 tetramers, receptive for loading with synthetic peptides by incubation. No such reagent is in fact available for this HLA-DR allele, one of the most frequent in the Caucasian population. Results We compared soluble MHC class II-immunoglobulin fusion proteins (HLA-DR*1101-Ig with soluble MHC class II protein fused with an optimised Bir site for enzymatic biotynilation (HLA-DR*1101-Bir, both produced in insect cells. The molecules were multimerised by binding fluorochrome-protein A or fluorochrome-streptavidin, respectively. We find that HLA-DR*1101-Bir molecules are superior to the HLA-DR*1101-Ig ones both in biochemical and functional terms. HLA-DR*1101-Bir molecules can be pulsed with at least three different promiscuous peptide epitopes, derived from Tetanus Toxoid, influenza HA and the tumour associated antigen MAGE-3 respectively, to stain specific CD4+ T cells. Both staining temperature and activation state of CD4+ T cells are critical for the binding of peptide-pulsed HLA-DR*1101-Bir to the cognate TCR. Conclusion It is therefore possible to generate a soluble recombinant HLA-DR*1101 backbone that is receptive for loading with different peptides to stain specific CD4+ T cells. As shown for other HLA-DR alleles, we confirm that not all the strategies to produce soluble HLA-DR*1101 multimers are equivalent.

  8. Substrate optimization and clinical validation of reporter peptides for MS-based protease profiling in serum specimens: a new approach for diagnosis of malignant disease.

    Science.gov (United States)

    Yepes, Diego; Jacob, Anette; Dauber, Marc; Costina, Victor; Hofheinz, Ralf; Neumaier, Michael; Findeisen, Peter

    2011-07-01

    The progression of many solid tumors is characterized by the release of tumor-associated proteases, such as cancer procoagulant, MMP2 and MMP7. Consequently, the detection of tumor-specific proteolytic activity in serum specimens has recently been proposed as a new diagnostic tool in oncology. However, tumor-associated proteases are highly diluted in serum specimens and it is challenging to identify substrates that are specifically cleaved. In this study, we describe the systematic optimization of a synthetic peptide substrate using a positional scanning synthetic combinatorial library (PS-SCL) approach. The initial reporter peptide (RP) comprises of the cleavage site, WKPYDAAD, that is part of the coagulation factor X, the natural substrate of the tumor-associated cysteine protease cancer procoagulant (EC 3.4.22.26). Specifically, the amino acid substitution of aspartatic acid (D) in position P1' against asparagine (N) improved the processing of respective RPs in serum specimens from patients with colorectal tumors compared to healthy controls. Proteolytic fragments of RPs accumulated during prolonged incubation with serum specimens and were quantified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Finally, the optimized RP with the cleaved motif WKPYNAAD was combined with the RPs, VPLSLTMG and IPVSLRSG, that were cleaved by the tumor-associated proteases, MMP2 and MMP7, respectively. The diagnostic accuracy of MS-based protease profiling was evaluated for this triplex RP mix in a cohort of 50 serum specimens equally divided into colorectal cancer patients and healthy control individuals. Multiparametric analysis showed an AUC value of 0.90 for the receiver operating characteristic curve and was superior to the classification accuracy of the single markers. Our results demonstrate that RPs for MS-based protease profiling can systematically be optimized with a PS-SCL. Furthermore, the combination of different RPs can

  9. High-throughput kinase assays with protein substrates using fluorescent polymer superquenching

    Directory of Open Access Journals (Sweden)

    Weatherford Wendy

    2005-05-01

    Full Text Available Abstract Background High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Results Using a modified QTL Lightspeed™ assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP, Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1. Phosphorylation of the proteins was detected by Protein Kinase Cα (PKCα and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4. Enzyme inhibition yielded IC50 values that were comparable to those obtained using

  10. High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.

    Science.gov (United States)

    Rininsland, Frauke; Stankewicz, Casey; Weatherford, Wendy; McBranch, Duncan

    2005-05-31

    High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that

  11. Regulation of the mesolimbic dopamine circuit by feeding peptides.

    Science.gov (United States)

    Liu, S; Borgland, S L

    2015-03-19

    Polypeptides produced in the gastrointestinal tract, stomach, adipocytes, pancreas and brain that influence food intake are referred to as 'feeding-related' peptides. Most peptides that influence feeding exert an inhibitory effect (anorexigenic peptides). In contrast, only a few exert a stimulating effect (orexigenic peptides), such as ghrelin. Homeostatic feeding refers to when food consumed matches energy deficits. However, in western society where access to palatable energy-dense food is nearly unlimited, food is mostly consumed for non-homeostatic reasons. Emerging evidence implicates the mesocorticolimbic circuitry, including dopamine neurons of the ventral tegmental area (VTA), as a key substrate for non-homeostatic feeding. VTA dopamine neurons encode cues that predict rewards and phasic release of dopamine in the ventral striatum motivates animals to forage for food. To elucidate how feeding-related peptides regulate reward pathways is of importance to reveal the mechanisms underlying non-homeostatic or hedonic feeding. Here, we review the current knowledge of how anorexigenic peptides and orexigenic peptides act within the VTA. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  12. Insights into Substrate Specificity and Metal Activation of Mammalian Tetrahedral Aspartyl Aminopeptidase

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yuanyuan; Farquhar, Erik R.; Chance, Mark R.; Palczewski, Krzysztof; Kiser, Philip D. (Case Western)

    2012-07-11

    Aminopeptidases are key enzymes involved in the regulation of signaling peptide activity. Here, we present a detailed biochemical and structural analysis of an evolutionary highly conserved aspartyl aminopeptidase called DNPEP. We show that this peptidase can cleave multiple physiologically relevant substrates, including angiotensins, and thus may play a key role in regulating neuron function. Using a combination of x-ray crystallography, x-ray absorption spectroscopy, and single particle electron microscopy analysis, we provide the first detailed structural analysis of DNPEP. We show that this enzyme possesses a binuclear zinc-active site in which one of the zinc ions is readily exchangeable with other divalent cations such as manganese, which strongly stimulates the enzymatic activity of the protein. The plasticity of this metal-binding site suggests a mechanism for regulation of DNPEP activity. We also demonstrate that DNPEP assembles into a functionally relevant tetrahedral complex that restricts access of peptide substrates to the active site. These structural data allow rationalization of the enzyme's preference for short peptide substrates with N-terminal acidic residues. This study provides a structural basis for understanding the physiology and bioinorganic chemistry of DNPEP and other M18 family aminopeptidases.

  13. Fiscal 1993 report on results of R and D on innovative technology for producing advanced biomaterial. Peptide applied carbon dioxide fixation/effective utilization technology (First volume); 1993 nendo senshin bio zairyo no sosei kako gijutsu no kenkyu kaihatsu seika hokokusho. 1. Peptide oyo nisanka tanso koteika yuko riyo gijutsu

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1994-03-01

    Technology is being developed for preparing functional materials by synthesizing new functional peptides in which non-natural amino acid needed for the functional manifestation is introduced, and by modifying the surface of a base plate such as silica glass by using such peptides. Activities were conducted in the three areas of (1) creation of functional molecules, (2) materialization technology, and (3) comprehensive investigation and research; the activities were carried out independently and parallelly in the first two areas. In (1), design technique for the structures and functions of peptides was developed, as were conformational control technique, synthesis of peptides having optical/electronic functions, peptide synthesis by an enzyme method, and R and D on introduction of non-natural amino acid into peptides; in (2), element technologies were developed such as substrate forming technique (pattern forming and thin film forming technology), substrate modification technique, development of reagent for binding peptide onto a substrate, and R and D on creation of biomaterials having molecular recognition function and its stabilization technique. In (3), progress control in promoting themes and a meeting for exchanging information were conducted, while survey on related element technologies was systematically and comprehensively carried out. (NEDO)

  14. Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhigang [Wayne State Univ., Detroit, MI (United States); Case Western Reserve Univ., Cleveland, OH (United States); Harbor Hospital Baltimore, MD (United States); Wang, Yong [Wayne State Univ., Detroit, MI (United States); Yedidi, Ravikiran S. [Wayne State Univ., Detroit, MI (United States); National Institutes of Health, Bethesda, MD (United States); Dewdney, Tamaria G. [Wayne State Univ., Detroit, MI (United States); Reiter, Samuel J. [Wayne State Univ., Detroit, MI (United States); Brunzelle, Joseph S. [Northwestern Univ. Feinberg School of Medicine, Chicago, IL (United States); Kovari, Iulia A. [Wayne State Univ., Detroit, MI (United States); Kovari, Ladislau C. [Wayne State Univ., Detroit, MI (United States)

    2012-12-19

    Success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. Additionally, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.

  15. Peptide inhibitors of botulinum neurotoxin by mRNA display

    International Nuclear Information System (INIS)

    Yiadom, Kwabena P.A.B.; Muhie, Seid; Yang, David C.H.

    2005-01-01

    Botulinum neurotoxins (BoNTs) are extremely toxic. The metalloproteases associated with the toxins cleave proteins essential for neurotransmitter secretion. Inhibitors of the metalloprotease are currently sought to control the toxicity of BoNTs. Toward that goal, we produced a synthetic cDNA for the expression and purification of the metalloprotease of BoNT/A in Escherichia coli as a biotin-ubiquitin fusion protein, and constructed a combinatorial peptide library to screen for BoNT/A light chain inhibitors using mRNA display. A protease assay was developed using immobilized intact SNAP-25 as the substrate. The new peptide inhibitors showed a 10-fold increase in affinity to BoNT/A light chain than the parent peptide. Interestingly, the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues. The results suggest that mRNA display may provide a general approach in developing peptide inhibitors of BoNTs

  16. Rational design and application of responsive α-helical peptide hydrogels

    Science.gov (United States)

    Banwell, Eleanor F.; Abelardo, Edgardo S.; Adams, Dave J.; Birchall, Martin A.; Corrigan, Adam; Donald, Athene M.; Kirkland, Mark; Serpell, Louise C.; Butler, Michael F.; Woolfson, Derek N.

    2009-01-01

    Biocompatible hydrogels have a wide variety of potential applications in biotechnology and medicine, such as the controlled delivery and release of cells, cosmetics and drugs; and as supports for cell growth and tissue engineering1. Rational peptide design and engineering are emerging as promising new routes to such functional biomaterials2-4. Here we present the first examples of rationally designed and fully characterized self-assembling hydrogels based on standard linear peptides with purely α-helical structures, which we call hydrogelating self-assembling fibres (hSAFs). These form spanning networks of α-helical fibrils that interact to give self-supporting physical hydrogels of >99% water content. The peptide sequences can be engineered to alter the underlying mechanism of gelation and, consequently, the hydrogel properties. Interestingly, for example, those with hydrogen-bonded networks melt upon heating, whereas those formed via hydrophobic interactions strengthen when warmed. The hSAFs are dual-peptide systems that only gel on mixing, which gives tight control over assembly5. These properties raise possibilities for using the hSAFs as substrates in cell culture. We have tested this in comparison with the widely used Matrigel substrate, and demonstrate that, like Matrigel, hSAFs support both growth and differentiation of rat adrenal pheochromocytoma cells for sustained periods in culture. PMID:19543314

  17. Self-assembly of fibronectin mimetic peptide-amphiphile nanofibers

    Science.gov (United States)

    Rexeisen, Emilie Lynn

    Many therapeutic strategies incorporate peptides into their designs to mimic the natural protein ligands found in vivo. A few examples are the short peptide sequences RGD and PHSRN that mimic the primary and synergy-binding domains of the extracellular matrix protein, fibronectin, which is recognized by the cell surface receptor, alpha5beta 1 integrin. Even though scaffold modification with biomimetic peptides remains one of the most promising approaches for tissue engineering, the use of these peptides in therapeutic tissue-engineered products and drug delivery systems available on the commercial market is limited because the peptides are not easily able to mimic the natural protein. The design of a peptide that can effectively target the alpha5beta1 integrin would greatly increase biomimetic scaffold therapeutic potential. A novel peptide containing both the RGD primary binding domain and PHSRN synergy-binding domain for fibronectin joined with the appropriate linker should bind alpha 5beta1 integrin more efficiently and lead to greater cell adhesion over RGD alone. Several fibronectin mimetic peptides were designed and coupled to dialkyl hydrocarbon tails to make peptide-amphiphiles. The peptides contained different linkers connecting the two binding domains and different spacers separating the hydrophobic tails from the hydrophilic headgroups. The peptide-amphiphiles were deposited on mica substrates using the Langmuir-Blodgett technique. Langmuir isotherms indicated that the peptide-amphiphiles that contained higher numbers of serine residues formed a more tightly packed monolayer, but the increased number of serines also made transferring the amphiphiles to the mica substrate more difficult. Atomic force microscopy (AFM) images of the bilayers showed that the headgroups might be bent, forming small divots in the surface. These divots may help expose the PHSRN synergy-binding domain. Parallel studies undertaken by fellow group members showed that human

  18. Designer Self-Assembling Peptide Nanofiber Scaffolds Containing Link Protein N-Terminal Peptide Induce Chondrogenesis of Rabbit Bone Marrow Stem Cells

    Directory of Open Access Journals (Sweden)

    Baichuan Wang

    2014-01-01

    Full Text Available Designer self-assembling peptide nanofiber hydrogel scaffolds have been considered as promising biomaterials for tissue engineering because of their excellent biocompatibility and biofunctionality. Our previous studies have shown that a novel designer functionalized self-assembling peptide nanofiber hydrogel scaffold (RLN/RADA16, LN-NS containing N-terminal peptide sequence of link protein (link N can promote nucleus pulposus cells (NPCs adhesion and three-dimensional (3D migration and stimulate biosynthesis of type II collagen and aggrecan by NPCs in vitro. The present study has extended these investigations to determine the effects of this functionalized LN-NS on bone marrow stem cells (BMSCs, a potential cell source for NP regeneration. Although the functionalized LN-NS cannot promote BMSCs proliferation, it significantly promotes BMSCs adhesion compared with that of the pure RADA16 hydrogel scaffold. Moreover, the functionalized LN-NS remarkably stimulates biosynthesis and deposition of type II collagen and aggrecan. These data demonstrate that the functionalized peptide nanofiber hydrogel scaffold containing link N peptide as a potential matrix substrate will be very useful in the NP tissue regeneration.

  19. Di/tri-peptide transporters as drug delivery targets

    DEFF Research Database (Denmark)

    Nielsen, C U; Brodin, Birger

    2003-01-01

    -dependent, and the transporters thus belong to the Proton-dependent Oligopeptide Transporter (POT)-family. The transporters are not drug targets per se, however due to their uniquely broad substrate specificity; they have proved to be relevant drug targets at the level of drug transport. Drug molecules such as oral active beta....../tri-peptide transporters from vesicular storages 3) changes in gene transcription/mRNA stability. The aim of the present review is to discuss physiological, patho-physiological and drug-induced regulation of di/tri-peptide transporter mediated transport....

  20. Associations between dopamine D4 receptor gene variation with both infidelity and sexual promiscuity.

    Science.gov (United States)

    Garcia, Justin R; MacKillop, James; Aller, Edward L; Merriwether, Ann M; Wilson, David Sloan; Lum, J Koji

    2010-11-30

    Human sexual behavior is highly variable both within and between populations. While sex-related characteristics and sexual behavior are central to evolutionary theory (sexual selection), little is known about the genetic bases of individual variation in sexual behavior. The variable number tandem repeats (VNTR) polymorphism in exon III of the human dopamine D4 receptor gene (DRD4) has been correlated with an array of behavioral phenotypes and may be predicatively responsible for variation in motivating some sexual behaviors, particularly promiscuity and infidelity. We administered an anonymous survey on personal history of sexual behavior and intimate relationships to 181 young adults. We also collected buccal wash samples and genotyped the DRD4 VNTR. Here we show that individuals with at least one 7-repeat allele (7R+) report a greater categorical rate of promiscuous sexual behavior (i.e., having ever had a "one-night stand") and report a more than 50% increase in instances of sexual infidelity. DRD4 VNTR genotype varies considerably within and among populations and has been subject to relatively recent, local selective pressures. Individual differences in sexual behavior are likely partially mediated by individual genetic variation in genes coding for motivation and reward in the brain. Conceptualizing these findings in terms of r/K selection theory suggests a mechanism for selective pressure for and against the 7R+ genotype that may explain the considerable global allelic variation for this polymorphism.

  1. Associations between Dopamine D4 Receptor Gene Variation with Both Infidelity and Sexual Promiscuity

    Science.gov (United States)

    Garcia, Justin R.; MacKillop, James; Aller, Edward L.; Merriwether, Ann M.; Wilson, David Sloan; Lum, J. Koji

    2010-01-01

    Background Human sexual behavior is highly variable both within and between populations. While sex-related characteristics and sexual behavior are central to evolutionary theory (sexual selection), little is known about the genetic bases of individual variation in sexual behavior. The variable number tandem repeats (VNTR) polymorphism in exon III of the human dopamine D4 receptor gene (DRD4) has been correlated with an array of behavioral phenotypes and may be predicatively responsible for variation in motivating some sexual behaviors, particularly promiscuity and infidelity. Methodology/Principal Findings We administered an anonymous survey on personal history of sexual behavior and intimate relationships to 181 young adults. We also collected buccal wash samples and genotyped the DRD4 VNTR. Here we show that individuals with at least one 7-repeat allele (7R+) report a greater categorical rate of promiscuous sexual behavior (i.e., having ever had a “one-night stand”) and report a more than 50% increase in instances of sexual infidelity. Conclusions/Significance DRD4 VNTR genotype varies considerably within and among populations and has been subject to relatively recent, local selective pressures. Individual differences in sexual behavior are likely partially mediated by individual genetic variation in genes coding for motivation and reward in the brain. Conceptualizing these findings in terms of r/K selection theory suggests a mechanism for selective pressure for and against the 7R+ genotype that may explain the considerable global allelic variation for this polymorphism. PMID:21152404

  2. Associations between dopamine D4 receptor gene variation with both infidelity and sexual promiscuity.

    Directory of Open Access Journals (Sweden)

    Justin R Garcia

    2010-11-01

    Full Text Available Human sexual behavior is highly variable both within and between populations. While sex-related characteristics and sexual behavior are central to evolutionary theory (sexual selection, little is known about the genetic bases of individual variation in sexual behavior. The variable number tandem repeats (VNTR polymorphism in exon III of the human dopamine D4 receptor gene (DRD4 has been correlated with an array of behavioral phenotypes and may be predicatively responsible for variation in motivating some sexual behaviors, particularly promiscuity and infidelity.We administered an anonymous survey on personal history of sexual behavior and intimate relationships to 181 young adults. We also collected buccal wash samples and genotyped the DRD4 VNTR. Here we show that individuals with at least one 7-repeat allele (7R+ report a greater categorical rate of promiscuous sexual behavior (i.e., having ever had a "one-night stand" and report a more than 50% increase in instances of sexual infidelity.DRD4 VNTR genotype varies considerably within and among populations and has been subject to relatively recent, local selective pressures. Individual differences in sexual behavior are likely partially mediated by individual genetic variation in genes coding for motivation and reward in the brain. Conceptualizing these findings in terms of r/K selection theory suggests a mechanism for selective pressure for and against the 7R+ genotype that may explain the considerable global allelic variation for this polymorphism.

  3. Substrate-Directed Catalytic Selective Chemical Reactions.

    Science.gov (United States)

    Sawano, Takahiro; Yamamoto, Hisashi

    2018-05-04

    The development of highly efficient reactions at only the desired position is one of the most important subjects in organic chemistry. Most of the reactions in current organic chemistry are reagent- or catalyst-controlled reactions, and the regio- and stereoselectivity of the reactions are determined by the inherent nature of the reagent or catalyst. In sharp contrast, substrate-directed reaction determines the selectivity of the reactions by the functional group on the substrate and can strictly distinguish sterically and electronically similar multiple reaction sites in the substrate. In this Perspective, three topics of substrate-directed reaction are mainly reviewed: (1) directing group-assisted epoxidation of alkenes, (2) ring-opening reactions of epoxides by various nucleophiles, and (3) catalytic peptide synthesis. Our newly developed synthetic methods with new ligands including hydroxamic acid derived ligands realized not only highly efficient reactions but also pinpointed reactions at the expected position, demonstrating the substrate-directed reaction as a powerful method to achieve the desired regio- and stereoselective functionalization of molecules from different viewpoints of reagent- or catalyst-controlled reactions.

  4. Signal peptide of eosinophil cationic protein is toxic to cells lacking signal peptide peptidase

    International Nuclear Information System (INIS)

    Wu, C.-M.; Chang, Margaret Dah-Tsyr

    2004-01-01

    Eosinophil cationic protein (ECP) is a toxin secreted by activated human eosinophils. The properties of mature ECP have been well studied but those of the signal peptide of ECP (ECPsp) are not clear. In this study, several chimeric proteins containing N-terminal fusion of ECPsp were generated, and introduced into Escherichia coli, Pichia pastoris, and human epidermoid carcinoma cell line A431 to study the function of ECPsp. We found that expression of ECPsp chimeric proteins inhibited the growth of E. coli and P. pastoris but not A431 cells. Primary sequence analysis and in vitro transcription/translation of ECPsp have revealed that it is a potential substrate for human signal peptide peptidase (hSPP), an intramembrane protease located in endoplasmic reticulum. In addition, knockdown of the hSPP mRNA expression in ECPsp-eGFP/A431 cells caused the growth inhibitory effect, whereas complementally expression of hSPP in P. pastoris system rescued the cell growth. Taken together, we have demonstrated that ECPsp is a toxic signal peptide, and expression of hSPP protects the cells from growth inhibition

  5. Determination of conformation and orientation of immobilized peptides and proteins at buried interfaces

    Science.gov (United States)

    Shen, Lei; Ulrich, Nathan W.; Mello, Charlene M.; Chen, Zhan

    2015-01-01

    Surface immobilized peptides/proteins have important applications such as antimicrobial coating and biosensing. We report a study of such peptides/proteins using sum frequency generation vibrational spectroscopy and ATR-FTIR. Immobilization on surfaces via physical adsorption and chemical coupling revealed that structures of chemically immobilized peptides are determined by immobilization sites, chemical environments, and substrate surfaces. In addition, controlling enzyme orientation by engineering the surface immobilization site demonstrated that structures can be well-correlated to measured chemical activity. This research facilitates the development of immobilized peptides/proteins with improved activities by optimizing their surface orientation and structure.

  6. Design, synthesis and evaluation of a potent substrate analog inhibitor identified by scanning Ala/Phe mutagenesis, mimicking substrate co-evolution, against multidrug-resistant HIV-1 protease

    Energy Technology Data Exchange (ETDEWEB)

    Yedidi, Ravikiran S. [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Muhuhi, Joseck M. [Department of Chemistry, Wayne State University, Detroit, MI 48202 (United States); Liu, Zhigang [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Bencze, Krisztina Z. [Department of Chemistry, Fort Hays State University, Hays, KS 67601 (United States); Koupparis, Kyriacos [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Department of Chemistry, Wayne State University, Detroit, MI 48202 (United States); O’Connor, Carrie E.; Kovari, Iulia A. [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Spaller, Mark R. [Department of Chemistry, Wayne State University, Detroit, MI 48202 (United States); Kovari, Ladislau C., E-mail: kovari@med.wayne.edu [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States)

    2013-09-06

    Highlights: •Inhibitors against MDR HIV-1 protease were designed, synthesized and evaluated. •Lead peptide (6a) showed potent inhibition (IC{sub 50}: 4.4 nM) of MDR HIV-1 protease. •(6a) Showed favorable binding isotherms against NL4-3 and MDR proteases. •(6a) Induced perturbations in the {sup 15}N-HSQC spectrum of MDR HIV-1 protease. •Molecular modeling suggested that (6a) may induce total flap closure inMDR protease. -- Abstract: Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: (1TW7)), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded active site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC{sub 50}: 4.4 nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6aagainst both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of {sup 15}N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV.

  7. Design, synthesis and evaluation of a potent substrate analog inhibitor identified by scanning Ala/Phe mutagenesis, mimicking substrate co-evolution, against multidrug-resistant HIV-1 protease

    International Nuclear Information System (INIS)

    Yedidi, Ravikiran S.; Muhuhi, Joseck M.; Liu, Zhigang; Bencze, Krisztina Z.; Koupparis, Kyriacos; O’Connor, Carrie E.; Kovari, Iulia A.; Spaller, Mark R.; Kovari, Ladislau C.

    2013-01-01

    Highlights: •Inhibitors against MDR HIV-1 protease were designed, synthesized and evaluated. •Lead peptide (6a) showed potent inhibition (IC 50 : 4.4 nM) of MDR HIV-1 protease. •(6a) Showed favorable binding isotherms against NL4-3 and MDR proteases. •(6a) Induced perturbations in the 15 N-HSQC spectrum of MDR HIV-1 protease. •Molecular modeling suggested that (6a) may induce total flap closure inMDR protease. -- Abstract: Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: (1TW7)), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded active site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC 50 : 4.4 nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6aagainst both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of 15 N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV

  8. MDR1 P-glycoprotein transports endogenous opioid peptides

    NARCIS (Netherlands)

    Oude Elferink, R. P.; Zadina, J.

    2001-01-01

    MDR1 P-glycoprotein is generally regarded as an efflux pump for amphipathic toxic compounds. The question remains, however, whether certain endogenous compounds are also substrates for this transporter. Certain peptides have been shown to interact with MDR1 Pgp as well and we have therefore

  9. Substrate Scope of O-Methyltransferase from Streptomyces peucetius for Biosynthesis of Diverse Natural Products Methoxides.

    Science.gov (United States)

    Parajuli, Prakash; Pandey, Ramesh Prasad; Nguyen, Thi Huyen Trang; Dhakal, Dipesh; Sohng, Jae Kyung

    2018-04-01

    Methylation is a common post-modification reaction that is observed during the biosynthesis of secondary metabolites produced by plants and microorganisms. Based on the sequence information from Streptomyces peucetius ATCC27952, a putative O-methyltransferase (OMT) gene SpOMT7740 was polymerase chain reaction amplified and cloned into E. coli BL21 (DE3) host to test the substrate promiscuity and conduct functional characterization. In vitro and in vivo reaction assays were carried out over various classes of substrates: flavonoids (flavonol, flavones, and isoflavonoid), chalcones, anthraquinones, anthracyclines, and sterol molecules, and the applications in synthesizing diverse classes of O-methoxy natural products were also illustrated. SpOMT7740 catalyzed the O-methylation reaction to form various natural and non-natural O-methoxides, includes 7-hydroxy-8-O-methoxy flavone, 3-O-methoxy flavone, three mono-, di-, and tri-O-methoxy genistein, mono-O-methoxy phloretin, mono-O-methoxy luteolin, 3-O-methoxy β-sitosterol, and O-methoxy anthraquinones (emodin and aloe emodin) and O-methoxy anthracycline (daunorubicin) exhibiting diverse substrate flexibility. Daunorubicin is a native secondary metabolite of S. peucetius. Among the compounds tested, 7,8-dihydroxyflavone was the best substrate for bioconversion to 7-hydroxy-8-O-methoxy flavone, and it was structurally elucidated. This enzyme showed a flexible catalysis over the given ranges of temperature, pH, and divalent cationic conditions for O-methylation.

  10. Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: Identification of peptides in the DNA binding domain

    International Nuclear Information System (INIS)

    Farrar, Y.J.K.; Evans, R.K.; Beach, C.M.; Coleman, M.S.

    1991-01-01

    Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32 P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32 P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp 221 -Lys 231 (peptide B8) and Cys 234 -Lys 249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an α-helical array of 39 angstrom which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase β that has been implicated in the binding of DNA template

  11. Structure/Function Analysis of Cotton-Based Peptide-Cellulose Conjugates: Spatiotemporal/Kinetic Assessment of Protease Aerogels Compared to Nanocrystalline and Paper Cellulose

    Directory of Open Access Journals (Sweden)

    J. Vincent Edwards

    2018-03-01

    Full Text Available Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis–Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity (Km of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased Km observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency (kcat/Km, attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and β-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding.

  12. Structure/Function Analysis of Cotton-Based Peptide-Cellulose Conjugates: Spatiotemporal/Kinetic Assessment of Protease Aerogels Compared to Nanocrystalline and Paper Cellulose

    Science.gov (United States)

    Edwards, J. Vincent; Fontenot, Krystal; Liebner, Falk; Pircher, Nicole Doyle nee; French, Alfred D.; Condon, Brian D.

    2018-01-01

    Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis–Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity (Km) of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased Km observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency (kcat/Km), attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and β-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding. PMID:29534033

  13. The emerging role of promiscuous 7TM receptors as chemosensors for food intake

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Johansen, Lars Dan; Bräuner-Osborne, Hans

    2010-01-01

    review, we describe the molecular mechanisms of nutrient-sensing of the calcium-sensing receptor (CaR), the G protein-coupled receptor family C, group 6, subtype A (GPRC6A), and the taste1 receptor T1R1/T1R3-sensing L-a-amino acids; the carbohydrate-sensing T1R2/T1R3 receptor; the proteolytic degradation......In recent years, several highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized of which many are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids (FFAs) and are expressed in taste tissue, the gastrointestinal...

  14. Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures

    Czech Academy of Sciences Publication Activity Database

    Zoll, Sebastian; Stanchev, Stancho; Began, Jakub; Škerle, Jan; Lepšík, Martin; Peclinovská, Lucie; Majer, Pavel; Stříšovský, Kvido

    2014-01-01

    Roč. 33, č. 20 (2014), s. 2408-2421 ISSN 0261-4189 R&D Projects: GA ČR GAP305/11/1886; GA MŠk(CZ) LK11206; GA MŠk LO1302; GA ČR GBP208/12/G016 Institutional support: RVO:61388963 Keywords : intramembrane protease * rhomboid family * rhomboid protease * structure * substrate recognition Subject RIV: CE - Biochemistry Impact factor: 10.434, year: 2014

  15. Biochemical Characterisation of Phage Pseudomurein Endoisopeptidases PeiW and PeiP Using Synthetic Peptides.

    Science.gov (United States)

    Schofield, Linley R; Beattie, Amy K; Tootill, Catherine M; Dey, Debjit; Ronimus, Ron S

    2015-01-01

    Pseudomurein endoisopeptidases cause lysis of the cell walls of methanogens by cleaving the isopeptide bond Ala-ε-Lys in the peptide chain of pseudomurein. PeiW and PeiP are two thermostable pseudomurein endoisopeptidases encoded by phage ΨM100 of Methanothermobacter wolfei and phages ΨM1 and ΨM2 of Methanothermobacter marburgensis, respectively. A continuous assay using synthetic peptide substrates was developed and used in the biochemical characterisation of recombinant PeiW and PeiP. The advantages of these synthetic peptide substrates over natural substrates are sensitivity, high purity, and characterisation and the fact that they are more easily obtained than natural substrates. In the presence of a reducing agent, purified PeiW and PeiP each showed similar activity under aerobic and anaerobic conditions. Both enzymes required a divalent metal for activity and showed greater thermostability in the presence of Ca(2+). PeiW and PeiP involve a cysteine residue in catalysis and have a monomeric native conformation. The kinetic parameters, K(M) and k(cat), were determined, and the ε-isopeptide bond between alanine and lysine was confirmed as the bond lysed by these enzymes in pseudomurein. The new assay may have wider applications for the general study of peptidases and the identification of specific methanogens susceptible to lysis by specific pseudomurein endoisopeptidases.

  16. Biochemical Characterisation of Phage Pseudomurein Endoisopeptidases PeiW and PeiP Using Synthetic Peptides

    Directory of Open Access Journals (Sweden)

    Linley R. Schofield

    2015-01-01

    Full Text Available Pseudomurein endoisopeptidases cause lysis of the cell walls of methanogens by cleaving the isopeptide bond Ala-ε-Lys in the peptide chain of pseudomurein. PeiW and PeiP are two thermostable pseudomurein endoisopeptidases encoded by phage ΨM100 of Methanothermobacter wolfei and phages ΨM1 and ΨM2 of Methanothermobacter marburgensis, respectively. A continuous assay using synthetic peptide substrates was developed and used in the biochemical characterisation of recombinant PeiW and PeiP. The advantages of these synthetic peptide substrates over natural substrates are sensitivity, high purity, and characterisation and the fact that they are more easily obtained than natural substrates. In the presence of a reducing agent, purified PeiW and PeiP each showed similar activity under aerobic and anaerobic conditions. Both enzymes required a divalent metal for activity and showed greater thermostability in the presence of Ca2+. PeiW and PeiP involve a cysteine residue in catalysis and have a monomeric native conformation. The kinetic parameters, KM and kcat, were determined, and the ε-isopeptide bond between alanine and lysine was confirmed as the bond lysed by these enzymes in pseudomurein. The new assay may have wider applications for the general study of peptidases and the identification of specific methanogens susceptible to lysis by specific pseudomurein endoisopeptidases.

  17. Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.

    Science.gov (United States)

    Ma, Xianyue; Cline, Kenneth

    2013-03-01

    Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC-cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.

  18. Peptide chemistry toolbox - Transforming natural peptides into peptide therapeutics.

    Science.gov (United States)

    Erak, Miloš; Bellmann-Sickert, Kathrin; Els-Heindl, Sylvia; Beck-Sickinger, Annette G

    2018-06-01

    The development of solid phase peptide synthesis has released tremendous opportunities for using synthetic peptides in medicinal applications. In the last decades, peptide therapeutics became an emerging market in pharmaceutical industry. The need for synthetic strategies in order to improve peptidic properties, such as longer half-life, higher bioavailability, increased potency and efficiency is accordingly rising. In this mini-review, we present a toolbox of modifications in peptide chemistry for overcoming the main drawbacks during the transition from natural peptides to peptide therapeutics. Modifications at the level of the peptide backbone, amino acid side chains and higher orders of structures are described. Furthermore, we are discussing the future of peptide therapeutics development and their impact on the pharmaceutical market. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. The evolutionary appearance of non-cyanogenic hydroxynitrile glucosides in the Lotus genus is accompanied by the substrate specialization of paralogous beta-glucosidases resulting from a crucial amino acid substitution

    DEFF Research Database (Denmark)

    Lai, Daniela; Abou Hachem, Maher; Robson, Fran

    2014-01-01

    has the dominant physiological role in rhodiocyanoside degradation. Structural modelling, site-directed mutagenesis and activity assays establish that a glycine residue (G211) in the aglycone binding site of BGD2 is essential for its ability to hydrolyse the endogenous cyanogenic glucosides...... with the Lotus corniculatus clade within the Lotus genus. This suggests the evolutionary scenario that substrate specialization for rhodiocyanosides evolved from a promiscuous activity of a progenitor cyanogenic beta-glucosidase, resembling BGD2, and required no more than a single amino acid substitution....

  20. Post-translational Introduction of D-Alanine into Ribosomally Synthesized Peptides by the Dehydroalanine Reductase NpnJ.

    Science.gov (United States)

    Yang, Xiao; van der Donk, Wilfred A

    2015-10-07

    Ribosomally synthesized peptides are generally limited to L-amino acid building blocks. Given the advantageous properties of peptides containing D-amino acids such as stabilization of certain turns and against proteolytic degradation, methods to introduce D-stereocenters are valuable. Here we report the first in vitro reconstitution and characterization of a dehydrogenase that carries out the asymmetric reduction of dehydroalanine. NpnJA reduces dehydroalanine to D-Ala using NAPDH as cosubstrate. The enzyme displays high substrate tolerance allowing introduction of D-Ala into a range of non-native substrates. In addition to the in vitro reactions, we describe five examples of using Escherichia coli as biosynthetic host for D-alanine introduction into ribosomal peptides. A deuterium-label-based coupled-enzyme assay was used to rapidly determine the stereochemistry of the newly installed alanine.

  1. Substrate specificity of low-molecular mass bacterial DD-peptidases.

    Science.gov (United States)

    Nemmara, Venkatesh V; Dzhekieva, Liudmila; Sarkar, Kumar Subarno; Adediran, S A; Duez, Colette; Nicholas, Robert A; Pratt, R F

    2011-11-22

    The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high-molecular mass (HMM) and low-molecular mass (LMM) enzymes. The latter group, which is subdivided into classes A-C (LMMA, -B, and -C, respectively), is believed to catalyze DD-carboxypeptidase and endopeptidase reactions in vivo. To date, the specificity of their reactions with particular elements of peptidoglycan structure has not, in general, been defined. This paper describes the steady-state kinetics of hydrolysis of a series of specific peptidoglycan-mimetic peptides, representing various elements of stem peptide structure, catalyzed by a range of LMM PBPs (the LMMA enzymes, Escherichia coli PBP5, Neisseria gonorrhoeae PBP4, and Streptococcus pneumoniae PBP3, and the LMMC enzymes, the Actinomadura R39 dd-peptidase, Bacillus subtilis PBP4a, and N. gonorrhoeae PBP3). The R39 enzyme (LMMC), like the previously studied Streptomyces R61 DD-peptidase (LMMB), specifically and rapidly hydrolyzes stem peptide fragments with a free N-terminus. In accord with this result, the crystal structures of the R61 and R39 enzymes display a binding site specific to the stem peptide N-terminus. These are water-soluble enzymes, however, with no known specific function in vivo. On the other hand, soluble versions of the remaining enzymes of those noted above, all of which are likely to be membrane-bound and/or associated in vivo and have been assigned particular roles in cell wall biosynthesis and maintenance, show little or no specificity for peptides containing elements of peptidoglycan structure. Peptidoglycan-mimetic boronate transition-state analogues do inhibit these enzymes but display notable specificity only for the LMMC enzymes, where, unlike peptide substrates, they may be able to effectively induce a specific active site structure. The manner in which LMMA (and HMM) DD

  2. Peptides Displayed as High Density Brush Polymers Resist Proteolysis and Retain Bioactivity

    Science.gov (United States)

    2015-01-01

    We describe a strategy for rendering peptides resistant to proteolysis by formulating them as high-density brush polymers. The utility of this approach is demonstrated by polymerizing well-established cell-penetrating peptides (CPPs) and showing that the resulting polymers are not only resistant to proteolysis but also maintain their ability to enter cells. The scope of this design concept is explored by studying the proteolytic resistance of brush polymers composed of peptides that are substrates for either thrombin or a metalloprotease. Finally, we demonstrate that the proteolytic susceptibility of peptide brush polymers can be tuned by adjusting the density of the polymer brush and offer in silico models to rationalize this finding. We contend that this strategy offers a plausible method of preparing peptides for in vivo use, where rapid digestion by proteases has traditionally restricted their utility. PMID:25314576

  3. Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates.

    Science.gov (United States)

    Kleifeld, Oded; Doucet, Alain; Prudova, Anna; auf dem Keller, Ulrich; Gioia, Magda; Kizhakkedathu, Jayachandran N; Overall, Christopher M

    2011-09-22

    Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and

  4. Carrier-mediated transport of peptides by the kidney

    International Nuclear Information System (INIS)

    Skopicki, H.A.

    1988-01-01

    Small peptide transport was characterized to determine if: (1) Multiple carriers are present in the luminal membrane of renal proximal tubular cells; (2) Carrier-mediated peptide transport is limited by size; and (3) Gentamicin inhibits carrier-mediated reabsorption of peptides. Uptake of glycyl-[ 3 H]proline (Gly-Pro) into renal brush border membrane vesicles demonstrated a dual affinity carrier system. Whether multiple carriers are present was further investigated by characterizing the uptake of [ 3 H]pyroglutamyl-histidine. To determine if carrier-mediated transport of peptides is limited by size of the molecule, uptake of the hydrolytically resistant tripeptide, [ 3 H]pryroglutamyl-histidyl-tryptophan (pGlu-His-Trp), and tetrapeptide, [ 3 H]pyroglutamyl-histidyl-tryptophyl-serine (pGlu-His-Trp-Ser) were assessed. These data indicate: multiple carriers exist on the luminal membrane of renal proximal tubular cells for the transport of dipeptides, and tripeptide pGlu-His-Trp and the tetrapeptide pGlu-His-Trp-Ser are not taken up by a carrier-mediated mechanism, suggesting that the carrier may be limited by the size of the substrate

  5. Characterization of CYP154F1 from Thermobifida fusca YX and Extension of Its Substrate Spectrum by Site-Directed Mutagenesis.

    Science.gov (United States)

    Rühlmann, Ansgar; Groth, Georg; Urlacher, Vlada B

    2018-03-02

    Previous studies on cytochrome P450 monooxygenases (CYP) from family 154 reported their substrate promiscuity and high activity. Hence, herein, the uncharacterized family member CYP154F1 is described. Screening of more than 100 organic compounds revealed that CYP154F1 preferably accepts small linear molecules with a carbon chain length of 8-10 atoms. In contrast to thoroughly characterized CYP154E1, CYP154F1 has a much narrower substrate spectrum and lower activity. A structural alignment of homology models of CYP154F1 and CYP154E1 revealed few differences in the active sites of both family members. By gradual mutagenesis of the CYP154F1 active site towards those of CYP154E1, a key residue accounting for the different activities of both enzymes was identified at position 234. Substitution of T234 for large hydrophobic amino acids led to up to tenfold higher conversion rates of small substrates, such as geraniol. Replacement of T234 by small hydrophobic amino acids, valine or alanine, resulted in mutants with extended substrate spectra. These mutants are able to convert some of the larger substrates of CYP154E1, such as (E)-stilbene and (+)-nootkatone. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Males and females gain differentially from sociality in a promiscuous fruit bat Cynopterus sphinx.

    Directory of Open Access Journals (Sweden)

    Kritika M Garg

    Full Text Available Sociality emerges when the benefits of group living outweigh its costs. While both males and females are capable of strong social ties, the evolutionary drivers for sociality and the benefits accrued maybe different for each sex. In this study, we investigate the differential reproductive success benefits of group membership that males and females might obtain in the promiscuous fruit bat Cynopterus sphinx. Individuals of this species live in flexible social groups called colonies. These colonies are labile and there is high turnover of individuals. However, colony males sire more offspring within the colony suggesting that being part of a colony may result in reproductive benefits for males. This also raises the possibility that long-term loyalty towards the colony may confer additional advantage in terms of higher reproductive success. We used ten seasons of genetic parentage data to estimate reproductive success and relatedness of individuals in the colony. We used recapture data to identify long and short-term residents in the colony as well as to obtain rates of recapture for males and females. Our results reveal that males have a significantly higher chance of becoming long-term residents (than females, and these long-term resident males gain twice the reproductive success compared to short-term resident males. We also observed that long-term resident females are related to each other and also achieve higher reproductive success than short-term resident females. In contrast, long-term resident males do not differ from short-term resident males in their levels of relatedness. Our results re-iterate the benefits of sociality even in species that are promiscuous and socially labile and possible benefits of maintaining a colony.

  7. Males and females gain differentially from sociality in a promiscuous fruit bat Cynopterus sphinx.

    Science.gov (United States)

    Garg, Kritika M; Chattopadhyay, Balaji; Swami Doss, D P; Kumar, A K Vinoth; Kandula, Sripathi; Ramakrishnan, Uma

    2015-01-01

    Sociality emerges when the benefits of group living outweigh its costs. While both males and females are capable of strong social ties, the evolutionary drivers for sociality and the benefits accrued maybe different for each sex. In this study, we investigate the differential reproductive success benefits of group membership that males and females might obtain in the promiscuous fruit bat Cynopterus sphinx. Individuals of this species live in flexible social groups called colonies. These colonies are labile and there is high turnover of individuals. However, colony males sire more offspring within the colony suggesting that being part of a colony may result in reproductive benefits for males. This also raises the possibility that long-term loyalty towards the colony may confer additional advantage in terms of higher reproductive success. We used ten seasons of genetic parentage data to estimate reproductive success and relatedness of individuals in the colony. We used recapture data to identify long and short-term residents in the colony as well as to obtain rates of recapture for males and females. Our results reveal that males have a significantly higher chance of becoming long-term residents (than females), and these long-term resident males gain twice the reproductive success compared to short-term resident males. We also observed that long-term resident females are related to each other and also achieve higher reproductive success than short-term resident females. In contrast, long-term resident males do not differ from short-term resident males in their levels of relatedness. Our results re-iterate the benefits of sociality even in species that are promiscuous and socially labile and possible benefits of maintaining a colony.

  8. Orally active-targeted drug delivery systems for proteins and peptides.

    Science.gov (United States)

    Li, Xiuying; Yu, Miaorong; Fan, Weiwei; Gan, Yong; Hovgaard, Lars; Yang, Mingshi

    2014-09-01

    In the past decade, extensive efforts have been devoted to designing 'active targeted' drug delivery systems (ATDDS) to improve oral absorption of proteins and peptides. Such ATDDS enhance cellular internalization and permeability of proteins and peptides via molecular recognition processes such as ligand-receptor or antigen-antibody interaction, and thus enhance drug absorption. This review focuses on recent advances with orally ATDDS, including ligand-protein conjugates, recombinant ligand-protein fusion proteins and ligand-modified carriers. In addition to traditional intestinal active transport systems of substrates and their corresponding receptors, transporters and carriers, new targets such as intercellular adhesion molecule-1 and β-integrin are also discussed. ATDDS can improve oral absorption of proteins and peptides. However, currently, no clinical studies on ATDDS for proteins and peptides are underway, perhaps due to the complexity and limited knowledge of transport mechanisms. Therefore, more research is warranted to optimize ATDDS efficiency.

  9. Phytochemicals perturb membranes and promiscuously alter protein function.

    Science.gov (United States)

    Ingólfsson, Helgi I; Thakur, Pratima; Herold, Karl F; Hobart, E Ashley; Ramsey, Nicole B; Periole, Xavier; de Jong, Djurre H; Zwama, Martijn; Yilmaz, Duygu; Hall, Katherine; Maretzky, Thorsten; Hemmings, Hugh C; Blobel, Carl; Marrink, Siewert J; Koçer, Armağan; Sack, Jon T; Andersen, Olaf S

    2014-08-15

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.

  10. Legume-rhizobia signal exchange: promiscuity and environmental effects

    Directory of Open Access Journals (Sweden)

    Mario Andrade Lira Junior

    2015-09-01

    Full Text Available Although signal exchange between legumes and their rhizobia is among the best-known examples of this biological process, most of the more characterized data comes from just a few legume species and environmental stresses. Although a relative wealth of information is available for some model legumes and some of the major pulses such as soybean, little is known about tropical legumes. This relative disparity in current knowledge is also apparent in the research on the effects of environmental stress on signal exchange; cool-climate stresses, such as low-soil temperature, comprise a relatively large body of research, whereas high-temperature stresses and drought are not nearly as well understood. Both tropical legumes and their environmental stress-induced effects are increasingly important due to global population growth (the demand for protein, climate change (increasing temperatures and more extreme climate behavior, and urbanization (and thus heavy metals. This knowledge gap for both legumes and their environmental stresses is compounded because whereas most temperate legume-rhizobia symbioses are relatively specific and cultivated under relatively stable environments, the converse is true for tropical legumes, which tend to be promiscuous and grow in highly variable conditions. This review will clarify some of this missing information and highlight fields in which further research would benefit our current knowledge.

  11. Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

    Directory of Open Access Journals (Sweden)

    B.L. Fernandes

    2000-07-01

    Full Text Available The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser was cleaved with a Km of 4.6 µM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp which was cleaved at a single bond (Phe-Ser with a Km of 13.6 µM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.

  12. A Two-Piece Derivative of a Group I Intron RNA as a Platform for Designing Self-Assembling RNA Templates to Promote Peptide Ligation

    Directory of Open Access Journals (Sweden)

    Takahiro Tanaka

    2012-01-01

    Full Text Available Multicomponent RNA-peptide complexes are attractive from the viewpoint of artificial design of functional biomacromolecular systems. We have developed self-folding and self-assembling RNAs that serve as templates to assist chemical ligation between two reactive peptides with RNA-binding capabilities. The design principle of previous templates, however, can be applied only to limited classes of RNA-binding peptides. In this study, we employed a two-piece derivative of a group I intron RNA from the Tetrahymena large subunit ribosomal RNA (LSU rRNA as a platform for new template RNAs. In this group I intron-based self-assembling platform, modules for the recognition of substrate peptides can be installed independently from modules holding the platform structure. The new self-assembling platform allows us to expand the repertoire of substrate peptides in template RNA design.

  13. Grafted Cross-Linked Polyolefin Substrates for Peptide Synthesis and Assays

    DEFF Research Database (Denmark)

    1999-01-01

    suited for use in solid-phase biosystems, notably bioassays, such as immunoassays, DNA hybridization assays or PCR amplification. The grafted chains may bear substituents which are such that the polymer-grafted cross-linked polyolefin substrate is swellable by water or aqueous media, in other words...

  14. Kinetic analysis of the mechanism and specificity of protein-disulfide isomerase using fluorescence-quenched peptides

    DEFF Research Database (Denmark)

    Westphal, V; Spetzler, J C; Meldal, M

    1998-01-01

    Protein-disulfide isomerase (PDI) is an abundant folding catalyst in the endoplasmic reticulum of eukaryotic cells. PDI introduces disulfide bonds into newly synthesized proteins and catalyzes disulfide bond isomerizations. We have synthesized a library of disulfide-linked fluorescence......-quenched peptides, individually linked to resin beads, for two purposes: 1) to probe PDI specificity, and 2) to identify simple, sensitive peptide substrates of PDI. Using this library, beads that became rapidly fluorescent by reduction by human PDI were selected. Amino acid sequencing of the bead-linked peptides...

  15. Fiscal 1997 R and D project on industrial science and technology under a consignment from NEDO. R and D of the synthesis and processing of advanced biomaterials (fixation and effective utilization of carbon dioxide using peptides); 1997 nendo Shin energy Sangyo Gijutsu Sogo Kaihatsu Kiko itaku. Senshin bio zairyo no sosei kako gijutsu no kenkyu kaihatsu (peptide oyo nisanka tanso koteika yuko riyo gijutsu) seika hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1998-03-01

    In fiscal 1997, study was made on formation of peptides with a biogenic function and molecular recognition function, and peptide materials. The existing automatic design technique of peptides including non-natural amino acids was applied to design of complementary peptides of proteins with known structures to optimize a development technique of ligand. Practical function of peptides as advanced receptor ligand was verified by applying the existing conformation control technique to stable holding of peptide active conformation. Creation of bio-materials, and development of preparation technology of bio-substrates and materializing technology of functional molecular materials were carried out. To construct an electron transfer system by self- organized body of peptides formed on a substrate, a peptide molecular device was prepared and verified using linkers. To use peptides as photoelectron functional molecule, metallized peptide with main helix structure was prepared. The possibility of molecular materials with a fixation function of CO2 was examined. A high-sensitivity bio-sensor was also mentioned. 75 refs., 130 figs., 25 tabs.

  16. Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae: BIOCHEMICAL, STRUCTURAL, AND EVOLUTIONARY INSIGHTS.

    Science.gov (United States)

    Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S; Flick, Robert; Wolf, Yuri I; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M; Koonin, Eugene V; Yakunin, Alexander F

    2015-07-24

    The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Kinome profiling of Arabidopsis using arrays of kinase consensus substrates

    Directory of Open Access Journals (Sweden)

    Pieterse Corné MJ

    2007-02-01

    Full Text Available Abstract Background Kinome profiling aims at the parallel analysis of kinase activities in a cell. Novel developed arrays containing consensus substrates for kinases are used to assess those kinase activities. The arrays described in this paper were already used to determine kinase activities in mammalian systems, but since substrates from many organisms are present we decided to test these arrays for the determination of kinase activities in the model plant species Arabidopsis thaliana. Results Kinome profiling using Arabidopsis cell extracts resulted in the labelling of many consensus peptides by kinases from the plant, indicating the usefulness of this kinome profiling tool for plants. Method development showed that fresh and frozen plant material could be used to make cell lysates containing active kinases. Dilution of the plant extract increased the signal to noise ratio and non-radioactive ATP enhances full development of spot intensities. Upon infection of Arabidopsis with an avirulent strain of the bacterial pathogen Pseudomonas syringae pv. tomato, we could detect differential kinase activities by measuring phosphorylation of consensus peptides. Conclusion We show that kinome profiling on arrays with consensus substrates can be used to monitor kinase activities in plants. In a case study we show that upon infection with avirulent P. syringae differential kinase activities can be found. The PepChip can for example be used to purify (unknown kinases that play a role in P. syringae infection. This paper shows that kinome profiling using arrays of consensus peptides is a valuable new tool to study signal-transduction in plants. It complements the available methods for genomics and proteomics research.

  18. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

    Science.gov (United States)

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

    2016-07-15

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior*

    Science.gov (United States)

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H.; Muyldermans, Serge; Declerck, Paul J.; Huang, Mingdong; Andreasen, Peter A.; Ngo, Jacky Chi Ki

    2016-01-01

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30–40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

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

    Science.gov (United States)

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

    2016-03-02

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

  1. Extraction and identification of α-amylase inhibitor peptides from Nephelium lappacheum and Nephelium mutabile seed protein using gastro-digestive enzymes.

    Science.gov (United States)

    Evaristus, Natashya Anak; Wan Abdullah, Wan Nadiah; Gan, Chee-Yuen

    2018-04-01

    The potential of N. lappacheum and N. mutabile seed as a source of α-amylase inhibitor peptides was explored based on the local traditional practice of using the seed. Different gastro-digestive enzymes (i.e. pepsin or chymotrypsin) or a sequential digestion were used to extract the peptides. The effects of digestion time and enzyme to substrate (E:S) ratio on the α-amylase inhibitory activity were investigated. Results showed that chymotrypsin was effective in producing the inhibitor peptides from rambutan seed protein at E:S ratio 1:20 for 1 h, whereas pepsin was more effective for pulasan seed protein under the same condition. A total of 20 and 31 novel inhibitor peptides were identified, respectively. These peptides could bind with the subsites of α-amylase (i.e. Trp58, Trp59, Tyr62, Asp96, Arg195, Asp197, Glu233, His299, Asp300, and His305) and formed a sliding barrier that preventing the formation of enzyme/substrate intermediate leading to lower α-amylase activity. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Tritium labeling of amino acids and peptides with liquid and solid tritium

    International Nuclear Information System (INIS)

    Peng, C.T.; Hua, R.L.; Souers, P.C.; Coronado, P.R.

    1988-01-01

    Amino acids and peptides were labeled with liquid and solid tritium at 21 K and 9 K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenyl-alanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when used as supports for dispersion of the substrate promote tritium labeling at 21 K. Our study shows that both liquid and solid tritium are potentially useful agents for labeling peptides and proteins. 11 refs., 1 fig., 3 tabs

  3. Tritium labeling of amino acids and peptides with liquid and solid tritium

    International Nuclear Information System (INIS)

    Souers, P.C.; Coronado, P.R.; Peng, C.T.; Hua, R.L.

    1988-01-01

    Amino acids and peptides were labeled with liquid and solid tritium at 21/degree/K and 9/degree/K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenylalanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when used as supports for dispersion of the substrate promote tritium labeling at 21 K. Our study shows that both liquid and solid tritiums are potentially useful agents for labeling peptides and proteins

  4. Software-aided approach to investigate peptide structure and metabolic susceptibility of amide bonds in peptide drugs based on high resolution mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Tatiana Radchenko

    Full Text Available Interest in using peptide molecules as therapeutic agents due to high selectivity and efficacy is increasing within the pharmaceutical industry. However, most peptide-derived drugs cannot be administered orally because of low bioavailability and instability in the gastrointestinal tract due to protease activity. Therefore, structural modifications peptides are required to improve their stability. For this purpose, several in-silico software tools have been developed such as PeptideCutter or PoPS, which aim to predict peptide cleavage sites for different proteases. Moreover, several databases exist where this information is collected and stored from public sources such as MEROPS and ExPASy ENZYME databases. These tools can help design a peptide drug with increased stability against proteolysis, though they are limited to natural amino acids or cannot process cyclic peptides, for example. We worked to develop a new methodology to analyze peptide structure and amide bond metabolic stability based on the peptide structure (linear/cyclic, natural/unnatural amino acids. This approach used liquid chromatography / high resolution, mass spectrometry to obtain the analytical data from in vitro incubations. We collected experimental data for a set (linear/cyclic, natural/unnatural amino acids of fourteen peptide drugs and four substrate peptides incubated with different proteolytic media: trypsin, chymotrypsin, pepsin, pancreatic elastase, dipeptidyl peptidase-4 and neprilysin. Mass spectrometry data was analyzed to find metabolites and determine their structures, then all the results were stored in a chemically aware manner, which allows us to compute the peptide bond susceptibility by using a frequency analysis of the metabolic-liable bonds. In total 132 metabolites were found from the various in vitro conditions tested resulting in 77 distinct cleavage sites. The most frequent observed cleavage sites agreed with those reported in the literature. The

  5. Psychosocial predictors of human papillomavirus vaccination intentions for young women 18 to 26: religiosity, morality, promiscuity, and cancer worry.

    Science.gov (United States)

    Krakow, Melinda M; Jensen, Jakob D; Carcioppolo, Nick; Weaver, Jeremy; Liu, Miao; Guntzviller, Lisa M

    2015-01-01

    To determine whether five psychosocial variables, namely, religiosity, morality, perceived promiscuity, cancer worry frequency, and cancer worry severity, predict young women's intentions to receive the human papillomavirus (HPV) vaccination. Female undergraduate students (n=408) completed an online survey. Questions pertaining to hypothesized predictors were analyzed through bivariate correlations and hierarchical regression equations. Regressions examined whether the five psychosocial variables of interest predicted intentions to vaccinate above and beyond controls. Proposed interactions among predictor variables were also tested. Study findings supported cancer worry as a direct predictor of HPV vaccination intention, and religiosity and sexual experience as moderators of the relationship between concerns of promiscuity reputation and intentions to vaccinate. One dimension of cancer worry (severity) emerged as a particularly robust predictor for this population. This study provides support for several important, yet understudied, factors contributing to HPV vaccination intentions among college-aged women: cancer worry severity and religiosity. Future research should continue to assess the predictive contributions of these variables and evaluate how messages and campaigns to increase HPV vaccination uptake can utilize religious involvement and worry about cancer to promote more effectively HPV vaccination as a cancer prevention strategy. Copyright © 2015 Jacobs Institute of Women's Health. Published by Elsevier Inc. All rights reserved.

  6. Kinetic and structural characterization of amyloid-β peptide hydrolysis by human angiotensin-1-converting enzyme.

    Science.gov (United States)

    Larmuth, Kate M; Masuyer, Geoffrey; Douglas, Ross G; Schwager, Sylva L; Acharya, K Ravi; Sturrock, Edward D

    2016-03-01

    Angiotensin-1-converting enzyme (ACE), a zinc metallopeptidase, consists of two homologous catalytic domains (N and C) with different substrate specificities. Here we report kinetic parameters of five different forms of human ACE with various amyloid beta (Aβ) substrates together with high resolution crystal structures of the N-domain in complex with Aβ fragments. For the physiological Aβ(1-16) peptide, a novel ACE cleavage site was found at His14-Gln15. Furthermore, Aβ(1-16) was preferentially cleaved by the individual N-domain; however, the presence of an inactive C-domain in full-length somatic ACE (sACE) greatly reduced enzyme activity and affected apparent selectivity. Two fluorogenic substrates, Aβ(4-10)Q and Aβ(4-10)Y, underwent endoproteolytic cleavage at the Asp7-Ser8 bond with all ACE constructs showing greater catalytic efficiency for Aβ(4-10)Y. Surprisingly, in contrast to Aβ(1-16) and Aβ(4-10)Q, sACE showed positive domain cooperativity and the double C-domain (CC-sACE) construct no cooperativity towards Aβ(4-10)Y. The structures of the Aβ peptide-ACE complexes revealed a common mode of peptide binding for both domains which principally targets the C-terminal P2' position to the S2' pocket and recognizes the main chain of the P1' peptide. It is likely that N-domain selectivity for the amyloid peptide is conferred through the N-domain specific S2' residue Thr358. Additionally, the N-domain can accommodate larger substrates through movement of the N-terminal helices, as suggested by the disorder of the hinge region in the crystal structures. Our findings are important for the design of domain selective inhibitors as the differences in domain selectivity are more pronounced with the truncated domains compared to the more physiological full-length forms. The atomic coordinates and structure factors for N-domain ACE with Aβ peptides 4-10 (5AM8), 10-16 (5AM9), 1-16 (5AMA), 35-42 (5AMB) and (4-10)Y (5AMC) complexes have been deposited in the

  7. A versatile method for the preparation of conjugates of peptides with DNA/PNA/analog by employing chemo-selective click reaction in water

    Science.gov (United States)

    Gogoi, Khirud; Mane, Meenakshi V.; Kunte, Sunita S.; Kumar, Vaijayanti A.

    2007-01-01

    The specific 1,3 dipolar Hüisgen cycloaddition reaction known as ‘click-reaction’ between azide and alkyne groups is employed for the synthesis of peptide–oligonucleotide conjugates. The peptide nucleic acids (PNA)/DNA and peptides may be appended either by azide or alkyne groups. The cycloaddition reaction between the azide and alkyne appended substrates allows the synthesis of the desired conjugates in high purity and yields irrespective of the sequence and functional groups on either of the two substrates. The versatile approach could also be employed to generate the conjugates of peptides with thioacetamido nucleic acid (TANA) analog. The click reaction is catalyzed by Cu (I) in either water or in organic medium. In water, ∼3-fold excess of the peptide-alkyne/azide drives the reaction to completion in 2 h with no side products. PMID:17981837

  8. Bioactive Peptides in Animal Food Products

    Directory of Open Access Journals (Sweden)

    Marzia Albenzio

    2017-05-01

    Full Text Available Proteins of animal origin represent physiologically active components in the human diet; they exert a direct action or constitute a substrate for enzymatic hydrolysis upon food processing and consumption. Bioactive peptides may descend from the hydrolysis by digestive enzymes, enzymes endogenous to raw food materials, and enzymes from microorganisms added during food processing. Milk proteins have different polymorphisms for each dairy species that influence the amount and the biochemical characteristics (e.g., amino acid chain, phosphorylation, and glycosylation of the protein. Milk from other species alternative to cow has been exploited for their role in children with cow milk allergy and in some infant pathologies, such as epilepsy, by monitoring the immune status. Different mechanisms concur for bioactive peptides generation from meat and meat products, and their functionality and application as functional ingredients have proven effects on consumer health. Animal food proteins are currently the main source of a range of biologically-active peptides which have gained special interest because they may also influence numerous physiological responses in the organism. The addition of probiotics to animal food products represent a strategy for the increase of molecules with health and functional properties.

  9. Plasmodium falciparum signal peptide peptidase cleaves malaria heat shock protein 101 (HSP101). Implications for gametocytogenesis

    International Nuclear Information System (INIS)

    Baldwin, Michael; Russo, Crystal; Li, Xuerong; Chishti, Athar H.

    2014-01-01

    Highlights: • PfSPP is an ER resident protease. • PfSPP is expressed both as a monomer and dimer. • The signal peptide of HSP101 is the first known substrate of PfSPP. • Reduced PfSPP activity may significantly affect ER homeostasis. - Abstract: Previously we described the identification of a Plasmodium falciparum signal peptide peptidase (PfSPP) functioning at the blood stage of malaria infection. Our studies also demonstrated that mammalian SPP inhibitors prevent malaria parasite growth at the late-ring/early trophozoite stage of intra-erythrocytic development. Consistent with its role in development, we tested the hypothesis that PfSPP functions at the endoplasmic reticulum of P.falciparum where it cleaves membrane-bound signal peptides generated following the enzyme activity of signal peptidase. The localization of PfSPP to the endoplasmic reticulum was confirmed by immunofluorescence microscopy and immunogold electron microscopy. Biochemical analysis indicated the existence of monomer and dimer forms of PfSPP in the parasite lysate. A comprehensive bioinformatics screen identified several candidate PfSPP substrates in the parasite genome. Using an established transfection based in vivo luminescence assay, malaria heat shock protein 101 (HSP101) was identified as a substrate of PfSPP, and partial inhibition of PfSPP correlated with the emergence of gametocytes. This finding unveils the first known substrate of PfSPP, and provides new perspectives for the function of intra-membrane proteolysis at the erythrocyte stage of malaria parasite life cycle

  10. Plasmodium falciparum signal peptide peptidase cleaves malaria heat shock protein 101 (HSP101). Implications for gametocytogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Baldwin, Michael; Russo, Crystal; Li, Xuerong [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Chishti, Athar H., E-mail: athar.chishti@tufts.edu [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Sackler School of Graduate Biomedical Sciences, Programs in Physiology, Pharmacology, and Microbiology, Tufts University School of Medicine, Boston, MA 02111 (United States)

    2014-08-08

    Highlights: • PfSPP is an ER resident protease. • PfSPP is expressed both as a monomer and dimer. • The signal peptide of HSP101 is the first known substrate of PfSPP. • Reduced PfSPP activity may significantly affect ER homeostasis. - Abstract: Previously we described the identification of a Plasmodium falciparum signal peptide peptidase (PfSPP) functioning at the blood stage of malaria infection. Our studies also demonstrated that mammalian SPP inhibitors prevent malaria parasite growth at the late-ring/early trophozoite stage of intra-erythrocytic development. Consistent with its role in development, we tested the hypothesis that PfSPP functions at the endoplasmic reticulum of P.falciparum where it cleaves membrane-bound signal peptides generated following the enzyme activity of signal peptidase. The localization of PfSPP to the endoplasmic reticulum was confirmed by immunofluorescence microscopy and immunogold electron microscopy. Biochemical analysis indicated the existence of monomer and dimer forms of PfSPP in the parasite lysate. A comprehensive bioinformatics screen identified several candidate PfSPP substrates in the parasite genome. Using an established transfection based in vivo luminescence assay, malaria heat shock protein 101 (HSP101) was identified as a substrate of PfSPP, and partial inhibition of PfSPP correlated with the emergence of gametocytes. This finding unveils the first known substrate of PfSPP, and provides new perspectives for the function of intra-membrane proteolysis at the erythrocyte stage of malaria parasite life cycle.

  11. An ecological analysis of the effects of deviant peer clustering on sexual promiscuity, problem behavior, and childbearing from early adolescence to adulthood: an enhancement of the life history framework.

    Science.gov (United States)

    Dishion, Thomas J; Ha, Thao; Véronneau, Marie-Hélène

    2012-05-01

    The authors propose that peer relationships should be included in a life history perspective on adolescent problem behavior. Longitudinal analyses were used to examine deviant peer clustering as the mediating link between attenuated family ties, peer marginalization, and social disadvantage in early adolescence and sexual promiscuity in middle adolescence and childbearing by early adulthood. Specifically, 998 youths, along with their families, were assessed at age 11 years and periodically through age 24 years. Structural equation modeling revealed that the peer-enhanced life history model provided a good fit to the longitudinal data, with deviant peer clustering strongly predicting adolescent sexual promiscuity and other correlated problem behaviors. Sexual promiscuity, as expected, also strongly predicted the number of children by ages 22-24 years. Consistent with a life history perspective, family social disadvantage directly predicted deviant peer clustering and number of children in early adulthood, controlling for all other variables in the model. These data suggest that deviant peer clustering is a core dimension of a fast life history strategy, with strong links to sexual activity and childbearing. The implications of these findings are discussed with respect to the need to integrate an evolutionary-based model of self-organized peer groups in developmental and intervention science.

  12. Analysis of the DosR regulon genes to select cytotoxic T lymphocyte epitope specific vaccine candidates using a reverse vaccinology approach

    Directory of Open Access Journals (Sweden)

    Kirti Pandey

    2016-01-01

    Conclusion: Our study has generated several promiscuous antigenic peptides capable of binding to major histocompatibility complex class I with high affinity. These epitopes can become part of a postexposure multivalent subunit vaccine upon experimental validation.

  13. Characterization and in vitro biological evaluation of mineral/osteogenic growth peptide nanocomposites synthesized biomimetically on titanium

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Cen; Kong, Xiangdong [Bio-X Center, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Zhang, Sheng-Min [Advanced Biomaterials and Tissue Engineering Center, Huazhong University of Science and Technology, Wuhan 430074 (China); Lee, In-Seop, E-mail: inseop@yonsei.ac.kr [Bio-X Center, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Institute of Natural Sciences, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2015-04-15

    Graphical abstract: - Highlights: • Mineral/OGP nanocomposite layers were synthesized biomimetically on Ti substrates. • Incorporated OGP affected the morphology and ultimate structure of mineral. • Incorporated OGP improved the MSCs adhesion, proliferation, and ALP activity. - Abstract: Nanocomposite layers of mineral/osteogenic growth peptide (OGP) were synthesized on calcium phosphate coated titanium substrates by immersing in calcium-phosphate buffer solution containing OGP. Peptide incorporated mineral was characterized by determining quantity loaded, effects on mineral morphology and structure. Also, the biological activity was investigated by cell adhesion, proliferation assay, and measurement of alkaline phosphatase (ALP) activity. X-ray photoelectron spectroscopy (XPS) and micro-bicinchoninic acid (BCA) assay revealed that OGP was successfully incorporated with mineral and the amount was increased with immersion time. Incorporated OGP changed the mineral morphology from sharp plate-like shape to more rounded one, and the octacalcium phosphate structure of the mineral was gradually transformed into apatite. With confocal microscopy to examine the incorporation of fluorescently labeled peptide, OGP was evenly distributed throughout mineral layers. Mineral/OGP nanocomposites promoted cell adhesion and proliferation, and also increased ALP activity of mesenchymal stem cells (MSCs). Results presented here indicated that the mineral/OGP nanocomposites formed on titanium substrates had the potential for applications in dental implants.

  14. Characterization and in vitro biological evaluation of mineral/osteogenic growth peptide nanocomposites synthesized biomimetically on titanium

    International Nuclear Information System (INIS)

    Chen, Cen; Kong, Xiangdong; Zhang, Sheng-Min; Lee, In-Seop

    2015-01-01

    Graphical abstract: - Highlights: • Mineral/OGP nanocomposite layers were synthesized biomimetically on Ti substrates. • Incorporated OGP affected the morphology and ultimate structure of mineral. • Incorporated OGP improved the MSCs adhesion, proliferation, and ALP activity. - Abstract: Nanocomposite layers of mineral/osteogenic growth peptide (OGP) were synthesized on calcium phosphate coated titanium substrates by immersing in calcium-phosphate buffer solution containing OGP. Peptide incorporated mineral was characterized by determining quantity loaded, effects on mineral morphology and structure. Also, the biological activity was investigated by cell adhesion, proliferation assay, and measurement of alkaline phosphatase (ALP) activity. X-ray photoelectron spectroscopy (XPS) and micro-bicinchoninic acid (BCA) assay revealed that OGP was successfully incorporated with mineral and the amount was increased with immersion time. Incorporated OGP changed the mineral morphology from sharp plate-like shape to more rounded one, and the octacalcium phosphate structure of the mineral was gradually transformed into apatite. With confocal microscopy to examine the incorporation of fluorescently labeled peptide, OGP was evenly distributed throughout mineral layers. Mineral/OGP nanocomposites promoted cell adhesion and proliferation, and also increased ALP activity of mesenchymal stem cells (MSCs). Results presented here indicated that the mineral/OGP nanocomposites formed on titanium substrates had the potential for applications in dental implants

  15. Caged Protein Prenyltransferase Substrates: Tools for Understanding Protein Prenylation

    Energy Technology Data Exchange (ETDEWEB)

    DeGraw, Amanda J.; Hast, Michael A.; Xu, Juhua; Mullen, Daniel; Beese, Lorena S.; Barany, George; Distefano, Mark D. (Duke); (UMM)

    2010-11-15

    Originally designed to block the prenylation of oncogenic Ras, inhibitors of protein farnesyltransferase currently in preclinical and clinical trials are showing efficacy in cancers with normal Ras. Blocking protein prenylation has also shown promise in the treatment of malaria, Chagas disease and progeria syndrome. A better understanding of the mechanism, targets and in vivo consequences of protein prenylation are needed to elucidate the mode of action of current PFTase (Protein Farnesyltransferase) inhibitors and to create more potent and selective compounds. Caged enzyme substrates are useful tools for understanding enzyme mechanism and biological function. Reported here is the synthesis and characterization of caged substrates of PFTase. The caged isoprenoid diphosphates are poor substrates prior to photolysis. The caged CAAX peptide is a true catalytically caged substrate of PFTase in that it is to not a substrate, yet is able to bind to the enzyme as established by inhibition studies and X-ray crystallography. Irradiation of the caged molecules with 350 nm light readily releases their cognate substrate and their photolysis products are benign. These properties highlight the utility of those analogs towards a variety of in vitro and in vivo applications.

  16. Chimeric peptides as modulators of CK2-dependent signaling: Mechanism of action and off-target effects.

    Science.gov (United States)

    Zanin, Sofia; Sandre, Michele; Cozza, Giorgio; Ottaviani, Daniele; Marin, Oriano; Pinna, Lorenzo A; Ruzzene, Maria

    2015-10-01

    Protein kinase CK2 is a tetrameric enzyme composed of two catalytic (α/α') and two regulatory (β) subunits. It has a global prosurvival function, especially in cancer, and represents an attractive therapeutic target. Most CK2 inhibitors available so far are ATP-competitive compounds; however, the possibility to block only the phosphorylation of few substrates has been recently explored, and a compound composed of a Tat cell-penetrating peptide and an active cyclic peptide, selected for its ability to bind to the CK2 substrate E7 protein of human papilloma virus, has been developed [Perea et al., Cancer Res. 2004; 64:7127-7129]. By using a similar chimeric peptide (CK2 modulatory chimeric peptide, CK2-MCP), we performed a study to dissect its molecular mechanism of action and the signaling pathways that it affects in cells. We found that it directly interacts with CK2 itself, counteracting the regulatory and stabilizing functions of the β subunit. Cell treatment with CK2-MCP induces a rapid decrease of the amount of CK2 subunits, as well as of other signaling proteins. Concomitant cell death is observed, more pronounced in tumor cells and not accompanied by apoptotic events. CK2 relocalizes to lysosomes, whose proteases are activated, while the proteasome machinery is inhibited. Several sequence variants of the chimeric peptide have been also synthesized, and their effects compared to those of the parental peptide. Intriguingly, the Tat moiety is essential not only for cell penetration but also for the in vitro efficacy of the peptide. We conclude that this class of chimeric peptides, in addition to altering some properties of CK2 holoenzyme, affects several other cellular targets, causing profound perturbations of cell biology. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. A highly conserved basidiomycete peptide synthetase produces a trimeric hydroxamate siderophore.

    Science.gov (United States)

    Brandenburger, Eileen; Gressler, Markus; Leonhardt, Robin; Lackner, Gerald; Habel, Andreas; Hertweck, Christian; Brock, Matthias; Hoffmeister, Dirk

    2017-08-25

    The model white-rot basidiomycete Ceriporiopsis ( Gelatoporia ) subvermispora B encodes putative natural product biosynthesis genes. Among them is the gene for the seven-domain nonribosomal peptide synthetase CsNPS2. It is a member of the as-yet uncharacterized fungal type VI siderophore synthetase family which is highly conserved and widely distributed among the basidiomycetes. These enzymes include only one adenylation (A) domain, i.e., one complete peptide synthetase module and two thiolation/condensation (T-C) di-domain partial modules which, together, constitute an AT 1 C 1 T 2 C 2 T 3 C 3 domain setup. The full-length CsNPS2 enzyme (274.5 kDa) was heterologously produced as polyhistidine fusion in Aspergillus niger as soluble and active protein. N 5 -acetyl- N 5 -hydroxy-l-ornithine (l-AHO) and N 5 - cis -anhydromevalonyl- N 5 -hydroxy-l-ornithine (l-AMHO) were accepted as substrates, as assessed in vitro using the substrate-dependent [ 32 P]ATP-pyrophosphate radioisotope exchange assay. Full-length holo -CsNPS2 catalyzed amide bond formation between three l-AHO molecules to release the linear l-AHO trimer, called basidioferrin, as product in vitro , which was verified by LC-HRESIMS. Phylogenetic analyses suggest that type VI family siderophore synthetases are widespread in mushrooms and have evolved in a common ancestor of basidiomycetes. Importance : The basidiomycete nonribosomal peptide synthetase CsNPS2 represents a member of a widely distributed but previously uninvestigated class (type VI) of fungal siderophore synthetases. Genes orthologous to CsNPS2 are highly conserved across various phylogenetic clades of the basidiomycetes. Hence, our work serves as a broadly applicable model for siderophore biosynthesis and iron metabolism in higher fungi. Also, our results on the amino acid substrate preference of CsNPS2 supports further understanding of the substrate selectivity of fungal adenylation domains. Methodologically, this report highlights the

  18. Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries.

    Science.gov (United States)

    El Zoeiby, Ahmed; Sanschagrin, François; Darveau, André; Brisson, Jean-Robert; Levesque, Roger C

    2003-03-01

    The machinery of peptidoglycan biosynthesis is an ideal site at which to look for novel antimicrobial targets. Phage display was used to develop novel peptide inhibitors for MurC, an essential enzyme involved in the early steps of biosynthesis of peptidoglycan monomer. We cloned and overexpressed the murA, -B and -C genes from Pseudomonas aeruginosa in the pET expression vector, adding a His-tag to their C termini. The three proteins were overproduced in Escherichia coli and purified to homogeneity in milligram quantities. MurA and -B were combinatorially used to synthesize the MurC substrate UDP-N-acetylmuramate, the identity of which was confirmed by mass spectrometry and nuclear magnetic resonance analysis. Two phage-display libraries were screened against MurC in order to identify peptide ligands to the enzyme. Three rounds of biopanning were carried out, successively increasing elution specificity from round 1 to 3. The third round was accomplished with both non-specific elution and competitive elution with each of the three MurC substrates, UDP-N-acetylmuramic acid (UNAM), ATP and L-alanine. The DNA of 10 phage, selected randomly from each group, was extracted and sequenced, and consensus peptide sequences were elucidated. Peptides were synthesized and tested for inhibition of the MurC-catalysed reaction, and two peptides were shown to be inhibitors of MurC activity with IC(50)s of 1.5 and 0.9 mM, respectively. The powerful selection technique of phage display allowed us to identify two peptide inhibitors of the essential bacterial enzyme MurC. The peptide sequences represent the basis for the synthesis of inhibitory peptidomimetic molecules.

  19. Functionalized D-form self-assembling peptide hydrogels for bone regeneration

    Directory of Open Access Journals (Sweden)

    He B

    2016-04-01

    Full Text Available Bin He,1 Yunsheng Ou,1 Ao Zhou,1 Shuo Chen,1 Weikang Zhao,1 Jinqiu Zhao,2 Hong Li,3 Yong Zhu,1 Zenghui Zhao,1 Dianming Jiang1 1Department of Orthopedics, 2Department of Infectious Diseases, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China; 3School of Physical Science and Technology, Sichuan University, Chengdu, People’s Republic of China Abstract: Bone defects are very common in orthopedics, and there is great need to develop suitable bone grafts for transplantation in vivo. However, current bone grafts still encounter some limitations, including limited availability, immune rejection, poor osteoinduction and osteoconduction, poor biocompatibility and degradation properties, etc. Self-assembling peptide nanofiber scaffolds have emerged as an important substrate for cell culture and bone regeneration. We report on the structural features (eg, Congo red staining, circular dichroism spectroscopy, transmission electron microscopy, and rheometry assays and osteogenic ability of D-RADA16-RGD peptide hydrogels (with or without basic fibroblast growth factor due to the better stability of peptide bonds formed by these peptides compared with those formed by L-form peptides, and use them to fill the femoral condyle defect of Sprague Dawley rat model. The bone morphology change, two-dimensional reconstructions using microcomputed tomography, quantification of the microcomputed tomography analyses as well as histological analyses have demonstrated that RGD-modified D-form peptide scaffolds are able to enhance extensive bone regeneration. Keywords: bone defect, functionalized D-form self-assembling peptide, D-RADA16-RGD, peptide hydrogel, bone regeneration

  20. Self-assembling peptide hydrogels immobilized on silicon surfaces

    International Nuclear Information System (INIS)

    Franchi, Stefano; Battocchio, Chiara; Galluzzi, Martina; Navisse, Emanuele; Zamuner, Annj; Dettin, Monica; Iucci, Giovanna

    2016-01-01

    The hydrogels of self-assembling ionic complementary peptides have collected in the scientific community increasing consensus as mimetics of the extracellular matrix that can offer 3D supports for cell growth or be vehicles for the delivery of stem cells or drugs. Such scaffolds have also been proposed as bone substitutes for small defects as they promote beneficial effects on human osteoblasts. In this context, our research deals with the introduction of a layer of self-assembling peptides on a silicon surface by covalent anchoring and subsequent physisorption. In this work, we present a spectroscopic investigation of the proposed bioactive scaffolds, carried out by surface-sensitive spectroscopic techniques such as XPS (X-ray photoelectron spectroscopy) and RAIRS (Reflection Absorption Infrared Spectroscopy) and by state-of-the-art synchrotron radiation methodologies such as angle dependent NEXAFS (Near Edge X-ray Absorption Fine Structure). XPS studies confirmed the change in the surface composition in agreement with the proposed enrichments, and led to assess the self-assembling peptide chemical stability. NEXAFS spectra, collected in angular dependent mode at the N K-edge, allowed to investigate the self-assembling behavior of the macromolecules, as well as to determine their molecular orientation on the substrate. Furthermore, Infrared Spectroscopy measurements demonstrated that the peptide maintains its secondary structure (β-sheet anti-parallel) after deposition on the silicon surface. The complementary information acquired by means of XPS, NEXAFS and RAIRS lead to hypothesize a “layer-by-layer” arrangement of the immobilized peptides, giving rise to an ordered 3D nanostructure. - Highlights: • A self-assembling peptide (SAP) was covalently immobilized of on a flat silicon surface. • A physisorbed SAP layer was grown on top of the covalently immobilized peptide layer. • Molecular order and orientation of the peptide overlayer on the flat silicon

  1. Self-assembling peptide hydrogels immobilized on silicon surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Franchi, Stefano; Battocchio, Chiara; Galluzzi, Martina; Navisse, Emanuele [Department of Sciences, University “Roma Tre”, Via della Vasca Navale 79, Roma, 00146 (Italy); Zamuner, Annj; Dettin, Monica [Department of Industrial Engineering, University of Padua, Via Marzolo, 9, Padua, 35131 (Italy); Iucci, Giovanna, E-mail: giovanna.iucci@uniroma3.it [Department of Sciences, University “Roma Tre”, Via della Vasca Navale 79, Roma, 00146 (Italy)

    2016-12-01

    The hydrogels of self-assembling ionic complementary peptides have collected in the scientific community increasing consensus as mimetics of the extracellular matrix that can offer 3D supports for cell growth or be vehicles for the delivery of stem cells or drugs. Such scaffolds have also been proposed as bone substitutes for small defects as they promote beneficial effects on human osteoblasts. In this context, our research deals with the introduction of a layer of self-assembling peptides on a silicon surface by covalent anchoring and subsequent physisorption. In this work, we present a spectroscopic investigation of the proposed bioactive scaffolds, carried out by surface-sensitive spectroscopic techniques such as XPS (X-ray photoelectron spectroscopy) and RAIRS (Reflection Absorption Infrared Spectroscopy) and by state-of-the-art synchrotron radiation methodologies such as angle dependent NEXAFS (Near Edge X-ray Absorption Fine Structure). XPS studies confirmed the change in the surface composition in agreement with the proposed enrichments, and led to assess the self-assembling peptide chemical stability. NEXAFS spectra, collected in angular dependent mode at the N K-edge, allowed to investigate the self-assembling behavior of the macromolecules, as well as to determine their molecular orientation on the substrate. Furthermore, Infrared Spectroscopy measurements demonstrated that the peptide maintains its secondary structure (β-sheet anti-parallel) after deposition on the silicon surface. The complementary information acquired by means of XPS, NEXAFS and RAIRS lead to hypothesize a “layer-by-layer” arrangement of the immobilized peptides, giving rise to an ordered 3D nanostructure. - Highlights: • A self-assembling peptide (SAP) was covalently immobilized of on a flat silicon surface. • A physisorbed SAP layer was grown on top of the covalently immobilized peptide layer. • Molecular order and orientation of the peptide overlayer on the flat silicon

  2. Structural basis of carbohydrate recognition by lectin II from Ulex europaeus, a protein with a promiscuous carbohydrate-binding site.

    Science.gov (United States)

    Loris, R; De Greve, H; Dao-Thi, M H; Messens, J; Imberty, A; Wyns, L

    2000-08-25

    Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins. The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework. Copyright 2000 Academic Press.

  3. Water-Floating Giant Nanosheets from Helical Peptide Pentamers

    Science.gov (United States)

    Lee, Jaehun; Nam, Ki Tae

    One of the important challenges in the development of protein-mimetic materials is to understand the sequence specific assembly behavior and the dynamic folding change. Conventional strategies to construct two dimensional nanostructures from the peptides have been limited to beta-sheet forming sequences in use of basic building blocks because of their natural tendency to form sheet like aggregations. Here we identified a new peptide sequence, YFCFY that can form dimers by the disulfide bridge, fold into helix and assemble into macroscopic flat sheet at the air/water interface. Because of large driving force for two dimensional assembly and high elastic modulus of the resulting sheet, the peptide assembly induces the flattening of initially round water droplet. Additionally, we found that stabilization of helix by the dimerization is a key determinant for maintaining macroscopic flatness over a few tens centimeter even with a uniform thickness below 10 nm. Furthermore, the capability to transfer 2D film from water droplet to other substrates allows for the multiple stacking of 2D peptide nanostructure, suggesting possible applications in the biomimetic catalysts, biosensor and 2D related electronic devices. This work was supported by Samsung Research Funding Center of Samsung Electronics under Project Number SRFC-MA1401-01.

  4. Biochemical Characterisation of Phage Pseudomurein Endoisopeptidases PeiW and PeiP Using Synthetic Peptides

    OpenAIRE

    Schofield, Linley R.; Beattie, Amy K.; Tootill, Catherine M.; Dey, Debjit; Ronimus, Ron S.

    2015-01-01

    Pseudomurein endoisopeptidases cause lysis of the cell walls of methanogens by cleaving the isopeptide bond Ala-ε-Lys in the peptide chain of pseudomurein. PeiW and PeiP are two thermostable pseudomurein endoisopeptidases encoded by phage ΨM100 of Methanothermobacter wolfei and phages ΨM1 and ΨM2 of Methanothermobacter marburgensis, respectively. A continuous assay using synthetic peptide substrates was developed and used in the biochemical characterisation of recombinant PeiW and PeiP. The a...

  5. The under-appreciated promiscuity of the epidermal growth factor receptor family.

    Directory of Open Access Journals (Sweden)

    Sean P Kennedy

    2016-08-01

    Full Text Available Each member of the epidermal growth factor receptor (EGFR family plays a key role in normal development, homeostasis and a variety of pathophysiological conditions, most notably in cancer. According to the prevailing dogma, these four receptor tyrosine kinases (RTKs; EGFR, ERBB2, ERBB3 and ERBB4 function exclusively through the formation of homodimers and heterodimers within the EGFR family. These combinatorial receptor interactions are known to generate increased interactome diversity and therefore influence signalling output, subcellular localization and function of the heterodimer. This molecular plasticity is also thought to play a role in the development of resistance towards targeted cancer therapies aimed at these known oncogenes. Interestingly, many studies now challenge this dogma and suggest that the potential for EGFR family receptors to interact with more distantly related RTKs is much greater than currently appreciated. Here we discuss how the promiscuity of these oncogenic receptors may lead to the formation of many unexpected receptor pairings and the significant implications for the efficiency of many targeted cancer therapies.

  6. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

    Directory of Open Access Journals (Sweden)

    James Smadbeck

    Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA–protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 mM, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2

  7. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

    Directory of Open Access Journals (Sweden)

    James Smadbeck

    Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA-protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 [Formula: see text]M, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly

  8. In silico panning for a non-competitive peptide inhibitor

    Directory of Open Access Journals (Sweden)

    Ikebukuro Kazunori

    2007-01-01

    Full Text Available Abstract Background Peptide ligands have tremendous therapeutic potential as efficacious drugs. Currently, more than 40 peptides are available in the market for a drug. However, since costly and time-consuming synthesis procedures represent a problem for high-throughput screening, novel procedures to reduce the time and labor involved in screening peptide ligands are required. We propose the novel approach of 'in silico panning' which consists of a two-stage screening, involving affinity selection by docking simulation and evolution of the peptide ligand using genetic algorithms (GAs. In silico panning was successfully applied to the selection of peptide inhibitor for water-soluble quinoprotein glucose dehydrogenase (PQQGDH. Results The evolution of peptide ligands for a target enzyme was achieved by combining a docking simulation with evolution of the peptide ligand using genetic algorithms (GAs, which mimic Darwinian evolution. Designation of the target area as next to the substrate-binding site of the enzyme in the docking simulation enabled the selection of a non-competitive inhibitor. In all, four rounds of selection were carried out on the computer; the distribution of the docking energy decreased gradually for each generation and improvements in the docking energy were observed over the four rounds of selection. One of the top three selected peptides with the lowest docking energy, 'SERG' showed an inhibitory effect with Ki value of 20 μM. PQQGDH activity, in terms of the Vmax value, was 3-fold lower than that of the wild-type enzyme in the presence of this peptide. The mechanism of the SERG blockage of the enzyme was identified as non-competitive inhibition. We confirmed the specific binding of the peptide, and its equilibrium dissociation constant (KD value was calculated as 60 μM by surface plasmon resonance (SPR analysis. Conclusion We demonstrate an effective methodology of in silico panning for the selection of a non

  9. Silver nanoparticles as matrix for laser desorption/ionization mass spectrometry of peptides

    International Nuclear Information System (INIS)

    Hua Lin; Chen Jianrong; Ge Liya; Tan, Swee Ngin

    2007-01-01

    Silver nanoparticle synthesized from chemical reduction has been successfully utilized as a matrix in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) of peptides. Acting as a substrate to adsorb analytes, as well as a transmission medium for UV laser, silver nanoparticle was found to assist in the desorption/ionization of peptides with little or no induced fragmentation. The size of the nanoparticle was typically in the range of 160 ± 20 nm. One of the key advantages of silver nanoparticle for peptides analysis is its simple step for on-probe sample preparation. In addition, it also minimizes the interferences of sodium dodecyl sulfate (SDS) surfactant background signal, resulting in cleaner mass spectra and more sensitive signal, when compared to α-cyano-4-hydroxycinnamic acid (CCA) matrix

  10. Characterization and in vitro biological evaluation of mineral/osteogenic growth peptide nanocomposites synthesized biomimetically on titanium

    Science.gov (United States)

    Chen, Cen; Kong, Xiangdong; Zhang, Sheng-Min; Lee, In-Seop

    2015-04-01

    Nanocomposite layers of mineral/osteogenic growth peptide (OGP) were synthesized on calcium phosphate coated titanium substrates by immersing in calcium-phosphate buffer solution containing OGP. Peptide incorporated mineral was characterized by determining quantity loaded, effects on mineral morphology and structure. Also, the biological activity was investigated by cell adhesion, proliferation assay, and measurement of alkaline phosphatase (ALP) activity. X-ray photoelectron spectroscopy (XPS) and micro-bicinchoninic acid (BCA) assay revealed that OGP was successfully incorporated with mineral and the amount was increased with immersion time. Incorporated OGP changed the mineral morphology from sharp plate-like shape to more rounded one, and the octacalcium phosphate structure of the mineral was gradually transformed into apatite. With confocal microscopy to examine the incorporation of fluorescently labeled peptide, OGP was evenly distributed throughout mineral layers. Mineral/OGP nanocomposites promoted cell adhesion and proliferation, and also increased ALP activity of mesenchymal stem cells (MSCs). Results presented here indicated that the mineral/OGP nanocomposites formed on titanium substrates had the potential for applications in dental implants.

  11. Combined Cell Culture-Biosensing Platform Using Vertically Aligned Patterned Peptide Nanofibers for Cellular Studies

    DEFF Research Database (Denmark)

    Taskin, Mehmet B.; Sasso, Luigi; Dimaki, Maria

    2013-01-01

    it possible to avoid a loss of sensitivity because of the diffusion of the sample. The obtained results showed that the peptide nanofibers were suitable as a cell culturing substrate for PC12 cells. The peptide nanofibers could be employed as an alternative biological material to increase the adherence......This Article presents the development of a combined cell culture–biosensing platform using vertically aligned self-assembled peptide nanofibers. Peptide nanofibers were patterned on a microchip containing gold microelectrodes to provide the cells with a 3D environment enabling them to grow...... and proliferate. Gold microelectrodes were functionalized with conductive polymers for the electrochemical detection of dopamine released from PC12 cells. The combined cell culture–biosensing platform assured a close proximity of the release site, the cells and the active surface of the sensor, thereby rendering...

  12. Hsc66 substrate specificity is directed toward a discrete region of the iron-sulfur cluster template protein IscU.

    Science.gov (United States)

    Hoff, Kevin G; Ta, Dennis T; Tapley, Tim L; Silberg, Jonathan J; Vickery, Larry E

    2002-07-26

    Hsc66 and Hsc20 comprise a specialized chaperone system important for the assembly of iron-sulfur clusters in Escherchia coli. Only a single substrate, the Fe/S template protein IscU, has been identified for the Hsc66/Hsc20 system, but the mechanism by which Hsc66 selectively binds IscU is unknown. We have investigated Hsc66 substrate specificity using phage display and a peptide array of IscU. Screening of a heptameric peptide phage display library revealed that Hsc66 prefers peptides with a centrally located Pro-Pro motif. Using a cellulose-bound peptide array of IscU we determined that Hsc66 interacts specifically with a region (residues 99-103, LPPVK) that is invariant among all IscU family members. A synthetic peptide (ELPPVKIHC) corresponding to IscU residues 98-106 behaves in a similar manner to native IscU, stimulating the ATPase activity of Hsc66 with similar affinity as IscU, preventing Hsc66 suppression of bovine rhodanese aggregation, and interacting with the peptide-binding domain of Hsc66. Unlike native IscU, however, the synthetic peptide is not bound by Hsc20 and does not synergistically stimulate Hsc66 ATPase activity with Hsc20. Our results indicate that Hsc66 and Hsc20 recognize distinct regions of IscU and further suggest that Hsc66 will not bind LPPVK motifs with high affinity in vivo unless they are in the context of native IscU and can be directed to Hsc66 by Hsc20.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-04-03

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

  14. Bacterial protease uses distinct thermodynamic signatures for substrate recognition.

    Science.gov (United States)

    Bezerra, Gustavo Arruda; Ohara-Nemoto, Yuko; Cornaciu, Irina; Fedosyuk, Sofiya; Hoffmann, Guillaume; Round, Adam; Márquez, José A; Nemoto, Takayuki K; Djinović-Carugo, Kristina

    2017-06-06

    Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis, the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable interest. Surprisingly, these two microorganisms do not ferment carbohydrates; rather they use proteinaceous substrates as carbon and energy sources. However, the underlying biochemical mechanisms of their energy metabolism remain unknown. Here, we show that dipeptidyl peptidase 11 (DPP11), a central metabolic enzyme in these bacteria, undergoes a conformational change upon peptide binding to distinguish substrates from end products. It binds substrates through an entropy-driven process and end products in an enthalpy-driven fashion. We show that increase in protein conformational entropy is the main-driving force for substrate binding via the unfolding of specific regions of the enzyme ("entropy reservoirs"). The relationship between our structural and thermodynamics data yields a distinct model for protein-protein interactions where protein conformational entropy modulates the binding free-energy. Further, our findings provide a framework for the structure-based design of specific DPP11 inhibitors.

  15. Phosphorylation sites of Arabidopsis MAP Kinase Substrate 1 (MKS1)

    DEFF Research Database (Denmark)

    Caspersen, M.B.; Qiu, J.-L.; Zhang, X.

    2007-01-01

    The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophore......The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified...... phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal...... at approximately 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine...

  16. Molecular basis of substrate promiscuity for the SAM-dependent O-methyltransferase NcsB1, involved in the biosynthesis of the enediyne antitumor antibiotic neocarzinostatin.

    Science.gov (United States)

    Cooke, Heather A; Guenther, Elizabeth L; Luo, Yinggang; Shen, Ben; Bruner, Steven D

    2009-10-13

    The small molecule component of chromoprotein enediyne antitumor antibiotics is biosynthesized through a convergent route, incorporating amino acid, polyketide, and carbohydrate building blocks around a central enediyne hydrocarbon core. The naphthoic acid moiety of the enediyne neocarzinostatin plays key roles in the biological activity of the natural product by interacting with both the carrier protein and duplex DNA at the site of action. We have previously described the in vitro characterization of an S-adenosylmethionine-dependent O-methyltransferase (NcsB1) in the neocarzinostatin biosynthetic pathway [Luo, Y., Lin, S., Zhang, J., Cooke, H. A., Bruner, S. D., and Shen, B. (2008) J. Biol. Chem. 283, 14694-14702]. Here we provide a structural basis for NcsB1 activity, illustrating that the enzyme shares an overall architecture with a large family of S-adenosylmethionine-dependent proteins. In addition, NcsB1 represents the first enzyme to be structurally characterized in the biosynthetic pathway of neocarzinostatin. By cocrystallizing the enzyme with various combinations of the cofactor and substrate analogues, details of the active site structure have been established. Changes in subdomain orientation were observed via comparison of structures in the presence and absence of substrate, suggesting that reorientation of the enzyme is involved in binding of the substrate. In addition, residues important for substrate discrimination were predicted and probed through site-directed mutagenesis and in vitro biochemical characterization.

  17. Three-Dimensional Graphene–RGD Peptide Nanoisland Composites That Enhance the Osteogenesis of Human Adipose-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Ee-Seul Kang

    2018-02-01

    Full Text Available Graphene derivatives have immense potential in stem cell research. Here, we report a three-dimensional graphene/arginine-glycine-aspartic acid (RGD peptide nanoisland composite effective in guiding the osteogenesis of human adipose-derived mesenchymal stem cells (ADSCs. Amine-modified silica nanoparticles (SiNPs were uniformly coated onto an indium tin oxide electrode (ITO, followed by graphene oxide (GO encapsulation and electrochemical deposition of gold nanoparticles. A RGD–MAP–C peptide, with a triple-branched repeating RGD sequence and a terminal cysteine, was self-assembled onto the gold nanoparticles, generating the final three-dimensional graphene–RGD peptide nanoisland composite. We generated substrates with various gold nanoparticle–RGD peptide cluster densities, and found that the platform with the maximal number of clusters was most suitable for ADSC adhesion and spreading. Remarkably, the same platform was also highly efficient at guiding ADSC osteogenesis compared with other substrates, based on gene expression (alkaline phosphatase (ALP, runt-related transcription factor 2, enzyme activity (ALP, and calcium deposition. ADSCs induced to differentiate into osteoblasts showed higher calcium accumulations after 14–21 days than when grown on typical GO-SiNP complexes, suggesting that the platform can accelerate ADSC osteoblastic differentiation. The results demonstrate that a three-dimensional graphene–RGD peptide nanoisland composite can efficiently derive osteoblasts from mesenchymal stem cells.

  18. In situ imaging and proteome profiling indicate andrographolide is a highly promiscuous compound

    Science.gov (United States)

    Li, Lin; Wijaya, Hadhi; Samanta, Sanjay; Lam, Yulin; Yao, Shao Q.

    2015-06-01

    Natural products represent an enormous source of pharmacologically useful compounds, and are often used as the starting point in modern drug discovery. Many biologically interesting natural products are however not being pursued as potential drug candidates, partly due to a lack of well-defined mechanism-of-action. Traditional in vitro methods for target identification of natural products based on affinity protein enrichment from crude cellular lysates cannot faithfully recapitulate protein-drug interactions in living cells. Reported herein are dual-purpose probes inspired by the natural product andrographolide, capable of both reaction-based, real-time bioimaging and in situ proteome profiling/target identification in live mammalian cells. Our results confirm that andrographolide is a highly promiscuous compound and engaged in covalent interactions with numerous previously unknown cellular targets in cell type-specific manner. We caution its potential therapeutic effects should be further investigated in detail.

  19. Structure and Functional Analysis of ClbQ, an Unusual Intermediate-Releasing Thioesterase from the Colibactin Biosynthetic Pathway.

    Science.gov (United States)

    Guntaka, Naga Sandhya; Healy, Alan R; Crawford, Jason M; Herzon, Seth B; Bruner, Steven D

    2017-10-20

    Colibactin is a genotoxic hybrid nonribosomal peptide/polyketide secondary metabolite produced by various pathogenic and probiotic bacteria residing in the human gut. The presence of colibactin metabolites has been correlated to colorectal cancer formation in several studies. The specific function of many gene products in the colibactin gene cluster can be predicted. However, the role of ClbQ, a type II editing thioesterase, has not been established. The importance of ClbQ has been demonstrated by genetic deletions that abolish colibactin cytotoxic activity, and recent studies suggest an atypical role in releasing pathway intermediates from the assembly line. Here we report the 2.0 Å crystal structure and biochemical characterization of ClbQ. Our data reveal that ClbQ exhibits greater catalytic efficiency toward acyl-thioester substrates as compared to precolibactin intermediates and does not discriminate among carrier proteins. Cyclized pyridone-containing colibactins, which are off-pathway derivatives, are not viable substrates for ClbQ, while linear precursors are, supporting a role of ClbQ in facilitating the promiscuous off-loading of premature precolibactin metabolites and novel insights into colibactin biosynthesis.

  20. Structure and Functional Analysis of ClbQ, an Unusual Intermediate-Releasing Thioesterase from the Colibactin Biosynthetic Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Guntaka, Naga Sandhya; Healy, Alan R.; Crawford, Jason M.; Herzon, Seth B.; Bruner, Steven D. (Yale); (Florida); (Yale-MED)

    2017-09-08

    Colibactin is a genotoxic hybrid nonribosomal peptide/polyketide secondary metabolite produced by various pathogenic and probiotic bacteria residing in the human gut. The presence of colibactin metabolites has been correlated to colorectal cancer formation in several studies. The specific function of many gene products in the colibactin gene cluster can be predicted. However, the role of ClbQ, a type II editing thioesterase, has not been established. The importance of ClbQ has been demonstrated by genetic deletions that abolish colibactin cytotoxic activity, and recent studies suggest an atypical role in releasing pathway intermediates from the assembly line. Here we report the 2.0 Å crystal structure and biochemical characterization of ClbQ. Our data reveal that ClbQ exhibits greater catalytic efficiency toward acyl-thioester substrates as compared to precolibactin intermediates and does not discriminate among carrier proteins. Cyclized pyridone-containing colibactins, which are off-pathway derivatives, are not viable substrates for ClbQ, while linear precursors are, supporting a role of ClbQ in facilitating the promiscuous off-loading of premature precolibactin metabolites and novel insights into colibactin biosynthesis.

  1. Antioxidant activity measurement and potential antioxidant peptides exploration from hydrolysates of novel continuous microwave-assisted enzymolysis of the Scomberomorus niphonius protein.

    Science.gov (United States)

    Huang, Yipeng; Ruan, Guihua; Qin, Zhijun; Li, Haiyun; Zheng, Yanjie

    2017-05-15

    A novel continuous microwave-assisted enzymatic digestion (cMAED) method is proposed for the digestion of protein from Scomberomorus niphonius to obtain potential antioxidant peptides. In this study, bromelain was found to have a high capacity for the digestion of the Scomberomorus niphonius protein. The following cMAED conditions were investigated: protease species, microwave power, temperature, bromelain content, acidity of the substrate solution, and incubation time. At 400W, 40°C, 1500U·g -1 bromelain, 20% substrate concentration, pH 6.0 and 5min incubation, the degree of hydrolysis and total antioxidant activity of the hydrolysates were 15.86% and 131.49μg·mL -1 , respectively. The peptide analyses showed that eight of the potential antioxidant peptide sequences, which ranged from 502.32 to 1080.55Da with 4-10 amino acid residues, had features typical of well-known antioxidant proteins. Thus, the new cMAED method can be useful to obtain potential antioxidant peptides from protein sources, such as Scomberomorus niphonius. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Structural and functional analysis of cyclin D1 reveals p27 and substrate inhibitor binding requirements.

    Science.gov (United States)

    Liu, Shu; Bolger, Joshua K; Kirkland, Lindsay O; Premnath, Padmavathy N; McInnes, Campbell

    2010-12-17

    An alternative strategy for inhibition of the cyclin dependent kinases (CDKs) in antitumor drug discovery is afforded through the substrate recruitment site on the cyclin positive regulatory subunit. Critical CDK substrates such as the Rb and E2F families must undergo cyclin groove binding before phosphorylation, and hence inhibitors of this interaction also block substrate specific kinase activity. This approach offers the potential to generate highly selective and cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7 and 9, commonly observed with ATP competitive compounds. While highly potent peptide and small molecule inhibitors of CDK2/cyclin A, E substrate recruitment have been reported, little information has been generated on the determinants of inhibitor binding to the cyclin groove of the CDK4/cyclin D1 complex. CDK4/cyclin D is a validated anticancer drug target and continues to be widely pursued in the development of new therapeutics based on cell cycle blockade. We have therefore investigated the structural basis for peptide binding to its cyclin groove and have examined the features contributing to potency and selectivity of inhibitors. Peptidic inhibitors of CDK4/cyclin D of pRb phosphorylation have been synthesized, and their complexes with CDK4/cyclin D1 crystal structures have been generated. Based on available structural information, comparisons of the cyclin grooves of cyclin A2 and D1 are presented and provide insights into the determinants for peptide binding and the basis for differential binding and inhibition. In addition, a complex structure has been generated in order to model the interactions of the CDKI, p27(KIP)¹, with cyclin D1. This information has been used to shed light onto the endogenous inhibition of CDK4 and also to identify unique aspects of cyclin D1 that can be exploited in the design of cyclin groove based CDK inhibitors. Peptidic and nonpeptidic compounds have been

  3. The Cysteine S-Alkylation Reaction as a Synthetic Method to Covalently Modify Peptide Sequences.

    Science.gov (United States)

    Calce, Enrica; De Luca, Stefania

    2017-01-05

    Synthetic methodologies to chemically modify peptide molecules have long been investigated for their impact in the field of chemical biology. They allow the introduction of biochemical probes useful for studying protein functions, for manipulating peptides with therapeutic potential, and for structure-activity relationship investigations. The commonly used approach was the derivatization of an amino acid side chain. In this regard, the cysteine, for its unique reactivity, has been widely employed as the substrate for such modifications. Herein, we report on methodologies developed to modify the cysteine thiol group through the S-alkylation reaction. Some procedures perform the alkylation of cysteine derivatives, in order to prepare building blocks to be used during the peptide synthesis, whilst some others selectively modify peptide sequences containing a cysteine residue with a free thiol group, both in solution and in the solid phase. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Fluorescent turn-on determination of the activity of peptidases using peptide templated gold nanoclusters

    International Nuclear Information System (INIS)

    Luo, Junjun; Wang, Liqiang; Zeng, Ke; Shen, Congcong; Qian, Pin; Yang, Minghui; Rasooly, Avraham; Qu, Fengli

    2016-01-01

    The fluorescence intensity of gold nanoclusters (AuNCs) is inversely related to the length of a peptide immobilized on its surface. This finding has been exploited to design a turn-on fluorescent method for the determination of the activity of peptidase. The β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) was chosen as a model peptidase. BACE1 cleaves the peptide substrates on AuNCs, and the fluorescence intensity of the AuNCs (at exCitation/emission wavelengths of 320/405 nm) carrying the rest of the cleaved peptide is significantly higher than that of the AuNCs with uncleaved peptide. Transmission electron microscopy revealed a decrease in the size of the AuNCs which is assumed cause fluorescence enhancement. The assay was applied to the determination of BACE1 activity in spiked cell lysates, and recoveries were between 96.9 and 104.0 %. (author)

  5. Peptide stabilized amphotericin B nanodisks

    Science.gov (United States)

    Tufteland, Megan; Pesavento, Joseph B.; Bermingham, Rachelle L.; Hoeprich, Paul D.; Ryan, Robert O.

    2007-01-01

    Nanometer scale apolipoprotein A-I stabilized phospholipid disk complexes (nanodisks; ND) have been formulated with the polyene antibiotic amphotericin B (AMB). The present studies were designed to evaluate if a peptide can substitute for the function of the apolipoprotein component of ND with respect to particle formation and stability. An 18-residue synthetic amphipathic α-helical peptide, termed 4F (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2), solubilized vesicles comprised of egg phosphatidylcholine (egg PC), dipentadecanoyl PC or dimyristoylphosphatidylcholine (DMPC) at rates greater than or equal to solubilization rates observed with human apolipoprotein A-I (apoA-I; 243 amino acids). Characterization studies revealed that interaction with DMPC induced a near doubling of 4F tryptophan fluorescence emission quantum yield (excitation 280 nm) and a ~7 nm blue shift in emission wavelength maximum. Inclusion of AMB in the vesicle substrate resulted in formation of 4F AMB-ND. Spectra of AMB containing particles revealed the antibiotic is a highly effective quencher of 4F tryptophan fluorescence emission, giving rise to a Ksv = 7.7 × 104. Negative stain electron microscopy revealed that AMB-ND prepared with 4F possessed a disk shaped morphology similar to ND prepared without AMB or prepared with apoA-I. In yeast and pathogenic fungi growth inhibition assays, 4F AMB-ND was as effective as apoA-I AMB-ND. The data indicate that AMB-ND generated using an amphipathic peptide in lieu of apoA-I form a discrete population of particles that possess potent biological activity. Given their intrinsic versatility, peptides may be preferred for scale up and clinical application of AMB-ND. PMID:17293004

  6. Sensitive Versatile Fluorogenic Transmembrane Peptide Substrates for Rhomboid Intramembrane Proteases

    Czech Academy of Sciences Publication Activity Database

    Tichá, Anežka; Stanchev, Stancho; Škerle, Jan; Began, Jakub; Ingr, M.; Švehlová, Kateřina; Polovinkin, L.; Růžička, Martin; Bednárová, Lucie; Hadravová, Romana; Poláchová, Edita; Rampírová, Petra; Březinová, Jana; Kašička, Václav; Majer, Pavel; Stříšovský, Kvido

    2017-01-01

    Roč. 292, č. 7 (2017), s. 2703-2713 ISSN 0021-9258 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302; GA ČR(CZ) GBP208/12/G016; GA ČR(CZ) GA15-01948S EU Projects: European Commission(XE) 304154 - Rhomboid substrates Grant - others:EMBO(DE) 2329 Institutional support: RVO:61388963 Keywords : protein secondary structure * membrane proteins * circular dichroism Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.125, year: 2016 http://www.jbc.org/content/292/7/2703.full

  7. Short Peptides Enhance Single Cell Adhesion and Viability onMicroarrays

    Energy Technology Data Exchange (ETDEWEB)

    Veiseh, Mandana; Veiseh, Omid; Martin, Michael C.; Asphahani,Fareid; Zhang, Miqin

    2007-01-19

    Single cell patterning holds important implications forbiology, biochemistry, biotechnology, medicine, and bioinformatics. Thechallenge for single cell patterning is to produce small islands hostingonly single cells and retaining their viability for a prolonged period oftime. This study demonstrated a surface engineering approach that uses acovalently bound short peptide as a mediator to pattern cells withimproved single cell adhesion and prolonged cellular viabilityon goldpatterned SiO2 substrates. The underlying hypothesis is that celladhesion is regulated bythe type, availability, and stability ofeffective cell adhesion peptides, and thus covalently bound shortpeptides would promote cell spreading and, thus, single cell adhesion andviability. The effectiveness of this approach and the underlyingmechanism for the increased probability of single cell adhesion andprolonged cell viability by short peptides were studied by comparingcellular behavior of human umbilical cord vein endothelial cells on threemodelsurfaces whose gold electrodes were immobilized with fibronectin,physically adsorbed Arg-Glu-Asp-Val-Tyr, and covalently boundLys-Arg-Glu-Asp-Val-Tyr, respectively. The surface chemistry and bindingproperties were characterized by reflectance Fourier transform infraredspectroscopy. Both short peptides were superior to fibronectin inproducing adhesion of only single cells, whereas the covalently boundpeptide also reduced apoptosis and necrosisof adhered cells. Controllingcell spreading by peptide binding domains to regulate apoptosis andviability represents a fundamental mechanism in cell-materialsinteraction and provides an effective strategy in engineering arrays ofsingle cells.

  8. Cardioprotection Resulting from Glucagon-Like Peptide-1 Administration Involves Shifting Metabolic Substrate Utilization to Increase Energy Efficiency in the Rat Heart.

    Science.gov (United States)

    Aravindhan, Karpagam; Bao, Weike; Harpel, Mark R; Willette, Robert N; Lepore, John J; Jucker, Beat M

    2015-01-01

    Previous studies have shown that glucagon-like peptide-1 (GLP-1) provides cardiovascular benefits independent of its role on peripheral glycemic control. However, the precise mechanism(s) by which GLP-1 treatment renders cardioprotection during myocardial ischemia remain unresolved. Here we examined the role for GLP-1 treatment on glucose and fatty acid metabolism in normal and ischemic rat hearts following a 30 min ischemia and 24 h reperfusion injury, and in isolated cardiomyocytes (CM). Relative carbohydrate and fat oxidation levels were measured in both normal and ischemic hearts using a 1-13C glucose clamp coupled with NMR-based isotopomer analysis, as well as in adult rat CMs by monitoring pH and O2 consumption in the presence of glucose or palmitate. In normal heart, GLP-1 increased glucose uptake (↑64%, pregions in response to this injury. In particular, a switch to anaerobic glycolysis in the ischemic area provides a compensatory substrate switch to overcome the energetic deficit in this region in the face of reduced tissue oxygenation, whereas a switch to more energetically favorable carbohydrate oxidation in more highly oxygenated remote regions supports maintaining cardiac contractility in a complementary manner.

  9. Activation of the DnaK-ClpB Complex is Regulated by the Properties of the Bound Substrate.

    Science.gov (United States)

    Fernández-Higuero, Jose Angel; Aguado, Alejandra; Perales-Calvo, Judit; Moro, Fernando; Muga, Arturo

    2018-04-11

    The chaperone ClpB in bacteria is responsible for the reactivation of aggregated proteins in collaboration with the DnaK system. Association of these chaperones at the aggregate surface stimulates ATP hydrolysis, which mediates substrate remodeling. However, a question that remains unanswered is whether the bichaperone complex can be selectively activated by substrates that require remodeling. We find that large aggregates or bulky, native-like substrates activates the complex, whereas a smaller, permanently unfolded protein or extended, short peptides fail to stimulate it. Our data also indicate that ClpB interacts differently with DnaK in the presence of aggregates or small peptides, displaying a higher affinity for aggregate-bound DnaK, and that DnaK-ClpB collaboration requires the coupled ATPase-dependent remodeling activities of both chaperones. Complex stimulation is mediated by residues at the β subdomain of DnaK substrate binding domain, which become accessible to the disaggregase when the lid is allosterically detached from the β subdomain. Complex activation also requires an active NBD2 and the integrity of the M domain-ring of ClpB. Disruption of the M-domain ring allows the unproductive stimulation of the DnaK-ClpB complex in solution. The ability of the DnaK-ClpB complex to discrimínate different substrate proteins might allow its activation when client proteins require remodeling.

  10. Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

    Science.gov (United States)

    Deuerling, Elke; Patzelt, Holger; Vorderwülbecke, Sonja; Rauch, Thomas; Kramer, Günter; Schaffitzel, Elke; Mogk, Axel; Schulze-Specking, Agnes; Langen, Hanno; Bukau, Bernd

    2003-03-01

    Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

  11. Characterizing Protease Specificity: How Many Substrates Do We Need?

    Directory of Open Access Journals (Sweden)

    Michael Schauperl

    Full Text Available Calculation of cleavage entropies allows to quantify, map and compare protease substrate specificity by an information entropy based approach. The metric intrinsically depends on the number of experimentally determined substrates (data points. Thus a statistical analysis of its numerical stability is crucial to estimate the systematic error made by estimating specificity based on a limited number of substrates. In this contribution, we show the mathematical basis for estimating the uncertainty in cleavage entropies. Sets of cleavage entropies are calculated using experimental cleavage data and modeled extreme cases. By analyzing the underlying mathematics and applying statistical tools, a linear dependence of the metric in respect to 1/n was found. This allows us to extrapolate the values to an infinite number of samples and to estimate the errors. Analyzing the errors, a minimum number of 30 substrates was found to be necessary to characterize substrate specificity, in terms of amino acid variability, for a protease (S4-S4' with an uncertainty of 5 percent. Therefore, we encourage experimental researchers in the protease field to record specificity profiles of novel proteases aiming to identify at least 30 peptide substrates of maximum sequence diversity. We expect a full characterization of protease specificity helpful to rationalize biological functions of proteases and to assist rational drug design.

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

    Science.gov (United States)

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

    2004-06-04

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

  13. Transport of peptidomimetic drugs by the intestinal Di/tri-peptide transporter, PepT1

    DEFF Research Database (Denmark)

    Brodin, Birger; Nielsen, Carsten Uhd; Steffansen, Bente

    2002-01-01

    The apical membrane of small intestinal enterocytes possess an uptake system for di- and tripeptides. The physiological function of the system is to transport small peptides resulting from digestion of dietary protein. Moreover, due to the broad substrate specificity of the system, it is also cap...

  14. Peptides and Anti-peptide Antibodies for Small and Medium Scale Peptide and Anti-peptide Affinity Microarrays: Antigenic Peptide Selection, Immobilization, and Processing.

    Science.gov (United States)

    Zhang, Fan; Briones, Andrea; Soloviev, Mikhail

    2016-01-01

    This chapter describes the principles of selection of antigenic peptides for the development of anti-peptide antibodies for use in microarray-based multiplex affinity assays and also with mass-spectrometry detection. The methods described here are mostly applicable to small to medium scale arrays. Although the same principles of peptide selection would be suitable for larger scale arrays (with 100+ features) the actual informatics software and printing methods may well be different. Because of the sheer number of proteins/peptides to be processed and analyzed dedicated software capable of processing all the proteins and an enterprise level array robotics may be necessary for larger scale efforts. This report aims to provide practical advice to those who develop or use arrays with up to ~100 different peptide or protein features.

  15. Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation

    Science.gov (United States)

    Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

    2016-11-01

    Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD+-dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some “loose-binding” substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

  16. Therapeutic peptides for cancer therapy. Part II - cell cycle inhibitory peptides and apoptosis-inducing peptides.

    Science.gov (United States)

    Raucher, Drazen; Moktan, Shama; Massodi, Iqbal; Bidwell, Gene L

    2009-10-01

    Therapeutic peptides have great potential as anticancer agents owing to their ease of rational design and target specificity. However, their utility in vivo is limited by low stability and poor tumor penetration. The authors review the development of peptide inhibitors with potential for cancer therapy. Peptides that arrest the cell cycle by mimicking CDK inhibitors or induce apoptosis directly are discussed. The authors searched Medline for articles concerning the development of therapeutic peptides and their delivery. Inhibition of cancer cell proliferation directly using peptides that arrest the cell cycle or induce apoptosis is a promising strategy. Peptides can be designed that interact very specifically with cyclins and/or cyclin-dependent kinases and with members of apoptotic cascades. Use of these peptides is not limited by their design, as a rational approach to peptide design is much less challenging than the design of small molecule inhibitors of specific protein-protein interactions. However, the limitations of peptide therapy lie in the poor pharmacokinetic properties of these large, often charged molecules. Therefore, overcoming the drug delivery hurdles could open the door for effective peptide therapy, thus making an entirely new class of molecules useful as anticancer drugs.

  17. Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.

    Science.gov (United States)

    Prudova, Anna; auf dem Keller, Ulrich; Butler, Georgina S; Overall, Christopher M

    2010-05-01

    Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses

  18. Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display

    OpenAIRE

    Zhang, Keya; Nelson, Kathryn M.; Bhuripanyo, Karan; Grimes, Kimberly D.; Zhao, Bo; Aldrich, Courtney C.; Yin, Jun

    2013-01-01

    The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in kcat/Km with nonnative sub...

  19. Microbial degradation rates of small peptides and amino acids in the oxygen minimum zone of Chilean coastal waters

    Science.gov (United States)

    Pantoja, Silvio; Rossel, Pamela; Castro, Rodrigo; Cuevas, L. Antonio; Daneri, Giovanni; Córdova, Candy

    2009-07-01

    We found similar microbial degradation rates of labile dissolved organic matter in oxic and suboxic waters off northern Chile. Rates of peptide hydrolysis and amino acid uptake in unconcentrated water samples were not low in the water column where oxygen concentration was depleted. Hydrolysis rates ranged from 65 to 160 nmol peptide L -1 h -1 in the top 20 m, 8-28 nmol peptide L -1 h -1 between 100 and 300 m (O 2-depleted zone), and 14-19 nmol peptide L -1 h -1 between 600 and 800 m. Dissolved free amino acid uptake rates were 9-26, 3-17, and 6 nmol L -1 h -1 at similar depth intervals. Since these findings are consistent with a model of comparable potential activity of microbes in degrading labile substrates of planktonic origin, we suggest, as do other authors, that differences in decomposition rates with high and low oxygen concentrations may be a matter of substrate lability. The comparison between hydrolysis and uptake rates indicates that microbial peptide hydrolysis occurs at similar or faster rates than amino acid uptake in the water column, and that the hydrolysis of peptides is not a rate-limiting step for the complete remineralization of labile macromolecules. Low O 2 waters process about 10 tons of peptide carbon per h, double the amount processed in surface-oxygenated water. In the oxygen minimum zone, we suggest that the C balance may be affected by the low lability of the dissolved organic matter when this is upwelled to the surface. An important fraction of dissolved organic matter is processed in the oxygen minimum layer, a prominent feature of the coastal ocean in the highly productive Humboldt Current System.

  20. Diversity of Francisella tularensis Schu4 antigens recognized by T lymphocytes after natural infections in humans: identification of candidate epitopes for inclusion in a rationally designed tularemia vaccine

    DEFF Research Database (Denmark)

    McMurry, Julie A; Gregory, Stephen H; Moise, Leonard

    2007-01-01

    The T lymphocyte antigens, which may have a role in protection against tularemia, were predicted by immunoinformatics analysis of Francisella tularensis Schu4. Twenty-seven class II putative promiscuous epitopes and 125 putative class I supertype epitopes were chosen for synthesis; peptides were...... responded to pools of 25 A2, A24, and B7 peptides, respectively. These data can aid in the development of novel epitope-based and subunit tularemia vaccines....

  1. Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs using PICS with proteome-derived peptide libraries.

    Directory of Open Access Journals (Sweden)

    Olivier Barré

    Full Text Available Type II transmembrane serine proteases (TTSPs are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS. Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin to simultaneously determine sequence preferences on the N-terminal non-prime (P and C-terminal prime (P' sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1' position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1' positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.

  2. Molecular dynamics simulations of protein-tyrosine phosphatase 1B: II. Substrate-enzyme interactions and dynamics

    DEFF Research Database (Denmark)

    Peters, Günther H.j.; Frimurer, T. M.; Andersen, J. N.

    2000-01-01

    Molecular dynamics simulations of protein tyrosine phosphatase 1B (PTP1B) complexed with the phosphorylated peptide substrate DADEpYL and the free substrate have been conducted to investigate 1) the physical forces involved in substrate-protein interactions, 2) the importance of enzyme...... to substrate binding. Based on essential dynamics analysis of the PTP1B/DADEpYL trajectory, it is shown that internal motions in the binding pocket occur in a subspace of only a few degrees of freedom. in particular, relatively large flexibilities are observed along several eigenvectors in the segments: Arg(24...... for catalysis. Analysis of the individual enzyme-substrate interaction energies revealed that mainly electrostatic forces contribute to binding. Indeed, calculation of the electrostatic field of the enzyme reveals that only the field surrounding the binding pocket is positive, while the remaining protein...

  3. Peptides, polypeptides and peptide-polymer hybrids as nucleic acid carriers.

    Science.gov (United States)

    Ahmed, Marya

    2017-10-24

    Cell penetrating peptides (CPPs), and protein transduction domains (PTDs) of viruses and other natural proteins serve as a template for the development of efficient peptide based gene delivery vectors. PTDs are sequences of acidic or basic amphipathic amino acids, with superior membrane trespassing efficacies. Gene delivery vectors derived from these natural, cationic and cationic amphipathic peptides, however, offer little flexibility in tailoring the physicochemical properties of single chain peptide based systems. Owing to significant advances in the field of peptide chemistry, synthetic mimics of natural peptides are often prepared and have been evaluated for their gene expression, as a function of amino acid functionalities, architecture and net cationic content of peptide chains. Moreover, chimeric single polypeptide chains are prepared by a combination of multiple small natural or synthetic peptides, which imparts distinct physiological properties to peptide based gene delivery therapeutics. In order to obtain multivalency and improve the gene delivery efficacies of low molecular weight cationic peptides, bioactive peptides are often incorporated into a polymeric architecture to obtain novel 'polymer-peptide hybrids' with improved gene delivery efficacies. Peptide modified polymers prepared by physical or chemical modifications exhibit enhanced endosomal escape, stimuli responsive degradation and targeting efficacies, as a function of physicochemical and biological activities of peptides attached onto a polymeric scaffold. The focus of this review is to provide comprehensive and step-wise progress in major natural and synthetic peptides, chimeric polypeptides, and peptide-polymer hybrids for nucleic acid delivery applications.

  4. Quantitative kinetics of proteolytic enzymes determined by a surface concentration-based assay using peptide arrays.

    Science.gov (United States)

    Jung, Se-Hui; Kong, Deok-Hoon; Park, Seoung-Woo; Kim, Young-Myeong; Ha, Kwon-Soo

    2012-08-21

    Peptide arrays have emerged as a key technology for drug discovery, diagnosis, and cell biology. Despite the promise of these arrays, applications of peptide arrays to quantitative analysis of enzyme kinetics have been limited due to the difficulty in obtaining quantitative information of enzymatic reaction products. In this study, we developed a new approach for the quantitative kinetics analysis of proteases using fluorescence-conjugated peptide arrays, a surface concentration-based assay with solid-phase peptide standards using dry-off measurements, and compared it with an applied concentration-based assay. For fabrication of the peptide arrays, substrate peptides of cMMP-3, caspase-3, caspase-9, and calpain-1 were functionalized with TAMRA and cysteine, and were immobilized onto amine-functionalized arrays using a heterobifunctional linker, N-[γ-maleimidobutyloxy]succinimide ester. The proteolytic activities of the four enzymes were quantitatively analyzed by calculating changes induced by enzymatic reactions in the concentrations of peptides bound to array surfaces. In addition, this assay was successfully applied for calculating the Michaelis constant (K(m,surf)) for the four enzymes. Thus, this new assay has a strong potential for use in the quantitative evaluation of proteases, and for drug discovery through kinetics studies including the determination of K(m) and V(max).

  5. Synthesis and characterisation of substrate-based peptides as inhibitors of histone demethylase KDM4C

    DEFF Research Database (Denmark)

    Nielsen, Simon D; Leurs, Ulrike; Bergner, Magnus

    2016-01-01

    different series of peptides were designed and synthesized. One series contained N-acylated H3K9 and two series introduced triazoles in this position via click chemistry to enable facile variation of headgroups. As the click reaction is compatible with free amino acids this was performed on an azido...

  6. Catalytic water co-existing with a product peptide in the active site of HIV-1 protease revealed by X-ray structure analysis.

    Science.gov (United States)

    Prashar, Vishal; Bihani, Subhash; Das, Amit; Ferrer, Jean-Luc; Hosur, Madhusoodan

    2009-11-17

    It is known that HIV-1 protease is an important target for design of antiviral compounds in the treatment of Acquired Immuno Deficiency Syndrome (AIDS). In this context, understanding the catalytic mechanism of the enzyme is of crucial importance as transition state structure directs inhibitor design. Most mechanistic proposals invoke nucleophilic attack on the scissile peptide bond by a water molecule. But such a water molecule coexisting with any ligand in the active site has not been found so far in the crystal structures. We report here the first observation of the coexistence in the active site, of a water molecule WAT1, along with the carboxyl terminal product (Q product) peptide. The product peptide has been generated in situ through cleavage of the full-length substrate. The N-terminal product (P product) has diffused out and is replaced by a set of water molecules while the Q product is still held in the active site through hydrogen bonds. The position of WAT1, which hydrogen bonds to both the catalytic aspartates, is different from when there is no substrate bound in the active site. We propose WAT1 to be the position from where catalytic water attacks the scissile peptide bond. Comparison of structures of HIV-1 protease complexed with the same oligopeptide substrate, but at pH 2.0 and at pH 7.0 shows interesting changes in the conformation and hydrogen bonding interactions from the catalytic aspartates. The structure is suggestive of the repositioning, during substrate binding, of the catalytic water for activation and subsequent nucleophilic attack. The structure could be a snap shot of the enzyme active site primed for the next round of catalysis. This structure further suggests that to achieve the goal of designing inhibitors mimicking the transition-state, the hydrogen-bonding pattern between WAT1 and the enzyme should be replicated.

  7. Fiscal 1994 report on results of R and D on innovative technology for producing advanced biomaterial. Technology of fixation and utilization of carbon dioxide using peptides; 1994 nendo senshin bio zairyo no sosei kako gijutsu no kenkyu kaihatsu seika hokokusho. Pepuchido oyo nisanka tanso koteika yuko riyo gijutsu

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-03-01

    R and D of a material was conducted that has a function of fixation of carbon dioxide for example, by synthesizing non-natural amino acids required for the manifestation of the function, incorporating the amino acids into new bio-functional peptides to be synthesized, and fixing the peptides on a substrate. Activities were carried out in three areas, which were (1) innovative technologies for producing functional molecules: R and D concerning structural and functional design of peptides, conformational control technique, preparation of peptides with photoelectric conversion function, peptide synthesis using enzyme, and method of incorporating non-natural amino acids into peptides, (2) R and D on materialization technologies of functional molecules: formation of film onto a substrate, pattern forming technique, substrate modification technique, peptide binding reagent or linker, and development of technologies for creation of biomaterials having molecular recognition function and their stabilization. and (3) comprehensive investigation and adjustment (operation of information exchange conference and adjustment of the progress). In (2), an examination was made on the effect of polymerization on functions, for example, by fixing molecular recognition peptides in monomers and polymers on a catalyst support. (NEDO)

  8. Cathepsin-Mediated Cleavage of Peptides from Peptide Amphiphiles Leads to Enhanced Intracellular Peptide Accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Acar, Handan [Institute; Department; Samaeekia, Ravand [Institute; Department; Schnorenberg, Mathew R. [Institute; Department; Medical; Sasmal, Dibyendu K. [Institute; Huang, Jun [Institute; Tirrell, Matthew V. [Institute; Institute; LaBelle, James L. [Department

    2017-08-24

    Peptides synthesized in the likeness of their native interaction domain(s) are natural choices to target protein protein interactions (PPIs) due to their fidelity of orthostatic contact points between binding partners. Despite therapeutic promise, intracellular delivery of biofunctional peptides at concentrations necessary for efficacy remains a formidable challenge. Peptide amphiphiles (PAs) provide a facile method of intracellular delivery and stabilization of bioactive peptides. PAs consisting of biofunctional peptide headgroups linked to hydrophobic alkyl lipid-like tails prevent peptide hydrolysis and proteolysis in circulation, and PA monomers are internalized via endocytosis. However, endocytotic sequestration and steric hindrance from the lipid tail are two major mechanisms that limit PA efficacy to target intracellular PPIs. To address these problems, we have constructed a PA platform consisting of cathepsin-B cleavable PAs in which a selective p53-based inhibitory peptide is cleaved from its lipid tail within endosomes, allowing for intracellular peptide accumulation and extracellular recycling of the lipid moiety. We monitor for cleavage and follow individual PA components in real time using a resonance energy transfer (FRET)-based tracking system. Using this platform, components in real time using a Forster we provide a better understanding and quantification of cellular internalization, trafficking, and endosomal cleavage of PAs and of the ultimate fates of each component.

  9. Revisiting and re-engineering the classical zinc finger peptide: consensus peptide-1 (CP-1).

    Science.gov (United States)

    Besold, Angelique N; Widger, Leland R; Namuswe, Frances; Michalek, Jamie L; Michel, Sarah L J; Goldberg, David P

    2016-04-01

    Zinc plays key structural and catalytic roles in biology. Structural zinc sites are often referred to as zinc finger (ZF) sites, and the classical ZF contains a Cys2His2 motif that is involved in coordinating Zn(II). An optimized Cys2His2 ZF, named consensus peptide 1 (CP-1), was identified more than 20 years ago using a limited set of sequenced proteins. We have reexamined the CP-1 sequence, using our current, much larger database of sequenced proteins that have been identified from high-throughput sequencing methods, and found the sequence to be largely unchanged. The CCHH ligand set of CP-1 was then altered to a CAHH motif to impart hydrolytic activity. This ligand set mimics the His2Cys ligand set of peptide deformylase (PDF), a hydrolytically active M(II)-centered (M = Zn or Fe) protein. The resultant peptide [CP-1(CAHH)] was evaluated for its ability to coordinate Zn(II) and Co(II) ions, adopt secondary structure, and promote hydrolysis. CP-1(CAHH) was found to coordinate Co(II) and Zn(II) and a pentacoordinate geometry for Co(II)-CP-1(CAHH) was implicated from UV-vis data. This suggests a His2Cys(H2O)2 environment at the metal center. The Zn(II)-bound CP-1(CAHH) was shown to adopt partial secondary structure by 1-D (1)H NMR spectroscopy. Both Zn(II)-CP-1(CAHH) and Co(II)-CP-1(CAHH) show good hydrolytic activity toward the test substrate 4-nitrophenyl acetate, exhibiting faster rates than most active synthetic Zn(II) complexes.

  10. Engineered Chimeric Peptides as Antimicrobial Surface Coating Agents toward Infection-Free Implants.

    Science.gov (United States)

    Yazici, Hilal; O'Neill, Mary B; Kacar, Turgay; Wilson, Brandon R; Oren, E Emre; Sarikaya, Mehmet; Tamerler, Candan

    2016-03-02

    Prevention of bacterial colonization and consequent biofilm formation remains a major challenge in implantable medical devices. Implant-associated infections are not only a major cause of implant failures but also their conventional treatment with antibiotics brings further complications due to the escalation in multidrug resistance to a variety of bacterial species. Owing to their unique properties, antimicrobial peptides (AMPs) have gained significant attention as effective agents to combat colonization of microorganisms. These peptides have been shown to exhibit a wide spectrum of activities with specificity to a target cell while having a low tendency for developing bacterial resistance. Engineering biomaterial surfaces that feature AMP properties, therefore, offer a promising approach to prevent implant infections. Here, we engineered a chimeric peptide with bifunctionality that both forms a robust solid-surface coating while presenting antimicrobial property. The individual domains of the chimeric peptides were evaluated for their solid-binding kinetics to titanium substrate as well as for their antimicrobial properties in solution. The antimicrobial efficacy of the chimeric peptide on the implant material was evaluated in vitro against infection by a variety of bacteria, including Streptococcus mutans, Staphylococcus. epidermidis, and Escherichia coli, which are commonly found in oral and orthopedic implant related surgeries. Our results demonstrate significant improvement in reducing bacterial colonization onto titanium surfaces below the detectable limit. Engineered chimeric peptides with freely displayed antimicrobial domains could be a potential solution for developing infection-free surfaces by engineering implant interfaces with highly reduced bacterial colonization property.

  11. Introducing enzyme selectivity as a quantitative parameter to describe the effects of substrate concentration on protein hydrolysis

    NARCIS (Netherlands)

    Butré, C.I.

    2014-01-01

    To understand the differences in peptide composition that result from variations in the conditions of enzymatic hydrolysis of proteins (e.g. substrate concentration) the mechanism of hydrolysis needs to be understood in detail. Therefore, methods and tools were developed to characterize and

  12. Probing the Catalytic Promiscuity of a Regio- and Stereospecific C-Glycosyltransferase from Mangifera indica.

    Science.gov (United States)

    Chen, Dawei; Chen, Ridao; Wang, Ruishan; Li, Jianhua; Xie, Kebo; Bian, Chuancai; Sun, Lili; Zhang, Xiaolin; Liu, Jimei; Yang, Lin; Ye, Fei; Yu, Xiaoming; Dai, Jungui

    2015-10-19

    The catalytic promiscuity of the novel benzophenone C-glycosyltransferase, MiCGT, which is involved in the biosynthesis of mangiferin from Mangifera indica, was explored. MiCGT exhibited a robust capability to regio- and stereospecific C-glycosylation of 35 structurally diverse druglike scaffolds and simple phenolics with UDP-glucose, and also formed O- and N-glycosides. Moreover, MiCGT was able to generate C-xylosides with UDP-xylose. The OGT-reversibility of MiCGT was also exploited to generate C-glucosides with simple sugar donor. Three aryl-C-glycosides exhibited potent SGLT2 inhibitory activities with IC50  values of 2.6×, 7.6×, and 7.6×10(-7)  M, respectively. These findings demonstrate for the first time the significant potential of an enzymatic approach to diversification through C-glycosidation of bioactive natural and unnatural products in drug discovery. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. An Ecological Analysis of the Effects of Deviant Peer Clustering on Sexual Promiscuity, Problem Behavior, and Childbearing from Early Adolescence to Adulthood: An Enhancement of the Life History Framework

    Science.gov (United States)

    Dishion, Thomas J.; Ha, Thao; Veronneau, Marie-Helene

    2012-01-01

    The authors propose that peer relationships should be included in a life history perspective on adolescent problem behavior. Longitudinal analyses were used to examine deviant peer clustering as the mediating link between attenuated family ties, peer marginalization, and social disadvantage in early adolescence and sexual promiscuity in middle…

  14. Prebiotic Peptide (Amide) Bond Synthesis Accelerated by Glycerol and Bicarbonate Under Neutral to Alkaline Dry-Down Conditions

    Science.gov (United States)

    Forsythe, J. G.; Weber, A. L.

    2017-07-01

    We report a new process for robust peptide bond synthesis in the pH 6–10 range that involves dry-down heating of amino acids in the presence of glycerol and bicarbonate (substrates: L-alanine, L-2-aminobutyric acid, β-alanine, isoserine).

  15. Studies of the stability of water-soluble polypeptoid helices and investigation of synthetic, biomimetic substrates for the development of a thermally triggered, enzymatically crosslinked hydrogel for biomedical applications

    Science.gov (United States)

    Sanborn, Tracy Joella

    Due to the unique 3D structures of proteins, these biopolymers are able to perform a myriad of vital functions and activities in vivo. Peptidomimetic oligomers are being synthesized to mimic the structure and function of natural peptides. We have examined the stability of secondary structure of a poly-N-substituted glycine (peptoid) and developed synthetic substrates for transglutaminase enzymes. We synthesized an amphipathic, helical, 36 residue peptoid to study the stability of peptoid secondary structure using circular dichroism. We saw no significant dependence of helical structure on concentration, solvent, or temperature. The extraordinary resistance of these peptoid helices to denaturation is consistent with a dominant role, of steric forces in their structural stabilization. The structured polypeptoids studied here have potential as robust mimics of helical polypeptides of therapeutic interest. The ability of transglutaminases to crosslink peptidomimetic substrates was also investigated. There is a medical need for robust, biocompatible hydrogels that can be rapidly crosslinked in situ, for application as surgical adhesives, bone-inductive materials, or for drug delivery. We have taken an enzymatic approach to the creation of a novel gelation system that fits these requirements, utilizing transglutaminase enzymes, thermo-responsive liposomes, and a biomimetic enzyme substrate based on a peptide-polymer conjugate. At room temperature, the hydrogel system is a solution. Upon heating to 37°C, the calcium-loaded liposomes release calcium that activates Factor XIII in the presence of thrombin, producing a gel within 9 minutes. Rheological studies demonstrated that the hydrogel behaves as a robust, elastic solid, while scanning electron microscopy studies revealed that the hydrogel has a very dense morphology overall. We also investigated the ability of transglutaminases to crosslink non-natural, peptoid-based substrates. The activity of five lysine

  16. Activation of phospholipase A2 by temporin B: Formation of antimicrobial peptide-enzyme amyloid-type cofibrils

    NARCIS (Netherlands)

    Code, Christian; Domanov, Y.A.; Killian, J.A.; Kinnunen, P.K.J.

    2009-01-01

    Phospholipases A2 have been shown to be activated in a concentration dependent manner by a number of antimicrobial peptides, including melittin, magainin 2, indolicidin, and temporins B and L. Here we used fluorescently labelled bee venom PLA2 (PLA2D) and the saturated phospholipid substrate

  17. Micropattern printing of adhesion, spreading, and migration peptides on poly(tetrafluoroethylene) films to promote endothelialization.

    Science.gov (United States)

    Gauvreau, Virginie; Laroche, Gaétan

    2005-01-01

    We report here the development of an original multistep micropatterning technique for printing peptides on surfaces, based on the ink-jet printer technology. Contrary to most micropatterning methods used nowadays, this technique is advantageous because it allows displaying 2D-arrays of multiple biomolecules. Moreover, this low cost procedure allies the advantages of computer-aided design with high flexibility and reproducibility. A Hewlett-Packard printer was modified to print peptide solutions, and Adobe Illustrator was used as the graphic-editing software to design high-resolution checkerboard-like micropatterns. In a first step, PTFE films were treated with ammonia plasma to introduce amino groups on the surface. These chemical functionalities were reacted with heterobifunctional cross-linker sulfo-succinimidyl 4-(N-maleimidomethyl)cycloexane-1-carboxylate (S-SMCC) to allow the subsequent surface covalent conjugation of various cysteine-modified peptides to the polymer substrate. These peptidic molecules containing RGD and WQPPRARI sequences were selected for their adhesive, spreading, and migrational properties toward endothelial cells. On one hand, our data demonstrated that the initial cell adhesion does not depend on the chemical structure and combination of the peptides covalently bonded either through conventional conjugation or micropatterning. On the other hand, spreading and migration of endothelial cells is clearly enhanced while coconjugating the GRGDS peptide in conjunction with WQPPRARI. This behavior is further improved by micropatterning these peptides on specific areas of the polymer surface.

  18. Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase

    DEFF Research Database (Denmark)

    Costa, Sara; Marek, Magdalena; Axelsen, Kristian Buhl

    2016-01-01

    and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues......) compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys(158) and Cys(172) has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications...

  19. Engineering the substrate specificity of the DhbE adenylation domain by yeast cell surface display.

    Science.gov (United States)

    Zhang, Keya; Nelson, Kathryn M; Bhuripanyo, Karan; Grimes, Kimberly D; Zhao, Bo; Aldrich, Courtney C; Yin, Jun

    2013-01-24

    The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in k(cat)/K(m) with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in k(cat)/K(m) values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the "nonribosomal code" of A-domains. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Intelligent "Peptide-Gathering Mechanical Arm" Tames Wild "Trojan-Horse" Peptides for the Controlled Delivery of Cancer Nanotherapeutics.

    Science.gov (United States)

    Shi, Nian-Qiu; Li, Yan; Zhang, Yong; Shen, Nan; Qi, Ling; Wang, Shu-Ran; Qi, Xian-Rong

    2017-12-06

    Cell-penetrating peptides (CPPs), also called "Trojan-Horse" peptides, have been used for facilitating intracellular delivery of numerous diverse cargoes and even nanocarriers. However, the lack of targeting specificity ("wildness" or nonselectivity) of CPP-nanocarriers remains an intractable challenge for many in vivo applications. In this work, we used an intelligent "peptide-gathering mechanical arm" (Int PMA) to curb CPPs' wildness and enhance the selectivity of R 9 -liposome-based cargo delivery for tumor targeting. The peptide NGR, serving as a cell-targeting peptide for anchoring, and peptide PLGLAG, serving as a substrate peptide for deanchoring, were embedded in the Int PMA motif. The Int PMA construct was designed to be sensitive to tumor microenvironmental stimuli, including aminopeptidase N (CD13) and matrix metalloproteinases (MMP-2/9). Moreover, Int PMA could be specifically recognized by tumor tissues via CD13-mediated anchoring and released for cell entry by MMP-2/9-mediated deanchoring. To test the Int PMA design, a series of experiments were conducted in vitro and in vivo. Functional conjugates Int PMA-R 9 -poly(ethylene glycol) (PEG) 2000 -distearoylphosphatidyl-ethanolamine (DSPE) and R 9 -PEG 2000 -DSPE were synthesized by Michael addition reaction and were characterized by thin-layer chromatography and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The Int PMA-R 9 -modified doxorubicin-loaded liposomes (Int PMA-R 9 -Lip-DOX) exhibited a proper particle diameter (approximately 155 nm) with in vitro sustained release characteristics. Cleavage assay showed that Int PMA-R 9 peptide molecules could be cleaved by MMP-2/9 for completion of deanchoring. Flow cytometry and confocal microscopy studies indicated that Int PMA-R 9 -Lip-DOX can respond to both endogenous and exogenous stimuli in the presence/absence of excess MMP-2/9 and MMP-2/9 inhibitor (GM6001) and effectively function under competitive receptor

  1. Field effect of screened charges: electrical detection of peptides and proteins by a thin-film resistor.

    Science.gov (United States)

    Lud, Simon Q; Nikolaides, Michael G; Haase, Ilka; Fischer, Markus; Bausch, Andreas R

    2006-02-13

    For many biotechnological applications the label-free detection of biomolecular interactions is becoming of outstanding importance. In this Article we report the direct electrical detection of small peptides and proteins by their intrinsic charges using a biofunctionalized thin-film resistor. The label-free selective and quantitative detection of small peptides and proteins is achieved using hydrophobized silicon-on-insulator (SOI) substrates functionalized with lipid membranes that incorporate metal-chelating lipids. The response of the nanometer-thin conducting silicon film to electrolyte screening effects is taken into account to determine quantitatively the charges of peptides. It is even possible to detect peptides with a single charge and to distinguish single charge variations of the analytes even in physiological electrolyte solutions. As the device is based on standard semiconductor technologies, parallelization and miniaturization of the SOI-based biosensor is achievable by standard CMOS technologies and thus a promising basis for high-throughput screening or biotechnological applications.

  2. Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: Application to proenkephalin processing enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Irvine, J.W.; Roberts, S.F.; Lindberg, I. (Louisiana State Univ. Medical Center, New Orleans (USA))

    1990-10-01

    A novel method is described for the zymographic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates such as ({sup 35}S)methionine-labeled proenkephalin or {sup 125}I-labeled proinsulin. After electrophoresis the enzyme is reactivated and cleaves the radiolabeled in situ substrate into smaller peptides. These small peptides are able to diffuse out of the gel, leaving clear areas against a dark background when visualized by autoradiography. The technique can be used to detect as little as 200 fg of trypsin using only 50 ng (1.25 microCi) of ({sup 35}S)proenkephalin. Soluble- and membrane-bound adrenal trypsin-like enzyme were isolated from bovine adrenal chromaffin granules. Both proteinases cleaved ({sup 35}S)methionine-labeled proenkephalin but not {sup 125}I-labeled proinsulin. Moreover, both had a Mr of approximately 30,000. The potential of this technique for general use is discussed. An additional method using the synthetic fluorogenic substrate t-butoxycarbonyl Glu-Lys-Lys aminomethylcoumarin is also described.

  3. Peptide array-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design.

    Science.gov (United States)

    Kato, Ryuji; Kaga, Chiaki; Kunimatsu, Mitoshi; Kobayashi, Takeshi; Honda, Hiroyuki

    2006-06-01

    Peptide array, the designable peptide library covalently synthesized on cellulose support, was applied to assay peptide-cell interaction, between solid-bound peptides and anchorage-dependant cells, to study objective peptide design. As a model case, cell-adhesive peptides that could enhance cell growth as tissue engineering scaffold material, was studied. On the peptide array, the relative cell-adhesion ratio of NIH/3T3 cells was 2.5-fold higher on the RGDS (Arg-Gly-Asp-Ser) peptide spot as compared to the spot with no peptide, thus indicating integrin-mediated peptide-cell interaction. Such strong cell adhesion mediated by the RGDS peptide was easily disrupted by single residue substitution on the peptide array, thus indicating that the sequence recognition accuracy of cells was strictly conserved in our optimized scheme. The observed cellular morphological extension with active actin stress-fiber on the RGD motif-containing peptide supported our strategy that peptide array-based interaction assay of solid-bound peptide and anchorage-dependant cells (PIASPAC) could provide quantitative data on biological peptide-cell interaction. The analysis of 180 peptides obtained from fibronectin type III domain (no. 1447-1629) yielded 18 novel cell-adhesive peptides without the RGD motif. Taken together with the novel candidates, representative rules of ineffective amino acid usage were obtained from non-effective candidate sequences for the effective designing of cell-adhesive peptides. On comparing the amino acid usage of the top 20 and last 20 peptides from the 180 peptides, the following four brief design rules were indicated: (i) Arg or Lys of positively charged amino acids (except His) could enhance cell adhesion, (ii) small hydrophilic amino acids are favored in cell-adhesion peptides, (iii) negatively charged amino acids and small amino acids (except Gly) could reduce cell adhesion, and (iv) Cys and Met could be excluded from the sequence combination since they have

  4. Divergence of substrate specificity and function in the Escherichia coli hotdog-fold thioesterase paralogs YdiI and YbdB.

    Science.gov (United States)

    Latham, John A; Chen, Danqi; Allen, Karen N; Dunaway-Mariano, Debra

    2014-07-29

    The work described in this paper, and its companion paper (Wu, R., Latham, J. A., Chen, D., Farelli, J., Zhao, H., Matthews, K. Allen, K. N., and Dunaway-Mariano, D. (2014) Structure and Catalysis in the Escherichia coli Hotdog-fold Thioesterase Paralogs YdiI and YbdB. Biochemistry, DOI: 10.1021/bi500334v), focuses on the evolution of a pair of paralogous hotdog-fold superfamily thioesterases of E. coli, YbdB and YdiI, which share a high level of sequence identity but perform different biological functions (viz., proofreader of 2,3-dihydroxybenzoyl-holoEntB in the enterobactin biosynthetic pathway and catalyst of the 1,4-dihydoxynapthoyl-CoA hydrolysis step in the menaquinone biosynthetic pathway, respectively). In vitro substrate activity screening of a library of thioester metabolites showed that YbdB displays high activity with benzoyl-holoEntB and benzoyl-CoA substrates, marginal activity with acyl-CoA thioesters, and no activity with 1,4-dihydoxynapthoyl-CoA. YdiI, on the other hand, showed a high level of activity with its physiological substrate, significant activity toward a wide range of acyl-CoA thioesters, and minimal activity toward benzoyl-holoEntB. These results were interpreted as evidence for substrate promiscuity that facilitates YbdB and YdiI evolvability, and divergence in substrate preference, which correlates with their assumed biological function. YdiI support of the menaquinone biosynthetic pathway was confirmed by demonstrating reduced anaerobic growth of the E. coli ydiI-knockout mutant (vs wild-type E. coli) on glucose in the presence of the electron acceptor fumarate. Bioinformatic analysis revealed that a small biological range exists for YbdB orthologs (i.e., limited to Enterobacteriales) relative to that of YdiI orthologs. The divergence in YbdB and YdiI substrate specificity detailed in this paper set the stage for their structural analyses reported in the companion paper.

  5. Albumin-derived peptides efficiently reduce renal uptake of radiolabelled peptides

    International Nuclear Information System (INIS)

    Vegt, Erik; Eek, Annemarie; Oyen, Wim J.G.; Gotthardt, Martin; Boerman, Otto C.; Jong, Marion de

    2010-01-01

    In peptide-receptor radionuclide therapy (PRRT), the maximum activity dose that can safely be administered is limited by high renal uptake and retention of radiolabelled peptides. The kidney radiation dose can be reduced by coinfusion of agents that competitively inhibit the reabsorption of radiolabelled peptides, such as positively charged amino acids, Gelofusine, or trypsinised albumin. The aim of this study was to identify more specific and potent inhibitors of the kidney reabsorption of radiolabelled peptides, based on albumin. Albumin was fragmented using cyanogen bromide and six albumin-derived peptides with different numbers of electric charges were selected and synthesised. The effect of albumin fragments (FRALB-C) and selected albumin-derived peptides on the internalisation of 111 In-albumin, 111 In-minigastrin, 111 In-exendin and 111 In-octreotide by megalin-expressing cells was assessed. In rats, the effect of Gelofusine and albumin-derived peptides on the renal uptake and biodistribution of 111 In-minigastrin, 111 In-exendin and 111 In-octreotide was determined. FRALB-C significantly reduced the uptake of all radiolabelled peptides in vitro. The albumin-derived peptides showed different potencies in reducing the uptake of 111 In-albumin, 111 In-exendin and 111 In-minigastrin in vitro. The most efficient albumin-derived peptide (peptide 6), was selected for in vivo testing. In rats, 5 mg of peptide 6 very efficiently inhibited the renal uptake of 111 In-minigastrin, by 88%. Uptake of 111 In-exendin and 111 In-octreotide was reduced by 26 and 33%, respectively. The albumin-derived peptide 6 efficiently inhibited the renal reabsorption of 111 In-minigastrin, 111 In-exendin and 111 In-octreotide and is a promising candidate for kidney protection in PRRT. (orig.)

  6. A substrate-optimized electrophoretic mobility shift assay for ADAM12

    DEFF Research Database (Denmark)

    Kotzsch, Alexander; Skovgaard, Tine; Buus, Uwe

    2014-01-01

    long been investigated as pharmaceutical drug targets; however, due to lack of potency and in vivo side effects, none of the small-molecule inhibitors discovered so far has made it beyond clinical testing. Ongoing research on novel selective inhibitors of ADAMs requires reliable biochemical assays...... to validate molecular probes from large-scale screening efforts. Here we describe an electrophoretic mobility shift assay for ADAM12 based on the identification of an optimized peptide substrate that is characterized by excellent performance and reproducibility....

  7. PSA-alpha-2-macroglobulin complex is enzymatically active in the serum of patients with advanced prostate cancer and can degrade circulating peptide hormones.

    Science.gov (United States)

    Kostova, Maya B; Brennen, William Nathaniel; Lopez, David; Anthony, Lizamma; Wang, Hao; Platz, Elizabeth; Denmeade, Samuel R

    2018-08-01

    Prostate cancer cells produce high levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the tumor microenvironment but is presumed to be enzymatically inactive in the blood due to complex formation with serum protease inhibitors α-1-antichymotrypsin and α-2-macroglobulin (A2M). PSA-A2M complexes cannot be measured by standard ELISA assays and are also rapidly cleared from the circulation. Thus the exact magnitude of PSA production by prostate cancer cells is not easily measured. The PSA complexed to A2M is unable to cleave proteins but maintains the ability to cleave small peptide substrates. Thus, in advanced prostate cancer, sufficient PSA-A2M may be in circulation to effect total A2M levels, levels of cytokines bound to A2M and hydrolyze small circulating peptide hormones. Total A2M levels in men with advanced prostate cancer and PSA levels above 1000 ng/mL were measured by ELISA and compared to controls. Additional ELISA assays were used to measure levels of IL-6 and TGF-beta which can bind to A2M. The ability of PSA-A2M complexes to hydrolyze protein and peptide substrates was analyzed ± PSA inhibitor. Enzymatic activity of PSA-A2M in serum of men with high PSA levels was also assayed. Serum A2M levels are inversely correlated with PSA levels in men with advanced prostate cancer. Il-6 Levels are significantly elevated in men with PSA >1000 ng/mL compared to controls with PSA PSA-A2M complex in serum of men with PSA levels >1000 ng/mL can hydrolyze small fluorescently labeled peptide substrates but not large proteins that are PSA substrates. PSA can hydrolyze small peptide hormones like PTHrP and osteocalcin. PSA complexed to A2M retains the ability to degrade PTHrP. In advanced prostate cancer with PSA levels >1000 ng/mL, sufficient PSA-A2M is present in circulation to produce enzymatic activity against circulating small peptide hormones. Sufficient PSA is produced in advanced prostate cancer to alter

  8. Multifunctional hybrid networks based on self assembling peptide sequences

    Science.gov (United States)

    Sathaye, Sameer

    The overall aim of this dissertation is to achieve a comprehensive correlation between the molecular level changes in primary amino acid sequences of amphiphilic beta-hairpin peptides and their consequent solution-assembly properties and bulk network hydrogel behavior. This has been accomplished using two broad approaches. In the first approach, amino acid substitutions were made to peptide sequence MAX1 such that the hydrophobic surfaces of the folded beta-hairpins from the peptides demonstrate shape specificity in hydrophobic interactions with other beta-hairpins during the assembly process, thereby causing changes to the peptide nanostructure and bulk rheological properties of hydrogels formed from the peptides. Steric lock and key complementary hydrophobic interactions were designed to occur between two beta-hairpin molecules of a single molecule, LNK1 during beta-sheet fibrillar assembly of LNK1. Experimental results from circular dichroism, transmission electron microscopy and oscillatory rheology collectively indicate that the molecular design of the LNK1 peptide can be assigned the cause of the drastically different behavior of the networks relative to MAX1. The results indicate elimination or significant reduction of fibrillar branching due to steric complementarity in LNK1 that does not exist in MAX1, thus supporting the original hypothesis. As an extension of the designed steric lock and key complementarity between two beta-hairpin molecules of the same peptide molecule. LNK1, three new pairs of peptide molecules LP1-KP1, LP2-KP2 and LP3-KP3 that resemble complementary 'wedge' and 'trough' shapes when folded into beta-hairpins were designed and studied. All six peptides individually and when blended with their corresponding shape complement formed fibrillar nanostructures with non-uniform thickness values. Loose packing in the assembled structures was observed in all the new peptides as compared to the uniform tight packing in MAX1 by SANS analysis. This

  9. Promiscuous prediction and conservancy analysis of CTL binding epitopes of HCV 3a viral proteome from Punjab Pakistan: an In Silico Approach

    Directory of Open Access Journals (Sweden)

    Idrees Muhammad

    2011-02-01

    Full Text Available Abstract Background HCV is a positive sense RNA virus affecting approximately 180 million people world wide and about 10 million Pakistani populations. HCV genotype 3a is the major cause of infection in Pakistani population. One of the major problems of HCV infection especially in the developing countries that limits the limits the antiviral therapy is the long term treatment, high dosage and side effects. Studies of antigenic epitopes of viral sequences of a specific origin can provide an effective way to overcome the mutation rate and to determine the promiscuous binders to be used for epitope based subunit vaccine design. An in silico approach was applied for the analysis of entire HCV proteome of Pakistani origin, aimed to identify the viral epitopes and their conservancy in HCV genotypes 1, 2 and 3 of diverse origin. Results Immunoinformatic tools were applied for the predictive analysis of HCV 3a antigenic epitopes of Pakistani origin. All the predicted epitopes were then subjected for their conservancy analysis in HCV genotypes 1, 2 and 3 of diverse origin (worldwide. Using freely available web servers, 150 MHC II epitopes were predicted as promiscuous binders against 51 subjected alleles. E2 protein represented the 20% of all the predicted MHC II epitopes. 75.33% of the predicted MHC II epitopes were (77-100% conserve in genotype 3; 47.33% and 40.66% in genotype 1 and 2 respectively. 69 MHC I epitopes were predicted as promiscuous binders against 47 subjected alleles. NS4b represented 26% of all the MHC I predicted epitopes. Significantly higher epitope conservancy was represented by genotype 3 i.e. 78.26% and 21.05% for genotype 1 and 2. Conclusions The study revealed comprehensive catalogue of potential HCV derived CTL epitopes from viral proteome of Pakistan origin. A considerable number of predicted epitopes were found to be conserved in different HCV genotype. However, the number of conserved epitopes in HCV genotype 3 was

  10. Interactions of Bio-Inspired Membranes with Peptides and Peptide-Mimetic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Michael Sebastiano

    2015-08-01

    Full Text Available Via Dissipative Particle Dynamics (DPD and implicit solvent coarse-grained (CG Molecular Dynamics (MD we examine the interaction of an amphiphilic cell-penetrating peptide PMLKE and its synthetic counterpart with a bio-inspired membrane. We use the DPD technique to investigate the interaction of peptide-mimetic nanoparticles, or nanopins, with a three-component membrane. The CG MD approach is used to investigate the interaction of a cell-penetrating peptide PMLKE with single-component membrane. We observe the spontaneous binding and subsequent insertion of peptide and nanopin in the membrane by using CG MD and DPD approaches, respectively. In addition, we find that the insertion of peptide and nanopins is mainly driven by the favorable enthalpic interactions between the hydrophobic components of the peptide, or nanopin, and the membrane. Our study provides insights into the mechanism underlying the interactions of amphiphilic peptide and peptide-mimetic nanoparticles with a membrane. The result of this study can be used to guide the functional integration of peptide and peptide-mimetic nanoparticles with a cell membrane.

  11. Acidity-Triggered Tumor Retention/Internalization of Chimeric Peptide for Enhanced Photodynamic Therapy and Real-Time Monitoring of Therapeutic Effects.

    Science.gov (United States)

    Han, Kai; Zhang, Wei-Yun; Ma, Zhao-Yu; Wang, Shi-Bo; Xu, Lu-Ming; Liu, Jia; Zhang, Xian-Zheng; Han, He-You

    2017-05-17

    Photodynamic therapy (PDT) holds great promise in tumor treatment. Nevertheless, it remains highly desirable to develop easy-to-fabricated PDT systems with improved tumor accumulation/internalization and timely therapeutic feedback. Here, we report a tumor-acidity-responsive chimeric peptide for enhanced PDT and noninvasive real-time apoptosis imaging. Both in vitro and in vivo studies revealed that a tumor mildly acidic microenvironment could trigger rapid protonation of carboxylate anions in chimeric peptide, which led to increased ζ potential, improved hydrophobicity, controlled size enlargement, and precise morphology switching from sphere to spherocylinder shape of the chimeric peptide. All of these factors realized superfast accumulation and prolonged retention in the tumor region, selective cellular internalization, and enhanced PDT against the tumor. Meanwhile, this chimeric peptide could further generate reactive oxygen species and initiate cell apoptosis during PDT. The subsequent formation of caspase-3 enzyme hydrolyzed the chimeric peptide, achieving a high signal/noise ratio and timely fluorescence feedback. Importantly, direct utilization of the acidity responsiveness of a biofunctional Asp-Glu-Val-Asp-Gly (DEVDG, caspase-3 enzyme substrate) peptide sequence dramatically simplified the preparation and increased the performance of the chimeric peptide furthest.

  12. [Plant signaling peptides. Cysteine-rich peptides].

    Science.gov (United States)

    Ostrowski, Maciej; Kowalczyk, Stanisław

    2015-01-01

    Recent bioinformatic and genetic analyses of several model plant genomes have revealed the existence of a highly abundant group of signaling peptides that are defined as cysteine-rich peptides (CRPs). CRPs are usually in size between 50 and 90 amino acid residues, they are positively charged, and they contain 4-16 cysteine residues that are important for the correct conformational folding. Despite the structural differences among CRP classes, members from each class have striking similarities in their molecular properties and function. The present review presents the recent progress in research on signaling peptides from several families including: EPF/EPFL, SP11/SCR, PrsS, RALF, LURE, and some other peptides belonging to CRP group. There is convincing evidence indicating multiple roles for these CRPs as signaling molecules during the plant life cycle, ranging from stomata development and patterning, self-incompatibility, pollen tube growth and guidance, reproductive processes, and nodule formation.

  13. Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay.

    Science.gov (United States)

    Duckworth, Benjamin P; Wilson, Daniel J; Aldrich, Courtney C

    2016-01-01

    Adenylation is a crucial enzymatic process in the biosynthesis of nonribosomal peptide synthetase (NRPS) derived natural products. Adenylation domains are considered the gatekeepers of NRPSs since they select, activate, and load the carboxylic acid substrate onto a downstream peptidyl carrier protein (PCP) domain of the NRPS. We describe a coupled continuous kinetic assay for NRPS adenylation domains that substitutes the PCP domain with hydroxylamine as the acceptor molecule. The pyrophosphate released from the first-half reaction is then measured using a two-enzyme coupling system, which detects conversion of the chromogenic substrate 7-methylthioguanosine (MesG) to 7-methylthioguanine. From profiling substrate specificity of unknown or engineered adenylation domains to studying chemical inhibition of adenylating enzymes, this robust assay will be of widespread utility in the broad field NRPS enzymology.

  14. A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

    Science.gov (United States)

    Wall, Jonathan S; Williams, Angela; Richey, Tina; Stuckey, Alan; Huang, Ying; Wooliver, Craig; Macy, Sallie; Heidel, Eric; Gupta, Neil; Lee, Angela; Rader, Brianna; Martin, Emily B; Kennel, Stephen J

    2013-01-01

    Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

  15. Human peptide transporters

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Brodin, Birger; Jørgensen, Flemming Steen

    2002-01-01

    Peptide transporters are epithelial solute carriers. Their functional role has been characterised in the small intestine and proximal tubules, where they are involved in absorption of dietary peptides and peptide reabsorption, respectively. Currently, two peptide transporters, PepT1 and PepT2, wh...

  16. Peptide dendrimers

    Czech Academy of Sciences Publication Activity Database

    Niederhafner, Petr; Šebestík, Jaroslav; Ježek, Jan

    2005-01-01

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

  17. Development and use of engineered peptide deformylase in chemoenzymatic peptide synthesis

    NARCIS (Netherlands)

    Di Toma, Claudia

    2012-01-01

    Deze thesis beschrijft het onderzoek naar potentieel van het gebruik van het peptide deformylase (PDF) in chemo enzymatische peptide synthese. PDF is geschikt voor selective N terminale deformylatie van bepaalde N-formyl-peptides zonder gelijktijdige hydrolyse van de peptide binding. Door de

  18. Catalytic water co-existing with a product peptide in the active site of HIV-1 protease revealed by X-ray structure analysis.

    Directory of Open Access Journals (Sweden)

    Vishal Prashar

    Full Text Available BACKGROUND: It is known that HIV-1 protease is an important target for design of antiviral compounds in the treatment of Acquired Immuno Deficiency Syndrome (AIDS. In this context, understanding the catalytic mechanism of the enzyme is of crucial importance as transition state structure directs inhibitor design. Most mechanistic proposals invoke nucleophilic attack on the scissile peptide bond by a water molecule. But such a water molecule coexisting with any ligand in the active site has not been found so far in the crystal structures. PRINCIPAL FINDINGS: We report here the first observation of the coexistence in the active site, of a water molecule WAT1, along with the carboxyl terminal product (Q product peptide. The product peptide has been generated in situ through cleavage of the full-length substrate. The N-terminal product (P product has diffused out and is replaced by a set of water molecules while the Q product is still held in the active site through hydrogen bonds. The position of WAT1, which hydrogen bonds to both the catalytic aspartates, is different from when there is no substrate bound in the active site. We propose WAT1 to be the position from where catalytic water attacks the scissile peptide bond. Comparison of structures of HIV-1 protease complexed with the same oligopeptide substrate, but at pH 2.0 and at pH 7.0 shows interesting changes in the conformation and hydrogen bonding interactions from the catalytic aspartates. CONCLUSIONS/SIGNIFICANCE: The structure is suggestive of the repositioning, during substrate binding, of the catalytic water for activation and subsequent nucleophilic attack. The structure could be a snap shot of the enzyme active site primed for the next round of catalysis. This structure further suggests that to achieve the goal of designing inhibitors mimicking the transition-state, the hydrogen-bonding pattern between WAT1 and the enzyme should be replicated.

  19. Construction of hybrid peptide synthetases by module and domain fusions.

    Science.gov (United States)

    Mootz, H D; Schwarzer, D; Marahiel, M A

    2000-05-23

    Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular organization provides a great potential for the rational design of novel compounds by recombination of the biosynthetic genes. Here we describe the extension of a dimodular system to trimodular ones based on whole-module fusion. The recombinant hybrid enzymes were purified to monitor product assembly in vitro. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide dPhe-Pro, to construct such hybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine and leucine, respectively, resulted in dimodular hybrid enzymes exhibiting the combined substrate specificities. The thioesterase domain was fused to the terminal module. Upon incubation of these dimodular enzymes with the first tyrocidine module, TycA, incorporating dPhe, the predicted tripeptides dPhe-Pro-Orn and dPhe-Pro-Leu were obtained at rates of 0.15 min(-1) and 2.1 min(-1). The internal thioesterase domain was necessary and sufficient to release the products from the hybrid enzymes and thereby facilitate a catalytic turnover. Our approach of whole-module fusion is based on an improved definition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inherent potential for the synthesis of novel, designed peptides.

  20. Genetic and biochemical analysis of peptide transport in Escherichia coli

    International Nuclear Information System (INIS)

    Andrews, J.C.

    1986-01-01

    E. coli peptide transport mutants have been isolated based on their resistance to toxic tripeptides. These genetic defects were found to map in two distinct chromosomal locations. The transport systems which require expression of the trp-linked opp genes and the oppE gene(s) for activity were shown to have different substrate preferences. Growth of E. coli in medium containing leucine results in increased entry of exogenously supplied tripeptides into the bacterial cell. This leucine-mediated elevation of peptide transport required expression of the trp-linked opp operon and was accompanied by increased sensitivity to toxic tripeptides, by an enhanced capacity to utilize nutritional peptides, and by an increase in both the velocity and apparent steady-state level of L-(U- 14 C)alanyl-L-alanyl-L-alanine accumulation for E. coli grown in leucine-containing medium relative to these parameters of peptide transport measured with bacteria grown in media lacking leucine. Direct measurement of opp operon expression by pulse-labeling experiments demonstrated that growth of E. coli in the presence of leucine resulted in increased synthesis of the oppA-encoded periplasmic binding protein. The transcriptional regulation of the trp-linked opp operon of E. coli was investigated using λ placMu51-generated lac operon fusions. Synthesis of β-galactosidase by strains harboring oppA-lac, oppB-lac, and oppD-lac fusions occurred at a basal level when the fusion-containing strains were grown in minimal medium

  1. Characterization of proteinases from the midgut of Rhipicephalus (Boophilus microplus involved in the generation of antimicrobial peptides

    Directory of Open Access Journals (Sweden)

    Craik Charles S

    2010-07-01

    Full Text Available Abstract Background Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins. A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. Results An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'. Conclusions BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

  2. Increased production of peptide deformylase eliminates retention of formylmethionine in bovine somatotropin overproduced in Escherichia coli.

    Science.gov (United States)

    Warren, W C; Bentle, K A; Schlittler, M R; Schwane, A C; O'Neil, J P; Bogosian, G

    1996-10-03

    In Escherichia coli and most other microorganisms, peptide synthesis is started at methionine start codons which are read only by N-formyl-methionine-tRNA. The formyl group is normally removed from the N-terminal Met residue of the peptide by peptide deformylase (PDF). However, it has been observed that overproduction of proteins in recombinant bacteria often yields protein products which are incompletely deformylated. Certain proteins could be poor substrates for PDF and exhibit incomplete deformylation, particularly when they are overproduced. Strains of E. coli which overproduce bovine somatotropin (BST) have a significant fraction of the BST with the formyl group retained. The PDF gene was isolated and positioned into a BST production vector in such a way that the BST and PDF genes were coexpressed. In strains containing this coexpression vector, the levels of PDF were increased and formylated BST was undetectable.

  3. In silico design, synthesis, and assays of specific substrates for proteinase 3: influence of fluorogenic and charged groups.

    Science.gov (United States)

    Narawane, Shailesh; Budnjo, Adnan; Grauffel, Cédric; Haug, Bengt Erik; Reuter, Nathalie

    2014-02-13

    Neutrophil serine proteases are specific regulators of the immune response, and proteinase 3 is a major target antigen in antineutrophil cytoplasmic antibody-associated vasculitis. FRET peptides containing 2-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) as fluorophore and quencher groups, respectively, have been widely used to probe proteases specificity. Using in silico design followed by enzymatic assays, we show that Abz and EDDnp significantly contribute to substrate hydrolysis by PR3. We also propose a new substrate specific for PR3.

  4. CD147 is a regulatory subunit of the gamma-secretase complex inAlzheimer's disease amyloid beta-peptide production

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Shuxia; Zhou, Hua; Walian, Peter J.; Jap, Bing K.

    2005-04-06

    {gamma}-secretase is a membrane protein complex that cleaves the {beta}-amyloid precursor protein (APP) within the transmembrane region, following prior processing by {beta}-secretase, producing amyloid {beta}-peptides (A{beta}{sub 40} and A{beta}{sub 42}). Errant production of A{beta}-peptides that substantially increases A{beta}{sub 42} production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1 (Psn-1), which contains the proteolytic active site, and three other membrane proteins, nicastrin (Nct), APH-1, and PEN-2 are required to form the core of the active {gamma}-secretase complex. Here, we report the purification of the native {gamma}-secretase complexes from HeLa cell membranes and the identification of an additional {gamma}-secretase complex subunit, CD147, a transmembrane glycoprotein with two immunoglobulin-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through co-immunoprecipitation studies of the purified protein from HeLa cells and solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of A{beta} peptides without changing the expression level of the other {gamma}-secretase components or APP substrates while CD147 overexpression had no statistically significant effect on amyloid {beta}-peptide production, other {gamma}-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the {gamma}-secretase complex directly down-modulates the production of A{beta}-peptides. {gamma}-secretase was first recognized through its role in the production of the A{beta} peptides that are pathogenic in Alzheimer's disease (AD) (1). {gamma}-secretase is a membrane protein complex with unusual aspartyl protease activity that cleaves a variety of type I membrane proteins

  5. New dendrimer - Peptide host - Guest complexes: Towards dendrimers as peptide carriers

    DEFF Research Database (Denmark)

    Boas, Ulrik; Sontjens, S.H.M.; Jensen, Knud Jørgen

    2002-01-01

    Adamantyl urea and adamantyl thiourea modified poly(propylene imine) dendrimers act as hosts for N-terminal tert-butoxycarbonyl (Boc)-protected peptides and form chloroform-soluble complexes. investigations with NMR spectroscopy show that the peptide is bound to the dendrimer by ionic interactions...... between the dendrimer outer shell tertiary amines and the C-terminal carboxylic acid of the peptide, and also through host-urea to peptide-amide hydrogen bonding. The hydrogen-bonding nature of the peptide dendrimer interactions was further confirmed by using Fourier transform IR spectroscopy, for which...... the NH- and CO-stretch signals of the peptide amide moieties shift towards lower wave-numbers upon complexation with the dendrimer. Spatial analysis of the complexes with NOESY spectroscopy generally shows close proximity of the N-terminal Boc group of the peptide to the peripheral adamantyl groups...

  6. Peptide-Mediated Platelet Capture at Gold Micropore Arrays.

    Science.gov (United States)

    Adamson, Kellie; Spain, Elaine; Prendergast, Una; Moran, Niamh; Forster, Robert J; Keyes, Tia E

    2016-11-30

    Ordered spherical cap gold cavity arrays with 5.4, 1.6, and 0.98 μm diameter apertures were explored as capture surfaces for human blood platelets to investigate the impact of surface geometry and chemical modification on platelet capture efficiency and their potential as platforms for surface enhanced Raman spectroscopy of single platelets. The substrates were chemically modified with single-constituent self-assembled monolayers (SAM) or mixed SAMs comprised of thiol-functionalized arginine-glycine-aspartic acid (RGD, a platelet integrin target) with or without 1-octanethiol (adhesion inhibitor). As expected, platelet adhesion was promoted and inhibited at RGD and alkanethiol modified surfaces, respectively. Platelet adhesion was reversible, and binding efficiency at the peptide modified substrates correlated inversely with pore diameter. Captured platelets underwent morphological change on capture, the extent of which depended on the topology of the underlying substrate. Regioselective capture of the platelets enabled study for the first time of the surface enhanced Raman spectroscopy of single blood platelets, yielding high quality Raman spectroscopy of individual platelets at 1.6 μm diameter pore arrays. Given the medical importance of blood platelets across a range of diseases from cancer to psychiatric illness, such approaches to platelet capture may provide a useful route to Raman spectroscopy for platelet related diagnostics.

  7. Gliadin peptides induce tissue transglutaminase activation and ER-stress through Ca2+ mobilization in Caco-2 cells.

    Directory of Open Access Journals (Sweden)

    Ivana Caputo

    Full Text Available BACKGROUND: Celiac disease (CD is an intestinal inflammatory condition that develops in genetically susceptible individuals after exposure to dietary wheat gliadin. The role of post-translational modifications of gliadin catalyzed by tissue transglutaminase (tTG seems to play a crucial role in CD. However, it remains to be established how and where tTG is activated in vivo. We have investigated whether gliadin peptides modulate intracellular Ca(2+ homeostasis and tTG activity. METHODS/PRINCIPAL FINDINGS: We studied Ca(2+ homeostasis in Caco-2 cells by single cell microfluorimetry. Under our conditions, A-gliadin peptides 31-43 and 57-68 rapidly mobilized Ca(2+ from intracellular stores. Specifically, peptide 31-43 mobilized Ca(2+ from the endoplasmic reticulum (ER and mitochondria, whereas peptide 57-68 mobilized Ca(2+ only from mitochondria. We also found that gliadin peptide-induced Ca(2+ mobilization activates the enzymatic function of intracellular tTG as revealed by in situ tTG activity using the tTG substrate pentylamine-biotin. Moreover, we demonstrate that peptide 31-43, but not peptide 57-68, induces an increase of tTG expression. Finally, we monitored the expression of glucose-regulated protein-78 and of CCAAT/enhancer binding protein-homologous protein, which are two biochemical markers of ER-stress, by real-time RT-PCR and western blot. We found that chronic administration of peptide 31-43, but not of peptide 57-68, induces the expression of both genes. CONCLUSIONS: By inducing Ca(2+ mobilization from the ER, peptide 31-43 could promote an ER-stress pathway that may be relevant in CD pathogenesis. Furthermore, peptides 31-43 and 57-68, by activating intracellular tTG, could alter inflammatory key regulators, and induce deamidation of immunogenic peptides and gliadin-tTG crosslinking in enterocytes and specialized antigen-presenting cells.

  8. HIV-1 protease-substrate coevolution in nelfinavir resistance.

    Science.gov (United States)

    Kolli, Madhavi; Ozen, Ayşegül; Kurt-Yilmaz, Nese; Schiffer, Celia A

    2014-07-01

    Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  9. Structural Basis for Substrate Recognition by the Ankyrin Repeat Domain of Human DHHC17 Palmitoyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Verardi, Raffaello; Kim, Jin-Sik; Ghirlando, Rodolfo; Banerjee, Anirban

    2017-09-01

    DHHC enzymes catalyze palmitoylation, a major post-translational modification that regulates a number of key cellular processes. There are up to 24 DHHCs in mammals and hundreds of substrate proteins that get palmitoylated. However, how DHHC enzymes engage with their substrates is still poorly understood. There is currently no structural information about the interaction between any DHHC enzyme and protein substrates. In this study we have investigated the structural and thermodynamic bases of interaction between the ankyrin repeat domain of human DHHC17 (ANK17) and Snap25b. We solved a high-resolution crystal structure of the complex between ANK17 and a peptide fragment of Snap25b. Through structure-guided mutagenesis, we discovered key residues in DHHC17 that are critically important for interaction with Snap25b. We further extended our finding by showing that the same residues are also crucial for the interaction of DHHC17 with Huntingtin, one of its most physiologically relevant substrates.

  10. Controlling noncovalent interactions between a lysine-rich α-helical peptide and self-assembled monolayers of alkanethiols on Au through functional group diversity

    Energy Technology Data Exchange (ETDEWEB)

    Raigoza, Annette F.; Onyirioha, Kristeen; Webb, Lauren J., E-mail: lwebb@cm.utexas.edu

    2017-02-28

    Highlights: • Functional variety in SAMs control covalent binding of proteins to surfaces. • Peptide density on Au(111) surfaces controlled by SAM functional groups. • Affinity between biomolecule and SAM surface follows a Langmuir isotherm. • Surface chemistry can mimic functional group diversity in proteins and peptides. - Abstract: Reliably attaching a structured biomolecule to an inorganic substrate would enable the preparation of surfaces that incorporate both biological and inorganic functions and structures. To this end, we have previously developed a procedure using the copper(I)-catalyzed click reaction to tether synthetic α-helical peptides carrying two alkyne groups to well-ordered alkanethiol self-assembled monolayers (SAM) on a Au(111) surface, in which the SAM is composed of a mixture of methyl and azide termination. Proteins, however, are composed of many diverse functional groups, and this composition directly effects protein structure, interactions, and reactivity. Here, we explore the utility of mixed SAMs with alternative terminating functional groups to tune and direct the reactivity of the surface through noncovalent peptide-surface interactions. We study both polar surfaces (OH-terminated) and charged surfaces (COOH- and NH{sub 3}-terminated, which are negatively and positively charged, respectively, under our reaction conditions). Surfaces were functionalized with a bipolar peptide composed of Lys and Leu residues that could express different interactions through either hydrophilic and/or charge (Lys) or hydrophobic (Leu) influences. X-ray photoelectron spectroscopy (XPS) and surface infrared spectroscopy were used to characterize surfaces at all stages of the peptide functionalization procedure. This strategy resulted in a high density of surface-bound α-helices without aggregation. Mixed SAMs that included a positively charged alkanethiol along with the azide-terminated thiol resulted in a more efficient reaction and better

  11. Controlling noncovalent interactions between a lysine-rich α-helical peptide and self-assembled monolayers of alkanethiols on Au through functional group diversity

    International Nuclear Information System (INIS)

    Raigoza, Annette F.; Onyirioha, Kristeen; Webb, Lauren J.

    2017-01-01

    Highlights: • Functional variety in SAMs control covalent binding of proteins to surfaces. • Peptide density on Au(111) surfaces controlled by SAM functional groups. • Affinity between biomolecule and SAM surface follows a Langmuir isotherm. • Surface chemistry can mimic functional group diversity in proteins and peptides. - Abstract: Reliably attaching a structured biomolecule to an inorganic substrate would enable the preparation of surfaces that incorporate both biological and inorganic functions and structures. To this end, we have previously developed a procedure using the copper(I)-catalyzed click reaction to tether synthetic α-helical peptides carrying two alkyne groups to well-ordered alkanethiol self-assembled monolayers (SAM) on a Au(111) surface, in which the SAM is composed of a mixture of methyl and azide termination. Proteins, however, are composed of many diverse functional groups, and this composition directly effects protein structure, interactions, and reactivity. Here, we explore the utility of mixed SAMs with alternative terminating functional groups to tune and direct the reactivity of the surface through noncovalent peptide-surface interactions. We study both polar surfaces (OH-terminated) and charged surfaces (COOH- and NH_3-terminated, which are negatively and positively charged, respectively, under our reaction conditions). Surfaces were functionalized with a bipolar peptide composed of Lys and Leu residues that could express different interactions through either hydrophilic and/or charge (Lys) or hydrophobic (Leu) influences. X-ray photoelectron spectroscopy (XPS) and surface infrared spectroscopy were used to characterize surfaces at all stages of the peptide functionalization procedure. This strategy resulted in a high density of surface-bound α-helices without aggregation. Mixed SAMs that included a positively charged alkanethiol along with the azide-terminated thiol resulted in a more efficient reaction and better alignment

  12. Protein kinase CK2 mutants defective in substrate recognition. Purification and kinetic analysis

    DEFF Research Database (Denmark)

    Sarno, S; Vaglio, P; Meggio, F

    1996-01-01

    Five mutants of protein kinase CK2 alpha subunit in which altogether 14 basic residues were singly to quadruply replaced by alanines (K74A,K75A,K76A,K77A; K79A, R80A,K83A; R191A,R195A,K198A; R228A; and R278A, K279A,R280A) have been purified to near homogeneity either as such or after addition...... of the recombinant beta subunit. By this latter procedure five mutated tetrameric holoenzymes were obtained as judged from their subunit composition, sedimentation coefficient on sucrose gradient ultracentrifugation, and increased activity toward a specific peptide substrate as compared with the isolated alpha......191A,R195A, K198A; K79A,R80A,K83A; and K74A,K75A, K76A,K77A are assayed with the peptides RRRADDSADDDD, RRRADDSDDADD, and RRRADDSDDDAA, respectively. In contrast, the phosphorylation efficiencies of the other substituted peptides decrease more markedly with these mutants than with CK2 wild type...

  13. Amide I SFG Spectral Line Width Probes the Lipid-Peptide and Peptide-Peptide Interactions at Cell Membrane In Situ and in Real Time.

    Science.gov (United States)

    Zhang, Baixiong; Tan, Junjun; Li, Chuanzhao; Zhang, Jiahui; Ye, Shuji

    2018-06-13

    The balance of lipid-peptide and peptide-peptide interactions at cell membrane is essential to a large variety of cellular processes. In this study, we have experimentally demonstrated for the first time that sum frequency generation vibrational spectroscopy can be used to probe the peptide-peptide and lipid-peptide interactions in cell membrane in situ and in real time by determination of the line width of amide I band of protein backbone. Using a "benchmark" model of α-helical WALP23, it is found that the dominated lipid-peptide interaction causes a narrow line width of the amide I band, whereas the peptide-peptide interaction can markedly broaden the line width. When WALP23 molecules insert into the lipid bilayer, a quite narrow line width of the amide I band is observed because of the lipid-peptide interaction. In contrast, when the peptide lies down on the bilayer surface, the line width of amide I band becomes very broad owing to the peptide-peptide interaction. In terms of the real-time change in the line width, the transition from peptide-peptide interaction to lipid-peptide interaction is monitored during the insertion of WALP23 into 1,2-dipalmitoyl- sn-glycero-3-phospho-(1'- rac-glycerol) (DPPG) lipid bilayer. The dephasing time of a pure α-helical WALP23 in 1-palmitoyl-2-oleoyl- sn-glycero-3-phospho-(1'- rac-glycerol) and DPPG bilayer is determined to be 2.2 and 0.64 ps, respectively. The peptide-peptide interaction can largely accelerate the dephasing time.

  14. Peptide Synthesis Method and Solid Support for Use in the Method

    DEFF Research Database (Denmark)

    1994-01-01

    A method for the solid-phase synthesis of peptides or proteins in high yield and high purity uses a solid support consisting of a functionalized polystyrene-grafted polymer substrate, the grafted polystyrene chains being substantially non-cross-linked and having a chain molecular weight, not incl...... is immersed in a solution of optionally substituted styrene monomer in an alcohol such as methanol, the volume percentage of styrene in the solution preferably being about 30% v/v, and subjected to gamma irradiation....

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

    Directory of Open Access Journals (Sweden)

    Punitha Velmurugan

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

  16. A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

    Directory of Open Access Journals (Sweden)

    Jonathan S Wall

    Full Text Available Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

  17. Taylor Dispersion Analysis as a promising tool for assessment of peptide-peptide interactions.

    Science.gov (United States)

    Høgstedt, Ulrich B; Schwach, Grégoire; van de Weert, Marco; Østergaard, Jesper

    2016-10-10

    Protein-protein and peptide-peptide (self-)interactions are of key importance in understanding the physiochemical behavior of proteins and peptides in solution. However, due to the small size of peptide molecules, characterization of these interactions is more challenging than for proteins. In this work, we show that protein-protein and peptide-peptide interactions can advantageously be investigated by measurement of the diffusion coefficient using Taylor Dispersion Analysis. Through comparison to Dynamic Light Scattering it was shown that Taylor Dispersion Analysis is well suited for the characterization of protein-protein interactions of solutions of α-lactalbumin and human serum albumin. The peptide-peptide interactions of three selected peptides were then investigated in a concentration range spanning from 0.5mg/ml up to 80mg/ml using Taylor Dispersion Analysis. The peptide-peptide interactions determination indicated that multibody interactions significantly affect the PPIs at concentration levels above 25mg/ml for the two charged peptides. Relative viscosity measurements, performed using the capillary based setup applied for Taylor Dispersion Analysis, showed that the viscosity of the peptide solutions increased with concentration. Our results indicate that a viscosity difference between run buffer and sample in Taylor Dispersion Analysis may result in overestimation of the measured diffusion coefficient. Thus, Taylor Dispersion Analysis provides a practical, but as yet primarily qualitative, approach to assessment of the colloidal stability of both peptide and protein formulations. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Diversity in peptide recognition by the SH2 domain of SH2B1.

    Science.gov (United States)

    McKercher, Marissa A; Guan, Xiaoyang; Tan, Zhongping; Wuttke, Deborah S

    2018-02-01

    SH2B1 is a multidomain protein that serves as a key adaptor to regulate numerous cellular events, such as insulin, leptin, and growth hormone signaling pathways. Many of these protein-protein interactions are mediated by the SH2 domain of SH2B1, which recognizes ligands containing a phosphorylated tyrosine (pY), including peptides derived from janus kinase 2, insulin receptor, and insulin receptor substrate-1 and -2. Specificity for the SH2 domain of SH2B1 is conferred in these ligands either by a hydrophobic or an acidic side chain at the +3 position C-terminal to the pY. This specificity for chemically disparate species suggests that SH2B1 relies on distinct thermodynamic or structural mechanisms to bind to peptides. Using binding and structural strategies, we have identified unique thermodynamic signatures for each peptide binding mode, and several SH2B1 residues, including K575 and R578, that play distinct roles in peptide binding. The high-resolution structure of the SH2 domain of SH2B1 further reveals conformationally plastic protein loops that may contribute to the ability of the protein to recognize dissimilar ligands. Together, numerous hydrophobic and electrostatic interactions, in addition to backbone conformational flexibility, permit the recognition of diverse peptides by SH2B1. An understanding of this expanded peptide recognition will allow for the identification of novel physiologically relevant SH2B1/peptide interactions, which can contribute to the design of obesity and diabetes pharmaceuticals to target the ligand-binding interface of SH2B1 with high specificity. © 2017 Wiley Periodicals, Inc.

  19. PTB domain-directed substrate targeting in a tyrosine kinase from the unicellular choanoflagellate Monosiga brevicollis.

    Directory of Open Access Journals (Sweden)

    Victoria Prieto-Echagüe

    2011-04-01

    Full Text Available Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition.

  20. Optimizing electrode-attached redox-peptide systems for kinetic characterization of protease action on immobilized substrates. Observation of dissimilar behavior of trypsin and thrombin enzymes.

    Science.gov (United States)

    Anne, Agnès; Chovin, Arnaud; Demaille, Christophe

    2012-06-12

    In this work, we experimentally address the issue of optimizing gold electrode attached ferrocene (Fc)-peptide systems for kinetic measurements of protease action. Considering human α-thrombin and bovine trypsin as proteases of interest, we show that the recurring problem of incomplete cleavage of the peptide layer by these enzymes can be solved by using ultraflat template-stripped gold, instead of polished polycrystalline gold, as the Fc-peptide bearing electrode material. We describe how these fragile surfaces can be mounted in a rotating disk configuration so that enzyme mass transfer no longer limits the overall measured cleavage kinetics. Finally, we demonstrate that, once the system has been optimized, in situ real-time cyclic voltammetry monitoring of the protease action can yield high-quality kinetic data, showing no sign of interfering effects. The cleavage progress curves then closely match the Langmuirian variation expected for a kinetically controlled surface process. Global fit of the progress curves yield accurate values of the peptide cleavage rate for both trypsin and thrombin. It is shown that, whereas trypsin action on the surface-attached peptide closely follows Michaelis-Menten kinetics, thrombin displays a specific and unexpected behavior characterized by a nearly enzyme-concentration-independent cleavage rate in the subnanomolar enzyme concentration range. The reason for this behavior has still to be clarified, but its occurrence may limit the sensitivity of thrombin sensors based on Fc-peptide layers.

  1. Cutting edge: identification of novel T cell epitopes in Lol p5a by computational prediction.

    Science.gov (United States)

    de Lalla, C; Sturniolo, T; Abbruzzese, L; Hammer, J; Sidoli, A; Sinigaglia, F; Panina-Bordignon, P

    1999-08-15

    Although atopic allergy affects Lol p5a allergen from rye grass. In vitro binding studies confirmed the promiscuous binding characteristics of these peptides. Moreover, most of the predicted ligands were novel T cell epitopes that were able to stimulate T cells from atopic patients. We generated a panel of Lol p5a-specific T cell clones, the majority of which recognized the peptides in a cross-reactive fashion. The computational prediction of DR ligands might thus allow the design of T cell epitopes with potential useful application in novel immunotherapy strategies.

  2. Antimicrobial Peptides in 2014

    Directory of Open Access Journals (Sweden)

    Guangshun Wang

    2015-03-01

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

  3. Osmoprotection of Bacillus subtilis through Import and Proteolysis of Proline-Containing Peptides

    Science.gov (United States)

    Zaprasis, Adrienne; Brill, Jeanette; Thüring, Marietta; Wünsche, Guido; Heun, Magnus; Barzantny, Helena; Hoffmann, Tamara

    2013-01-01

    Bacillus subtilis can attain cellular protection against the detrimental effects of high osmolarity through osmotically induced de novo synthesis and uptake of the compatible solute l-proline. We have now found that B. subtilis can also exploit exogenously provided proline-containing peptides of various lengths and compositions as osmoprotectants. Osmoprotection by these types of peptides is generally dependent on their import via the peptide transport systems (Dpp, Opp, App, and DtpT) operating in B. subtilis and relies on their hydrolysis to liberate proline. The effectiveness with which proline-containing peptides confer osmoprotection varies considerably, and this can be correlated with the amount of the liberated and subsequently accumulated free proline by the osmotically stressed cell. Through gene disruption experiments, growth studies, and the quantification of the intracellular proline pool, we have identified the PapA (YqhT) and PapB (YkvY) peptidases as responsible for the hydrolysis of various types of Xaa-Pro dipeptides and Xaa-Pro-Xaa tripeptides. The PapA and PapB peptidases possess overlapping substrate specificities. In contrast, osmoprotection by peptides of various lengths and compositions with a proline residue positioned at their N terminus was not affected by defects in the PapA and PapB peptidases. Taken together, our data provide new insight into the physiology of the osmotic stress response of B. subtilis. They illustrate the flexibility of this ubiquitously distributed microorganism to effectively exploit environmental resources in its acclimatization to sustained high-osmolarity surroundings through the accumulation of compatible solutes. PMID:23144141

  4. Kinetics of Single-Enzyme Reactions on Vesicles: Role of Substrate Aggregation

    Science.gov (United States)

    Zhdanov, Vladimir P.

    2015-03-01

    Enzymatic reactions occurring in vivo on lipid membranes can be influenced by various factors including macromolecular crowding in general and substrate aggregation in particular. In academic studies, the role of these factors can experimentally be clarified by tracking single-enzyme kinetics occurring on individual lipid vesicles. To extend the conceptual basis for such experiments, we analyze herein the corresponding kinetics mathematically with emphasis on the role of substrate aggregation. In general, the aggregation may occur on different length scales. Small aggregates may e.g. contain a few proteins or peptides while large aggregates may be mesoscopic as in the case of lipid domains which can be formed in the membranes composed of different lipids. We present a kinetic model describing comprehensively the effect of aggregation of the former type on the dependence of the reaction rate on substrate membrane concentration. The results obtained with physically reasonable parameters indicate that the aggregation-related deviations from the conventional Michaelis-Menten kinetics may be appreciable. Special Issue Comments: This theoretical article is focused on single-enzyme reactions occurring in parallel with substrate aggregation on individual vesicles. This subject is related to a few Special Issue articles concerning enzyme dynamics6,7 and function8 and mathematical aspects of stochastic kinetics.9

  5. Biomimetic oligosaccharide and peptide surfactant polymers designed for cardiovascular biomaterials

    Science.gov (United States)

    Ruegsegger, Mark Andrew

    A common problem associated with cardiovascular devices is surface induced thrombosis initiated by the rapid, non-specific adsorption of plasma proteins onto the biomaterial surface. Control of the initial protein adsorption is crucial to achieve the desired longevity of the implanted biomaterial. The cell membrane glycocalyx acts as a non-thrombogenic interface through passive (dense oligosaccharide structures) and active (ligand/receptor interactions) mechanisms. This thesis is designed to investigate biomimicry of the cell glycocalyx to minimize non-specific protein adsorption and promote specific ligand/receptor interactions. Biomimetic macromolecules were designed through the molecular-scale engineering of polymer surfactants, utilizing a poly(vinyl amine) (PVAm) backbone to which hydrophilic (dextran, maltose, peptide) and hydrophobic alkyl (hexanoyl or hexanal) chains are simultaneously attached. The structure was controlled through the molar feed ratio of hydrophobic-to-hydrophilic groups, which also provided control of the solution and surface-active properties. To mimic passive properties, a series of oligomaltose surfactants were synthesized with increasing saccharide length (n = 2, 7, 15 where n is number of glucose units) to investigate the effect of coating height on protein adsorption. The surfactants were characterized by infra red (IR) and nuclear magnetic resonance (NMR) spectroscopies for structural properties and atomic force microscopy (AFM) and contact angle goniometry for surface activity. Protein adsorption under dynamic flow (5 dyn/cm2) was reduced by 85%--95% over the bare hydrophobic substrate; platelet adhesion dropped by ˜80% compared to glass. Peptide ligands were incorporated into the oligosaccharide surfactant to promote functional activity of the passive coating. The surfactants were synthesized to contain 0%, 25%, 50%, 75%, and 100% peptide ligand density and were stable on hydrophobic surfaces. The peptide surface density was

  6. Evaluating Factor XIII Specificity for Glutamine-Containing Substrates Using a MALDI-TOF Mass Spectrometry Assay

    Science.gov (United States)

    Doiphode, Prakash G.; Malovichko, Marina V.; Mouapi, Kelly Njine; Maurer, Muriel C.

    2014-01-01

    Activated Factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetics assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, S. Aureus fibronectin binding protein A, and thrombin activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character and the P-2 and P-3 to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase. PMID:24751466

  7. Fiscal 1993 report on results of R and D on innovative technology for producing advanced biomaterial. Peptide applied carbon dioxide fixation/effective utilization technology (Second volume: comprehensive investigation and research); 1993 nendo senshin bio zairyo no sosei kako gijutsu no kenkyu kaihatsu seika hokokusho (sogo chosa kenkyu). 2. Peptide oyo nisanka tanso koteika yuko riyo gijutsu

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1994-03-01

    Technology was developed for preparing functional materials by synthesizing new functional peptides in which non-natural amino acid needed for the functional manifestation was introduced, and by fixing the peptides to the surface of a base plate such as silica glass or modifying the surface with them. As the related technological investigation, the following six areas were surveyed. (1) Structural design of peptides, (2) synthesis of peptides by chemical and enzyme methods, (3) synthesis of non-natural amino acid and its introduction into peptides, (4) structural analysis of peptides, (5) physiological activity of peptides, and (6) materialization and functional manifestation of peptides. With the exception of the area (5) consisting of one advanced research case, other areas were constituted of analysis, general remarks and research cases. The area (6) was constituted of materialization technique 1 and 2, film function and peptide, catalytic action 1 and 2, and one research case; the materialization technique was defined as a technique for modifying the surface of a synthetic resin, metallic or silica glass substrate with functional peptides; and an explanation was given to the present state of the fixation, pattern formation and integration technologies. (NEDO)

  8. Connecting peptide (c-peptide) and the duration of diabetes mellitus ...

    African Journals Online (AJOL)

    Objective: C-peptide is derived from proinsulin and it is secreted in equimolar concentration with insulin. Plasma C-peptide is more stable than insulin and it provides an indirect measure of insulin secretory reserve and beta cell function. To determine relationship between C-peptide and duration of diabetes mellitus, age, ...

  9. Peptide-Carrier Conjugation

    DEFF Research Database (Denmark)

    Hansen, Paul Robert

    2015-01-01

    To produce antibodies against synthetic peptides it is necessary to couple them to a protein carrier. This chapter provides a nonspecialist overview of peptide-carrier conjugation. Furthermore, a protocol for coupling cysteine-containing peptides to bovine serum albumin is outlined....

  10. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns

    DEFF Research Database (Denmark)

    Larsen, Martin Røssel; Thingholm, Tine E; Jensen, Ole N

    2005-01-01

    based on TiO2microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased...... the selectivity of the enrichment of phosphorylated peptides by TiO2. We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS...... was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented....

  11. A microbially derived tyrosine-sulfated peptide mimics a plant peptide hormone.

    Science.gov (United States)

    Pruitt, Rory N; Joe, Anna; Zhang, Weiguo; Feng, Wei; Stewart, Valley; Schwessinger, Benjamin; Dinneny, José R; Ronald, Pamela C

    2017-07-01

    The biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo) produces a sulfated peptide named RaxX, which shares similarity to peptides in the PSY (plant peptide containing sulfated tyrosine) family. We hypothesize that RaxX mimics the growth-stimulating activity of PSY peptides. Root length was measured in Arabidopsis and rice treated with synthetic RaxX peptides. We also used comparative genomic analyses and reactive oxygen species burst assays to evaluate the activity of RaxX and PSY peptides. Here we found that a synthetic sulfated RaxX derivative comprising 13 residues (RaxX13-sY), highly conserved between RaxX and PSY, induces root growth in Arabidopsis and rice in a manner similar to that triggered by PSY. We identified residues that are required for activation of immunity mediated by the rice XA21 receptor but that are not essential for root growth induced by PSY. Finally, we showed that a Xanthomonas strain lacking raxX is impaired in virulence. These findings suggest that RaxX serves as a molecular mimic of PSY peptides to facilitate Xoo infection and that XA21 has evolved the ability to recognize and respond specifically to the microbial form of the peptide. © 2017 UT-Battelle LLC. New Phytologist © 2017 New Phytologist Trust.

  12. Tumor penetrating peptides

    Directory of Open Access Journals (Sweden)

    Tambet eTeesalu

    2013-08-01

    Full Text Available Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC, contains the integrin-binding RGD motif. RGD mediates tumor homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular zip code of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is

  13. Characterization of bioactive RGD peptide immobilized onto poly(acrylic acid) thin films by plasma polymerization

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Hyun Suk; Ko, Yeong Mu; Shim, Jae Won [Department of Dental Materials, School of Dentistry, MRC Center, Chosun University, Gwangju (Korea, Republic of); Lim, Yun Kyong; Kook, Joong-Ki [Department of Oral Biochemistry, School of Dentistry, Chosun University, Gwangju (Korea, Republic of); Cho, Dong-Lyun [School of Applied Chemical Engineering and Center for Functional Nano Fine Chemicals, Chonnam National University, Gwangju (Korea, Republic of); Kim, Byung Hoon, E-mail: kim5055@chosun.ac.kr [Department of Dental Materials, School of Dentistry, MRC Center, Chosun University, Gwangju (Korea, Republic of)

    2010-11-01

    Plasma surface modification can be used to improve the surface properties of commercial pure Ti by creating functional groups to produce bioactive materials with different surface topography. In this study, a titanium surface was modified with acrylic acid (AA) using a plasma treatment and immobilized with bioactive arginine-glycine-aspartic acid (RGD) peptide, which may accelerate the tissue integration of bone implants. Both terminals containing the -NH{sub 2} of RGD peptide sequence and -COOH of poly(acrylic acid) (PAA) thin film were combined with a covalent bond in the presence of 1-ethyl-3-3-dimethylaminopropyl carbodiimide (EDC). The chemical structure and morphology of AA film and RGD immobilized surface were investigated by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR), atomic force microscopy (AFM), and scanning electron microscopy (SEM). All chemical analysis showed full coverage of the Ti substrate with the PAA thin film containing COOH groups and the RGD peptide. The MC3T3-E1 cells were cultured on each specimen, and the cell alkaline phosphatase (ALP) activity were examined. The surface-immobilized RGD peptide has a significantly increased the ALP activity of MC3T3-E1 cells. These results suggest that the RGD peptide immobilization on the titanium surface has an effect on osteoblastic differentiation of MC3T3-E1 cells and potential use in osteo-conductive bone implants.

  14. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2003-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  15. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  16. PeptideAtlas

    Data.gov (United States)

    U.S. Department of Health & Human Services — PeptideAtlas is a multi-organism, publicly accessible compendium of peptides identified in a large set of tandem mass spectrometry proteomics experiments. Mass...

  17. Synthetic peptide vaccines: palmitoylation of peptide antigens by a thioester bond increases immunogenicity

    DEFF Research Database (Denmark)

    Beekman, N.J.C.M.; Schaaper, W.M.M.; Tesser, G.I.

    1997-01-01

    Synthetic peptides have frequently been used to immunize animals. However, peptides less than about 20 to 30 amino acids long are poor immunogens. In general, to increase its immunogenicity, the presentation of the peptide should be improved, and molecular weight needs to be increased. Many...... or an amide bond. It was found that these S-palmitoylated peptides were much more immunogenic than N-palmitoylated peptides and at least similar to KLH-conjugated peptides with respect to appearance and magnitude of induced antibodies (canine parvovirus) or immunocastration effect (gonadotropin...

  18. The contribution of VHL substrate binding and HIF1-alpha to the phenotype of VHL loss in renal cell carcinoma.

    Science.gov (United States)

    Maranchie, Jodi K; Vasselli, James R; Riss, Joseph; Bonifacino, Juan S; Linehan, W Marston; Klausner, Richard D

    2002-04-01

    Clear-cell renal carcinoma is associated with inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. VHL is the substrate recognition subunit of an E3 ligase, known to target the alpha subunits of the HIF heterodimeric transcription factor for ubiquitin-mediated degradation under normoxic conditions. We demonstrate that competitive inhibition of the VHL substrate recognition site with a peptide derived from the oxygen degradation domain of HIF1alpha recapitulates the tumorigenic phenotype of VHL-deficient tumor cells. These studies prove that VHL substrate recognition is essential to the tumor suppressor function of VHL. We further demonstrate that normoxic stabilization of HIF1alpha alone, while capable of mimicking some aspects of VHL loss, is not sufficient to reproduce tumorigenesis, indicating that it is not the critical oncogenic substrate of VHL.

  19. Convenience polyandry or convenience polygyny? Costly sex under female control in a promiscuous primate.

    Science.gov (United States)

    Huchard, Elise; Canale, Cindy I; Le Gros, Chloé; Perret, Martine; Henry, Pierre-Yves; Kappeler, Peter M

    2012-04-07

    Classic sex roles depict females as choosy, but polyandry is widespread. Empirical attempts to understand the evolution of polyandry have often focused on its adaptive value to females, whereas 'convenience polyandry' might simply decrease the costs of sexual harassment. We tested whether constraint-free female strategies favour promiscuity over mating selectivity through an original experimental design. We investigated variation in mating behaviour in response to a reversible alteration of sexual dimorphism in body mass in the grey mouse lemur, a small primate where female brief sexual receptivity allows quantifying polyandry. We manipulated body condition in captive females, predicting that convenience polyandry would increase when females are weaker than males, thus less likely to resist their solicitations. Our results rather support the alternative hypothesis of 'adaptive polyandry': females in better condition are more polyandrous. Furthermore, we reveal that multiple mating incurs significant energetic costs, which are strikingly symmetrical between the sexes. Our study shows that mouse lemur females exert tight control over mating and actively seek multiple mates. The benefits of remating are nevertheless not offset by its costs in low-condition females, suggesting that polyandry is a flexible strategy yielding moderate fitness benefits in this small mammal.

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

    Science.gov (United States)

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

    2001-04-01

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

  1. Diversity-oriented peptide stapling

    DEFF Research Database (Denmark)

    Tran, Thu Phuong; Larsen, Christian Ørnbøl; Røndbjerg, Tobias

    2017-01-01

    as a powerful method for peptide stapling. However, to date CuAAC stapling has not provided a simple method for obtaining peptides that are easily diversified further. In the present study, we report a new diversity-oriented peptide stapling (DOPS) methodology based on CuAAC chemistry. Stapling of peptides...

  2. Effect of a Fusion Peptide by Covalent Conjugation of a Mitochondrial Cell-Penetrating Peptide and a Glutathione Analog Peptide

    Directory of Open Access Journals (Sweden)

    Carmine Pasquale Cerrato

    2017-06-01

    Full Text Available Previously, we designed and synthesized a library of mitochondrial antioxidative cell-penetrating peptides (mtCPPs superior to the parent peptide, SS31, to protect mitochondria from oxidative damage. A library of antioxidative glutathione analogs called glutathione peptides (UPFs, exceptional in hydroxyl radical elimination compared with glutathione, were also designed and synthesized. Here, a follow-up study is described, investigating the effects of the most promising members from both libraries on reactive oxidative species scavenging ability. None of the peptides influenced cell viability at the concentrations used. Fluorescence microscopy studies showed that the fluorescein-mtCPP1-UPF25 (mtgCPP internalized into cells, and spectrofluorometric analysis determined the presence and extent of peptide into different cell compartments. mtgCPP has superior antioxidative activity compared with mtCPP1 and UPF25 against H2O2 insult, preventing ROS formation by 2- and 3-fold, respectively. Moreover, we neither observed effects on mitochondrial membrane potential nor production of ATP. These data indicate that mtgCPP is targeting mitochondria, protecting them from oxidative damage, while also being present in the cytosol. Our hypothesis is based on a synergistic effect resulting from the fused peptide. The mitochondrial peptide segment is targeting mitochondria, whereas the glutathione analog peptide segment is active in the cytosol, resulting in increased scavenging ability.

  3. Peptide-membrane interactions of arginine-tryptophan peptides probed using quartz crystal microbalance with dissipation monitoring.

    KAUST Repository

    Rydberg, Hanna A

    2014-04-18

    Membrane-active peptides include peptides that can cross cellular membranes and deliver macromolecular cargo as well as peptides that inhibit bacterial growth. Some of these peptides can act as both transporters and antibacterial agents. It is desirable to combine the knowledge from these two different fields of membrane-active peptides into design of new peptides with tailored actions, as transporters of cargo or as antibacterial substances, targeting specific membranes. We have previously shown that the position of the amino acid tryptophan in the peptide sequence of three arginine-tryptophan peptides affects their uptake and intracellular localization in live mammalian cells, as well as their ability to inhibit bacterial growth. Here, we use quartz crystal microbalance with dissipation monitoring to assess the induced changes caused by binding of the three peptides to supported model membranes composed of POPC, POPC/POPG, POPC/POPG/cholesterol or POPC/lactosyl PE. Our results indicate that the tryptophan position in the peptide sequence affects the way these peptides interact with the different model membranes and that the presence of cholesterol in particular seems to affect the membrane interaction of the peptide with an even distribution of tryptophans in the peptide sequence. These results give mechanistic insight into the function of these peptides and may aid in the design of membrane-active peptides with specified cellular targets and actions.

  4. Peptide-membrane interactions of arginine-tryptophan peptides probed using quartz crystal microbalance with dissipation monitoring.

    KAUST Repository

    Rydberg, Hanna A; Kunze, Angelika; Carlsson, Nils; Altgä rde, Noomi; Svedhem, Sofia; Nordé n, Bengt

    2014-01-01

    Membrane-active peptides include peptides that can cross cellular membranes and deliver macromolecular cargo as well as peptides that inhibit bacterial growth. Some of these peptides can act as both transporters and antibacterial agents. It is desirable to combine the knowledge from these two different fields of membrane-active peptides into design of new peptides with tailored actions, as transporters of cargo or as antibacterial substances, targeting specific membranes. We have previously shown that the position of the amino acid tryptophan in the peptide sequence of three arginine-tryptophan peptides affects their uptake and intracellular localization in live mammalian cells, as well as their ability to inhibit bacterial growth. Here, we use quartz crystal microbalance with dissipation monitoring to assess the induced changes caused by binding of the three peptides to supported model membranes composed of POPC, POPC/POPG, POPC/POPG/cholesterol or POPC/lactosyl PE. Our results indicate that the tryptophan position in the peptide sequence affects the way these peptides interact with the different model membranes and that the presence of cholesterol in particular seems to affect the membrane interaction of the peptide with an even distribution of tryptophans in the peptide sequence. These results give mechanistic insight into the function of these peptides and may aid in the design of membrane-active peptides with specified cellular targets and actions.

  5. Phosphorylated peptides occur in a non-helical portion of the tail of a catch muscle myosin

    International Nuclear Information System (INIS)

    Castellani, L.; Elliott, B.W. Jr.; Cohen, C.

    1987-01-01

    Myosin from a molluscan catch muscle (the Anterior Byssus Retractor (ABRM) of Mytilus edulis) is unusual in being phosphorylated in the rod by an endogenous heavy-chain kinase. This phosphorylation enhances myosin solubility at low ionic strength and induces molecular folding of the myosin tail. Papain and chymotryptic cleavage of this myosin, phosphorylated with [γ- 32 P]ATP, indicates that the phosphorylated residues are associated with the carboxy-terminal end of the light meromyosin. Ion-exchange and reverse-phase HPLC of radiolabeled chymotryptic peptides allow the isolation of two different peptides with high specific activity. One of these peptides is rich in lysine and arginine residues, a finding consistent with the observation that basic residues often determine the substrate specificity of protein kinases. The second peptide contains proline residues. Taken together, these results suggest that, as in the case of Acanthamoeba myosin, phosphorylation occurs in a nonhelical portion of the rod that may also control solubility. Identification of the residues that are phosphorylated and their location in the rod may reveal how the phosphorylation-dependent changes observed in the myosin in vitro are related to changes in intermolecular interactions in the thick filaments in vivo

  6. Grafting of a peptide probe for Prostate-Specific Antigen detection using diazonium electroreduction and click chemistry.

    Science.gov (United States)

    Strzemińska, I; Sainte Rose Fanchine, S; Anquetin, G; Reisberg, S; Noël, V; Pham, M C; Piro, B

    2016-07-15

    The main objective of this work was to validate a label-free electrochemical method of protein detection using peptides as capture probes. As a proof-of-concept, we used a 7 amino acids sequence (HSSKLQL) specific for Prostate Specific Antigen. We investigated various electrografting conditions of two anilines (2-[(4-aminophenyl)sulfanyl]-8-hydroxy-1,4-naphthoquinone and 4-azidoaniline) further converted in situ into their corresponding diazonium salts on glassy carbon electrodes. It was demonstrated that the best method to obtain a mixed layer is the simultaneous electroreduction of the two diazonium salts. 4-azidoaniline was used to covalently immobilize the ethynyl-functionalized peptide probe by click coupling, and the hydroxynaphthoquinone derivative plays the role of electrochemical transducer of the peptide-protein recognition. The proteolytic activity of PSA towards a small peptide substrate carrying streptavidin at its distal end was also investigated to design an original sensing architecture leading to a reagentless, label free, and "signal-on" PSA sensor. Without optimization, the limit of quantification can be estimated in the nM to pM range. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Lisa K. Denzin

    2013-12-01

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

  8. Automated solid-phase peptide synthesis to obtain therapeutic peptides

    Directory of Open Access Journals (Sweden)

    Veronika Mäde

    2014-05-01

    Full Text Available The great versatility and the inherent high affinities of peptides for their respective targets have led to tremendous progress for therapeutic applications in the last years. In order to increase the drugability of these frequently unstable and rapidly cleared molecules, chemical modifications are of great interest. Automated solid-phase peptide synthesis (SPPS offers a suitable technology to produce chemically engineered peptides. This review concentrates on the application of SPPS by Fmoc/t-Bu protecting-group strategy, which is most commonly used. Critical issues and suggestions for the synthesis are covered. The development of automated methods from conventional to essentially improved microwave-assisted instruments is discussed. In order to improve pharmacokinetic properties of peptides, lipidation and PEGylation are described as covalent conjugation methods, which can be applied by a combination of automated and manual synthesis approaches. The synthesis and application of SPPS is described for neuropeptide Y receptor analogs as an example for bioactive hormones. The applied strategies represent innovative and potent methods for the development of novel peptide drug candidates that can be manufactured with optimized automated synthesis technologies.

  9. Surface chemical immobilization of bioactive peptides on synthetic polymers for cardiac tissue engineering.

    Science.gov (United States)

    Rosellini, Elisabetta; Cristallini, Caterina; Guerra, Giulio D; Barbani, Niccoletta

    2015-01-01

    The aim of this work was the development of new synthetic polymeric systems, functionalized by surface chemical modification with bioactive peptides, for myocardial tissue engineering. Polycaprolactone and a poly(ester-ether-ester) block copolymer synthesized in our lab, polycaprolactone-poly(ethylene oxide)-polycaprolactone (PCL-PEO-PCL), were used as the substrates to be modified. Two pentapeptides, H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS) from fibronectin and H-Tyr-Ile-Gly-Ser-Arg-OH (YIGSR) from laminin, were used for the functionalization. Polymeric membranes were obtained by casting from solutions and then functionalized by means of alkaline hydrolysis and subsequent coupling of the bioactive molecules through 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride/N-hydroxysuccinimide chemistry. The hydrolysis conditions, in terms of hydrolysis time, temperature, and sodium hydroxide concentration, were optimized for the two materials. The occurrence of the coupling reaction was demonstrated by infrared spectroscopy, as the presence on the functionalized materials of the absorption peaks typical of the two peptides. The peptide surface density was determined by chromatographic analysis and the distribution was studied by infrared chemical imaging. The results showed a nearly homogeneous peptide distribution, with a density above the minimum value necessary to promote cell adhesion. Preliminary in vitro cell culture studies demonstrated that the introduction of the bioactive molecules had a positive effect on improving C2C12 myoblasts growth on the synthetic materials.

  10. Production of peptide antisera specific for mouse and rat proinsulin C-peptide 2

    DEFF Research Database (Denmark)

    Blume, N; Madsen, O D; Kofod, Hans

    1990-01-01

    for antibody binding to the immunizing antigen. Antisera to C-peptide 2, stained islet beta-cells on mouse and rat, but not monkey pancreas sections in immunocytochemical analysis. Preabsorption to the synthetic C-peptide 2, but not the synthetic mouse and rat C-peptide 1 abolished staining. In conclusion we......Mice and rats have two functional non-allelic insulin genes. By using a synthetic peptide representing a common sequence in mouse and rat C-peptide 2 as antigen, we have produced rabbit antisera specific for an epitope which is not present in mouse or rat C-peptide 1. Long-term immunization did...... not seem to increase the end point titre as tested in direct ELISA. The specificity of the antiserum was determined by competitive ELISA and histochemistry on pancreas sections. Only the synthetic C-peptide 2, but not the homologous synthetic C-peptide 1 from mouse and rat competed efficiently in ELISA...

  11. Therapeutic peptides for cancer therapy. Part I - peptide inhibitors of signal transduction cascades.

    Science.gov (United States)

    Bidwell, Gene L; Raucher, Drazen

    2009-10-01

    Therapeutic peptides have great potential as anticancer agents owing to their ease of rational design and target specificity. However, their utility in vivo is limited by low stability and poor tumor penetration. The authors review the development of peptide inhibitors with potential for cancer therapy. Peptides that inhibit signal transduction cascades are discussed. The authors searched Medline for articles concerning the development of therapeutic peptides and their delivery. Given our current knowledge of protein sequences, structures and interaction interfaces, therapeutic peptides that inhibit interactions of interest are easily designed. These peptides are advantageous because they are highly specific for the interaction of interest, and they are much more easily developed than small molecule inhibitors of the same interactions. The main hurdle to application of peptides for cancer therapy is their poor pharmacokinetic and biodistribution parameters. Therefore, successful development of peptide delivery vectors could potentially make possible the use of this new and very promising class of anticancer agents.

  12. Production of an antimicrobial peptide derived from slaughterhouse by-product and its potential application on meat as preservative.

    Science.gov (United States)

    Przybylski, Rémi; Firdaous, Loubna; Châtaigné, Gabrielle; Dhulster, Pascal; Nedjar, Naïma

    2016-11-15

    Bovine cruor, a slaughterhouse by-product, contains mainly hemoglobin, broadly described as a rich source of antimicrobial peptides. In the current context of food safety, bioactive peptides could be of interest as preservatives in the distribution of food products. The aim of this work was to study the α137-141 fragment of hemoglobin (Thr-Ser-Lys-Tyr-Arg), a small (653Da) and hydrophilic antimicrobial peptide. Its production was fast, with more 65% finally produced at 24h already produced after 30min of hydrolysis with pepsin. Moreover, increasing substrate concentration (from 1 to 8% (w/v)) resulted in a proportional augmentation of α137-141 production (to 807.95±41.03mgL(-1)). The α137-141 application on meat as preservative (0.5%, w/w) reduced the lipid oxidation about 60% to delay meat rancidity. The α137-141 peptide also inhibited the microbial growths under refrigeration during 14days. These antimicrobial effects were close to those of the butylated hydroxytoluene (BHT). Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Purification and use of E. coli peptide deformylase for peptide deprotection in chemoenzymatic peptide synthesis

    NARCIS (Netherlands)

    Di Toma, Claudia; Sonke, Theo; Quaedflieg, Peter J.; Janssen, Dick B.

    Peptide deformylases (PDFs) catalyze the removal of the formyl group from the N-terminal methionine residue in nascent polypeptide chains in prokaryotes. Its deformylation activity makes PDF an attractive candidate for the biocatalytic deprotection of formylated peptides that are used in

  14. Antioxidant activity of yoghurt peptides: Part 2 – Characterisationof peptide fractions

    DEFF Research Database (Denmark)

    Farvin, Sabeena; Baron, Caroline; Nielsen, Nina Skall

    2010-01-01

    the peptides identified contained at least one proline residue. Some of the identified peptides included the hydrophobic amino acid residues Val or Leu at the N-terminus and Pro, His or Tyr in the amino acid sequence, which is characteristic of antioxidant peptides. In addition, the yoghurt contained...

  15. Ligand-regulated peptide aptamers.

    Science.gov (United States)

    Miller, Russell A

    2009-01-01

    The peptide aptamer approach employs high-throughput selection to identify members of a randomized peptide library displayed from a scaffold protein by virtue of their interaction with a target molecule. Extending this approach, we have developed a peptide aptamer scaffold protein that can impart small-molecule control over the aptamer-target interaction. This ligand-regulated peptide (LiRP) scaffold, consisting of the protein domains FKBP12, FRB, and GST, binds to the cell-permeable small-molecule rapamycin and the binding of this molecule can prevent the interaction of the randomizable linker region connecting FKBP12 with FRB. Here we present a detailed protocol for the creation of a peptide aptamer plasmid library, selection of peptide aptamers using the LiRP scaffold in a yeast two-hybrid system, and the screening of those peptide aptamers for a ligand-regulated interaction.

  16. Phosphorylation-induced conformational changes in short peptides probed by fluorescence resonance energy transfer in the 10A domain.

    Science.gov (United States)

    Sahoo, Harekrushna; Nau, Werner M

    2007-03-26

    Phosphorylation-induced conformational changes in short polypeptides were probed by a fluorescence resonance energy transfer (FRET) method by employing a short-distance FRET pair (R(0) approximately 10 A) based on tryptophan as natural donor and a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as synthetic acceptor. Two substrates for kinases, LeuArgArgTrpSerLeuGly-Dbo (peptide I) and TrpLysArgThrLeuArgArg-Dbo (peptide II), were investigated, with serine and threonine, respectively, as phosphorylation sites. Steady-state and time-resolved fluorescence experiments in H(2)O revealed a decrease in FRET efficiency for peptide I and an increase for peptide II; this suggested that the effective distances between donor and acceptor increased and decreased, respectively. The same trends and similar absolute variations in effective donor-acceptor distances were observed in propylene glycol, a less polar and highly viscous solvent; this suggested that the variations are due to intrinsic structural preferences. Fitting of the time-resolved decay traces according to a distribution function model (Gaussian distribution) provided the mean donor-acceptor distances, which showed an increase upon phosphorylation for peptide I (from 9.7 to 10.5 A) and a decrease for peptide II (from 10.9 to 9.3 A) in H(2)O. The broadness (half-width) of the distributions, which provides a measure of the rigidity of the peptides, remained similar upon phosphorylation of peptide I (3.0 versus 3.1 A), but decreased for peptide II (from 3.1 to 0.73 A in H(2)O); this suggests a more compact, structured conformation upon phosphorylation of the latter peptide. The elongation of the peptide backbone (by ca. 0.7 A) for peptide I is attributed to an increase in steric demand upon phosphorylation, which favors an extended conformation. The contraction (by ca. 1.4 A) and structural rigidification of peptide II is attributed to attractive Coulombic interactions and hydrogen bonding between the

  17. Preparation, Characterization and Activity of a Peptide-Cellulosic Aerogel Protease Sensor from Cotton

    Directory of Open Access Journals (Sweden)

    J. Vincent Edwards

    2016-10-01

    Full Text Available Nanocellulosic aerogels (NA provide a lightweight biocompatible material with structural properties, like interconnected high porosity and specific surface area, suitable for biosensor design. We report here the preparation, characterization and activity of peptide-nanocellulose aerogels (PepNA made from unprocessed cotton and designed with protease detection activity. Low-density cellulosic aerogels were prepared from greige cotton by employing calcium thiocyanate octahydrate/lithium chloride as a direct cellulose dissolving medium. Subsequent casting, coagulation, solvent exchange and supercritical carbon dioxide drying afforded homogeneous cellulose II aerogels of fibrous morphology. The cotton-based aerogel had a porosity of 99% largely dominated by mesopores (2–50 nm and an internal surface of 163 m2·g−1. A fluorescent tripeptide-substrate (succinyl-alanine-proline-alanine-4-amino-7-methyl-coumarin was tethered to NA by (1 esterification of cellulose C6 surface hydroxyl groups with glycidyl-fluorenylmethyloxycarbonyl (FMOC, (2 deprotection and (3 coupling of the immobilized glycine with the tripeptide. Characterization of the NA and PepNA included techniques, such as elemental analysis, mass spectral analysis, attenuated total reflectance infrared imaging, nitrogen adsorption, scanning electron microscopy and bioactivity studies. The degree of substitution of the peptide analog attached to the anhydroglucose units of PepNA was 0.015. The findings from mass spectral analysis and attenuated total reflectance infrared imaging indicated that the peptide substrate was immobilized on to the surface of the NA. Nitrogen adsorption revealed a high specific surface area and a highly porous system, which supports the open porous structure observed from scanning electron microscopy images. Bioactivity studies of PepNA revealed a detection sensitivity of 0.13 units/milliliter for human neutrophil elastase, a diagnostic biomarker for inflammatory

  18. Antimicrobial Peptides in Reptiles

    Science.gov (United States)

    van Hoek, Monique L.

    2014-01-01

    Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development. PMID:24918867

  19. Driving engineering of novel antimicrobial peptides from simulations of peptide-micelle interactions

    DEFF Research Database (Denmark)

    Khandelia, Himanshu; Langham, Allison A; Kaznessis, Yiannis N

    2006-01-01

    Simulations of antimicrobial peptides in membrane mimics can provide the high resolution, atomistic picture that is necessary to decipher which sequence and structure components are responsible for activity and toxicity. With such detailed insight, engineering new sequences that are active but non...... peptides and their interaction with membrane mimics. In this article, we discuss the promise and the challenges of widely used models and detail our recent work on peptide-micelle simulations as an attractive alternative to peptide-bilayer simulations. We detail our results with two large structural...... classes of peptides, helical and beta-sheet and demonstrate how simulations can assist in engineering of novel antimicrobials with therapeutic potential....

  20. Label-Free Fluorescent Detection of Trypsin Activity Based on DNA-Stabilized Silver Nanocluster-Peptide Conjugates

    Directory of Open Access Journals (Sweden)

    Cai-Xia Zhuo

    2016-11-01

    Full Text Available Trypsin is important during the regulation of pancreatic exocrine function. The detection of trypsin activity is currently limited because of the need for the substrate to be labeled with a fluorescent tag. A label-free fluorescent method has been developed to monitor trypsin activity. The designed peptide probe consists of six arginine molecules and a cysteine terminus and can be conjugated to DNA-stabilized silver nanoclusters (DNA-AgNCs by Ag-S bonding to enhance fluorescence. The peptide probe can also be adsorbed to the surface of graphene oxide (GO, thus resulting in the fluorescence quenching of DNA-AgNCs-peptide conjugate because of Förster resonance energy transfer. Once trypsin had degraded the peptide probe into amino acid residues, the DNA-AgNCs were released from the surface of GO, and the enhanced fluorescence of DNA-AgNCs was restored. Trypsin can be determined with a linear range of 0.0–50.0 ng/mL with a concentration as low as 1 ng/mL. This label-free method is simple and sensitive and has been successfully used for the determination of trypsin in serum. The method can also be modified to detect other proteases.

  1. Helicobacter pylori CagA Inhibits PAR1-MARK Family Kinases by Mimicking Host Substrates

    Energy Technology Data Exchange (ETDEWEB)

    Nesic, D.; Miller, M; Quinkert, Z; Stein, M; Chait, B; Stebbins, C

    2010-01-01

    The CagA protein of Helicobacter pylori interacts with numerous cellular factors and is associated with increased virulence and risk of gastric carcinoma. We present here the cocrystal structure of a subdomain of CagA with the human kinase PAR1b/MARK2, revealing that a CagA peptide mimics substrates of this kinase family, resembling eukaryotic protein kinase inhibitors. Mutagenesis of conserved residues central to this interaction renders CagA inactive as an inhibitor of MARK2.

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

    Science.gov (United States)

    Grohe, Bernd

    2017-08-01

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

  3. Drugs, ionophoric peptides, and steroids as substrates of the yeast multidrug transporter Pdr5p

    NARCIS (Netherlands)

    Kolaczkowski, M; vanderRest, M; CybularzKolaczkowska, A; Soumillion, JP; Konings, WN; Goffeau, A

    1996-01-01

    Pdr5p is the yeast Saccharomyces cerevisiae ATP-binding cassette transporter conferring resistance to several unrelated drugs. Its high overproduction in Pdr1p transcription factor mutants allows us to study the molecular mechanism of multidrug transport and substrate specificity. We have developed

  4. Flanking signal and mature peptide residues influence signal peptide cleavage

    Directory of Open Access Journals (Sweden)

    Ranganathan Shoba

    2008-12-01

    Full Text Available Abstract Background Signal peptides (SPs mediate the targeting of secretory precursor proteins to the correct subcellular compartments in prokaryotes and eukaryotes. Identifying these transient peptides is crucial to the medical, food and beverage and biotechnology industries yet our understanding of these peptides remains limited. This paper examines the most common type of signal peptides cleavable by the endoprotease signal peptidase I (SPase I, and the residues flanking the cleavage sites of three groups of signal peptide sequences, namely (i eukaryotes (Euk (ii Gram-positive (Gram+ bacteria, and (iii Gram-negative (Gram- bacteria. Results In this study, 2352 secretory peptide sequences from a variety of organisms with amino-terminal SPs are extracted from the manually curated SPdb database for analysis based on physicochemical properties such as pI, aliphatic index, GRAVY score, hydrophobicity, net charge and position-specific residue preferences. Our findings show that the three groups share several similarities in general, but they display distinctive features upon examination in terms of their amino acid compositions and frequencies, and various physico-chemical properties. Thus, analysis or prediction of their sequences should be separated and treated as distinct groups. Conclusion We conclude that the peptide segment recognized by SPase I extends to the start of the mature protein to a limited extent, upon our survey of the amino acid residues surrounding the cleavage processing site. These flanking residues possibly influence the cleavage processing and contribute to non-canonical cleavage sites. Our findings are applicable in defining more accurate prediction tools for recognition and identification of cleavage site of SPs.

  5. Analysis of peptide uptake and location of root hair-promoting peptide accumulation in plant roots.

    Science.gov (United States)

    Matsumiya, Yoshiki; Taniguchi, Rikiya; Kubo, Motoki

    2012-03-01

    Peptide uptake by plant roots from degraded soybean-meal products was analyzed in Brassica rapa and Solanum lycopersicum. B. rapa absorbed about 40% of the initial water volume, whereas peptide concentration was decreased by 75% after 24 h. Analysis by reversed-phase HPLC showed that number of peptides was absorbed by the roots during soaking in degraded soybean-meal products for 24 h. Carboxyfluorescein-labeled root hair-promoting peptide was synthesized, and its localization, movement, and accumulation in roots were investigated. The peptide appeared to be absorbed by root hairs and then moved to trichoblasts. Furthermore, the peptide was moved from trichoblasts to atrichoblasts after 24 h. The peptide was accumulated in epidermal cells, suggesting that the peptide may have a function in both trichoblasts and atrichoblasts. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

  6. Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach

    Directory of Open Access Journals (Sweden)

    Magdalena Opalińska

    2017-11-01

    Full Text Available Maintenance of functional mitochondria is vital for optimal cell performance and survival. This is accomplished by distinct mechanisms, of which preservation of mitochondrial protein homeostasis fulfills a pivotal role. In plants, inner membrane-embedded i-AAA protease, FTSH4, contributes to the mitochondrial proteome surveillance. Owing to the limited knowledge of FTSH4’s in vivo substrates, very little is known about the pathways and mechanisms directly controlled by this protease. Here, we applied substrate trapping coupled with mass spectrometry-based peptide identification in order to extend the list of FTSH4’s physiological substrates and interaction partners. Our analyses revealed, among several putative targets of FTSH4, novel (mitochondrial pyruvate carrier 4 (MPC4 and Pam18-2 and known (Tim17-2 substrates of this protease. Furthermore, we demonstrate that FTSH4 degrades oxidatively damaged proteins in mitochondria. Our report provides new insights into the function of FTSH4 in the maintenance of plant mitochondrial proteome.

  7. Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach.

    Science.gov (United States)

    Opalińska, Magdalena; Parys, Katarzyna; Jańska, Hanna

    2017-11-18

    Maintenance of functional mitochondria is vital for optimal cell performance and survival. This is accomplished by distinct mechanisms, of which preservation of mitochondrial protein homeostasis fulfills a pivotal role. In plants, inner membrane-embedded i -AAA protease, FTSH4, contributes to the mitochondrial proteome surveillance. Owing to the limited knowledge of FTSH4's in vivo substrates, very little is known about the pathways and mechanisms directly controlled by this protease. Here, we applied substrate trapping coupled with mass spectrometry-based peptide identification in order to extend the list of FTSH4's physiological substrates and interaction partners. Our analyses revealed, among several putative targets of FTSH4, novel (mitochondrial pyruvate carrier 4 (MPC4) and Pam18-2) and known (Tim17-2) substrates of this protease. Furthermore, we demonstrate that FTSH4 degrades oxidatively damaged proteins in mitochondria. Our report provides new insights into the function of FTSH4 in the maintenance of plant mitochondrial proteome.

  8. Laminin Peptide-Immobilized Hydrogels Modulate Valve Endothelial Cell Hemostatic Regulation.

    Directory of Open Access Journals (Sweden)

    Liezl Rae Balaoing

    Full Text Available Valve endothelial cells (VEC have unique phenotypic responses relative to other types of vascular endothelial cells and have highly sensitive hemostatic functions affected by changes in valve tissues. Furthermore, effects of environmental factors on VEC hemostatic function has not been characterized. This work used a poly(ethylene glycol diacrylate (PEGDA hydrogel platform to evaluate the effects of substrate stiffness and cell adhesive ligands on VEC phenotype and expression of hemostatic genes. Hydrogels of molecular weights (MWs 3.4, 8, and 20 kDa were polymerized into platforms of different rigidities and thiol-modified cell adhesive peptides were covalently bound to acrylate groups on the hydrogel surfaces. The peptide RKRLQVQLSIRT (RKR is a syndecan-1 binding ligand derived from laminin, a trimeric protein and a basement membrane matrix component. Conversely, RGDS is an integrin binding peptide found in many extracellular matrix (ECM proteins including fibronectin, fibrinogen, and von Willebrand factor (VWF. VECs adhered to and formed a stable monolayer on all RKR-coated hydrogel-MW combinations. RGDS-coated platforms supported VEC adhesion and growth on RGDS-3.4 kDa and RGDS-8 kDa hydrogels. VECs cultured on the softer RKR-8 kDa and RKR-20 kDa hydrogel platforms had significantly higher gene expression for all anti-thrombotic (ADAMTS-13, tissue factor pathway inhibitor, and tissue plasminogen activator and thrombotic (VWF, tissue factor, and P-selectin proteins than VECs cultured on RGDS-coated hydrogels and tissue culture polystyrene controls. Stimulated VECs promoted greater platelet adhesion than non-stimulated VECs on their respective culture condition; yet stimulated VECs on RGDS-3.4 kDa gels were not as responsive to stimulation relative to the RKR-gel groups. Thus, the syndecan binding, laminin-derived peptide promoted stable VEC adhesion on the softer hydrogels and maintained VEC phenotype and natural hemostatic function. In

  9. Insulin C-peptide test

    Science.gov (United States)

    C-peptide ... the test depends on the reason for the C-peptide measurement. Ask your health care provider if ... C-peptide is measured to tell the difference between insulin the body produces and insulin someone injects ...

  10. Double-Stranded Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2001-01-01

    A novel class of compounds, known as peptide nucleic acids, form double-stranded structures with one another and with ssDNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker.......A novel class of compounds, known as peptide nucleic acids, form double-stranded structures with one another and with ssDNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  11. Application of synthetic peptides for detection of anti-citrullinated peptide antibodies

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Holm, Bettina Eide; Slot, Ole

    2016-01-01

    Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and represent an important tool for the serological diagnosis of RA. In this study, we describe ACPA reactivity to overlapping citrullinated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-derived peptides...... (n=40), systemic lupus erythematosus (n=20), Sjögren's syndrome (n=40)) were screened for antibody reactivity. Antibodies to a panel of five citrullinated EBNA-1 peptides were found in 67% of RA sera, exclusively of the IgG isotype, while 53% of the patient sera reacted with a single peptide......, ARGGSRERARGRGRG-Cit-GEKR, accounting for more than half of the ACPA reactivity alone. Moreover, these antibodies were detected in 10% of CCP2-negative RA sera. In addition, 47% of the RA sera reacted with two or three citrullinated EBNA-1 peptides from the selected peptide panel. Furthermore, a negative...

  12. Novel production method of innovative antiangiogenic and antitumor small peptides in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Setrerrahmane S

    2017-11-01

    Full Text Available Sarra Setrerrahmane,1 Jian Yu,1 Jingchao Hao,1,2 Heng Zheng,3 Hanmei Xu1,3 1The Engineering Research Center of Peptide Drug Discovery and Development, China Pharmaceutical University, Nanjing, Jiangsu, 2College of Pharmacy & the Provincial Key Laboratory of Natural Drug and Pharmacology, Kunming, Yunnan, 3State Key Laboratory of Natural Medicines, Ministry of Education, China Pharmaceutical University, Nanjing, Jiangsu, People’s Republic of China Background: Developing innovative drugs with potent efficacy, specificity, and high safety remains an ongoing task in antitumor therapy development. In the last few years, peptide drugs have become attractive agents in cancer therapy. HM-3, mainly with antiangiogenic effect, and AP25, with an additional antiproliferative effect, are two peptides designed in our laboratory targeting αvβ3 and α5β1 integrins, respectively. The low molecular weight of the two peptides renders their recombinant expression very difficult, and the complicated structure of AP25 makes its chemical synthesis restricted, which presents a big challenge for its development.Methods: Bifunctional peptides designed by the ligation of HM-3 and AP25, using linkers with different flexibility, were prepared using recombinant DNA technology in Escherichia coli. The fusion peptides were expressed in a modified auto-induction medium based on a mixture of glucose, glycerol, and lactose as carbon substrates and NH4+ as nitrogen source without any amino acid or other elements. Subsequently, the antiangiogenic, antiproliferative, and cell adhesion assays were conducted to evaluate the bioactivity of the two fusion peptides.Results: The peptides were successfully expressed in a soluble form without any induction, which allows the culture to reach higher cell density before protein expression occurs. Human umbilical vein endothelial cell migration assay and chick embryo chorioallantoic membrane assay showed, at low doses, a significantly

  13. Design and characterization of hirulogs: A novel class of bivalent peptide inhibitors of thrombin

    Energy Technology Data Exchange (ETDEWEB)

    Maraganore, J.M.; Bourdon, P.; Jablonski, J.; Ramachandran, K.L. (Biogen, Inc., Cambridge, MA (USA)); Fenton, J.W. II (New York State Department of Health, Albany (USA))

    1990-07-31

    A novel class of synthetic peptides has been designed that inhibit the thrombin catalytic site and exhibit specificity for the anion-binding exosite (ABE) of {alpha}-thrombin. These peptides, called hirulogs, consist of (i) an active-site specificity sequence with a restricted Arg-Pro scissile bond, (ii) a polymeric linker of glycyl residues from 6 to 18 {angstrom} in length, and (iii) an ABE recognition sequence such as that in the hirudin C-terminus. Hirulog-1 ((D-Phe)-Pro-Arg-Pro-(Gly){sub 4}-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Tyr-Leu) inhibits the thrombin-catalyzed hydrolysis of a tripeptide p-nitroanilide substrate with K{sub i} = 2.3 nM. In contrast, the synthetic C-terminal hirudin peptide S-Hir{sub 53-64}, which binds to the thrombin ABE, blocked the fibrinogen clotting activity of the enzyme with K{sub i} = 144 nM but failed to inhibit the hydrolysis of p-nitroanilide substrates at concentrations as high as 1 mM. Hirulog-1, but not S-Hir{sub 53-64}, was found to inhibit the incorporation of ({sup 14}C)diisopropyl fluorophosphate in thrombin. Hirulog-1 appears specific for thrombin as it lacks inhibitory activities toward human factor Xa, human plasmin, and bovine trypsin at inhibitor:enzyme concentrations 3 orders of magnitude higher than those required to inhibit thrombin. The optimal inhibitory activity of hirulog-1 depends upon all three components of its structure. Comparison of anticoagulant activities of hirulog-1, hirudin, and S-Hir{sub 53-64} showed that the synthetic hirulog-1 is 2-fold more potent than hirudin and 100-fold more active than S-Hir{sub 53-64} in increasing the activated partial thromboplastin time of normal human plasma.

  14. Optimization of process parameters for the production of collagen peptides from fish skin (Epinephelus malabaricus) using response surface methodology and its characterization.

    Science.gov (United States)

    Hema, G S; Joshy, C G; Shyni, K; Chatterjee, Niladri S; Ninan, George; Mathew, Suseela

    2017-02-01

    The study optimized the hydrolysis conditions for the production of fish collagen peptides from skin of Malabar grouper ( Epinephelus malabaricus ) using response surface methodology. The hydrolysis was done with enzymes pepsin, papain and protease from bovine pancreas. Effects of process parameters viz: pH, temperature, enzyme substrate ratio and hydrolysis time of the three different enzymes on degree of hydrolysis were investigated. The optimum response of degree of hydrolysis was estimated to be 10, 20 and 28% respectively for pepsin, papain and protease. The functional properties of the product developed were analysed which showed changes in the properties from proteins to peptides. SDS-PAGE combined with MALDI TOF method was successfully applied to determine the molecular weight distribution of the hydrolysate. The electrophoretic pattern indicated that the molecular weights of peptides formed due to hydrolysis were nearly 2 kDa. MALDI TOF spectral analysis showed the developed hydrolysate contains peptides having molecular weight in the range below 2 kDa.

  15. The synthesis and coupling of photoreactive collagen-based peptides to restore integrin reactivity to an inert substrate, chemically-crosslinked collagen

    Science.gov (United States)

    Malcor, Jean-Daniel; Bax, Daniel; Hamaia, Samir W.; Davidenko, Natalia; Best, Serena M.; Cameron, Ruth E.; Farndale, Richard W.; Bihan, Dominique

    2016-01-01

    Collagen is frequently advocated as a scaffold for use in regenerative medicine. Increasing the mechanical stability of a collagen scaffold is widely achieved by cross-linking using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). However, this treatment consumes the carboxylate-containing amino acid sidechains that are crucial for recognition by the cell-surface integrins, abolishing cell adhesion. Here, we restore cell reactivity to a cross-linked type I collagen film by covalently linking synthetic triple-helical peptides (THPs), mimicking the structure of collagen. These THPs are ligands containing an active cell-recognition motif, GFOGER, a high-affinity binding site for the collagen-binding integrins. We end-stapled peptide strands containing GFOGER by coupling a short diglutamate-containing peptide to their N-terminus, improving the thermal stability of the resulting THP. A photoreactive Diazirine group was grafted onto the end-stapled THP to allow covalent linkage to the collagen film upon UV activation. Such GFOGER-derivatized collagen films showed restored affinity for the ligand-binding I domain of integrin α2β1, and increased integrin-dependent cell attachment and spreading of HT1080 and Rugli cell lines, expressing integrins α2β1 and α1β1, respectively. The method we describe has wide application, beyond collagen films or scaffolds, since the photoreactive diazirine will react with many organic carbon skeletons. PMID:26854392

  16. Primary structure and conformational analysis of peptide methionine-tyrosine, a peptide related to neuropeptide Y and peptide YY isolated from lamprey intestine

    DEFF Research Database (Denmark)

    Conlon, J M; Bjørnholm, B; Jørgensen, Flemming Steen

    1991-01-01

    A peptide belonging to the pancreatic-polypeptide-fold family of regulatory peptides has been isolated from the intestine of an Agnathan, the sea lamprey (Petromyzon marinus). The primary structure of the peptide (termed peptide methionine-tyrosine) was established as Met-Pro-Pro-Lys-Pro-Asp-Asn-...... in a preferred structure in which the conformation of the beta-turn between the two helical domains (residues 9-14) is appreciably different....

  17. Improving Peptide Applications Using Nanotechnology.

    Science.gov (United States)

    Narayanaswamy, Radhika; Wang, Tao; Torchilin, Vladimir P

    2016-01-01

    Peptides are being successfully used in various fields including therapy and drug delivery. With advancement in nanotechnology and targeted delivery carrier systems, suitable modification of peptides has enabled achievement of many desirable goals over-riding some of the major disadvantages associated with the delivery of peptides in vivo. Conjugation or physical encapsulation of peptides to various nanocarriers, such as liposomes, micelles and solid-lipid nanoparticles, has improved their in vivo performance multi-fold. The amenability of peptides to modification in chemistry and functionalization with suitable nanocarriers are very relevant aspects in their use and have led to the use of 'smart' nanoparticles with suitable linker chemistries that favor peptide targeting or release at the desired sites, minimizing off-target effects. This review focuses on how nanotechnology has been used to improve the number of peptide applications. The paper also focuses on the chemistry behind peptide conjugation to nanocarriers, the commonly employed linker chemistries and the several improvements that have already been achieved in the areas of peptide use with the help of nanotechnology.

  18. Cell penetrating peptides to dissect host-pathogen protein-protein interactions in Theileria -transformed leukocytes

    KAUST Repository

    Haidar, Malak

    2017-09-08

    One powerful application of cell penetrating peptides is the delivery into cells of molecules that function as specific competitors or inhibitors of protein-protein interactions. Ablating defined protein-protein interactions is a refined way to explore their contribution to a particular cellular phenotype in a given disease context. Cell-penetrating peptides can be synthetically constrained through various chemical modifications that stabilize a given structural fold with the potential to improve competitive binding to specific targets. Theileria-transformed leukocytes display high PKA activity, but PKAis an enzyme that plays key roles in multiple cellular processes; consequently genetic ablation of kinase activity gives rise to a myriad of confounding phenotypes. By contrast, ablation of a specific kinase-substrate interaction has the potential to give more refined information and we illustrate this here by describing how surgically ablating PKA interactions with BAD gives precise information on the type of glycolysis performed by Theileria-transformed leukocytes. In addition, we provide two other examples of how ablating specific protein-protein interactions in Theileria-infected leukocytes leads to precise phenotypes and argue that constrained penetrating peptides have great therapeutic potential to combat infectious diseases in general.

  19. Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity

    Directory of Open Access Journals (Sweden)

    Rachel S. Lee

    2011-01-01

    Full Text Available The AKT protooncogene mediates many cellular processes involved in normal development and disease states such as cancer. The three structurally similar isoforms: AKT1, AKT2, and AKT3 exhibit both functional redundancy and isoform-specific functions; however the basis for their differential signalling remains unclear. Here we show that in vitro, purified AKT3 is ∼47-fold more active than AKT1 at phosphorylating peptide and protein substrates. Despite these marked variations in specific activity between the individual isoforms, a comprehensive analysis of phosphorylation of validated AKT substrates indicated only subtle differences in signalling via individual isoforms in vivo. Therefore, we hypothesise, at least in this model system, that relative tissue/cellular abundance, rather than specific activity, plays the dominant role in determining AKT substrate specificity in situ.

  20. Peptide and Peptide-Dependent Motions in MHC Proteins: Immunological Implications and Biophysical Underpinnings

    Directory of Open Access Journals (Sweden)

    Cory M. Ayres

    2017-08-01

    Full Text Available Structural biology of peptides presented by class I and class II MHC proteins has transformed immunology, impacting our understanding of fundamental immune mechanisms and allowing researchers to rationalize immunogenicity and design novel vaccines. However, proteins are not static structures as often inferred from crystallographic structures. Their components move and breathe individually and collectively over a range of timescales. Peptides bound within MHC peptide-binding grooves are no exception and their motions have been shown to impact recognition by T cell and other receptors in ways that influence function. Furthermore, peptides tune the motions of MHC proteins themselves, which impacts recognition of peptide/MHC complexes by other proteins. Here, we review the motional properties of peptides in MHC binding grooves and discuss how peptide properties can influence MHC motions. We briefly review theoretical concepts about protein motion and highlight key data that illustrate immunological consequences. We focus primarily on class I systems due to greater availability of data, but segue into class II systems as the concepts and consequences overlap. We suggest that characterization of the dynamic “energy landscapes” of peptide/MHC complexes and the resulting functional consequences is one of the next frontiers in structural immunology.

  1. Antioxidant, ACE-Inhibitory and antibacterial activities of Kluyveromyces marxianus protein hydrolysates and their peptide fractions

    Directory of Open Access Journals (Sweden)

    Mahta Mirzaeia

    2016-07-01

    Full Text Available Background: There has been evidence that proteins are potentially excellent source of antioxidants, antihypertensive and antimicrobial peptides, and that enzymatic hydrolysis is an effective method to release these peptides from protein molecules. The functional properties of protein hydrolysates depends on the protein substrate, the specificity of the enzymes, the conditions used during proteolysis, degree of hydrolysis, and the nature of peptides released including molecular weight, amino acid composition, and hydrophobicity. Context and purpose of this study: The biomass of Kluyveromyces marxianus was considered as a source of ACE inhibitory, antioxidant and antimicrobial peptides. Results: Autolysis and enzymatic hydrolysis were completed respectively, after 96 h and 5 h. Overall, trypsin (18.52% DH and chymotrypsin (21.59% DH treatments were successful in releasing antioxidant and ACE inhibitory peptides. Autolysate sample (39.51% DH demonstrated poor antioxidant and ACE inhibitory activity compared to trypsin and chymotrypsin hydrolysates. The chymotrypsin 3-5 kDa (301.6±22.81 μM TE/mg protein and trypsin < 3 kDa (280.16±39.16 μM TE/mg protein permeate peptide fractions showed the highest DPPH radical scavenging activity. The trypsin <3 kDa permeate peptide fraction showed the highest ABTS radical scavenging (1691.1±48.68 μM TE/mg protein and ACE inhibitory (IC50=0.03±0.001 mg/mL activities. The fraction (MW=5-10 kD obtained after autolysis treatment showed antibacterial activity against St. aureus and Lis. monocytogenes in well diffusion screening. The minimum inhibitory concentration (MIC value was 13.3 mg/mLagainst St. aureus and Lis. monocytogenes calculated by turbidimetric assay and it showed bactericidal activity against St. aureus at 21.3 mg/mL protein concentration. Conclusions: Altogether, the results of this study reveal that K. marxianus proteins contain specific peptides in their sequences which can be released by

  2. Biosynthesis of cardiac natriuretic peptides

    DEFF Research Database (Denmark)

    Goetze, Jens Peter

    2010-01-01

    Cardiac-derived peptide hormones were identified more than 25 years ago. An astonishing amount of clinical studies have established cardiac natriuretic peptides and their molecular precursors as useful markers of heart disease. In contrast to the clinical applications, the biogenesis of cardiac...... peptides has only been elucidated during the last decade. The cellular synthesis including amino acid modifications and proteolytic cleavages has proven considerably more complex than initially perceived. Consequently, the elimination phase of the peptide products in circulation is not yet well....... An inefficient post-translational prohormone maturation will also affect the biology of the cardiac natriuretic peptide system. This review aims at summarizing the myocardial synthesis of natriuretic peptides focusing on B-type natriuretic peptide, where new data has disclosed cardiac myocytes as highly...

  3. Acetone-Linked Peptides: A Convergent Approach for Peptide Macrocyclization and Labeling.

    Science.gov (United States)

    Assem, Naila; Ferreira, David J; Wolan, Dennis W; Dawson, Philip E

    2015-07-20

    Macrocyclization is a broadly applied approach for overcoming the intrinsically disordered nature of linear peptides. Herein, it is shown that dichloroacetone (DCA) enhances helical secondary structures when introduced between peptide nucleophiles, such as thiols, to yield an acetone-linked bridge (ACE). Aside from stabilizing helical structures, the ketone moiety embedded in the linker can be modified with diverse molecular tags by oxime ligation. Insights into the structure of the tether were obtained through co-crystallization of a constrained S-peptide in complex with RNAse S. The scope of the acetone-linked peptides was further explored through the generation of N-terminus to side chain macrocycles and a new approach for generating fused macrocycles (bicycles). Together, these studies suggest that acetone linking is generally applicable to peptide macrocycles with a specific utility in the synthesis of stabilized helices that incorporate functional tags. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Preparation of peptide thioesters through fmoc-based solid-phase peptide synthesis by using amino thioesters

    DEFF Research Database (Denmark)

    Stuhr-Hansen, N.; Wilbek, T.S.; Strømgaard, K.

    2013-01-01

    protected peptide thioester, which was globally deprotected to afford the desired unprotected peptide thioester. The method is compatible with labile groups such as phosphoryl and glycosyl moieties. The synthesis of peptide alkyl thioesters by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis...

  5. Pathways to Structure-Property Relationships of Peptide-Materials Interfaces: Challenges in Predicting Molecular Structures.

    Science.gov (United States)

    Walsh, Tiffany R

    2017-07-18

    challenges in their successful application to model the biotic-abiotic interface, related to several factors. For instance, simulations require a plausible description of the chemistry and the physics of the interface, which comprises two very different states of matter, in the presence of liquid water. Also, it is essential that the conformational ensemble be comprehensively characterized under these conditions; this is especially challenging because intrinsically disordered peptides do not typically admit one single structure or set of structures. Moreover, a plausible structural model of the substrate is required, which may require a high level of detail, even for single-element materials such as Au surfaces or graphene. Developing and applying strategies to make credible predictions of the conformational ensemble of adsorbed peptides and using these to construct structure-property relationships of these interfaces have been the goals of our efforts. We have made substantial progress in developing interatomic potentials for these interfaces and adapting advanced conformational sampling approaches for these purposes. This Account summarizes our progress in the development and deployment of interfacial force fields and molecular simulation techniques for the purpose of elucidating these insights at biomolecule-materials interfaces, using examples from our laboratories ranging from noble-metal interfaces to graphitic substrates (including carbon nanotubes and graphene) and oxide materials (such as titania). In addition to the well-established application areas of plasmonic materials, biosensing, and the production of medical implant materials, we outline new directions for this field that have the potential to bring new advances in areas such as energy materials and regenerative medicine.

  6. Production and characterization of peptide antibodies

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Hansen, Paul Robert; Houen, Gunnar

    2012-01-01

    Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies...... are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors......, including solid-phase peptide-carrier conjugation and peptide-carrier conjugation in solution. Upon immunization, adjuvants such as Al(OH)(3) are added together with the immunogenic peptide-carrier conjugate, which usually leads to high-titred antisera. Following immunization and peptide antibody...

  7. Effects of Trimetaphosphate on Abiotic Formation and Hydrolysis of Peptides

    Directory of Open Access Journals (Sweden)

    Izabela K. Sibilska

    2017-11-01

    Full Text Available The primordial Earth probably had most of the factors needed for the emergence and development of life. It is believed that it had not only water, but also simple inorganic and organic materials. While studies since the 1950s on the origins of organic matter have established key roles for amino acids, conditions that would have promoted their condensation to make polymers, such as peptides or proteins, have yet to be fully defined. The condensation of amino acids in a water-rich environment is not thermodynamically favored. Therefore, the efficient formation of peptides requires the presence of a catalyst or the activation of a substrate. In living cells, the biosynthesis of proteins is assisted by enzymes and requires adenosine triphosphate (ATP, a relatively complex organic polyphosphate, which serves as an energy source. Outside the living organism, simpler inorganic polyphosphates can form active aminoacyl–phosphate anhydrides, which suggests the broader potential of phosphorus for enabling the polymerization of amino acids. However, this has yet to be demonstrated. To address this gap, aqueous solutions containing a simple dipeptide, diglycine, and a simple polyphosphate, trimetaphosphate, were dried, and reaction products were analyzed by high performance liquid chromatography and mass spectrometry (HPLC-MS. Different reaction environments, which were defined by the initial solution composition, pH, temperature, and incubation time, were found to affect the distribution and yield of products. Our results collectively provide strong evidence for reactions that both condense and hydrolyze peptides. It is noteworthy that the co-occurrence of reactions that form and cleave peptides are a central feature of Kauffman’s theory for the emergence of autocatalytic sets, which is a key step in the chemical origins of life.

  8. Mycoplasma hyopneumoniae in vitro peptidase activities: identification and cleavage of kallikrein-kinin system-like substrates.

    Science.gov (United States)

    Moitinho-Silva, Lucas; Kondo, Marcia Y; Oliveira, Lilian C G; Okamoto, Debora N; Paes, Jéssica A; Machado, Mauricio F M; Veronez, Camila L; Motta, Guacyara; Andrade, Sheila S; Juliano, Maria A; Ferreira, Henrique B; Juliano, Luiz; Gouvea, Iuri E

    2013-05-03

    Bacterial proteases are important for metabolic processes and pathogenesis in host organisms. The bacterial swine pathogen Mycoplasma hyopneumoniae has 15 putative protease-encoding genes annotated, but none of them have been functionally characterized. To identify and characterize peptidases that could be relevant for infection of swine hosts, we investigated the peptidase activity present in the pathogenic 7448 strain of M. hyopneumoniae. Combinatorial libraries of fluorescence resonance energy transfer peptides, specific inhibitors and pH profiling were used to screen and characterize endopeptidase, aminopeptidase and carboxypeptidase activities in cell lysates. One metalloendopeptidase, one serine endopeptidase, and one aminopeptidase were detected. The detected metalloendopeptidase activity, prominent at neutral and basic pH ranges, was due to a thimet oligopeptidase family member (M3 family), likely an oligoendopeptidase F (PepF), which cleaved the peptide Abz-GFSPFRQ-EDDnp at the F-S bond. A chymotrypsin-like serine endopeptidase activity, possibly a subtilisin-like serine protease, was prominent at higher pH levels, and was characterized by its preference for a Phe residue at the P1 position of the substrate. The aminopeptidase P (APP) activity showed a similar profile to that of human membrane-bound APP. Genes coding for these three peptidases were identified and their transcription was confirmed in the 7448 strain. Furthermore, M. hyopneumoniae cell lysate peptidases showed effects on kallikrein-kinin system-like substrates, such as bradykinin-derived substrates and human high molecular weight kininogen. The M. hyopneumoniae peptidase activities, here characterized for the first time, may be important for bacterial survival strategies and thus represent possible targets for drug development against M. hyopneumoniae swine infections. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Peptide ligands for targeting the extracellular domain of EGFR: Comparison between linear and cyclic peptides.

    Science.gov (United States)

    Williams, Tyrslai M; Sable, Rushikesh; Singh, Sitanshu; Vicente, Maria Graca H; Jois, Seetharama D

    2018-02-01

    Colorectal cancer (CRC) is the third most common solid internal malignancy among cancers. Early detection of cancer is key to increasing the survival rate of colorectal cancer patients. Overexpression of the EGFR protein is associated with CRC. We have designed a series of peptides that are highly specific for the extracellular domain of EGFR, based on our earlier studies on linear peptides. The previously reported linear peptide LARLLT, known to bind to EGFR, was modified with the goals of increasing its stability and its specificity toward EGFR. Peptide modifications, including D-amino acid substitution, cyclization, and chain reversal, were investigated. In addition, to facilitate labeling of the peptide with a fluorescent dye, an additional lysine residue was introduced onto the linear (KLARLLT) and cyclic peptides cyclo(KLARLLT) (Cyclo.L1). The lysine residue was also converted into an azide group in both a linear and reversed cyclic peptide sequences cyclo(K(N3)larllt) (Cyclo.L1.1) to allow for subsequent "click" conjugation. The cyclic peptides showed enhanced binding to EGFR by SPR. NMR and molecular modeling studies suggest that the peptides acquire a β-turn structure in solution. In vitro stability studies in human serum show that the cyclic peptide is more stable than the linear peptide. © 2017 John Wiley & Sons A/S.

  10. Peptide Nucleic Acids (PNA)

    DEFF Research Database (Denmark)

    2002-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  11. Peptide Nucleic Acid Synthons

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  12. Oxidative damage to collagen and related substrates by metal ion/hydrogen peroxide systems

    DEFF Research Database (Denmark)

    Hawkins, C L; Davies, Michael Jonathan

    1997-01-01

    . In this study electron paramagnetic resonance spectroscopy with spin trapping has been used to identify radicals formed on collagen and related materials by metal ion-H2O2 mixtures. Attack of the hydroxyl radical, from a Fe(II)-H2O2 redox couple, on collagen peptides gave signals from both side chain (.CHR...... are similar to those from the alpha-carbon site of peptides and the side-chain of lysine. Enzymatic digestion of the large, protein-derived, species releases similar low-molecular-weight adducts. The metal ion employed has a dramatic effect on the species observed. With Cu(I)-H2O2 or Cu(II)-H2O2 instead of Fe(II)-H......2O2, evidence has been obtained for: i) altered sites of attack and fragmentation, ii) C-terminal decarboxylation, and iii) hydrogen abstraction at N-terminal alpha-carbon sites. This altered behaviour is believed to be due to the binding of copper ions to some substrates and hence site...

  13. Collagenolytic Matrix Metalloproteinase Activities toward Peptomeric Triple-Helical Substrates.

    Science.gov (United States)

    Stawikowski, Maciej J; Stawikowska, Roma; Fields, Gregg B

    2015-05-19

    Although collagenolytic matrix metalloproteinases (MMPs) possess common domain organizations, there are subtle differences in their processing of collagenous triple-helical substrates. In this study, we have incorporated peptoid residues into collagen model triple-helical peptides and examined MMP activities toward these peptomeric chimeras. Several different peptoid residues were incorporated into triple-helical substrates at subsites P3, P1, P1', and P10' individually or in combination, and the effects of the peptoid residues were evaluated on the activities of full-length MMP-1, MMP-8, MMP-13, and MMP-14/MT1-MMP. Most peptomers showed little discrimination between MMPs. However, a peptomer containing N-methyl Gly (sarcosine) in the P1' subsite and N-isobutyl Gly (NLeu) in the P10' subsite was hydrolyzed efficiently only by MMP-13 [nomenclature relative to the α1(I)772-786 sequence]. Cleavage site analysis showed hydrolysis at the Gly-Gln bond, indicating a shifted binding of the triple helix compared to the parent sequence. Favorable hydrolysis by MMP-13 was not due to sequence specificity or instability of the substrate triple helix but rather was based on the specific interactions of the P7' peptoid residue with the MMP-13 hemopexin-like domain. A fluorescence resonance energy transfer triple-helical peptomer was constructed and found to be readily processed by MMP-13, not cleaved by MMP-1 and MMP-8, and weakly hydrolyzed by MT1-MMP. The influence of the triple-helical structure containing peptoid residues on the interaction between MMP subsites and individual substrate residues may provide additional information about the mechanism of collagenolysis, the understanding of collagen specificity, and the design of selective MMP probes.

  14. Solid-phase peptide synthesis

    DEFF Research Database (Denmark)

    Jensen, Knud Jørgen

    2013-01-01

    This chapter provides an introduction to and overview of peptide chemistry with a focus on solid-phase peptide synthesis. The background, the most common reagents, and some mechanisms are presented. This chapter also points to the different chapters and puts them into perspective.......This chapter provides an introduction to and overview of peptide chemistry with a focus on solid-phase peptide synthesis. The background, the most common reagents, and some mechanisms are presented. This chapter also points to the different chapters and puts them into perspective....

  15. Taylor Dispersion Analysis as a promising tool for assessment of peptide-peptide interactions

    DEFF Research Database (Denmark)

    Høgstedt, Ulrich B; Schwach, Grégoire; van de Weert, Marco

    2016-01-01

    solutions increased with concentration. Our results indicate that a viscosity difference between run buffer and sample in Taylor Dispersion Analysis may result in overestimation of the measured diffusion coefficient. Thus, Taylor Dispersion Analysis provides a practical, but as yet primarily qualitative......Protein-protein and peptide-peptide (self-)interactions are of key importance in understanding the physiochemical behavior of proteins and peptides in solution. However, due to the small size of peptide molecules, characterization of these interactions is more challenging than for proteins...

  16. Aggregation and toxicity of amyloid-beta peptide in relation to peptide sequence variation

    OpenAIRE

    Vandersteen, A.

    2012-01-01

    Generally, aggregation of the amyloid-ß peptide is considered the cause of neuronal death in Alzheimer disease. The heterogenous Aß peptide occurs in various lengths in vivo: Aß40 and Aß42 are the predominant forms while both shorter and longer peptides exist. Aß40 and shorter isoforms are less aggregation-prone and hence considered less dangerous than Aß42 and longer isoforms, which are more aggregation-prone. Up to now research mainly focussed on the predominant Aß peptides and their indivi...

  17. Breaking the Dogma of Aldolase Specificity: Simple Aliphatic Ketones and Aldehydes are Nucleophiles for Fructose-6-phosphate Aldolase.

    Science.gov (United States)

    Roldán, Raquel; Sanchez-Moreno, Israel; Scheidt, Thomas; Hélaine, Virgil; Lemaire, Marielle; Parella, Teodor; Clapés, Pere; Fessner, Wolf-Dieter; Guérard-Hélaine, Christine

    2017-04-11

    d-Fructose-6-phosphate aldolase (FSA) was probed for extended nucleophile promiscuity by using a series of fluorogenic substrates to reveal retro-aldol activity. Four nucleophiles ethanal, propanone, butanone, and cyclopentanone were subsequently confirmed to be non-natural substrates in the synthesis direction using the wild-type enzyme and its D6H variant. This exceptional widening of the nucleophile substrate scope offers a rapid entry, in good yields and high stereoselectivity, to less oxygenated alkyl ketones and aldehydes, which was hitherto impossible. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. A cocoa peptide protects Caenorhabditis elegans from oxidative stress and β-amyloid peptide toxicity.

    Directory of Open Access Journals (Sweden)

    Patricia Martorell

    Full Text Available BACKGROUND: Cocoa and cocoa-based products contain different compounds with beneficial properties for human health. Polyphenols are the most frequently studied, and display antioxidant properties. Moreover, protein content is a very interesting source of antioxidant bioactive peptides, which can be used therapeutically for the prevention of age-related diseases. METHODOLOGY/PRINCIPAL FINDINGS: A bioactive peptide, 13L (DNYDNSAGKWWVT, was obtained from a hydrolyzed cocoa by-product by chromatography. The in vitro inhibition of prolyl endopeptidase (PEP was used as screening method to select the suitable fraction for peptide identification. Functional analysis of 13L peptide was achieved using the transgenic Caenorhabditis elegans strain CL4176 expressing the human Aβ₁₋₄₂ peptide as a pre-clinical in vivo model for Alzheimer's disease. Among the peptides isolated, peptide 13L (1 µg/mL showed the highest antioxidant activity (P≤0.001 in the wild-type strain (N2. Furthermore, 13L produced a significant delay in body paralysis in strain CL4176, especially in the 24-47 h period after Aβ₁₋₄₂ peptide induction (P≤0.0001. This observation is in accordance with the reduction of Aβ deposits in CL4176 by western blot. Finally, transcriptomic analysis in wild-type nematodes treated with 13L revealed modulation of the proteosomal and synaptic functions as the main metabolic targets of the peptide. CONCLUSIONS/SIGNIFICANCE: These findings suggest that the cocoa 13L peptide has antioxidant activity and may reduce Aβ deposition in a C. elegans model of Alzheimer's disease; and therefore has a putative therapeutic potential for prevention of age-related diseases. Further studies in murine models and humans will be essential to analyze the effectiveness of the 13L peptide in higher animals.

  19. Radiolabelled peptides for oncological diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Laverman, Peter; Boerman, Otto C.; Oyen, Wim J.G. [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, Nijmegen (Netherlands); Sosabowski, Jane K. [Queen Mary University of London, Centre for Molecular Oncology, Barts Cancer Institute, London (United Kingdom)

    2012-02-15

    Radiolabelled receptor-binding peptides targeting receptors (over)expressed on tumour cells are widely under investigation for tumour diagnosis and therapy. The concept of using radiolabelled receptor-binding peptides to target receptor-expressing tissues in vivo has stimulated a large body of research in nuclear medicine. The {sup 111}In-labelled somatostatin analogue octreotide (OctreoScan trademark) is the most successful radiopeptide for tumour imaging, and was the first to be approved for diagnostic use. Based on the success of these studies, other receptor-targeting peptides such as cholecystokinin/gastrin analogues, glucagon-like peptide-1, bombesin (BN), chemokine receptor CXCR4 targeting peptides, and RGD peptides are currently under development or undergoing clinical trials. In this review, we discuss some of these peptides and their analogues, with regard to their potential for radionuclide imaging of tumours. (orig.)

  20. Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Deacon, Ashley M.; Wilson, Ian A.

    2015-09-15

    ABSTRACT

    Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.

    IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural

  1. Peptide Vaccines for Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Rory C. F. De Brito

    2018-05-01

    Full Text Available Due to an increase in the incidence of leishmaniases worldwide, the development of new strategies such as prophylactic vaccines to prevent infection and decrease the disease have become a high priority. Classic vaccines against leishmaniases were based on live or attenuated parasites or their subunits. Nevertheless, the use of whole parasite or their subunits for vaccine production has numerous disadvantages. Therefore, the use of Leishmania peptides to design more specific vaccines against leishmaniases seems promising. Moreover, peptides have several benefits in comparison with other kinds of antigens, for instance, good stability, absence of potentially damaging materials, antigen low complexity, and low-cost to scale up. By contrast, peptides are poor immunogenic alone, and they need to be delivered correctly. In this context, several approaches described in this review are useful to solve these drawbacks. Approaches, such as, peptides in combination with potent adjuvants, cellular vaccinations, adenovirus, polyepitopes, or DNA vaccines have been used to develop peptide-based vaccines. Recent advancements in peptide vaccine design, chimeric, or polypeptide vaccines and nanovaccines based on particles attached or formulated with antigenic components or peptides have been increasingly employed to drive a specific immune response. In this review, we briefly summarize the old, current, and future stands on peptide-based vaccines, describing the disadvantages and benefits associated with them. We also propose possible approaches to overcome the related weaknesses of synthetic vaccines and suggest future guidelines for their development.

  2. Peptide Vaccines for Leishmaniasis.

    Science.gov (United States)

    De Brito, Rory C F; Cardoso, Jamille M De O; Reis, Levi E S; Vieira, Joao F; Mathias, Fernando A S; Roatt, Bruno M; Aguiar-Soares, Rodrigo Dian D O; Ruiz, Jeronimo C; Resende, Daniela de M; Reis, Alexandre B

    2018-01-01

    Due to an increase in the incidence of leishmaniases worldwide, the development of new strategies such as prophylactic vaccines to prevent infection and decrease the disease have become a high priority. Classic vaccines against leishmaniases were based on live or attenuated parasites or their subunits. Nevertheless, the use of whole parasite or their subunits for vaccine production has numerous disadvantages. Therefore, the use of Leishmania peptides to design more specific vaccines against leishmaniases seems promising. Moreover, peptides have several benefits in comparison with other kinds of antigens, for instance, good stability, absence of potentially damaging materials, antigen low complexity, and low-cost to scale up. By contrast, peptides are poor immunogenic alone, and they need to be delivered correctly. In this context, several approaches described in this review are useful to solve these drawbacks. Approaches, such as, peptides in combination with potent adjuvants, cellular vaccinations, adenovirus, polyepitopes, or DNA vaccines have been used to develop peptide-based vaccines. Recent advancements in peptide vaccine design, chimeric, or polypeptide vaccines and nanovaccines based on particles attached or formulated with antigenic components or peptides have been increasingly employed to drive a specific immune response. In this review, we briefly summarize the old, current, and future stands on peptide-based vaccines, describing the disadvantages and benefits associated with them. We also propose possible approaches to overcome the related weaknesses of synthetic vaccines and suggest future guidelines for their development.

  3. Fasting plasma C-peptide, glucagon stimulated plasma C-peptide, and urinary C-peptide in relation to clinical type of diabetes

    DEFF Research Database (Denmark)

    Gjessing, H J; Matzen, L E; Faber, O K

    1989-01-01

    with a fasting plasma C-peptide value less than 0.20 nmol/l, a glucagon stimulated plasma C-peptide value less than 0.32 nmol/l, and a urinary C-peptide value less than 3.1 nmol/l, or less than 0.54 nmol/mmol creatinine/24 h, or less than 5.4 nmol/24 h mainly were Type 1 diabetic patients; while patients with C...

  4. Fbs1 protects the malfolded glycoproteins from the attack of peptide:N-glycanase

    International Nuclear Information System (INIS)

    Yamaguchi, Yoshiki; Hirao, Takeshi; Sakata, Eri; Kamiya, Yukiko; Kurimoto, Eiji; Yoshida, Yukiko; Suzuki, Tadashi; Tanaka, Keiji; Kato, Koichi

    2007-01-01

    Fbs1 is a cytosolic lectin putatively operating as a chaperone as well as a substrate-recognition subunit of the SCF Fbs1 ubiquitin ligase complex. To provide structural and functional basis of preferential binding of Fbs1 to unfolded glycoproteins, we herein characterize the interaction of Fbs1 with a heptapeptide carrying Man 3 GlcNAc 2 by nuclear magnetic resonance (NMR) spectroscopy and other biochemical methods. Inspection of the NMR data obtained by use of the isotopically labeled glycopeptide indicated that Fbs1 interacts with sugar-peptide junctions, which are shielded in native glycoprotein, in many cases, but become accessible to Fbs1 in unfolded glycoproteins. Furthermore, Fbs1 was shown to inhibit deglycosylation of denatured ribonuclease B by a cytosolic peptide:N-glycanase (PNGase). On the basis of these data, we suggest that Fbs1 captures malfolded glycoproteins, protecting them from the attack of PNGase, during the chaperoning or ubiquitinating operation in the cytosol

  5. Peptide Functionalized Gold Nanorods for the Sensitive Detection of a Cardiac Biomarker Using Plasmonic Paper Devices (Postprint)

    Science.gov (United States)

    2015-11-10

    Albumin to saturate the non-specific binding sites on the paper substrate prior to troponin exposure. For testing the biosensor, troponin of various...AFRL-RX-WP-JA-2016-0191 PEPTIDE FUNCTIONALIZED GOLD NANORODS FOR THE SENSITIVE DETECTION OF A CARDIAC BIOMARKER USING PLASMONIC PAPER ...SENSITIVE DETECTION OF A CARDIAC BIOMARKER USING PLASMONIC PAPER DEVICES (POSTPRINT) 5a. CONTRACT NUMBER FA8650-15-D-5405-0001 5b. GRANT NUMBER 5c

  6. PH dependent adhesive peptides

    Science.gov (United States)

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  7. Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1.

    Science.gov (United States)

    Feng, Tao; Nyffenegger, Christian; Højrup, Peter; Vidal-Melgosa, Silvia; Yan, Kok-Phen; Fangel, Jonatan Ulrik; Meyer, Anne S; Kirpekar, Finn; Willats, William G; Mikkelsen, Jørn D

    2014-12-01

    Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage patterns. Prt1 is an autoprocessing protease with an N-terminal signal pre-peptide and a pro-peptide which has to be removed in order to activate the protease. The sequential cleavage of the N-terminus was confirmed by mass spectrometry (MS) fingerprinting and N-terminus analysis. The optimal reaction conditions for the activity of Prt1 on azocasein were at pH 6.0, 50 °C. At these reaction conditions, K M was 1.81 mg/mL and k cat was 1.82 × 10(7) U M(-1). The enzyme was relatively stable at 50 °C with a half-life of 20 min. Ethylenediaminetetraacetic acid (EDTA) treatment abolished activity; Zn(2+) addition caused regain of the activity, but Zn(2+)addition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant extensin. Analysis of the cleavage pattern of different substrates after treatment with Prt1 indicated that the protease had a substrate cleavage preference for proline in substrate residue position P1 followed by a hydrophobic residue in residue position P1' at the cleavage point. The activity of Prt1 against plant cell wall structural proteins suggests that this enzyme might become an important new addition to the toolbox of cell-wall-degrading enzymes for biomass processing.

  8. Deciphering the Arginine-binding preferences at the substrate-binding groove of Ser/Thr kinases by computational surface mapping.

    Directory of Open Access Journals (Sweden)

    Avraham Ben-Shimon

    2011-11-01

    Full Text Available Protein kinases are key signaling enzymes that catalyze the transfer of γ-phosphate from an ATP molecule to a phospho-accepting residue in the substrate. Unraveling the molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor is important for understanding kinase specificities toward their substrates and for designing substrate-like peptidic inhibitors. We applied ANCHORSmap, a new fragment-based computational approach for mapping amino acid side chains on protein surfaces, to predict and characterize the preference of kinases toward Arginine binding. We focus on positions P-2 and P-5, commonly occupied by Arginine (Arg in substrates of basophilic Ser/Thr kinases. The method accurately identified all the P-2/P-5 Arg binding sites previously determined by X-ray crystallography and produced Arg preferences that corresponded to those experimentally found by peptide arrays. The predicted Arg-binding positions and their associated pockets were analyzed in terms of shape, physicochemical properties, amino acid composition, and in-silico mutagenesis, providing structural rationalization for previously unexplained trends in kinase preferences toward Arg moieties. This methodology sheds light on several kinases that were described in the literature as having non-trivial preferences for Arg, and provides some surprising departures from the prevailing views regarding residues that determine kinase specificity toward Arg. In particular, we found that the preference for a P-5 Arg is not necessarily governed by the 170/230 acidic pair, as was previously assumed, but by several different pairs of acidic residues, selected from positions 133, 169, and 230 (PKA numbering. The acidic residue at position 230 serves as a pivotal element in recognizing Arg from both the P-2 and P-5 positions.

  9. Enzymatic characterization of peptidic materials isolated from aqueous solutions of ammonium cyanide (pH 9) and hydrocyanic acid (pH 6) exposed to ionizing radiation.

    Science.gov (United States)

    Niketic, V; Draganić, Z; Nesković, S; Draganić, I

    1982-01-01

    The enzymatic digestion of some radiolytically produced peptidic materials was examined. The substrates were compounds isolated from 0.1 molar solutions of NH4CN (pH 9) and HCN (pH 6), after their exposure to gamma rays from a 60Co source (15-20 Mrad doses). Commercial proteolytic enzymes pronase and aminopeptidase M were used. The examined materials were of composite nature and proteolytic action was systematically observed after their subsequent purification. In some fractions the effect was found to be positive with up to 30% of peptide bonds cleaved with respect to the amino acid content. These findings support our previous conclusions on the free radical induced formation of peptidic backbones without the intervention of amino acids. Some side effects were also noted which might be of interest in observations on enzymatic cleavage of other composite peptidic materials of abiotic origin.

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

    Science.gov (United States)

    Duffy, Fergal J; O'Donovan, Darragh; Devocelle, Marc; Moran, Niamh; O'Connell, David J; Shields, Denis C

    2015-03-23

    Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.

  11. The non-peptidic part determines the internalization mechanism and intracellular trafficking of peptide amphiphiles.

    Directory of Open Access Journals (Sweden)

    Dimitris Missirlis

    Full Text Available BACKGROUND: Peptide amphiphiles (PAs are a class of amphiphilic molecules able to self-assemble into nanomaterials that have shown efficient in vivo targeted delivery. Understanding the interactions of PAs with cells and the mechanisms of their internalization and intracellular trafficking is critical in their further development for therapeutic delivery applications. METHODOLOGY/PRINCIPAL FINDINGS: PAs of a novel, cell- and tissue-penetrating peptide were synthesized possessing two different lipophilic tail architectures and their interactions with prostate cancer cells were studied in vitro. Cell uptake of peptides was greatly enhanced post-modification. Internalization occurred via lipid-raft mediated endocytosis and was common for the two analogs studied. On the contrary, we identified the non-peptidic part as the determining factor of differences between intracellular trafficking and retention of PAs. PAs composed of di-stearyl lipid tails linked through poly(ethylene glycol to the peptide exhibited higher exocytosis rates and employed different recycling pathways compared to ones consisting of di-palmitic-coupled peptides. As a result, cell association of the former PAs decreased with time. CONCLUSIONS/SIGNIFICANCE: Control over peptide intracellular localization and retention is possible by appropriate modification with synthetic hydrophobic tails. We propose this as a strategy to design improved peptide-based delivery systems.

  12. Synthesis and antioxidant activity of peptide-based ebselen analogues.

    Science.gov (United States)

    Satheeshkumar, Kandhan; Mugesh, Govindasamy

    2011-04-18

    A series of di- and tripeptide-based ebselen analogues has been synthesized. The compounds were characterized by (1)H, (13)C, and (77)Se NMR spectroscopy and mass spectral techniques. The glutathione peroxidase (GPx)-like antioxidant activity has been studied by using H(2)O(2) , tert-butyl hydroperoxide (tBuOOH), and cumene hydroperoxide (Cum-OOH) as substrates, and glutathione (GSH) as a cosubstrate. Although all the peptide-based compounds have a selenazole ring similar to that of ebselen, the GPx activity of these compounds highly depends on the nature of the peptide moiety attached to the nitrogen atom of the selenazole ring. It was observed that the introduction of a phenylalanine (Phe) amino acid residue in the N-terminal reduces the activity in all three peroxide systems. On the other hand, the introduction of aliphatic amino acid residues such as valine (Val) significantly enhances the GPx activity of the ebselen analogues. The difference in the catalytic activity of dipeptide-based ebselen derivatives can be ascribed mainly to the change in the reactivity of these compounds toward GSH and peroxide. Although the presence of the Val-Ala-CO(2) Me moiety facilitates the formation of a catalytically active selenol species, the reaction of ebselen analogues that has a Phe-Ile-CO(2) Me residue with GSH does not generate the corresponding selenol. To understand the antioxidant activity of the peptide-based ebselen analogues in the absence of GSH, these compounds were studied for their ability to inhibit peroxynitrite (PN)-mediated nitration of bovine serum albumin (BSA) and oxidation of dihydrorhodamine 123. In contrast to the GPx activity, the PN-scavenging activity of the Phe-based peptide analogues was found to be comparable to that of the Val-based compounds. However, the introduction of an additional Phe residue to the ebselen analogue that had a Val-Ala dipeptide significantly reduced the potency of the parent compound in PN-mediated nitration. Copyright

  13. Sensitive electrospray mass spectrometry analysis of one-bead-one-compound peptide libraries labeled by quaternary ammonium salts.

    Science.gov (United States)

    Bąchor, Remigiusz; Cydzik, Marzena; Rudowska, Magdalena; Kluczyk, Alicja; Stefanowicz, Piotr; Szewczuk, Zbigniew

    2012-08-01

    A rapid and straightforward method for high-throughput analysis of single resin beads from one-bead-one-compound combinatorial libraries with high resolution electrospray ionization tandem mass spectrometry (HR ESI-MS/MS) is presented. The application of an efficient method of peptide derivatization by quaternary ammonium salts (QAS) formation increases ionization efficiency and reduces the detection limit, allowing analysis of trace amounts of compounds by ESI-MS. Peptides, synthesized on solid support, contain a new cleavable linker composed of a Peg spacer (9-aza-3,6,12,15-tetraoxa-10-on-heptadecanoic acid), lysine with ɛ-amino group marked by the N,N,N-triethylglycine salt, and methionine, which makes possible the selective cleavage by cyanogen bromide. Even a small portion of peptides derivatized by QAS cleaved from a single resin bead is sufficient for sequencing by HR ESI-MS/MS experiments. The developed strategy was applied to a small training library of α chymotrypsin substrates. The obtained results confirm the applicability of the proposed method in combinatorial chemistry.

  14. Expression of the cationic antimicrobial peptide lactoferricin fused with the anionic peptide in Escherichia coli.

    Science.gov (United States)

    Kim, Ha-Kun; Chun, Dae-Sik; Kim, Joon-Sik; Yun, Cheol-Ho; Lee, Ju-Hoon; Hong, Soon-Kwang; Kang, Dae-Kyung

    2006-09-01

    Direct expression of lactoferricin, an antimicrobial peptide, is lethal to Escherichia coli. For the efficient production of lactoferricin in E. coli, we developed an expression system in which the gene for the lysine- and arginine-rich cationic lactoferricin was fused to an anionic peptide gene to neutralize the basic property of lactoferricin, and successfully overexpressed the concatemeric fusion gene in E. coli. The lactoferricin gene was linked to a modified magainin intervening sequence gene by a recombinational polymerase chain reaction, thus producing an acidic peptide-lactoferricin fusion gene. The monomeric acidic peptide-lactoferricin fusion gene was multimerized and expressed in E. coli BL21(DE3) upon induction with isopropyl-beta-D-thiogalactopyranoside. The expression levels of the fusion peptide reached the maximum at the tetramer, while further increases in the copy number of the fusion gene substantially reduced the peptide expression level. The fusion peptides were isolated and cleaved to generate the separate lactoferricin and acidic peptide. About 60 mg of pure recombinant lactoferricin was obtained from 1 L of E. coli culture. The purified recombinant lactoferricin was found to have a molecular weight similar to that of chemically synthesized lactoferricin. The recombinant lactoferricin showed antimicrobial activity and disrupted bacterial membrane permeability, as the native lactoferricin peptide does.

  15. Synthesis of peptide .alpha.-thioesters

    Science.gov (United States)

    Camarero, Julio A [Livermore, CA; Mitchell, Alexander R [Livermore, CA; De Yoreo, James J [Clayton, CA

    2008-08-19

    Disclosed herein is a new method for the solid phase peptide synthesis (SPPS) of C-terminal peptide .alpha. thioesters using Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazine linker, which is totally stable to conditions required for Fmoc-SPPS. When the peptide synthesis has been completed, activation of the linker is achieved by mild oxidation. The oxidation step converts the acyl-hydrazine group into a highly reactive acyl-diazene intermediate which reacts with an .alpha.-amino acid alkylthioester (H-AA-SR) to yield the corresponding peptide .alpha.-thioester in good yield. A variety of peptide thioesters, cyclic peptides and a fully functional Src homology 3 (SH3) protein domain have been successfully prepared.

  16. Legume-rhizobium symbiotic promiscuity and effectiveness do not affect plant invasiveness.

    Science.gov (United States)

    Keet, Jan-Hendrik; Ellis, Allan G; Hui, Cang; Le Roux, Johannes J

    2017-06-01

    The ability to fix atmospheric nitrogen is thought to play an important role in the invasion success of legumes. Interactions between legumes and nitrogen-fixing bacteria (rhizobia) span a continuum of specialization, and promiscuous legumes are thought to have higher chances of forming effective symbioses in novel ranges. Using Australian Acacia species in South Africa, it was hypothesized that widespread and highly invasive species will be more generalist in their rhizobial symbiotic requirements and more effective in fixing atmospheric nitrogen compared with localized and less invasive species. To test these hypotheses, eight localized and 11 widespread acacias were examined using next-generation sequencing data for the nodulation gene, nodC , to compare the identity, species richness, diversity and compositional similarity of rhizobia associated with these acacias. Stable isotope analysis was also used to determine levels of nitrogen obtained from the atmosphere via symbiotic nitrogen fixation. No differences were found in richness, diversity and community composition between localized and widespread acacias. Similarly, widespread and localized acacias did not differ in their ability to fix atmospheric nitrogen. However, for some species by site comparisons, significant differences in δ15N isotopic signatures were found, indicating differential symbiotic effectiveness between these species at specific localities. Overall, the results support recent findings that root nodule rhizobial diversity and community composition do not differ between acacias that vary in their invasiveness. Differential invasiveness of acacias in South Africa is probably linked to attributes such as differences in propagule pressure, reasons for (e.g. forestry vs. ornamental) and extent of, plantings in the country. © The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  17. Peptides in melanoma therapy.

    Science.gov (United States)

    Mocellin, Simone

    2012-01-01

    Peptides derived from tumor associated antigens can be utilized to elicit a therapeutically effective immune response against melanoma in experimental models. However, patient vaccination with peptides - although it is often followed by the induction of melanoma- specific T lymphocytes - is rarely associated with tumor response of clinical relevance. In this review I summarize the principles of peptide design as well as the results so far obtained in the clinical setting while treating cutaneous melanoma by means of this active immunotherapy strategy. I also discuss some immunological and methodological issues that might be helpful for the successful development of peptide-based vaccines.

  18. Computer-Aided Design of Antimicrobial Peptides

    DEFF Research Database (Denmark)

    Fjell, Christopher D.; Hancock, Robert E.W.; Jenssen, Håvard

    2010-01-01

    in antimicrobial activity. Consequently, the majority of peptides put into clinical trials have failed at some point, underlining the importance of a thorough peptide optimization. An important tool in peptide design and optimization is quantitative structure-activity relationship (QSAR) analysis, correlating...... chemical parameters with biological activities of the peptide, using statistical methods. In this review we will discuss two different in silico strategies of computer-aided antibacterial peptide design, a linear correlation model build as an extension of traditional principal component analysis (PCA......) and a non-linear artificial neural network model. Studies on structurally diverse peptides, have concluded that the PCA derived model are able to guide the antibacterial peptide design in a meaningful way, however requiring rather a high homology between the peptides in the test-set and the in silico...

  19. Ligand-regulated peptides: a general approach for modulating protein-peptide interactions with small molecules.

    Science.gov (United States)

    Binkowski, Brock F; Miller, Russell A; Belshaw, Peter J

    2005-07-01

    We engineered a novel ligand-regulated peptide (LiRP) system where the binding activity of intracellular peptides is controlled by a cell-permeable small molecule. In the absence of ligand, peptides expressed as fusions in an FKBP-peptide-FRB-GST LiRP scaffold protein are free to interact with target proteins. In the presence of the ligand rapamycin, or the nonimmunosuppressive rapamycin derivative AP23102, the scaffold protein undergoes a conformational change that prevents the interaction of the peptide with the target protein. The modular design of the scaffold enables the creation of LiRPs through rational design or selection from combinatorial peptide libraries. Using these methods, we identified LiRPs that interact with three independent targets: retinoblastoma protein, c-Src, and the AMP-activated protein kinase. The LiRP system should provide a general method to temporally and spatially regulate protein function in cells and organisms.

  20. Mycobacteria attenuate nociceptive responses by formyl peptide receptor triggered opioid peptide release from neutrophils.

    Directory of Open Access Journals (Sweden)

    Heike L Rittner

    2009-04-01

    Full Text Available In inflammation, pain is regulated by a balance of pro- and analgesic mediators. Analgesic mediators include opioid peptides which are secreted by neutrophils at the site of inflammation, leading to activation of opioid receptors on peripheral sensory neurons. In humans, local opioids and opioid peptides significantly downregulate postoperative as well as arthritic pain. In rats, inflammatory pain is induced by intraplantar injection of heat inactivated Mycobacterium butyricum, a component of complete Freund's adjuvant. We hypothesized that mycobacterially derived formyl peptide receptor (FPR and/or toll like receptor (TLR agonists could activate neutrophils, leading to opioid peptide release and inhibition of inflammatory pain. In complete Freund's adjuvant-induced inflammation, thermal and mechanical nociceptive thresholds of the paw were quantified (Hargreaves and Randall-Selitto methods, respectively. Withdrawal time to heat was decreased following systemic neutrophil depletion as well as local injection of opioid receptor antagonists or anti-opioid peptide (i.e. Met-enkephalin, beta-endorphin antibodies indicating an increase in pain. In vitro, opioid peptide release from human and rat neutrophils was measured by radioimmunoassay. Met-enkephalin release was triggered by Mycobacterium butyricum and formyl peptides but not by TLR-2 or TLR-4 agonists. Mycobacterium butyricum induced a rise in intracellular calcium as determined by FURA loading and calcium imaging. Opioid peptide release was blocked by intracellular calcium chelation as well as phosphoinositol-3-kinase inhibition. The FPR antagonists Boc-FLFLF and cyclosporine H reduced opioid peptide release in vitro and increased inflammatory pain in vivo while TLR 2/4 did not appear to be involved. In summary, mycobacteria activate FPR on neutrophils, resulting in tonic secretion of opioid peptides from neutrophils and in a decrease in inflammatory pain. Future therapeutic strategies may aim

  1. Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays.

    Science.gov (United States)

    Kanie, Kei; Kondo, Yuto; Owaki, Junki; Ikeda, Yurika; Narita, Yuji; Kato, Ryuji; Honda, Hiroyuki

    2016-11-19

    The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM) provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV), an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I), and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides.

  2. Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays

    Directory of Open Access Journals (Sweden)

    Kei Kanie

    2016-11-01

    Full Text Available The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV, an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I, and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides.

  3. [Distiller Yeasts Producing Antibacterial Peptides].

    Science.gov (United States)

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

    2015-01-01

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

  4. Bioavailability and transport of peptides and peptide drugs into the brain.

    Science.gov (United States)

    Egleton, R D; Davis, T P

    1997-01-01

    Rational drug design and the targeting of specific organs has become a reality in modern drug development, with the emergence of molecular biology and receptor chemistry as powerful tools for the pharmacologist. A greater understanding of peptide function as one of the major extracellular message systems has made neuropeptides an important target in neuropharmaceutical drug design. The major obstacle to targeting the brain with therapeutics is the presence of the blood-brain barrier (BBB), which controls the concentration and entry of solutes into the central nervous system. Peptides are generally polar in nature, do not easily cross the blood-brain barrier by diffusion, and except for a small number do not have specific transport systems. Peptides can also undergo metabolic deactivation by peptidases of the blood, brain and the endothelial cells that comprise the BBB. In this review, we discuss a number of the recent strategies which have been used to promote peptide stability and peptide entry into the brain. In addition, we approach the subject of targeting specific transport systems that can be found on the brain endothelial cells, and describe the limitations of the methodologies that are currently used to study brain entry of neuropharmaceuticals.

  5. Promiscuity and selectivity of small-molecule inhibitors across TAM receptor tyrosine kinases in pediatric leukemia.

    Science.gov (United States)

    Liu, Mao-Hua; Chen, Shi-Bing; Yu, Juan; Liu, Cheng-Jun; Zhang, Xiao-Jing

    2017-08-01

    The TAM receptor tyrosine kinase family member Mer has been recognized as an attractive therapeutic target for pediatric leukemia. Beside Mer the family contains other two kinases, namely, Tyro3 and Axl, which are highly homologues with Mer and thus most existing small-molecule inhibitors show moderate or high promiscuity across the three kinases. Here, the structural basis and energetic property of selective binding of small-molecule inhibitors to the three kinases were investigated at molecular level. It is found that the selectivity is primarily determined by the size, shape and configuration of kinase's ATP-binding site; the Mer and Axl possess a small, closed active pocket as compared to the bulky, open pocket of Tyro3. The location and conformation of active-site residues of Mer and Axl are highly consistent, suggesting that small-molecule inhibitors generally have a low Mer-over-Axl selectivity and a high Mer-over-Tyro3 selectivity. We demonstrated that the difference in ATP binding potency to the three kinases is also responsible for inhibitor selectivity. We also found that the long-range interactions and allosteric effect arising from rest of the kinase's active site can indirectly influence inhibitor binding and selectivity. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Peptide array-based screening of human mesenchymal stem cell-adhesive peptides derived from fibronectin type III domain

    International Nuclear Information System (INIS)

    Okochi, Mina; Nomura, Shigeyuki; Kaga, Chiaki; Honda, Hiroyuki

    2008-01-01

    Human mesenchymal stem cell-adhesive peptides were screened based on the amino acid sequence of fibronectin type III domain 8-11 (FN-III 8-11 ) using a peptide array synthesized by the Fmoc-chemistry. Using hexameric peptide library of FN-III 8-11 scan, we identified the ALNGR (Ala-Leu-Asn-Gly-Arg) peptide that induced cell adhesion as well as RGDS (Arg-Gly-Asp-Ser) peptide. After incubation for 2 h, approximately 68% of inoculated cells adhere to the ALNGR peptide disk. Adhesion inhibition assay with integrin antibodies showed that the ALNGR peptide interacts with integrin β1 but not with αvβ3, indicating that the receptors for ALNGR are different from RGDS. Additionally, the ALNGR peptide expressed cell specificities for adhesion: cell adhesion was promoted for fibroblasts but not for keratinocytes or endotherial cells. The ALNGR peptide induced cell adhesion and promoted cell proliferation without changing its property. It is therefore useful for the construction of functional biomaterials

  7. The Equine PeptideAtlas

    DEFF Research Database (Denmark)

    Bundgaard, Louise; Jacobsen, Stine; Sørensen, Mette Aamand

    2014-01-01

    Progress in MS-based methods for veterinary research and diagnostics is lagging behind compared to the human research, and proteome data of domestic animals is still not well represented in open source data repositories. This is particularly true for the equine species. Here we present a first...... Equine PeptideAtlas encompassing high-resolution tandem MS analyses of 51 samples representing a selection of equine tissues and body fluids from healthy and diseased animals. The raw data were processed through the Trans-Proteomic Pipeline to yield high quality identification of proteins and peptides....... The current release comprises 24 131 distinct peptides representing 2636 canonical proteins observed at false discovery rates of 0.2% at the peptide level and 1.4% at the protein level. Data from the Equine PeptideAtlas are available for experimental planning, validation of new datasets, and as a proteomic...

  8. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds known as peptide nucleic acids, bind complementary DNA and RNA strands, and generally do so more strongly than the corresponding DNA or RNA strands while exhibiting increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from...

  9. Relationships between Cargo, Cell Penetrating Peptides and Cell Type for Uptake of Non-Covalent Complexes into Live Cells

    Directory of Open Access Journals (Sweden)

    Andrea-Anneliese Keller

    2013-02-01

    Full Text Available Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing or conjugating them to cell penetrating peptides or by formation of non-covalent complexes. The latter is often preferred because of easy handling, uptake efficiency and auto-release of cargo into the live cell. In our studies complexes are formed with labeled or readily detectable cargoes for qualitative and quantitative estimation of their internalization. Properties and behavior of adhesion and suspension vertebrate cells as well as the protozoa Leishmania tarentolae are investigated with respect to proteolytic activity, uptake efficiency, intracellular localization and cytotoxicity. Our results show that peptide stability to membrane-bound, secreted or intracellular proteases varies between different CPPs and that the suitability of individual CPPs for a particular cargo in complex formation by non-covalent interactions requires detailed studies. Cells vary in their sensitivity to increasing concentrations of CPPs. Thus, most cells can be efficiently transduced with peptides, proteins and nucleotides with intracellular concentrations in the low micromole range. For each cargo, cell type and CPP the optimal conditions must be determined separately.

  10. Peptide Integrated Optics.

    Science.gov (United States)

    Handelman, Amir; Lapshina, Nadezda; Apter, Boris; Rosenman, Gil

    2018-02-01

    Bio-nanophotonics is a wide field in which advanced optical materials, biomedicine, fundamental optics, and nanotechnology are combined and result in the development of biomedical optical chips. Silk fibers or synthetic bioabsorbable polymers are the main light-guiding components. In this work, an advanced concept of integrated bio-optics is proposed, which is based on bioinspired peptide optical materials exhibiting wide optical transparency, nonlinear and electrooptical properties, and effective passive and active waveguiding. Developed new technology combining bottom-up controlled deposition of peptide planar wafers of a large area and top-down focus ion beam lithography provides direct fabrication of peptide optical integrated circuits. Finding a deep modification of peptide optical properties by reconformation of biological secondary structure from native phase to β-sheet architecture is followed by the appearance of visible fluorescence and unexpected transition from a native passive optical waveguiding to an active one. Original biocompatibility, switchable regimes of waveguiding, and multifunctional nonlinear optical properties make these new peptide planar optical materials attractive for application in emerging technology of lab-on-biochips, combining biomedical photonic and electronic circuits toward medical diagnosis, light-activated therapy, and health monitoring. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Collagen like peptide bioconjugates for targeted drug delivery applications

    Science.gov (United States)

    Luo, Tianzhi

    the coil/globule conformational transition of the PDEGMEMA building block above its LCST with stabilization of the nanostructures by the hydrophilic CLP. To the best of our knowledge, this is the first report on such assembled nanostructures from collagen-like peptide containing copolymers. Due to the strong propensity for CLPs to bind to natural collagen via strand invasion processes, these nanosized vesicles may be used as drug carriers for targeted delivery. In addition to synthetic polymers, the collagen like peptide is then conjugated with a thermoresponsive elastin-like peptide (ELP). The resulting ELP-CLP diblock conjugates show a remarkable reduction in the inverse transition temperature of the ELP domain, attributed to the anchoring effect of the CLP triple helix. The lower transition temperature of the conjugate enables facile formation of well-defined vesicles at physiological temperature and the unexpected resolubilization of the vesicles at elevated temperatures upon unfolding of the CLP domain. Given the ability of CLPs to modify collagens, this work provides not only a simple and versatile avenue for controlling the inverse transition behavior of elastin-like peptides, but also suggest future opportunities for these thermoresponsive nanostructures in biologically relevant environments. In the last section, the potential of using the ELP-CLP nanoparticles as drug delivery vehicles for targeting collagen containing matrices is evaluated. A sustained release of clinically relevant amount of encapsulated modelled drug is achieved within three weeks, followed by a thermally controlled burst release. As expected, the ELP-CLP nanoparticles show strong retention on collagen substrate, via specific binding through collagen triple helix hybridization. Additionally, cell viability and proliferation studies using fibroblasts and chondrocytes suggest the nanoparticles are non-cytotoxic. Additionally, almost no TNF-alpha expression from macrophages is observed

  12. New dendrimer - peptide host - guest complexes : towards dendrimers as peptide carriers

    NARCIS (Netherlands)

    Boas, U.; Sontjens, S.H.M.; Jensen, K.J.; Christensen, J.B.; Meijer, E.W.

    2002-01-01

    Adamantyl urea and adamantyl thiourea modified poly(propylene imine) dendrimers act as hosts for N-terminal tert-butoxycarbonyl (Boc)-protected peptides and form chloroform-soluble complexes. investigations with NMR spectroscopy show that the peptide is bound to the dendrimer by ionic interactions

  13. Effective modification of cell death-inducing intracellular peptides by means of a photo-cleavable peptide array-based screening system.

    Science.gov (United States)

    Kozaki, Ikko; Shimizu, Kazunori; Honda, Hiroyuki

    2017-08-01

    Intracellular functional peptides that play a significant role inside cells have been receiving a lot of attention as regulators of cellular activity. Previously, we proposed a novel screening system for intracellular functional peptides; it combined a photo-cleavable peptide array system with cell-penetrating peptides (CPPs). Various peptides can be delivered into cells and intracellular functions of the peptides can be assayed by means of our system. The aim of the present study was to demonstrate that the proposed screening system can be used for assessing the intracellular activity of peptides. The cell death-inducing peptide (LNLISKLF) identified in a mitochondria-targeting domain (MTD) of the Noxa protein served as an original peptide sequence for screening of peptides with higher activity via modification of the peptide sequence. We obtained 4 peptides with higher activity, in which we substituted serine (S) at the fifth position with phenylalanine (F), valine (V), tryptophan (W), or tyrosine (Y). During analysis of the mechanism of action, the modified peptides induced an increase in intracellular calcium concentration, which was caused by the treatment with the original peptide. Higher capacity for cell death induction by the modified peptides may be caused by increased hydrophobicity or an increased number of aromatic residues. Thus, the present work suggests that the intracellular activity of peptides can be assessed using the proposed screening system. It could be used for identifying intracellular functional peptides with higher activity through comprehensive screening. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  14. PeptideNavigator: An interactive tool for exploring large and complex data sets generated during peptide-based drug design projects.

    Science.gov (United States)

    Diller, Kyle I; Bayden, Alexander S; Audie, Joseph; Diller, David J

    2018-01-01

    There is growing interest in peptide-based drug design and discovery. Due to their relatively large size, polymeric nature, and chemical complexity, the design of peptide-based drugs presents an interesting "big data" challenge. Here, we describe an interactive computational environment, PeptideNavigator, for naturally exploring the tremendous amount of information generated during a peptide drug design project. The purpose of PeptideNavigator is the presentation of large and complex experimental and computational data sets, particularly 3D data, so as to enable multidisciplinary scientists to make optimal decisions during a peptide drug discovery project. PeptideNavigator provides users with numerous viewing options, such as scatter plots, sequence views, and sequence frequency diagrams. These views allow for the collective visualization and exploration of many peptides and their properties, ultimately enabling the user to focus on a small number of peptides of interest. To drill down into the details of individual peptides, PeptideNavigator provides users with a Ramachandran plot viewer and a fully featured 3D visualization tool. Each view is linked, allowing the user to seamlessly navigate from collective views of large peptide data sets to the details of individual peptides with promising property profiles. Two case studies, based on MHC-1A activating peptides and MDM2 scaffold design, are presented to demonstrate the utility of PeptideNavigator in the context of disparate peptide-design projects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Dendroaspis natriuretic peptide binds to the natriuretic peptide clearance receptor

    International Nuclear Information System (INIS)

    Johns, Douglas G.; Ao, Zhaohui; Heidrich, Bradley J.; Hunsberger, Gerald E.; Graham, Taylor; Payne, Lisa; Elshourbagy, Nabil; Lu, Quinn; Aiyar, Nambi; Douglas, Stephen A.

    2007-01-01

    Dendroaspis natriuretic peptide (DNP) is a newly-described natriuretic peptide which lowers blood pressure via vasodilation. The natriuretic peptide clearance receptor (NPR-C) removes natriuretic peptides from the circulation, but whether DNP interacts with human NPR-C directly is unknown. The purpose of this study was to test the hypothesis that DNP binds to NPR-C. ANP, BNP, CNP, and the NPR-C ligands AP-811 and cANP(4-23) displaced [ 125 I]-ANP from NPR-C with pM-to-nM K i values. DNP displaced [ 125 I]-ANP from NPR-C with nM potency, which represents the first direct demonstration of binding of DNP to human NPR-C. DNP showed high pM affinity for the GC-A receptor and no affinity for GC-B (K i > 1000 nM). DNP was nearly 10-fold more potent than ANP at stimulating cGMP production in GC-A expressing cells. Blockade of NPR-C might represent a novel therapeutic approach in augmenting the known beneficial actions of DNP in cardiovascular diseases such as hypertension and heart failure

  16. Matrix-assisted peptide synthesis on nanoparticles.

    Science.gov (United States)

    Khandadash, Raz; Machtey, Victoria; Weiss, Aryeh; Byk, Gerardo

    2014-09-01

    We report a new method for multistep peptide synthesis on polymeric nanoparticles of differing sizes. Polymeric nanoparticles were functionalized via their temporary embedment into a magnetic inorganic matrix that allows multistep peptide synthesis. The matrix is removed at the end of the process for obtaining nanoparticles functionalized with peptides. The matrix-assisted synthesis on nanoparticles was proved by generating various biologically relevant peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

  17. Bromine isotopic signature facilitates de novo sequencing of peptides in free-radical-initiated peptide sequencing (FRIPS) mass spectrometry.

    Science.gov (United States)

    Nam, Jungjoo; Kwon, Hyuksu; Jang, Inae; Jeon, Aeran; Moon, Jingyu; Lee, Sun Young; Kang, Dukjin; Han, Sang Yun; Moon, Bongjin; Oh, Han Bin

    2015-02-01

    We recently showed that free-radical-initiated peptide sequencing mass spectrometry (FRIPS MS) assisted by the remarkable thermochemical stability of (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO) is another attractive radical-driven peptide fragmentation MS tool. Facile homolytic cleavage of the bond between the benzylic carbon and the oxygen of the TEMPO moiety in o-TEMPO-Bz-C(O)-peptide and the high reactivity of the benzylic radical species generated in •Bz-C(O)-peptide are key elements leading to extensive radical-driven peptide backbone fragmentation. In the present study, we demonstrate that the incorporation of bromine into the benzene ring, i.e. o-TEMPO-Bz(Br)-C(O)-peptide, allows unambiguous distinction of the N-terminal peptide fragments from the C-terminal fragments through the unique bromine doublet isotopic signature. Furthermore, bromine substitution does not alter the overall radical-driven peptide backbone dissociation pathways of o-TEMPO-Bz-C(O)-peptide. From a practical perspective, the presence of the bromine isotopic signature in the N-terminal peptide fragments in TEMPO-assisted FRIPS MS represents a useful and cost-effective opportunity for de novo peptide sequencing. Copyright © 2015 John Wiley & Sons, Ltd.

  18. Substrate stiffness promotes latent TGF-β1 activation in hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Pang, Mingshu; Teng, Yao; Huang, Jianyong; Yuan, Yuan; Lin, Feng; Xiong, Chunyang

    2017-01-01

    Hepatocellular carcinoma (HCC) was usually coupled with increased stiffness of the extracellular matrix (ECM) and elevated level of transforming growth factor-β1 (TGF-β1). However, the mechanism by which substrate rigidity modulated TGF-β1 signaling transduction remained unknown. This paper investigated the molecular mechanism of how matrix stiffness regulating TGF-β1 signaling in HCC cells. By means of stiffness tunable collagen I-coated polyacrylamide (PA) gels, we found that the expressions of β1 integrin, p-FAK Y397 and p-Smad2 upregulated on stiffer gels as well as the content of TGF-β1 in culture media of HCC cells, which were inhibited by RGD blocking peptides, Y-27632 (ROCK inhibitor) or Blebbistatin (myosin II inhibitor). Cellular traction force was also significantly higher when plated on stiffer substrates but dramatically decreased after treatment with Y-27632 or Blebbistatin. Furthermore, the upregulation of p-Smad2 in the HCC cells on stiffer PA gels induced by exogenetic latent TGF-β1 was downregulated in the presence of RGD peptides. The nuclear translocation of Smad2 induced by latent TGF-β1 was inhibited by Y-27632 or Blebbistatin. Our results suggested that the extracellular matrix stiffness regulated latent TGF-β1 activation by cytoskeletal tension in HCC cells, showing that matrix stiffness was a key regulator involving the TGF-β1 activity in HCC cells. The current study presented a mechanism of how hepatocirrhosis developed into liver cancer. - Highlights: • TGF-β1 signaling pathway regulated by ECM stiffness was studied in hepatocellular carcinoma. • Matrix stiffness promoted latent TGF-β1 activation via β1 integrin-FAK-Rho GTPase pathway. • A mechanism of how hepatocirrhosis developed into liver cancer was presented.

  19. Suppressing IL-36-driven inflammation using peptide pseudosubstrates for neutrophil proteases.

    Science.gov (United States)

    Sullivan, Graeme P; Henry, Conor M; Clancy, Danielle M; Mametnabiev, Tazhir; Belotcerkovskaya, Ekaterina; Davidovich, Pavel; Sura-Trueba, Sylvia; Garabadzhiu, Alexander V; Martin, Seamus J

    2018-03-07

    Sterile inflammation is initiated by molecules released from necrotic cells, called damage-associated molecular patterns (DAMPs). Members of the extended IL-1 cytokine family are important DAMPs, are typically only released through necrosis, and require limited proteolytic processing for activation. The IL-1 family cytokines, IL-36α, IL-36β, and IL-36γ, are expressed as inactive precursors and have been implicated as key initiators of psoriatic-type skin inflammation. We have recently found that IL-36 family cytokines are proteolytically processed and activated by the neutrophil granule-derived proteases, elastase, and cathepsin G. Inhibitors of IL-36 processing may therefore have utility as anti-inflammatory agents through suppressing activation of the latter cytokines. We have identified peptide-based pseudosubstrates for cathepsin G and elastase, based on optimal substrate cleavage motifs, which can antagonize activation of all three IL-36 family cytokines by the latter proteases. Human psoriatic skin plaques displayed elevated IL-36β processing activity that could be antagonized by peptide pseudosubstrates specific for cathepsin G. Thus, antagonists of neutrophil-derived proteases may have therapeutic potential for blocking activation of IL-36 family cytokines in inflammatory conditions such as psoriasis.

  20. Synthesis of peptide thioacids at neutral pH using bis(2-sulfanylethyl)amido peptide precursors.

    Science.gov (United States)

    Pira, Silvain L; Boll, Emmanuelle; Melnyk, Oleg

    2013-10-18

    Reaction of bis(2-sulfanylethyl)amido (SEA) peptides with triisopropylsilylthiol in water at neutral pH yields peptide thiocarboxylates. An alkylthioester derived from β-alanine was used to trap the released bis(2-sulfanylethyl)amine and displace the equilibrium toward the peptide thiocarboxylate.

  1. Power electronics substrate for direct substrate cooling

    Science.gov (United States)

    Le, Khiet [Mission Viejo, CA; Ward, Terence G [Redondo Beach, CA; Mann, Brooks S [Redondo Beach, CA; Yankoski, Edward P [Corona, CA; Smith, Gregory S [Woodland Hills, CA

    2012-05-01

    Systems and apparatus are provided for power electronics substrates adapted for direct substrate cooling. A power electronics substrate comprises a first surface configured to have electrical circuitry disposed thereon, a second surface, and a plurality of physical features on the second surface. The physical features are configured to promote a turbulent boundary layer in a coolant impinged upon the second surface.

  2. Photodissociative Cross-Linking of Non-covalent Peptide-Peptide Ion Complexes in the Gas Phase

    Science.gov (United States)

    Nguyen, Huong T. H.; Andrikopoulos, Prokopis C.; Rulíšek, Lubomír; Shaffer, Christopher J.; Tureček, František

    2018-05-01

    We report a gas-phase UV photodissociation study investigating non-covalent interactions between neutral hydrophobic pentapeptides and peptide ions incorporating a diazirine-tagged photoleucine residue. Phenylalanine (Phe) and proline (Pro) were chosen as the conformation-affecting residues that were incorporated into a small library of neutral pentapeptides. Gas-phase ion-molecule complexes of these peptides with photo-labeled pentapeptides were subjected to photodissociation. Selective photocleavage of the diazirine ring at 355 nm formed short-lived carbene intermediates that underwent cross-linking by insertion into H-X bonds of the target peptide. The cross-link positions were established from collision-induced dissociation tandem mass spectra (CID-MS3) providing sequence information on the covalent adducts. Effects of the amino acid residue (Pro or Phe) and its position in the target peptide sequence were evaluated. For proline-containing peptides, interactions resulting in covalent cross-links in these complexes became more prominent as proline was moved towards the C-terminus of the target peptide sequence. The photocross-linking yields of phenylalanine-containing peptides depended on the position of both phenylalanine and photoleucine. Density functional theory calculations were used to assign structures of low-energy conformers of the (GLPMG + GLL*LK + H)+ complex. Born-Oppenheimer molecular dynamics trajectory calculations were used to capture the thermal motion in the complexes within 100 ps and determine close contacts between the incipient carbene and the H-X bonds in the target peptide. This provided atomic-level resolution of potential cross-links that aided spectra interpretation and was in agreement with experimental data. [Figure not available: see fulltext.

  3. Lack of interaction between the peptidomimetic substrates captopril and cephradine.

    Science.gov (United States)

    Foster, David R; Yee, Shiyin; Bleske, Barry E; Carver, Peggy L; Shea, Michael J; Menon, Sujatha S; Ramachandran, Chandrasekharan; Welage, Lynda S; Amidon, Gordon L

    2009-03-01

    Intestinal peptide transporters, including hPEPT1, facilitate the absorption of cephalosporins and angiotensin-converting enzyme inhibitors, and have been investigated as a means to improve oral drug absorption. Renal peptide transporters including hPEPT2, may also facilitate renal reabsorption of such compounds. In vitro and animal studies suggest that co-administration of peptidomimetic compounds may alter oral pharmacokinetics, although this has not been well studied in humans. The purpose of this study was to determine whether co-administration of the hPEPT substrates captopril and cephradine alters the oral pharmacokinetics of either agent. Nine healthy male volunteers received a single oral 25-mg dose of captopril, a single oral 500-mg dose of cephradine, or concurrent ingestion of captopril and cephradine in a cross-over manner. Venous blood samples were taken and captopril and cephradine pharmacokinetics were determined using noncompartmental analyses. No significant differences were observed in captopril or cephradine pharmacokinetics when administered together as compared to each agent alone (a marginal decrease in C(max) was observed for both captopril and cephradine during co-administration [5-15%]; however, differences were not statistically significant). The results of our study suggest that hPEPT1 and hPEPT2 are unlikely to contribute to clinically important drug interactions in humans.

  4. Peptides and polypeptides as scaffolds for optoelectronics and biomaterials applications

    Science.gov (United States)

    Charati, Manoj B.

    effects on peptide conformation. pi-orbital interactions at the molecular level were observed to be very sensitive to intermolecular distance and orientation of the chromophores attached to the alpha-helical peptide templates. When the methylstilbene or Oxa-PPV molecules were arranged on the same side of the helix with intermolecular spacing of 6A, the chromophores interacted strongly with each other forming excimers. Such interactions were absent when the molecules were arranged on the opposite side of the helix. These peptide-templated systems therefore offer enormous opportunities for the elucidation of complex photophysical phenomena that occur in relatively aggregated morphologies of conjugated species, but under dilute solution conditions in which the number of chromphores in the aggregate can be manipulated. Part 2. Synthesis and characterization of biocompatible polypeptide elastomer. Lately, the significance of mechanical forces and biological cues involved in tissue remodeling are highly valued; thus the capacity of a biomaterial to present a fitting mechanical and biological environment for optimal tissue generation has become a key parameter for biomaterial design. In addition to having suitable mechanical properties, materials used for these applications need to be biologically active, i.e. trigger dynamic interactions with cells and stimulate explicit cell and tissue responses. Thus, we have designed a resilin-based modular biomaterial incorporating both mechanically and biologically active domains to sense and aptly respond to the bio-mechanical demand or changes in their environment. The use of resilin-like polypeptides offers access to a class of hydrophilic elastomers with excellent resilience and high frequency responsiveness, which can be used for encapsulating hydrophilic drugs like proteins for drug delivery, and provides hydrophilic extracellular matrix mimicking cell adhesive and enzyme degradable substrate for tissue engineering. Hence, we have

  5. Rapid degradation of D- and L-succinimide-containing peptides by a post-proline endopeptidase from human erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Momand, J.; Clarke, S.

    1987-12-01

    The authors have been interested in the metabolic fate of proteins containing aspartyl succinimide (Asu) residues. These residues can be derived from the spontaneous rearrangement of Asp and Asn residues and from the spontaneous demethylation of enzymatically methylated L-isoAsp and D-Asp residues. Incubation of the synthetic hexapeptide N-Ac-Val-Tyr-Pro-Asu-Gly-Ala with the cytosolic fraction of human erythrocytes resulted in rapid cleavage of the prolyl-aspartyl succinimide bond producing the tripeptide N-Ac-Val-Try-Pro. The rate of this reaction is equal for both L- and D-Asu-containing peptides and is 10-fold greater that the rate of cleavage of a corresponding peptide containing a normal Pro-Asp linkage. When the aspartyl succinimide ring was replaced with an isoaspartyl residue, the cleavage rate was about 5 times that of the normal Pro-Asp peptide. The tripeptide-producing activity copurified on DEAE-cellulose chromatography with an activity that cleaves N-carbobenzoxy-Gly-Pro-4-methylcoumarin-7-amide, a post-proline endopeptidase substrate. These two activities were both inhibited by an antiserum to rat brain post-proline endopeptidase, and it appears that they are catalyzed by the same enzyme. This enzyme has a molecular weight of approximately 80,000 and is covalently labeled and inhibited by (/sup 3/H) diisopropyl fluorophosphate. The facile cleavage of the succinimide- and isoaspartyl-containing peptides by this post-proline endopeptidase suggests that it may play a role in the metabolism of peptides containing altered aspartyl residues.

  6. Maize Bioactive Peptides against Cancer

    Science.gov (United States)

    Díaz-Gómez, Jorge L.; Castorena-Torres, Fabiola; Preciado-Ortiz, Ricardo E.; García-Lara, Silverio

    2017-06-01

    Cancer is one of the main chronic degenerative diseases worldwide. In recent years, consumption of whole-grain cereals and their derived food products has been associated with reduction risks of various types of cancer. Cereals main biomolecules includes proteins, peptides, and amino acids present in different quantities within the grain. The nutraceutical properties associated with peptides exerts biological functions that promote health and prevent this disease. In this review, we report the current status and advances on maize peptides regarding bioactive properties that have been reported such as antioxidant, antihypertensive, hepatoprotective, and anti-tumour activities. We also highlighted its biological potential through which maize bioactive peptides exert anti-cancer activity. Finally, we analyse and emphasize the possible areas of application for maize peptides.

  7. The emerging role of promiscuous 7TM receptors as chemosensors for food intake.

    Science.gov (United States)

    Wellendorph, Petrine; Johansen, Lars Dan; Bräuner-Osborne, Hans

    2010-01-01

    In recent years, several highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized of which many are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids (FFAs) and are expressed in taste tissue, the gastrointestinal (GI) tract, endocrine glands, adipose tissue, and/or kidney. This has led to the hypothesis that these receptors may act as sensors of food intake modulating, for example, release of incretin hormones from the gut, insulin/glucagon from the pancreas, and leptin from adipose tissue. In the present review, we describe the molecular mechanisms of nutrient-sensing of the calcium-sensing receptor (CaR), the G protein-coupled receptor family C, group 6, subtype A (GPRC6A), and the taste1 receptor T1R1/T1R3-sensing L-α-amino acids; the carbohydrate-sensing T1R2/T1R3 receptor; the proteolytic degradation product sensor GPR93 (also termed GPR92); and the FFA sensing receptors FFA1, FFA2, FFA3, GPR84, and GPR120. Due to their omnipresent nature, the natural ligands have had limited usability in pharmacological/physiological studies which has hampered the elucidation of the physiological function and therapeutic prospect of their receptors. However, an increasing number of subtype-selective ligands and/or receptor knockout mice are being developed which at least for some of the receptors have validated them as promising drug targets in, for example, type II diabetes. Copyright © 2010 Elsevier Inc. All rights reserved.

  8. Photo-oxidation of histidine peptides yields high concentrations of unstable peroxides

    International Nuclear Information System (INIS)

    Policarpio, V.V.; Hawkins, C.L.; Davies, M.J.

    2003-01-01

    Oxidation of proteins by UV, and visible light in the presence of sensitizers, results in side chain modification as well as aggregation and fragmentation. In particular, singlet oxygen has been reported to oxidize Met, Trp, Tyr, Cys and His side chains in a selective manner. In this study the oxidation of histidine and its derivatives, and His-containing peptides is examined using a range of sensitizers, to determine whether peroxides are major intermediates, and the mechanism of formation of these species. Visible light-sensitised oxidation of Gly-His-Gly in the presence of oxygen and rose bengal gives unstable substrate-derived peroxides with the peroxide yield increasing with increasing photolysis time. Similar behaviour was detected with other photosensitizers, though the peroxide yields varied with the sensitizer at identical concentrations with rose bengal > aluminium phthalocyanine > hematoporphyrin IX > zinc phthalocyanine > tetrakisporphine. The peroxide yield was decreased in the presence of azide and enhanced when deuterium oxide was employed as the solvent, consistent with peroxide formation being singlet oxygen mediated. Experiments using anoxic conditions gave low yields of peroxides confirming the oxygen-dependence of these reactions. HPLC analysis showed rapid loss of the parent peptide, with subsequent formation of both stable and unstable products; these are currently being characterized by MS and NMR. Similar behavior has been observed with other His derivatives. The yield of singlet oxygen formed in these reactions has been estimated using a bleaching assay (N, N-dimethyl-4-nitrosoaniline). Quantification of singlet oxygen formation and Gly-His-Gly derived peroxide during rose bengal-mediated photooxidation indicated a conversion efficiency of the initial singlet oxygen into substrate-derived peroxides of ca. 75% indicating that peroxide formation is a highly efficient and major reaction pathway

  9. Insect Peptides - Perspectives in Human Diseases Treatment.

    Science.gov (United States)

    Chowanski, Szymon; Adamski, Zbigniew; Lubawy, Jan; Marciniak, Pawel; Pacholska-Bogalska, Joanna; Slocinska, Malgorzata; Spochacz, Marta; Szymczak, Monika; Urbanski, Arkadiusz; Walkowiak-Nowicka, Karolina; Rosinski, Grzegorz

    2017-01-01

    Insects are the largest and the most widely distributed group of animals in the world. Their diversity is a source of incredible variety of different mechanisms of life processes regulation. There are many agents that regulate immunology, reproduction, growth and development or metabolism. Hence, it seems that insects may be a source of numerous substances useful in human diseases treatment. Especially important in the regulation of insect physiology are peptides, like neuropeptides, peptide hormones or antimicrobial peptides. There are two main aspects where they can be helpful, 1) Peptides isolated from insects may become potential drugs in therapy of different diseases, 2) A lot of insect peptide hormones show structural or functional homology to mammalian peptide hormones and the comparative studies may give a new look on human disorders. In our review we focused on three group of insect derived peptides: 1) immune-active peptides, 2) peptide hormones and 3) peptides present in venoms. In our review we try to show the considerable potential of insect peptides in searching for new solutions for mammalian diseases treatment. We summarise the knowledge about properties of insect peptides against different virulent agents, anti-inflammatory or anti-nociceptive properties as well as compare insect and mammalian/vertebrate peptide endocrine system to indicate usefulness of knowledge about insect peptide hormones in drug design. The field of possible using of insect delivered peptide to therapy of various human diseases is still not sufficiently explored. Undoubtedly, more attention should be paid to insects due to searching new drugs. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  10. Peptide imprinted receptors for the determination of the small cell lung cancer associated biomarker progastrin releasing peptide

    DEFF Research Database (Denmark)

    Qader, A. A.; Urraca, J.; Torsetnes, S. B.

    2014-01-01

    Peptide imprinted polymers were developed for detection of progastrin releasing peptide (ProGRP); a low abundant blood based biomarker for small cell lung cancer. The polymers targeted the proteotypic nona-peptide sequence NLLGLIEAK and were used for selective enrichment of the proteotypic peptide...... prior to LCMS based quantification. Peptide imprinted polymers with the best affinity characteristics were first identified from a 96-polymer combinatorial library. The effects of functional monomers, crosslinker, porogen, and template on adsorption capacity and selectivity for NLLGLIEAK were...

  11. Study on the C-peptide radioimmunoassay with synthetized connecting peptide

    Energy Technology Data Exchange (ETDEWEB)

    Nakagawa, S; Sasaki, T; Nakayama, H; Watanabe, T; Aoki, S [Hokkaido Univ., Sapporo (Japan). School of Medicine

    1976-01-01

    A method of C-peptide radioimmunoassay with the synthetized connecting peptide by Yanaihara was tested for the determination of serum C-peptide immunoreactivity (CPR) in normal people and in diabetics with or without insulin treatment. The CPR value obtained by this method was not interfered with by the presence of serum proteins or by the insulin of people with or without insulin treatment judged by the dilution test and the recovery test. The normal fasting CPR was 2.80 +- 0.78 ng/ml with the synthetized C-peptide as a standard. The CPR value increased and reached a maximum 90 minutes after the ingestion of 50 g of glucose. The increase after the glucose loading reduced corresponding to the severity of diabetes, and some juvenile-onset diabetes showed no response. Adult-type diabetics under insulin treatment, however, showed weak but significant CPR response. The increment of CPR and immunoreactive insulin after glucose loading in normal people and non-treated diabetics was well correlated (..gamma..=0.8262). Judged from the above mentioned results, CPR determination in insulin-treated diabetics was thought to be a useful method for the assessment of the insulin-secreting ability of beta-cells of the pancreas.

  12. Requirements for superoxide-dependent tyrosine hydroperoxide formation in peptides

    DEFF Research Database (Denmark)

    Winterbourn, Christine C; Parsons-Mair, Helena N; Gebicki, Silvia

    2004-01-01

    Superoxide reacts rapidly with other radicals, but these reactions have received little attention in the context of oxidative stress. For tyrosyl radicals, reaction with superoxide is 3-fold faster than dimerization, and forms the addition product tyrosine hydroperoxide. We have explored structural...... requirements for hydroperoxide formation using tyrosine analogues and di- and tri-peptides. Superoxide and phenoxyl radicals were generated using xanthine oxidase, peroxidase and the respective tyrosine derivative, or by gamma-radiation. Peroxides were measured using FeSO4/Xylenol Orange. Tyrosine and tyramine...... formed stable hydroperoxides, but N-acetyltyrosine and p-hydroxyphenylacetic acid did not, demonstrating a requirement for a free amino group. Using [14C]tyrosine, the hydroperoxide and dityrosine were formed at a molar ratio of 1.8:1. Studies with pre-formed hydroperoxides, and measurements of substrate...

  13. A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

    Science.gov (United States)

    Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

    1996-01-01

    Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

  14. Tidbits for the synthesis of bis(2-sulfanylethyl)amido (SEA) polystyrene resin, SEA peptides and peptide thioesters.

    Science.gov (United States)

    Ollivier, Nathalie; Raibaut, Laurent; Blanpain, Annick; Desmet, Rémi; Dheur, Julien; Mhidia, Reda; Boll, Emmanuelle; Drobecq, Hervé; Pira, Silvain L; Melnyk, Oleg

    2014-02-01

    Protein total chemical synthesis enables the atom-by-atom control of the protein structure and therefore has a great potential for studying protein function. Native chemical ligation of C-terminal peptide thioesters with N-terminal cysteinyl peptides and related methodologies are central to the field of protein total synthesis. Consequently, methods enabling the facile synthesis of peptide thioesters using Fmoc-SPPS are of great value. Herein, we provide a detailed protocol for the preparation of bis(2-sulfanylethyl)amino polystyrene resin as a starting point for the synthesis of C-terminal bis(2-sulfanylethyl)amido peptides and of peptide thioesters derived from 3-mercaptopropionic acid. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

  15. Measurement of ex vivo ELISpot interferon-gamma recall responses to Plasmodium falciparum AMA1 and CSP in Ghanaian adults with natural exposure to malaria.

    Science.gov (United States)

    Ganeshan, Harini; Kusi, Kwadwo A; Anum, Dorothy; Hollingdale, Michael R; Peters, Bjoern; Kim, Yohan; Tetteh, John K A; Ofori, Michael F; Gyan, Ben A; Koram, Kwadwo A; Huang, Jun; Belmonte, Maria; Banania, Jo Glenna; Dodoo, Daniel; Villasante, Eileen; Sedegah, Martha

    2016-02-01

    Malaria eradication requires a concerted approach involving all available control tools, and an effective vaccine would complement these efforts. An effective malaria vaccine should be able to induce protective immune responses in a genetically diverse population. Identification of immunodominant T cell epitopes will assist in determining if candidate vaccines will be immunogenic in malaria-endemic areas. This study therefore investigated whether class I-restricted T cell epitopes of two leading malaria vaccine antigens, Plasmodium falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1), could recall T cell interferon-γ responses from naturally exposed subjects using ex vivo ELISpot assays. Thirty-five subjects aged between 24 and 43 years were recruited from a malaria-endemic urban community of Ghana in 2011, and their peripheral blood mononuclear cells (PBMCs) were tested in ELISpot IFN-γ assays against overlapping 15mer peptide pools spanning the entire CSP and AMA1 antigens, and 9-10mer peptide epitope mixtures that included previously identified and/or predicted human leukocyte antigen (HLA) class 1-restricted epitopes from same two antigens. For CSP, 26 % of subjects responded to at least one of the nine 15mer peptide pools whilst 17 % responded to at least one of the five 9-10mer HLA-restricted epitope mixtures. For AMA1, 63 % of subjects responded to at least one of the 12 AMA1 15mer peptide pools and 51 % responded to at least one of the six 9-10mer HLA-restricted epitope mixtures. Following analysis of data from the two sets of peptide pools, along with bioinformatics predictions of class I-restricted epitopes and the HLA supertypes expressed by a subset of study subjects, peptide pools that may contain epitopes recognized by multiple HLA supertypes were identified. Collectively, these results suggest that natural transmission elicits ELISpot IFN-γ activities to class 1-restricted epitopes that are largely HLA-promiscuous. These

  16. Radiopharmaceutical development of radiolabelled peptides

    Energy Technology Data Exchange (ETDEWEB)

    Fani, Melpomeni; Maecke, Helmut R. [University Hospital Freiburg, Department of Nuclear Medicine, Freiburg (Germany)

    2012-02-15

    Receptor targeting with radiolabelled peptides has become very important in nuclear medicine and oncology in the past few years. The overexpression of many peptide receptors in numerous cancers, compared to their relatively low density in physiological organs, represents the molecular basis for in vivo imaging and targeted radionuclide therapy with radiolabelled peptide-based probes. The prototypes are analogs of somatostatin which are routinely used in the clinic. More recent developments include somatostatin analogs with a broader receptor subtype profile or with antagonistic properties. Many other peptide families such as bombesin, cholecystokinin/gastrin, glucagon-like peptide-1 (GLP-1)/exendin, arginine-glycine-aspartic acid (RGD) etc. have been explored during the last few years and quite a number of potential radiolabelled probes have been derived from them. On the other hand, a variety of strategies and optimized protocols for efficient labelling of peptides with clinically relevant radionuclides such as {sup 99m}Tc, M{sup 3+} radiometals ({sup 111}In, {sup 86/90}Y, {sup 177}Lu, {sup 67/68}Ga), {sup 64/67}Cu, {sup 18}F or radioisotopes of iodine have been developed. The labelling approaches include direct labelling, the use of bifunctional chelators or prosthetic groups. The choice of the labelling approach is driven by the nature and the chemical properties of the radionuclide. Additionally, chemical strategies, including modification of the amino acid sequence and introduction of linkers/spacers with different characteristics, have been explored for the improvement of the overall performance of the radiopeptides, e.g. metabolic stability and pharmacokinetics. Herein, we discuss the development of peptides as radiopharmaceuticals starting from the choice of the labelling method and the conditions to the design and optimization of the peptide probe, as well as some recent developments, focusing on a selected list of peptide families, including somatostatin

  17. Different target surfaces for the analysis of peptides, peptide mixtures and peptide mass fingerprints by AP-MALDI ion trap-mass spectrometry.

    Science.gov (United States)

    Pittenauer, Ernst; Kassler, Alexander; Haubner, Roland; Allmaier, Günter

    2011-06-10

    The desorption/ionization behavior of individual peptides, an equimolare peptide mixture and a tryptic digest was investigated by AP-MALDI-IT-MS using four different target materials (gold-covered stainless steel (SS), titanium nitride-covered SS, hand-polished SS, and microdiamond-covered hardmetal) under identical conditions. Gold-covered as well as polished SS targets yielded comparable mass spectra for peptides and peptide mixture in the low pMol-range. The first target exhibited superior data down to the 10fMol-range. In contrast, titanium nitride-covered SS and microdiamond-covered hardmetal AP-MALDI-targets yielded poor sensitivity. These observations could be correlated with the surface roughness of the targets determined by 3D-confocal-white-light-microscopy. The roughest surfaces were found for titanium nitride-covered SS and microdiamond-covered hardmetal material showing both poor MS sensitivity. A less rough surface could be determined for the hand-polished SS target and the smoothest surface was found for the gold-covered target yielding the best sensitivity of all surfaces. These differences in the roughness having a strong impact on the ultimate sensitivity obtainable for peptide samples could be corroborated by electron microscopy. A peptide mixture covering a wide range of molecular weights and a tryptic protein digest (from 2-DE) exhibit the same behavior. This clearly indicates that the smooth gold-covered SS target is the surface of choice in AP-MALDI MS proteomics. Copyright © 2010. Published by Elsevier B.V.

  18. Human C-peptide. Pt. 1

    International Nuclear Information System (INIS)

    Beischer, W.; Keller, L.; Maas, M.; Schiefer, E.; Pfeiffer, E.F.

    1976-01-01

    Synthetic human C-peptide bearing a tyrosine group at its amino end is labelled with 125 iodine using chloramin T or hydrogen peroxide and lactoperoxidase. The results of the two methods are compared. Antiserum to synthetic human C-peptide (without tyrosine), which was partially coupled to rabbit albumin, is raised in guinea pigs and goats. Goats show to be superior to guinea pips concerning antibody production. The so-called 'hook effect' phenomenon is observed when setting up the standard curves for the radioimmunoassay. Monotonically decreasing standard curves are obtained on dilution of antiserum with a high antibody titer which was produced by repeated immunization in goats. Free C-peptide and C-peptide bound to antiserum are separated using the anion exchange resin amberlite. Using this separation technique we excluded unspecific binding of labelled C-peptide to protein fractions in serum of diabetics. The sensitivity of our radioimmunoassay is approx. 0.3 ng C-peptide/ml serum. Intra- and interassay variability are below 10%. Human proinsulin is the only substance found to crossreact with the antiserum. (orig.) [de

  19. Material Binding Peptides for Nanotechnology

    Directory of Open Access Journals (Sweden)

    Urartu Ozgur Safak Seker

    2011-02-01

    Full Text Available Remarkable progress has been made to date in the discovery of material binding peptides and their utilization in nanotechnology, which has brought new challenges and opportunities. Nowadays phage display is a versatile tool, important for the selection of ligands for proteins and peptides. This combinatorial approach has also been adapted over the past decade to select material-specific peptides. Screening and selection of such phage displayed material binding peptides has attracted great interest, in particular because of their use in nanotechnology. Phage display selected peptides are either synthesized independently or expressed on phage coat protein. Selected phage particles are subsequently utilized in the synthesis of nanoparticles, in the assembly of nanostructures on inorganic surfaces, and oriented protein immobilization as fusion partners of proteins. In this paper, we present an overview on the research conducted on this area. In this review we not only focus on the selection process, but also on molecular binding characterization and utilization of peptides as molecular linkers, molecular assemblers and material synthesizers.

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

    Science.gov (United States)

    Zorzi, Alessandro; Deyle, Kaycie; Heinis, Christian

    2017-06-01

    Cyclic peptides combine several favorable properties such as good binding affinity, target selectivity and low toxicity that make them an attractive modality for the development of therapeutics. Over 40 cyclic peptide drugs are currently in clinical use and around one new cyclic peptide drug enters the market every year on average. The vast majority of clinically approved cyclic peptides are derived from natural products, such as antimicrobials or human peptide hormones. New powerful techniques based on rational design and in vitro evolution have enabled the de novo development of cyclic peptide ligands to targets for which nature does not offer solutions. A look at the cyclic peptides currently under clinical evaluation shows that several have been developed using such techniques. This new source for cyclic peptide ligands introduces a freshness to the field, and it is likely that de novo developed cyclic peptides will be in clinical use in the near future. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Sequencing Cyclic Peptides by Multistage Mass Spectrometry

    Science.gov (United States)

    Mohimani, Hosein; Yang, Yu-Liang; Liu, Wei-Ting; Hsieh, Pei-Wen; Dorrestein, Pieter C.; Pevzner, Pavel A.

    2012-01-01

    Some of the most effective antibiotics (e.g., Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. While hundreds of biomedically important cyclic peptides have been sequenced, the computational techniques for sequencing cyclic peptides are still in their infancy. Previous methods for sequencing peptide antibiotics and other cyclic peptides are based on Nuclear Magnetic Resonance spectroscopy, and require large amount (miligrams) of purified materials that, for most compounds, are not possible to obtain. Recently, development of mass spectrometry based methods has provided some hope for accurate sequencing of cyclic peptides using picograms of materials. In this paper we develop a method for sequencing of cyclic peptides by multistage mass spectrometry, and show its advantages over single stage mass spectrometry. The method is tested on known and new cyclic peptides from Bacillus brevis, Dianthus superbus and Streptomyces griseus, as well as a new family of cyclic peptides produced by marine bacteria. PMID:21751357

  2. A Plasmodium Promiscuous T Cell Epitope Delivered within the Ad5 Hexon Protein Enhances the Protective Efficacy of a Protein Based Malaria Vaccine.

    Directory of Open Access Journals (Sweden)

    Jairo Andres Fonseca

    Full Text Available A malaria vaccine is a public health priority. In order to produce an effective vaccine, a multistage approach targeting both the blood and the liver stage infection is desirable. The vaccine candidates also need to induce balanced immune responses including antibodies, CD4+ and CD8+ T cells. Protein-based subunit vaccines like RTS,S are able to induce strong antibody response but poor cellular reactivity. Adenoviral vectors have been effective inducing protective CD8+ T cell responses in several models including malaria; nonetheless this vaccine platform exhibits a limited induction of humoral immune responses. Two approaches have been used to improve the humoral immunogenicity of recombinant adenovirus vectors, the use of heterologous prime-boost regimens with recombinant proteins or the genetic modification of the hypervariable regions (HVR of the capsid protein hexon to express B cell epitopes of interest. In this study, we describe the development of capsid modified Ad5 vectors that express a promiscuous Plasmodium yoelii T helper epitope denominated PyT53 within the hexon HVR2 region. Several regimens were tested in mice to determine the relevance of the hexon modification in enhancing protective immune responses induced by the previously described protein-based multi-stage experimental vaccine PyCMP. A heterologous prime-boost immunization regime that combines a hexon modified vector with transgenic expression of PyCMP followed by protein immunizations resulted in the induction of robust antibody and cellular immune responses in comparison to a similar regimen that includes a vector with unmodified hexon. These differences in immunogenicity translated into a better protective efficacy against both the hepatic and red blood cell stages of P. yoelii. To our knowledge, this is the first time that a hexon modification is used to deliver a promiscuous T cell epitope. Our data support the use of such modification to enhance the immunogenicity

  3. Prediction of promiscuous p-glycoprotein inhibition using a novel machine learning scheme.

    Directory of Open Access Journals (Sweden)

    Max K Leong

    Full Text Available BACKGROUND: P-glycoprotein (P-gp is an ATP-dependent membrane transporter that plays a pivotal role in eliminating xenobiotics by active extrusion of xenobiotics from the cell. Multidrug resistance (MDR is highly associated with the over-expression of P-gp by cells, resulting in increased efflux of chemotherapeutical agents and reduction of intracellular drug accumulation. It is of clinical importance to develop a P-gp inhibition predictive model in the process of drug discovery and development. METHODOLOGY/PRINCIPAL FINDINGS: An in silico model was derived to predict the inhibition of P-gp using the newly invented pharmacophore ensemble/support vector machine (PhE/SVM scheme based on the data compiled from the literature. The predictions by the PhE/SVM model were found to be in good agreement with the observed values for those structurally diverse molecules in the training set (n = 31, r(2 = 0.89, q(2 = 0.86, RMSE = 0.40, s = 0.28, the test set (n = 88, r(2 = 0.87, RMSE = 0.39, s = 0.25 and the outlier set (n = 11, r(2 = 0.96, RMSE = 0.10, s = 0.05. The generated PhE/SVM model also showed high accuracy when subjected to those validation criteria generally adopted to gauge the predictivity of a theoretical model. CONCLUSIONS/SIGNIFICANCE: This accurate, fast and robust PhE/SVM model that can take into account the promiscuous nature of P-gp can be applied to predict the P-gp inhibition of structurally diverse compounds that otherwise cannot be done by any other methods in a high-throughput fashion to facilitate drug discovery and development by designing drug candidates with better metabolism profile.

  4. Shifting to structures in physics and biology: a prophylactic for promiscuous realism.

    Science.gov (United States)

    French, Steven

    2011-06-01

    Within the philosophy of science, the realism debate has been revitalised by the development of forms of structural realism. These urge a shift in focus from the object oriented ontologies that come and go through the history of science to the structures that remain through theory change. Such views have typically been elaborated in the context of theories of physics and are motivated by, first of all, the presence within such theories of mathematical equations that allow straightforward representation of the relevant structures; and secondly, the implications of such theories for the individuality and identity of putative objects. My aim in this paper is to explore the possibility of extending such views to biological theories. An obvious concern is that within the context of the latter it is typically insisted that we cannot find the kinds of highly mathematised structures that structural realism can point to in physics. I shall indicate how the model-theoretic approach to theories might help allay such concerns. Furthermore, issues of identity and individuality also arise within biology. Thus Dupré has recently noted that there exists a 'General Problem of Biological Individuality' which relates to the issue of how one divides 'massively integrated and interconnected' systems into discrete components. In response Dupré advocates a form of 'Promiscuous Realism' that holds, for example, that there is no unique way of dividing the phylogenetic tree into kinds. Instead I shall urge serious consideration of those aspects of the work of Dupré and others that lean towards a structuralist interpretation. By doing so I hope to suggest possible ways in which a structuralist stance might be extended to biology. Copyright © 2010 Elsevier Ltd. All rights reserved.

  5. Vascular targeting with peptide libraries

    Energy Technology Data Exchange (ETDEWEB)

    Pasqualini, R. [La Jolla Cancer Research Center The Burnham Inst., La Jolla CA (United States)

    1999-06-01

    The authors have developed an 'in vivo' selection system in which phage capable of selective homing to different tissues are recovered from a phage display peptide library following intravenous administration. Using this strategy, they have isolate several organ and tumor-homing peptides. They have shown that each of those peptides binds of different receptors that are selectively expressed on the vasculature of the target tissue. The tumor-homing peptides bind to receptors that are up regulated in tumor angiogenic vasculature. Targeted delivery of doxorubicin to angiogenic vasculature using these peptides in animals models decrease toxicity and increased the therapeutic efficacy of the drug. Vascular targeting may facilitate the development of other treatment strategies that rely on inhibition of angio genesis and lead to advances to extend the potential for targeting of drugs, genes and radionuclides in the context of many diseases.

  6. Natriuretic peptides and cerebral hemodynamics

    DEFF Research Database (Denmark)

    Guo, Song; Barringer, Filippa; Zois, Nora Elisabeth

    2014-01-01

    Natriuretic peptides have emerged as important diagnostic and prognostic tools for cardiovascular disease. Plasma measurement of the bioactive peptides as well as precursor-derived fragments is a sensitive tool in assessing heart failure. In heart failure, the peptides are used as treatment...... in decompensated disease. In contrast, their biological effects on the cerebral hemodynamics are poorly understood. In this mini-review, we summarize the hemodynamic effects of the natriuretic peptides with a focus on the cerebral hemodynamics. In addition, we will discuss its potential implications in diseases...... where alteration of the cerebral hemodynamics plays a role such as migraine and acute brain injury including stroke. We conclude that a possible role of the peptides is feasible as evaluated from animal and in vitro studies, but more research is needed in humans to determine the precise response...

  7. Selective peptide bond hydrolysis of cysteine peptides in the presence of Ni(II) ions.

    Science.gov (United States)

    Protas, Anna Maria; Bonna, Arkadiusz; Kopera, Edyta; Bal, Wojciech

    2011-01-01

    Recently, we described a sequence-specific R1-(Ser/Thr) peptide bond hydrolysis reaction in peptides of a general sequence R1-(Ser/Thr)-Xaa-His-Zaa-R, which occurs in the presence of Ni(II) ions [A. Krężel, E. Kopera, A. M. Protas, A. Wysłouch-Cieszyńska, J. Poznański, W. Bal, J. Am. Chem. Soc. 132 (2010) 3355-3366]. In this study we explored the possibility of substituting the Ser/Thr and the His residues, necessary for the reaction to occur according to the Ni(II)-assisted acyl shift reaction mechanism, with Cys residues. We tested this concept by synthesizing three homologous peptides: R1-Ser-Arg-Cys-Trp-R2, R1-Cys-Arg-His-Trp-R2, and R1-Cys-Arg-Cys-Trp-R2, and the R1-Ser-Arg-His-Trp-R2 peptide as comparator (R1 and R2 were CH3CO-Gly-Ala and Lys-Phe-Leu-NH2, respectively). We studied their hydrolysis in the presence of Ni(II) ions, under anaerobic conditions and in the presence of TCEP as a thiol group antioxidant. We measured hydrolysis rates using HPLC and identified products of reaction using electrospray mass spectrometry. Potentiometry and UV-vis spectroscopy were used to assess Ni(II) complexation. We demonstrated that Ni(II) is not compatible with the Cys substitution of the Ser/Thr acyl acceptor residue, but the substitution of the Ni(II) binding His residue with a Cys yields a peptide susceptible to Ni(II)-related hydrolysis. The relatively high activity of the R1-Ser-Arg-Cys-Trp-R2 peptide at pH 7.0 suggests that this peptide and its Cys-containing analogs might be useful in practical applications of Ni(II)-dependent peptide bond hydrolysis. Copyright © 2010 Elsevier Inc. All rights reserved.

  8. Chemical methods for peptide and protein production.

    Science.gov (United States)

    Chandrudu, Saranya; Simerska, Pavla; Toth, Istvan

    2013-04-12

    Since the invention of solid phase synthetic methods by Merrifield in 1963, the number of research groups focusing on peptide synthesis has grown exponentially. However, the original step-by-step synthesis had limitations: the purity of the final product decreased with the number of coupling steps. After the development of Boc and Fmoc protecting groups, novel amino acid protecting groups and new techniques were introduced to provide high quality and quantity peptide products. Fragment condensation was a popular method for peptide production in the 1980s, but unfortunately the rate of racemization and reaction difficulties proved less than ideal. Kent and co-workers revolutionized peptide coupling by introducing the chemoselective reaction of unprotected peptides, called native chemical ligation. Subsequently, research has focused on the development of novel ligating techniques including the famous click reaction, ligation of peptide hydrazides, and the recently reported α-ketoacid-hydroxylamine ligations with 5-oxaproline. Several companies have been formed all over the world to prepare high quality Good Manufacturing Practice peptide products on a multi-kilogram scale. This review describes the advances in peptide chemistry including the variety of synthetic peptide methods currently available and the broad application of peptides in medicinal chemistry.

  9. Toxins and antimicrobial peptides: interactions with membranes

    Science.gov (United States)

    Schlamadinger, Diana E.; Gable, Jonathan E.; Kim, Judy E.

    2009-08-01

    The innate immunity to pathogenic invasion of organisms in the plant and animal kingdoms relies upon cationic antimicrobial peptides (AMPs) as the first line of defense. In addition to these natural peptide antibiotics, similar cationic peptides, such as the bee venom toxin melittin, act as nonspecific toxins. Molecular details of AMP and peptide toxin action are not known, but the universal function of these peptides to disrupt cell membranes of pathogenic bacteria (AMPs) or a diverse set of eukaryotes and prokaryotes (melittin) is widely accepted. Here, we have utilized spectroscopic techniques to elucidate peptide-membrane interactions of alpha-helical human and mouse AMPs of the cathelicidin family as well as the peptide toxin melittin. The activity of these natural peptides and their engineered analogs was studied on eukaryotic and prokaryotic membrane mimics consisting of resistant pathogens.

  10. Natriuretic peptides in cardiometabolic regulation and disease

    DEFF Research Database (Denmark)

    Zois, Nora E; Bartels, Emil D; Hunter, Ingrid

    2014-01-01

    decade. Dysregulation of the natriuretic peptide system has been associated with obesity, glucose intolerance, type 2 diabetes mellitus, and essential hypertension. Moreover, the natriuretic peptides have been implicated in the protection against atherosclerosis, thrombosis, and myocardial ischaemia. All...... these conditions can coexist and potentially lead to heart failure, a syndrome associated with a functional natriuretic peptide deficiency despite high circulating concentrations of immunoreactive peptides. Therefore, dysregulation of the natriuretic peptide system, a 'natriuretic handicap', might be an important...... factor in the initiation and progression of metabolic dysfunction and its accompanying cardiovascular complications. This Review provides a summary of the natriuretic peptide system and its involvement in these cardiometabolic conditions. We propose that these peptides might have an integrating role...

  11. Peptide aldehyde inhibitors of bacterial peptide deformylases.

    Science.gov (United States)

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

    1999-07-15

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

  12. Peptide-LNA oligonucleotide conjugates

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Hansen, Lykke Haastrup; Vester, Birte

    2013-01-01

    properties, peptides were introduced into oligonucleotides via a 2'-alkyne-2'-amino-LNA scaffold. Derivatives of methionine- and leucine-enkephalins were chosen as model peptides of mixed amino acid content, which were singly and doubly incorporated into LNA/DNA strands using highly efficient copper......(i)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry. DNA/RNA target binding affinity and selectivity of the resulting POCs were improved in comparison to LNA/DNA mixmers and unmodified DNA controls. This clearly demonstrates that internal attachment of peptides to oligonucleotides can significantly...

  13. Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

    Science.gov (United States)

    Sahin, Deniz; Taflan, Sevket Onur; Yartas, Gizem; Ashktorab, Hassan; Smoot, Duane T

    2018-04-25

    Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency of traditional techniques both in diagnosis and therapy of the disease makes the development of alternative and novel techniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be used to increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of this study is screening and identification of individual peptides specifically targeted to human gastric cancer cells using a phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptide sequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cells were used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide library were applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were established by enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clones after five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequencies of each amino acid in best binding clones to determine positively overexpressed amino acids for designing novel peptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific binding on MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acid frequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45 cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, but more importantly, data extracted from eluted phage clones may be used to design theoretical peptides with better binding properties than even experimentally selected ones

  14. Peptide YY receptors in the brain

    International Nuclear Information System (INIS)

    Inui, A.; Oya, M.; Okita, M.

    1988-01-01

    Radiolabelled ligand binding studies demonstrated that specific receptors for peptide YY are present in the porcine as well as the canine brains. Peptide YY was bound to brain tissue membranes via high-affinity (dissociation constant, 1.39 X 10(-10)M) and low-affinity (dissociation constant, 3.72 X 10(-8)M) components. The binding sites showed a high specificity for peptide YY and neuropeptide Y, but not for pancreatic polypeptide or structurally unrelated peptides. The specific activity of peptide YY binding was highest in the hippocampus, followed by the pituitary gland, the hypothalamus, and the amygdala of the porcine brain, this pattern being similarly observed in the canine brain. The results suggest that peptide YY and neuropeptide Y may regulate the function of these regions of the brain through interaction with a common receptor site

  15. Intracellular Signalling by C-Peptide

    Directory of Open Access Journals (Sweden)

    Claire E. Hills

    2008-01-01

    Full Text Available C-peptide, a cleavage product of the proinsulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. Recent findings demonstrate both a physiological and protective role of C-peptide when administered to individuals with type I diabetes. Data indicate that C-peptide appears to bind in nanomolar concentrations to a cell surface receptor which is most likely to be G-protein coupled. Binding of C-peptide initiates multiple cellular effects, evoking a rise in intracellular calcium, increased PI-3-kinase activity, stimulation of the Na+/K+ ATPase, increased eNOS transcription, and activation of the MAPK signalling pathway. These cell signalling effects have been studied in multiple cell types from multiple tissues. Overall these observations raise the possibility that C-peptide may serve as a potential therapeutic agent for the treatment or prevention of long-term complications associated with diabetes.

  16. A novel chimeric peptide with antimicrobial activity.

    Science.gov (United States)

    Alaybeyoglu, Begum; Akbulut, Berna Sariyar; Ozkirimli, Elif

    2015-04-01

    Beta-lactamase-mediated bacterial drug resistance exacerbates the prognosis of infectious diseases, which are sometimes treated with co-administration of beta-lactam type antibiotics and beta-lactamase inhibitors. Antimicrobial peptides are promising broad-spectrum alternatives to conventional antibiotics in this era of evolving bacterial resistance. Peptides based on the Ala46-Tyr51 beta-hairpin loop of beta-lactamase inhibitory protein (BLIP) have been previously shown to inhibit beta-lactamase. Here, our goal was to modify this peptide for improved beta-lactamase inhibition and cellular uptake. Motivated by the cell-penetrating pVEC sequence, which includes a hydrophobic stretch at its N-terminus, our approach involved the addition of LLIIL residues to the inhibitory peptide N-terminus to facilitate uptake. Activity measurements of the peptide based on the 45-53 loop of BLIP for enhanced inhibition verified that the peptide was a competitive beta-lactamase inhibitor with a K(i) value of 58 μM. Incubation of beta-lactam-resistant cells with peptide decreased the number of viable cells, while it had no effect on beta-lactamase-free cells, indicating that this peptide had antimicrobial activity via beta-lactamase inhibition. To elucidate the molecular mechanism by which this peptide moves across the membrane, steered molecular dynamics simulations were carried out. We propose that addition of hydrophobic residues to the N-terminus of the peptide affords a promising strategy in the design of novel antimicrobial peptides not only against beta-lactamase but also for other intracellular targets. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

  17. Peptides: Production, bioactivity, functionality, and applications

    DEFF Research Database (Denmark)

    Hajfathalian, Mona; Ghelichi, Sakhi; García Moreno, Pedro Jesús

    2017-01-01

    Production of peptides with various effects from proteins of different sources continues to receive academic attention. Researchers of different disciplines are putting increasing efforts to produce bioactive and functional peptides from different sources such as plants, animals, and food industry...... by-products. The aim of this review is to introduce production methods of hydrolysates and peptides and provide a comprehensive overview of their bioactivity in terms of their effects on immune, cardiovascular, nervous, and gastrointestinal systems. Moreover, functional and antioxidant properties...... of hydrolysates and isolated peptides are reviewed. Finally, industrial and commercial applications of bioactive peptides including their use in nutrition and production of pharmaceuticals and nutraceuticals are discussed....

  18. Profiling Y561-dependent and -independent substrates of CSF-1R in epithelial cells.

    Directory of Open Access Journals (Sweden)

    Melodie L Knowlton

    2010-10-01

    Full Text Available Receptor tyrosine kinases (RTKs activate multiple downstream cytosolic tyrosine kinases following ligand stimulation. SRC family kinases (SFKs, which are recruited to activated RTKs through SH2 domain interactions with RTK autophosphorylation sites, are targets of many subfamilies of RTKs. To date, there has not been a systematic analysis of the downstream substrates of such receptor-activated SFKs. Here, we conducted quantitative mass spectrometry utilizing stable isotope labeling (SILAC analysis to profile candidate SRC-substrates induced by the CSF-1R tyrosine kinase by comparing the phosphotyrosine-containing peptides from cells expressing either CSF-1R or a mutant form of this RTK that is unable to bind to SFKs. This analysis identified previously uncharacterized changes in tyrosine phosphorylation induced by CSF-1R in mammary epithelial cells as well as a set of candidate substrates dependent on SRC recruitment to CSF-1R. Many of these candidates may be direct SRC targets as the amino acids flanking the phosphorylation sites in these proteins are similar to known SRC kinase phosphorylation motifs. The putative SRC-dependent proteins include known SRC substrates as well as previously unrecognized SRC targets. The collection of substrates includes proteins involved in multiple cellular processes including cell-cell adhesion, endocytosis, and signal transduction. Analyses of phosphoproteomic data from breast and lung cancer patient samples identified a subset of the SRC-dependent phosphorylation sites as being strongly correlated with SRC activation, which represent candidate markers of SRC activation downstream of receptor tyrosine kinases in human tumors. In summary, our data reveal quantitative site-specific changes in tyrosine phosphorylation induced by CSF-1R activation in epithelial cells and identify many candidate SRC-dependent substrates phosphorylated downstream of an RTK.

  19. Chemical Methods for Peptide and Protein Production

    Directory of Open Access Journals (Sweden)

    Istvan Toth

    2013-04-01

    Full Text Available Since the invention of solid phase synthetic methods by Merrifield in 1963, the number of research groups focusing on peptide synthesis has grown exponentially. However, the original step-by-step synthesis had limitations: the purity of the final product decreased with the number of coupling steps. After the development of Boc and Fmoc protecting groups, novel amino acid protecting groups and new techniques were introduced to provide high quality and quantity peptide products. Fragment condensation was a popular method for peptide production in the 1980s, but unfortunately the rate of racemization and reaction difficulties proved less than ideal. Kent and co-workers revolutionized peptide coupling by introducing the chemoselective reaction of unprotected peptides, called native chemical ligation. Subsequently, research has focused on the development of novel ligating techniques including the famous click reaction, ligation of peptide hydrazides, and the recently reported a-ketoacid-hydroxylamine ligations with 5-oxaproline. Several companies have been formed all over the world to prepare high quality Good Manufacturing Practice peptide products on a multi-kilogram scale. This review describes the advances in peptide chemistry including the variety of synthetic peptide methods currently available and the broad application of peptides in medicinal chemistry.

  20. OK, thanks! A new mutualism between Chlamydomonas and methylobacteria facilitates growth on amino acids and peptides.

    Science.gov (United States)

    Calatrava, Victoria; Hom, Erik F Y; Llamas, Ángel; Fernández, Emilio; Galván, Aurora

    2018-04-01

    Nitrogen is a key nutrient for land plants and phytoplankton in terrestrial and aquatic ecosystems. The model alga Chlamydomonas reinhardtii can grow efficiently on several inorganic nitrogen sources (e.g. ammonium, nitrate, nitrite) as well as many amino acids. In this study, we show that Chlamydomonas is unable to use proline, hydroxyproline and peptides that contain these amino acids. However, we discovered that algal growth on these substrates is supported in association with Methylobacterium spp., and that a mutualistic carbon-nitrogen metabolic exchange between Chlamydomonas and Methylobacterium spp. is established. Specifically, the mineralization of these amino acids and peptides by Methylobacterium spp. produces ammonium that can be assimilated by Chlamydomonas, and CO2 photosynthetically fixed by Chlamydomonas yields glycerol that can be assimilated by Methylobacterium. As Chlamydomonas is an algal ancestor to land plants and Methylobacterium is a plant growth-promoting bacterium, this new model of mutualism may facilitate insights into the ecology and evolution of plant-bacterial interactions and design principles of synthetic ecology.