Method of treating endothelial dysfunction comprising administration of a thrombin peptide derivative

Endothelial dysfunction (ED) is associated with a number of diseases and disorders. Agonists of the non-proteolytically activated thrombin receptor can be used in methods to treat ED or ED-related diseases and disorders.Board of Regents, University of Texas Syste

The neuropeptide transcriptome of a model echinoderm, the sea urchin Strongylocentrotus purpuratus

The work reported here was supported by a grant from the University of London Central Research Fun

Discovery of a second SALMFamide gene in the sea urchin Strongylocentrotus purpuratus reveals that L-type and F-type SALMFamide neuropeptides coexist in an echinoderm species

NOTICE: this is the author’s version of a work that was accepted for publication in MARINE GENOMICS. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in MARINE GENOMICS, [VOL 3, ISSUE 2, (2010)] DOI: 10.1016/j.margen.2010.08.00

Regulation of Peptide Hormones.

C-Terminal amidated peptide hormones regulate numerous physiological process and are associated with many pathological conditions. Their C-terminal amidation is exclusively catalysed by the enzyme peptidylglycine alpha-amidating monooxygenase (PAM), which has two subunits: peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5). In the present study, the substrate specificity and inhibition of the PAM sourced from DMS53 human small cell lung cancer cells were investigated. A peptide consensus sequence N-Ac-Gly-(S)-Pro-Gln-(S)-Arg-(S)-Phe-Gly-COOH was constructed from the human amidated peptide hormone database, and a library of peptide substrates was designed by varying the amino acids located at the penultimate and antepenultimate positions from the C-terminus. These peptides were synthesised by solid phase peptide synthesis and studied in a competitive PHM binding assay. The results indicated that human glycine-extended hormone precursors having -Arg-Phe-Gly-COOH are likely to bind to PHM effectively in living bodies with IC50 values around twenty micro molar and those having -Asp-Phe-Gly-COOH and -Ala-Phe-Gly-COOH are likely to bind with IC50 values around seventy micro molar. The results also show that antepenultimate amino acids affect binding to PHM but to a lesser degree than the effect of penultimate amino acids. In a prodrug study, the anti-cancer drug Bexarotene was synthetically extended with a glycine to obtain a derivative that was expected to be cleaved by PAM and then deliver a cytotoxic drug. However, the derivative compound showed poor binding to PHM at the millimolar level, but had a similar cytotoxic activity against DMS53 cells to its parent agent. Unlike Bexarotene, a previous study showed that the glycine derivative of Temozolomide (another anti-cancer drug) has strong binding to PHM at micromolar concentration, but somehow does not convert to Temozolomide by PAM. One possible reason is that the glycine derivative possessing an imidazole moiety could inhibit PHM by metal chelation. To investigate this, a structurally-similar molecule N-Ac-(S)-His-Gly-COOH was tested and found to be processed by PAM and not undergo copper coordination. This implies that the glycine derivative of Temozolomide is unlikely to chelate the copper of the PHM active site. To investigate PHM and PAL activity simultaneously, a new PAM assay with a direct, rapid and sensitive HPLC-MS system was established. With this new assay the PAL inhibitor N-Ac-Phe-pyruvate and analogues were evaluated in terms of PAM inhibition through isolated enzyme competitive assays. This demonstrated that N-Ac-Phe-pyruvate is a PAL inhibitor at the nanomolar level against the medium PAM from DMS53 cells and deprotonation of its enol form results in a decrease of PAL inhibition. By using the established HPLC-MS detection system, the intermediate of the C-terminal amidation of calcitonin-Gly (CTG), alpha-hydroxyglycine-extended calcitonin (HO-CTG), was detected in the culture medium of DMS53 cells. This is the first time HO-CTG produced by cells has been detected. With and without a PAL inhibitor, the concentration of the HO-CTG in the medium sample was found to be more than two orders of magnitude lower than those of the corresponding substrate and amidated product, suggesting the PAL catalysis is much faster than PHM catalysis, and PHM catalysis is the rate-limiting step of amidation of calcitonin in human cells

Small Changes in the Primary Structure of Transportan 10 Alter the Thermodynamics and Kinetics of its Interaction with Phospholipid Vesicles

ABSTRACT: The kinetics and thermodynamics of binding of transportan 10 (tp10) and four of its variants to phospholipid vesicles, and the kinetics of peptide-induced dye efflux, were compared. Tp10 is a 21-residue, amphipathic, cationic, cell-penetrating peptide similar to helical antimicrobial peptides. The tp10 variants examined include amidated and free peptides, and replacements of tyrosine by tryptophan. Carboxy-terminal amidation or substitution of tryptophan for tyrosine enhance binding and activity. The Gibbs energies of peptide binding to membranes determined experimentally and calculated from the interfacial hydrophobicity scale are in good agreement. The Gibbs energy for insertion into the bilayer core was calculated using hydrophobicity scales of residue transfer from water to octanol and to the membrane/ water interface. Peptide-induced efflux becomes faster as the Gibbs energies for binding and insertion of the tp10 variants decrease. If anionic lipids are included, binding and efflux rate increase, as expected because all tp10 variants are cationic and an electrostatic component is added. Whether the most important effect of peptide amidation is the change in charge or an enhancement of helical structure, however, still needs to be established. Nevertheless, it is clear that the changes in efflux rate reflect the differences in the thermodynamics of binding and insertion of the free and amidated peptide groups. We have recently reported a detailed investigation (1) o

In Vitro Activities of Kissorphin, a Novel Hexapeptide KiSS-1 Derivative, in Neuronal Cells.

The primary products of the metastasis-suppressor KiSS-1 gene are the kisspeptin (KP) peptides that stimulate gonadotrophin-releasing-hormone (GnRH) release via GPR-54 receptor activation. Recent studies have suggested that the KP-10 peptide also activates neuropeptide FF (NPFF) receptors. The aim of the current study was to determine the activities of the KiSS-1 derivative kissorphin (KSO), which contains the first six amino acids of the KP-10 peptide, is C-terminally amidated, and shares amino acid similarities with the biologically active NPFF 3-8 sequence. The KSO peptide inhibited forskolin-stimulated cyclic adenosine monophosphate (cAMP) production in ND7/23 neuroblastoma cells via an action that could be inhibited by the NPFF receptor antagonist RF9. Release of GnRH by LA-N-1 neuroblastoma cells was not altered by the KSO peptide. In ND7/23 neuroblastoma cells, the KSO peptide was able to reduce forskolin neuroprotection against H(2)O(2) toxicity. The KSO peptide was also able to prevent prostaglandin E2-induced apoptosis in rat cortical neurons. The NPFF receptor antagonist RF9 could inhibit these actions of the KSO peptide in oxidative stress and apoptosis models. In conclusion, the kissorphin peptide, comprising the amino acid sequence Tyr-Asn-Trp-Asn-Ser-Phe-NH(2), has NPFF-like biological activity without showing any GnRH releasing activity and inhibits forskolin-activated cAMP release

Endothelium-mediated action of analogues of the endogenous neuropeptide kyotorphin (tyrosil-arginine) : mechanistic insights from permeation and effects on microcirculation

© 2016 American Chemical SocietyKyotorphin (KTP) is an endogenous peptide with analgesic properties when administered into the central nervous system (CNS). Its amidated form (l-Tyr-l-Arg-NH2; KTP-NH2) has improved analgesic efficacy after systemic administration, suggesting blood-brain barrier (BBB) crossing. KTP-NH2 also has anti-inflammatory action impacting on microcirculation. In this work, selected derivatives of KTP-NH2 were synthesized to improve lipophilicity and resistance to enzymatic degradation while introducing only minor changes in the chemical structure: N-terminal methylation and/or use of d amino acid residues. Intravital microscopy data show that KTP-NH2 having a d-Tyr residue, KTP-NH2-DL, efficiently decreases the number of leukocyte rolling in a murine model of inflammation induced by bacterial lipopolysaccharide (LPS): down to 46% after 30 min with 96 μM KTP-NH2-DL. The same molecule has lower ability to permeate membranes (relative permeability of 0.38) and no significant activity in a behavioral test which evaluates thermal nociception (hot-plate test). On the contrary, methylated isomers at 96 μM increase leukocyte rolling up to nearly 5-fold after 30 min, suggesting a proinflammatory activity. They have maximal ability to permeate membranes (relative permeability of 0.8) and induce long-lasting antinociception.FCT-MCTES is acknowledged for PhD fellowship SFRH/BD/52225/2013 to J.P. Marie Skłodowska-Curie Research and Innovation Staff Exchange (RISE) is acknowledged for funding: call H2020-MSCA-RISE-2014, Grant agreement 644167, 2015-2019.info:eu-repo/semantics/publishedVersio

The Evolution and Diversity of SALMFamide Neuropeptides

The SALMFamides are a family of neuropeptides that act as muscle relaxants in echinoderms. Two types of SALMFamides have been identified: L-type (e.g. the starfish neuropeptides S1 and S2) with the C-terminal motif LxFamide (x is variable) and F-type with the C-terminal motif FxFamide. In the sea urchin Strongylocentrotus purpuratus (class Echinoidea) there are two SALMFamide genes, one encoding L-type SALMFamides and a second encoding F-type SALMFamides, but hitherto it was not known if this applies to other echinoderms. Here we report the identification of SALMFamide genes in the sea cucumber Apostichopus japonicus (class Holothuroidea) and the starfish Patiria miniata (class Asteroidea). In both species there are two SALMFamide genes: one gene encoding L-type SALMFamides (e.g. S1 in P. miniata) and a second gene encoding F-type SALMFamides plus one or more L-type SALMFamides (e.g. S2-like peptide in P. miniata). Thus, the ancestry of the two SALMFamide gene types traces back to the common ancestor of echinoids, holothurians and asteroids, although it is not clear if the occurrence of L-type peptides in F-type SALMFamide precursors is an ancestral or derived character. The gene sequences also reveal a remarkable diversity of SALMFamide neuropeptides. Originally just two peptides (S1 and S2) were isolated from starfish but now we find that in P. miniata, for example, there are sixteen putative SALMFamide neuropeptides. Thus, the SALMFamides would be a good model system for experimental analysis of the physiological significance of neuropeptide "cocktails" derived from the same precursor protein

Structure and function of sea urchin neuropeptides

PhDThe subject of this thesis is the identification and functional characterization of sea urchin neuropeptides. Neuropeptides are important mediators of neural signalling in all known animals with a nervous system, including bilaterians, ctenophorans, and cnidarians. Sea urchin neuropeptides are of particular interest for three significant reasons; echinoderms have a radically different secondarily-derived pentaradial body structure, sea urchins have served as model organisms for research into embryonic development, and thirdly because the genome of a sea urchin, the purple sea urchin Strongylocentrotus purpuratus (Stimpson, 1857) has been sequenced (Sodergren et al., 2006). Only one family of neuropeptides, the SALMFamides, has previously been characterized in all classes of the phylum Echinodermata. The thesis reports the identification of putative neuropeptide GPCRs and at least seven novel sea urchin neuropeptide genes using genomic and Expressed Sequence Tag (EST) analysis. The novel sea urchin neuropeptides identified include putative homologues of vasotocin, the sea cucumber neuropeptide NGIWYamide, thyrotropin-releasing hormone, gonadotropin-releasing hormone, and calcitonin. A further three peptide precursor genes encoding peptides lacking strong homology to any known peptides were also identified and the peptides have been named GKamides and Pedal Peptide-like Neuropeptides. Two of the peptide precursor genes, those encoding peptides homologous to vasotocin and NGIWYamide, also each encode neurophysin domains, which have previously only been identified in association with vasopressin/oxytocin-like peptides. Biochemical and pharmacological techniques were employed to investigate the occurrence and functions of the putative neuropeptides identified. These included mass spectroscopy and in vitro bioassays, the former to detect the putative novel neuropeptides identified in this study and the latter to investigate bioactivity of the peptides in sea urchins. The thesis provides evidence of the neural expression and bioactivity of novel sea urchin neuropeptides and contributes to our understanding of the role of neuropeptides in echinoderm physiology and behaviour.University of London Central Research Fun