Ghrelin axis genes, peptides and receptors : recent findings and future challenges

The ghrelin axis consists of the gene products of the ghrelin gene (GHRL), and their receptors, including the classical ghrelin receptor GHSR. While it is well-known that the ghrelin gene encodes the 28 amino acid ghrelin peptide hormone, it is now also clear that the locus encodes a range of other bioactive molecules, including novel peptides and non-coding RNAs. For many of these molecules, the physiological functions and cognate receptor(s) remain to be determined. Emerging research techniques, including proteogenomics, are likely to reveal further ghrelin axis-derived molecules. Studies of the role of ghrelin axis genes, peptides and receptors, therefore, promises to be a fruitful area of basic and clinical research in years to come.

Stereochemical studies on cyclic peptides: Detailed energy minimization studies on hydrogen bonded all-trans cyclic pentapeptide backbones

Conformational studies have been carried out on hydrogenbonded all-trans cyclic pentapeptide backbone. Application of a combination of grid search and energy minimization on this system has resulted in obtaining 23 minimum energy conformations, which are characterized by unique patterns of hydrogen bonding comprising of β- and γ-turns. A study of the minimum energy conformationsvis-a-vis non-planar deviation of the peptide units reveals that non-planarity is an inherent feature in many cases. A study on conformational clustering of minimum energy conformations shows that the minimum energy conformations fall into 6 distinct conformational families. Preliminary comparison with available X-ray structures of cyclic pentapeptide indicates that only some of the minimum energy conformations have formed crystal structures. The set of minimum energy conformations worked out in the present study can form a consolidated database of prototypes for hydrogen bonded backbone and be useful for modelling cyclic pentapeptides both synthetic and bioactive in nature.

Two families of antimicrobial peptides from wasp (Vespa magnifica) venom

The hornet possesses highly toxic venom, which is rich in toxin, enzymes and biologically active peptides. Many bioactive substances were identified from wasp venom. Two families of antimicrobial peptides were purified and characterized from the venom of

Liver fluke (Fasciola hepatica)-derived peptides activate rat peritoneal mast cells

Short peptides with sequences derived from those found in the tegumental antigen of Fasciola hepatica have been synthesised. Incubation of some of these peptides with rat peritoneal mast cells resulted in the degranulation of the cells as measured by a histamine release assay. This activity was shown to be associated with the proline-lysine-proline motif, which is responsible for the induction of mast cell degranulation by the mammalian bioactive peptide substance P. Studies on the mode of action of the fluke-derived peptide indicated that it was operating through the same biochemical pathways as substance P. The implications of these findings for the development of immune responses during parasite infections are discussed. (C) 2003 Elsevier Science B.V. All rights reserved.

Nigrocin-2 peptides from Chinese Odorrana frogs – integration of UPLC/MS/MS with molecular cloning in amphibian skin peptidome analysis

Peptidomics is a powerful set of tools for the identification, structural elucidation and discovery of novel regulatory peptides and for monitoring the degradation pathways of structurally and catalytically important proteins. Amphibian skin secretions, arising from specialized granular glands, often contain complex peptidomes containing many components of entirely novel structure and unique site-substituted analogues of known peptide families. Following the discovery that the granular gland transcriptome is present in such secretions in a PCR-amenable form, we designed a strategy for peptide structural characterization involving the integration of ‘shotgun’ cloning of cDNAs encoding peptide precursors, deduction of putative bioactive peptide structures, and confirmation of these structures using tandem MS/MS sequencing. Here, we illustrate this strategy by means of elucidation of the primary structures of nigrocin-2 homologues from the defensive skin secretions of four species of Chinese Odorrana frogs, O. schmackeri, O. livida, O. hejiangensis and O. versabilis. Synthetic replicates of the peptides were found to possess antimicrobial activity. Nigrocin-2 peptides occur widely in the skin secretions of Asian ranid frogs and in those of the Odorrana group, and are particularly well-represented and of diverse structure in some species. Integration of the molecular analytical technologies described provides a means for rapid structural characterization of novel peptides from complex natural libraries in the absence of systematic online database information.

Schistosome I/Lamides - A new family of bioactive helminth neuropeptides

Here we report the identification of a new family of helminth neuropeptides with members in both nematodes and flatworms, and include preliminary cell biological and functional characterisation of one of the peptides from the trematode parasite of humans, Schistosoma mansoni. Bioinformatics and Rapid Amplification of cDNA Ends (RACE)-PCR were used to identify the completes. mansoni neuropeptide precursor gene Sm-npp-1, which encodes three pentapeptides bearing the motif (A/G)FVR(I/L).NH2. Similar peptides were identified in three other flatworm species and in 15 nematode species. Quantitative PCR (qPCR) and immunocytochemical (ICC) analyses showed that Sm-npp-1 is constitutively expressed in larval and adult worms. ICC and confocal microscopy were employed to localise one of the schistosome NPP-1 peptides (GFVRIamide) in adult worms and schistosomules; antibodies labelled a pair of neurones in the cerebral ganglia that extend posteriorly along the main nerve cords. GFVRIamide displayed no detectable co-localisation with FMRFamide-like peptides (FLPs), nor was it detectable in muscle innervation. Exogenously applied peptide had a significant inhibitory effect on the mobility of whole adult worm pairs at 10(-5) M (n = 9). Finally, we explored Sm-npp-1 function in schistosomules using RNA interference (RNAi); we successfully achieved specific knockdown of the Sm-npp-1 transcript (54.46 +/- 10.41% knockdown, n = 3), but did not detect any clear, aberrant mobility or morphological phenotypes. NPP-1-like peptides are a new family of helminth peptides with a cell-specific expression pattern distinct from FLPs and a modulatory effect on schistosome muscular activity. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Antimicrobial/cytolytic peptides from the venom of the North African scorpion, Androctonus amoreuxi: Biochemical and functional characterization of natural peptides and a single site-substituted analog

The venoms of scorpions are complex cocktails of polypeptide toxins that fall into two structural categories: those that contain cysteinyl residues with associated disulfide bridges and those that do not. As the majority of lethal toxins acting upon ion channels fall into the first category, most research has been focused there. Here we report the identification and structural characterization of two novel 18-mer antimicrobial peptides from the venom of the North African scorpion, Androctonus amoreuxi. Named AamAP1 and AamAP2, both peptides are C-terminally amidated and differ in primary structure at just two sites: Leu?Pro at position 2 and Phe?Ile at position 17. Synthetic replicates of both peptides exhibited a broad-spectrum of antimicrobial activity against a Gram-positive bacterium (Staphylococcus aureus), a Gram-negative bacterium (Escherichia coli) and a yeast (Candida albicans), at concentrations ranging between 20µM and 150µM. In this concentration range, both peptides produced significant degrees of hemolysis. A synthetic replicate of AamAP1 containing a single substitution (His?Lys) at position 8, generated a peptide (AamAP-S1) with enhanced antimicrobial potency (3-5µM) against the three test organisms and within this concentration range, hemolytic effects were negligible. In addition, this His?Lys variant exhibited potent growth inhibitory activity (ID(50) 25-40µm) against several human cancer cell lines and endothelial cells that was absent in both natural peptides. Natural bioactive peptide libraries, such as those that occur in scorpion venoms, thus constitute a unique source of novel lead compounds with drug development potential whose biological properties can be readily manipulated by simple synthetic chemical means.

Antimicrobial peptides and alytesin are co-secreted from the venom of the Midwife toad, Alytes maurus (Alytidae, Anura): Implications for the evolution of frog skin defensive secretions

The skin secretions of frogs and toads (Anura) have long been a known source of a vast abundance of bioactive substances. In the past decade, transcriptome data of the granular glands of anuran skin has given new impetus to investigations of the putative constituent peptides. Alytes obstetricans was recently investigated and novel peptides with antimicrobial activity were isolated and functionally characterised. However, genetic data for the evolutionarily ancient lineage to which Alytes belongs (midwife toads; Alytidae) remains unavailable.

Here we present the first such genetic data for Alytidae, derived via the granular gland transcriptome of a closely-related species of midwife toad, Alytes maurus. First, we present nucleotide sequences of the entire peptide precursors for four novel antimicrobial peptides (AMPs). The two precursors resemble those from Bombinatoridae in both their structural architecture and amino acid sequence. Each precursor comprises two AMPs as tandem repeats, with a member of the alyteserin-1 family (alyteserin-1Ma: GFKEVLKADLGSLVKGIAAHVAN-NH2 or alyteserin-1Mb: GFKEVLKAGLGSLVKGIPAHVAN-NH2) followed by its corresponding member from the alyteserin-2 family (alyteserin-2Ma: FIGKLISAASGLLSHL-NH2 or alyteserin-2Mb: ILGAIIPLVSGLLSHL-NH2). Synthetic replicates of the four AMPs possessed minimal inhibitory concentrations (MICs) ranging from 9.5 to 300 µM, with the most potent being alyteserin-2Ma. Second, we also cloned the cDNA encoding an alytesin precursor, with the active alytesin exhibiting high sequence identity to bombesin-related peptides from other frogs. All putative mature peptide sequences were confirmed to be present in the skin secretion via LC/MS.

The close structural resemblance of the alyteserin genes that we isolated for A. maurus with those of Bombina provide independent molecular evidence for a close evolutionary relationship between these genera as well as more support for the convergent evolution of the AMP system within anurans. In contrast to the more evolutionarily conserved nature of neuropeptides (including alytesin, which we also isolated), the more variable nature of the AMP system together with the sporadic distribution of AMPs among anuran amphibians fuels in part our hypothesis that the latter system was co-opted secondarily to fulfil a function in the innate immune system, having originally evolved for defence against potential macropredators.

Functional characterization on invertebrate and vertebrate tissues of tachykinin peptides from octopus venoms

It has been previously shown that octopus venoms contain novel tachykinin peptides that despite being isolated from an invertebrate, contain the motifs characteristic of vertebrate tachykinin peptides rather than being more like conventional invertebrate tachykinin peptides. Therefore, in this study we examined the effect of three variants of octopus venom tachykinin peptides on invertebrate and vertebrate tissues. While there were differential potencies between the three peptides, their relative effects were uniquely consistent between invertebrate and vertebrae tissue assays. The most potent form (OCT-TK-III) was not only the most anionically charged but also was the most structurally stable. These results not only reveal that the interaction of tachykinin peptides is more complex than previous structure–function theories envisioned, but also reinforce the fundamental premise that animal venoms are rich resources of novel bioactive molecules, which are useful investigational ligands and some of which may be useful as lead compounds for drug design and development.

Synthesis of unusual alpha-amino acids and study of the effect of their incorporation into antimicrobial peptides. Total synthesis of biactive marine natural products and analogues thereof

The principle theme of this thesis was the synthesis of bioactive compounds. To this end, this work was focus on two main projects. The first one, which was carried out in the Department of Chemistry of the University of Girona under the supervision of Dr Montserrat Heras, concerned the synthesis of new unnatural amino acids bearing a pyrimidine ring within their side chain for incorporation into the antimicrobial peptide BP100 following a rational design in order to improve its biological profile. On the other hand, the second chapter of this thesis was developed in collaboration with the Laboratoire de Chimie Organique (ESPCI-ParisTech, Paris, France) under the guidance of Pr Janine Cossy and Dr Arseniyadis. This chapter was centered on the total synthesis of three marine natural products with complex structures and interesting biological activities: acremolide B, (–) bitungolide F and lyngbouilloside.

Production of angiotensin converting enzyme inhibitory peptides from β-lactoglobulin and casein derived peptides: an integrative approach

Angiotensin I-converting enzyme (ACE) inhibition is one of the mechanisms by which reduction in blood pressure is exerted. Whey proteins are a rich source of ACE inhibitory peptides and have shown a blood pressure reduction effect i.e. antihypertensive activity. The aim of this work was to develop a simplified process using a combination of adsorption and microfiltration steps for the production of hydrolysates from whey with high ACE inhibitory activity and potency; the latter was measured as the IC50, which is the peptide concentration required to reduce ACE activity by half. This process integrates the selective separation of β-lactoglobulin and casein derived peptides (CDP) from rennet whey and their hydrolysis, which results in partially pure, less complex hydrolysates with high bioactive potency. Hydrolysis was carried out with protease N ‘Amano’ in a thermostatically controlled membrane reactor operated in a batch mode. By applying the integrative approach it was possible to produce from the same feedstock two different hydrolysates that exhibited high ACE inhibition. One hydrolysate was mainly composed of casein-derived peptides with IC50= 285 μg/mL. In this hydrolysate we identified the well known potent ACE-I and anti-hypertensive tri-peptide Ile-Pro-Pro (IPP) and another novel octa-peptide Gln-Asp-Lys-Thr-Glu-Ile-Pro-Thr (QDKTEIPT). The second hydrolysate was mainly composed of β-lactoglobulin derived peptides with IC50=128 µg/mL. This hydrolysate contained a tetra-peptide (Ile-Ile-Ala-Glu) IIAE as one of the two major peptides. A further advantage to this process is that enzyme activity was substantially increased as enzyme product inhibition was reduced.

Hemopressins and other hemoglobin-derived peptides in mouse brain: comparison between brain, blood, and heart peptidome and regulation in Cpefat/fat mice

P>Many hemoglobin-derived peptides are present in mouse brain, and several of these have bioactive properties including the hemopressins, a related series of peptides that bind to cannabinoid CB1 receptors. Although hemoglobin is a major component of red blood cells, it is also present in neurons and glia. To examine whether the hemoglobin-derived peptides in brain are similar to those present in blood and heart, we used a peptidomics approach involving mass spectrometry. Many hemoglobin-derived peptides are found only in brain and not in blood, whereas all hemoglobin-derived peptides found in heart were also seen in blood. Thus, it is likely that the majority of the hemoglobin-derived peptides detected in brain are produced from brain hemoglobin and not erythrocytes. We also examined if the hemopressins and other major hemoglobin-derived peptides were regulated in the Cpefat/fat mouse; previously these mice were reported to have elevated levels of several hemoglobin-derived peptides. Many, but not all of the hemoglobin-derived peptides were elevated in several brain regions of the Cpefat/fat mouse. Taken together, these findings suggest that the post-translational processing of alpha and beta hemoglobin into the hemopressins, as well as other peptides, is up-regulated in some but not all Cpefat/fat mouse brain regions.

Intracellular peptides as natural regulators of cell signaling

Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20-80 mu M) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R-CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns, several proteins involved in GPCR signaling were identified, including alpha-adaptin A and dynamin 1. These results suggest that before their complete degradation, intracellular peptides similar to those generated by proteasomes can actively affect cell signaling, probably representing additional bioactive molecules within cells.

Synthesis of Modified Amino Acids and Insertion in Peptides and Mimetics. Structural Aspects and Impact on Biological Activity.

I studied the effects exerted by the modifications on structures and biological activities of the compounds so obtained. I prepared peptide analogues containing unusual amino acids such as halogenated, alkylated (S)- or (R)-tryptophans, useful for the synthesis of mimetics of the endogenous opioid peptide endomorphin-1, or 2-oxo-1,3-oxazolidine-4-carboxylic acids, utilized as pseudo-prolines having a clear all-trans configuration of the preceding peptide bond. The latter gave access to a series of constrained peptidomimetics with potential interest in medicinal chemistry and in the field of the foldamers. In particular, I have dedicated much efforts to the preparation of cyclopentapeptides containing D-configured, alfa-, or beta-aminoacids, and also of cyclotetrapeptides including the retro-inverso modification. The conformational analyses confirmed that these cyclic compounds can be utilized as rigid scaffolds mimicking gamma- or beta-turns, allowing to generate new molecular and 3D diversity. Much work has been dedicated to the structural analysis in solution and in the receptor-bound state, fundamental for giving a rationale to the experimentally determined bioactivity, as well as for predicting the activity of virtual compounds (in silico pre-screen). The conformational analyses in solution has been done mostly by NMR (2D gCosy, Roesy, VT, molecular dynamics, etc.). A special section is dedicated to the prediction of plausible poses of the ligands when bound to the receptors by Molecular Docking. This computational method proved to be a powerful tool for the investigation of ligand-receptor interactions, and for the design of selective agonists and antagonists. Another practical use of cyclic peptidomimetics was the synthesis and biological evaluation of cyclic analogues of endomorphin-1 lacking in a protonable amino group. The studies revealed that a inverse type II beta-turn on D-Trp-Phe constituted the bioactive conformation.