Purification, conformational analysis and cytotoxic activities of host-defense peptides from the Tungara frog Engystomops pustulosus (Leptodactylidae; Leiuperinae).

The amphibian family Leptodactylidae is divided into three sub-families: Leiuperinae, Leptodactylinae, and Paratelmatobiinae. Host-defense peptides (HDPs) present in the skins of frogs belonging to the Leptodactylinae have been studied extensively, but information is limited  regarding peptides from Leiuperinae species. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Tungara frog Engystomops pustulosus (Leiuperinae) collected in Trinidad led to the isolation and structural characterization of previously undescribed pustulosin-1 (FWKADVKEIG KKLAAKLAEELAKKLGEQ), [Q28E] pustulosin-1 (pustulosin-2), and pustulosin-3 (DWKETAKELLKKIGAKVAQVISDKLNPAPQ). The primary structures of these peptides do not resemble those of previously described frog skin HDPs. In addition, the secretions contained tigerinin-1EP (GCKTYLIEPPVCT) with structural similarity to the tigerinins previously identified in skin secretions from frogs from the family Dicroglossidae. Pustulosin-1 and -3 adopted extended α-helical conformations in 25% trifluoroethanol-water and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). Pustulosin-1 and -3 displayed cytotoxic activity against a range of human tumor-derived cell lines (A549, MDA-MB-231, and HT29), but their therapeutic potential for development into anti-cancer agents is limited by their comparable cytotoxic activity against non-neoplastic human umbilical vein endothelial cells. The peptides also displayed weak antimicrobial activity against Escherichia coli (MIC = 125 µM) but were inactive against Staphylococcus aureus. Tigerinin-1EP was inactive against both the tumor-derived cells and bacteria.

Characterization of ß-turns by electronic circular dichroism spectroscopy: a coupled molecular dynamics and time-dependent density functional theory computational study.

Electronic circular dichroism is one of the most used spectroscopic techniques for peptide and protein structural characterization. However, while valuable experimental spectra exist for α-helix, ß-sheet and random coil secondary structures, previous studies showed important discrepancies for ß-turns, limiting their use as a reference for structural studies. In this paper, we simulated circular dichroism spectra for the best-characterized ß-turns in peptides, namely types I, II, I' and II'. In particular, by combining classical molecular dynamics simulations and state-of-the-art quantum time-dependent density functional theory (with the polarizable embedding multiscale model) computations, two common electronic circular dichroism patterns were found for couples of ß-turn types (namely, type I/type II' and type II/type I'), at first for a minimal di-peptide model (Ace-Ala-Ala-NHMe), but also for all sequences tested with non-aromatic residues in the central positions. On the other hand, as expected, aromatic substitution causes important perturbations to the previously found ECD patterns. Finally, by applying suitable approximations, these patterns were subsequently rationalized based on the exciton chirality rule. All these results provide useful predictions and pave the way for a possible experimental characterization of ß-turns based on circular dichroism spectroscopy.

Immunomodulatory, insulinotropic, and cytotoxic activities of phylloseptins and plasticin-TR from the Trinidanian leaf frog Phyllomedusa trinitatis.

The aim of the study was to determine the in vitro immunomodulatory, cytotoxic, and insulin-releasing activities of seven phylloseptin-TR peptides and plasticin-TR, first isolated from the frog Phyllomedusa trinitatis. The most cationic peptides, phylloseptin-1.1TR and phylloseptin-3.1TR, showed greatest cytotoxic potency against A549, MDA-MB231, and HT-29 human tumor-derived cells and against mouse erythrocytes. Phylloseptin-4TR was the most hydrophobic and the most effective peptide at inhibiting production of the proinflammatory cytokines TNF-α and IL-1ß by mouse peritoneal cells but was without effect on production of the antiinflammatory cytokine IL-10. Phylloseptin-2.1TR and phylloseptin-3.3TR were the most effective at stimulating the production of IL-10. The noncytotoxic peptide, plasticin-TR, inhibited production of TNF-α and IL-1ß but was without effect on IL-10 production. The results of CD spectroscopy suggest that the different properties of plasticin-TR compared with the immunostimulatory activities of the previously characterized plasticin-L1 from Leptodactylus laticeps may arise from greater ability of plasticin-TR to oligomerize and adopt a stable helical conformation in a membrane-mimetic environment. All peptides stimulated release of insulin from BRIN-BD11 rat clonal ß cells with phylloseptin-3.2TR being the most potent and effective and phylloseptin-2.1TR the least effective suggesting that insulinotropic potency correlates inversely with helicity. The study has provided insight into structure-activity relationships among the phylloseptins. The combination of immunomodulatory and insulinotropic activities together with low cytotoxicity suggests that phylloseptin-3.3TR and plasticin-TR may represent templates for the development of agents for use in antiinflammatory and type 2 diabetes therapies.

Revisiting absorption and electronic circular dichroism spectra of cholesterol in solution: a joint experimental and theoretical study.

Cholesterol is doubtless one of the most studied bio-molecules, which unfortunately features low emitting properties, precluding its in vivo study by fluorescence experiments. The design of fluorescent analogues of cholesterol is thus an appealing challenge in biochemistry, which simultaneously requires minor changes in its chemical structure (to retain main biological properties) and considerable enhancement of light emission. To this aim, the photochemical behaviour of the native molecule has to be deeply understood. In this work, we focused our attention on the electronic absorption of cholesterol in several common organic solutions, combining experimental (through ultraviolet-visible and electronic circular dichroism spectroscopy) and theoretical approaches (at the time-dependent density functional theory level) in order to solve the important discrepancies previously reported in the literature on the maximum absorption wavelengths and on the nature (Rydberg and/or π → π*) of the associated electronic transition.

Determination of Multimodal Isotopic Distributions: The Case of a (15)N Labeled Protein Produced into Hairy Roots.

Isotopic labeling is widely used in various fields like proteomics, metabolomics, fluxomics, as well as in NMR structural studies, but it requires an efficient determination of the isotopic enrichment. Mass spectrometry is the method of choice for such analysis. However, when complex expression systems like hairy roots are used for production, multiple populations of labeled proteins may be obtained. If the isotopic incorporation determination is actually well-known for unimodal distributions, the multimodal distributions have scarcely been investigated. Actually, only a few approaches allow the determination of the different labeled population proportions from multimodal distributions. Furthermore, they cannot be used when the number of the populations and their respective isotope ratios are unknown. The present study implements a new strategy to measure the (15)N labeled populations inside a multimodal distribution knowing only the peptide sequence and peak intensities from mass spectrometry analyses. Noteworthy, it could be applied to other elements, like carbon and hydrogen, and extended to a larger range of biomolecules.

A Joint Experimental and Theoretical Study on the Structural and Spectroscopic Properties of the Piv-Pro-d-Ser-NHMe Peptide.

In this paper, we investigate the secondary structure of the Piv-Pro-d-Ser-NHMe peptide by means of nuclear magnetic resonance (NMR) and electronic circular dichroism (ECD) experiments, in conjunction with theoretical simulations based on molecular dynamics and time-dependent density functional theory calculations including polarizable embedding to account for solvent effects. The various experimental and theoretical protocols are assessed and validated, and are shown to provide a consistent description of the turn structure adopted by this peptide in solution. In addition, a simple fitting procedure is proposed to make the simulated and experimental ECD almost perfectly match. This full methodology is finally tested on another small peptide, enlightening its efficiency and robustness.

Fluorinated pseudopeptide analogues of the neuropeptide 26RFa: synthesis, biological, and structural studies.

A series of four fluorinated dipeptide analogues each containing a fluoro-olefin moiety as peptide bond surrogate has been designed and synthesized. These motifs have been successfully introduced into the bioactive C-terminal heptapeptide of the neuropeptide 26RFa by conventional SPPS. We then evaluated the ability of the generated pseudopeptides to increase [Ca²âº](i) in GPR103-transfected cells. For these fluorinated analogues, greater stability in human serum was observed. Their conformations were also investigated, leading to the valuable identification of differences depending on the position of the fluoro-olefin moiety in the sequence.

Purification, Conformational Analysis and Cytotoxic Activities of Host-Defense Peptides from the Giant Gladiator Treefrog Boana boans (Hylidae: Hylinae).

Frogs from the extensive amphibian family Hylidae are a rich source of peptides with therapeutic potential. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Giant Gladiator Treefrog Boana boans (Hylidae: Hylinae) collected in Trinidad led to the isolation and structural characterization of five host-defense peptides with limited structural similarity to figainin 2 and picturin peptides from other frog species belonging to the genus Boana. In addition, the skin secretions contained high concentrations of tryptophyllin-BN (WRPFPFL) in both C-terminally α-amidated and non-amidated forms. Figainin 2BN (FLGVALKLGKVLG KALLPLASSLLHSQ) and picturin 1BN (GIFKDTLKKVVAAVLTTVADNIHPK) adopt α-helical conformations in trifluroethanol-water mixtures and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). The CD data also indicate contributions from turn structures. Both peptides and picturin 2BN (GLMDMLKKVGKVALT VAKSALLP) inhibited the growth of clinically relevant Gram-negative and Gram-positive bacteria with MIC values in the range 7.8-62.5 µM. Figainin 2BN was potently cytotoxic to A549, MDA-MB-231 and HT-29 human tumor-derived cells (LC50 = 7-14 µM) but displayed comparable potency against non-neoplastic HUVEC cells (LC50 = 15 µM) indicative of lack of selectivity for cancer cells.

Development of a root exudate collection protocol for metabolomics analysis using Nuclear Magnetic Resonance.

Large amounts of root exudates are released by plant roots into the soil. Due to their importance in regulating the rhizosphere properties, it is necessary to unravel the precise composition and function of exudates at the root-soil interface. However, obtaining root exudates without inducing artefacts is a difficult task. To analyse the low molecular weight molecules secreted by pea roots, a protocol of root exudate collection was developed to perform a metabolomics analysis using Nuclear Magnetic Resonance (NMR). To date a few NMR studies are dedicated to root exudates. Plant culture, exudates collection and sample preparation methods had thus to be adapted to the NMR approach. Here, pea seedlings were hydroponically grown. The obtained NMR fingerprints show that osmotic stress increases the quantity of the exudates but not their diversity. We therefore selected a protocol reducing the harvest time and using an ionic solvent and applied it to the analysis of faba bean exudates. NMR analysis of the metabolic profiles allowed to discriminate between pea and faba bean according to their exudate composition. This protocol is therefore very promising for studying the composition of root exudates from different plant species as well as their evolution in response to different environmental conditions or pathophysiological events.

Synthesis, conformational analysis and biological properties of a dicarba derivative of the antimicrobial peptide, brevinin-1BYa.

Brevinin-1BYa (FLPILASLAAKFGPKLFCLVTKKC), first isolated from skin secretions of the frog Rana boylii, displays broad-spectrum antimicrobial activity and potent haemolytic activity. This study investigates the effects on conformation and biological activity of replacement of the intramolecular disulphide bridge in the peptide by a non-reducible dicarba bond. Dicarba-brevinin-1BYa was prepared by microwave irradiation of [Agl(18),Agl(24)]-brevinin-1BYa (Agl = allylglycine) in the presence of a second generation Grubbs' catalyst. Circular dichroism spectroscopy in 50% trifluoroethanol-water indicated that the degree of α-helicity of the dicarba derivative (22%) was less than that of brevinin-1BYa (27%) but comparable to that of the acyclic derivative [Ser(18),Ser(24)]-brevinin-1BYa (23%). Dicarba-brevinin-1BYa showed a two-fold increase in potency against reference strains of Escherichia coli, Staphylococcus aureus, and Candida albicans compared with the native peptide and displayed potent bactericidal activity against clinical isolates of methicillin-resistant S. aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MIC in the range 1-8 µM). Compared with brevinin-1BYa and [Ser(18),Ser(24)]-brevinin-1BYa, the dicarba derivative was associated with increased cytotoxicity against human erythrocytes (2.5-fold), MDA-MB-231 breast carcinoma cells (1.3-fold) and HepG2 hepatoma-derived cells (1.5-fold).

Impaired SorLA maturation and trafficking as a new mechanism for SORL1 missense variants in Alzheimer disease.

The SorLA protein, encoded by the SORL1 gene, is a major player in Alzheimer's disease (AD) pathophysiology. Functional and genetic studies demonstrated that SorLA deficiency results in increased production of Aß peptides, and thus a higher risk of AD. A large number of SORL1 missense variants have been identified in AD patients, but their functional consequences remain largely undefined. Here, we identified a new pathophysiological mechanism, by which rare SORL1 missense variants identified in AD patients result in altered maturation and trafficking of the SorLA protein. An initial screening, based on the overexpression of 70 SorLA variants in HEK293 cells, revealed that 15 of them (S114R, R332W, G543E, S564G, S577P, R654W, R729W, D806N, Y934C, D1535N, D1545E, P1654L, Y1816C, W1862C, P1914S) induced a maturation and trafficking-deficient phenotype. Three of these variants (R332W, S577P, and R654W) and two maturation-competent variants (S124R and N371T) were further studied in details in CRISPR/Cas9-modified hiPSCs. When expressed at endogenous levels, the R332W, S577P, and R654W SorLA variants also showed a maturation defective profile. We further demonstrated that these variants were largely retained in the endoplasmic reticulum, resulting in a reduction in the delivery of SorLA mature protein to the plasma membrane and to the endosomal system. Importantly, expression of the R332W and R654W variants in hiPSCs was associated with a clear increase of Aß secretion, demonstrating a loss-of-function effect of these SorLA variants regarding this ultimate readout, and a direct link with AD pathophysiology. Furthermore, structural analysis of the impact of missense variants on SorLA protein suggested that impaired cellular trafficking of SorLA protein could be due to subtle variations of the protein 3D structure resulting from changes in the interatomic interactions.

Metabolic and Anti-Inflammatory Protective Properties of Human Enriched Serum Following Artichoke Leaf Extract Absorption: Results from an Innovative Ex Vivo Clinical Trial.

The aging of our population is accompanied by an increased prevalence of chronic diseases. Among those, liver, joint and adipose tissue-related pathologies have a major socio-economic impact. They share common origins as they result from a dysregulation of the inflammatory and metabolic status. Plant-derived nutrients and especially polyphenols, exert a large range of beneficial effects in the prevention of chronic diseases but require clinically validated approaches for optimized care management. In this study, we designed an innovative clinical approach considering the metabolites produced by the digestive tract following the ingestion of an artichoke leaf extract. Human serum, enriched with metabolites deriving from the extract, was collected and incubated with human hepatocytes, human primary chondrocytes and adipocytes to determine the biological activity of the extract. Changes in cellular behavior demonstrated that the artichoke leaf extract protects hepatocytes from lipotoxic stress, prevents adipocytes differentiation and hyperplasia, and exerts chondroprotective properties in an inflammatory context. These data validate the beneficial health properties of an artichoke leaf extract at the clinical level and provide both insights and further evidence that plant-derived nutrients and especially polyphenols from artichoke may represent a relevant alternative for nutritional strategies addressing chronic disease issues.

In vivo and in vitro structure-activity relationships and structural conformation of Kisspeptin-10-related peptides.

Kisspeptins, the natural ligands of the G protein-coupled receptor KISS1R, comprise a family of related peptides derived from the proteolytic processing of a common precursor encoded by the KISS1 gene. Among those, Kisspeptin-10 (Kp-10) contains the basic residues to retain full functional activity and exhibits higher receptor affinity and biopotency than longer forms of the peptide. Although kisspeptins were first characterized by their ability to inhibit tumor metastasis, recent studies have revealed that the KISS1/KISS1R system plays an essential role in the neuroendocrine control of the reproductive axis. In this context, development and functional analysis of Kp-10 analogs may help in the search for new agonists and antagonists as valuable tools to manipulate the KISS1/KISS1R system and hence fertility. We report herein functional and structural analyses of a series of Ala-substituted rat kp-10 analogs, involving [Ca(2+)](i) responses in rat kiss1r-transfected Chinese hamster ovary cells, dynamic luteinizing hormone (LH) responses in vivo, and NMR structural studies. In vitro assays revealed that Ala substitutions in positions 6 or 10 of kp-10 resulted in a significant increase in EC(50) values (>6.46 x 10(-6) M versus 1.54 to 2.6 x 10(-8) M for rat and human Kp-10, respectively) and a substantial decrease in the proportion of responsive cells coupled to a marked increase in the time required to reach maximal response. In vivo assays showed that Ala(6) substitution diminished and Ala(10) substitution eliminated LH secretory responses, whereas coadministration of each analog failed to affect the LH-releasing ability of kp-10. Molecular modeling under NMR restraints revealed that kp-10 exhibits a helicoidal structure between the Asn(4) and Tyr(10) residues, with mixed alpha- and 3(10)-helix characteristics. Ala(6) substitution induced limited destabilization of the helix around the position of the substitution. Ala(10) substitution was found to totally disrupt the helical structure in the C-terminal region of the molecule. Taken together, our results indicate that positions 6 and 10 are critical for kp-10 action at kiss1r and suggest that modifications in these positions could lead to the generation of new kisspeptin agonists and/or antagonists with altered functional and perhaps binding properties. Furthermore, they emphasize the importance of using combined, multidisciplinary approaches, including in vivo studies, to reliably evaluate structure function properties of novel kisspeptin analogs.

Insulinotropic activity of the host-defense peptide frenatin 2D: Conformational, structure-function and mechanistic studies.

Of four naturally occurring frenatin peptides tested, frenatin 2D (DLLGTLGNLPLPFI.NH2) from Discoglossus sardus was the most potent and effective in producing concentration-dependent stimulation of insulin release from BRIN-BD11 rat clonal ß-cells without displaying cytotoxicity. The peptide also stimulated insulin release from 1.1B4 human-derived clonal ß-cells and isolated mouse islets and improved glucose tolerance concomitant with increased circulating insulin concentrations in mice following intraperitoneal administration. The insulinotropic activity of frenatin 2D was not associated with membrane depolarization or an increase in intracellular [Ca2+] but incubation of the peptide (1 µM) with BRIN-BD11 cells produced a modest, but significant (P < 0.05), increase in cAMP production. Stimulation of insulin release was abolished in protein kinase A-downregulated cells but maintained in protein kinase C-downregulated cells. Circular dichroism studies showed that, in the presence of dodecylphosphocholine micelles, frenatin 2D exhibited a helical content of 35% and a turn content of 28%. Substitution of the Thr5, Asn8, Pro10, and Ile14 residues in frenatin-2D by Trp and interchange of Pro12 and Phe13 led to loss of insulinotropic activity but the [D1W] and [G7W] analogues were as potent and effective as the native peptide. Frenatin 2D (1 µM) also stimulated proliferation of BRIN-BD11 cells and provided significant protection of the cells against cytokine-induced apoptosis. It is concluded that the insulinotropic activity of frenatin 2D is mediated predominantly, if not exclusively, by the KATP channel-independent pathway.

Biological and structural analysis of truncated analogs of PACAP27.

The affinity toward the PAC1 receptor, the biological activity, and the alpha-helical content of several truncated PACAP27 analogs were measured. We first evaluated the pharmacological and structural parameters of C-terminal shortened PACAP fragments, from PACAP(1-23) to PACAP(1-19). All carboxy-truncated derivatives demonstrated circular dichroism spectra typical of a helical conformation. On the other hand, progressive shortening of the C-terminal domain gradually decreases the potency of PACAP to bind and to activate the PAC1 receptor. This decrease in biological activity was mainly attributed to the removal of residues that seem to interact directly with the receptor rather than to a destabilization of the C-terminal helical conformation. We also investigated the pharmacological and conformational characteristics of several hybrid PACAP27 derivatives containing an aliphatic molecular spacer connecting the N-terminal domain to the C-terminal region. However, this strategy revealed that none of these discontinuous analogs showed any significant affinity toward the PAC1 receptor, even if some of them exhibited circular dichroism spectra corresponding to an alpha-helical structure. This study suggests that several domains of PACAP27 are involved in the interaction with the PAC1 receptor and that the presence of the helical conformation is not a sufficient feature for receptor activation.

Solution structure of urotensin-II receptor extracellular loop III and characterization of its interaction with urotensin-II.

Urotensin-II (U-II) is a vasoactive hormone that acts through a G-protein-coupled receptor named UT. Recently, we have shown, using the surface plasmon resonance technology that human U-II (hU-II) interacts with the hUT(281-300) fragment, a segment containing the extracellular loop III (EC-III) and short extensions of the transmembrane domains VI and VII (TM-VI and TM-VII). To further investigate the interaction of UT receptor with U-II, we have determined the solution structure of hUT(281-300) by high-resolution NMR and molecular modeling and we have examined, also using NMR, the binding with hU-II at residue level. In the presence of dodecylphosphocholine micelles, hUT(281-300) exhibited a type III beta-turn (Q285-L288), followed by an -helical structure (A289-L299), the latter including a stretch of transmembrane helix VII. Upon addition of hU-II, significant chemical shift perturbations were observed for residues located just on the N-terminal side of the beta-turn (end of TM-VI/beginning of EC-III) and on one face of the -helix (end of EC-III/beginning of TM-VII). These data, in conjunction with intermolecular NOEs, suggest that the initiation site of EC-III, as well as the upstream portion of helix VII, would be involved in agonist binding and allow to propose points of interaction in the ligand-receptor complex.

Structure-activity relationships of urotensin II and URP.

Urotensin II (U-II) and urotensin II-related peptide (URP) are the endogenous ligands for the orphan G-protein-coupled receptor GPR14 now renamed UT. At the periphery, U-II and/or URP exert a wide range of biological effects on cardiovascular tissues, airway smooth muscles, kidney and endocrine glands, while central administration of U-II elicits various behavioral and cardiovascular responses. There is also evidence that U-II and/or URP may be involved in a number of pathological conditions including heart failure, atherosclerosis, renal dysfunction and diabetes. Because of the potential involvement of the urotensinergic system in various physiopathological processes, there is need for the rational design of potent and selective ligands for the UT receptor. Structure-activity relationship studies have shown that the minimal sequence required to retain full biological activity is the conserved U-II(4-11) domain, in particular the Cys5 and Cys10 residues involved in the disulfide bridge, and the Phe6, Lys8 and Tyr9 residues. Free alpha-amino group and C-terminal COOH group are not necessary for the biological activity, and modifications of these radicals may even increase the stability of the analogs. Punctual substitution of native amino acids, notably Phe6 and Trp7, by particular residues generates analogs with antagonistic properties. These studies, which provide crucial information regarding the structural and conformational requirements for ligand-receptor interactions, will be of considerable importance for the design of novel UT ligands with increased selectivity, potency and stability, that may eventually lead to the development of innovative drugs.

Solution structure of the region 51-160 of human KIN17 reveals an atypical winged helix domain.

Human KIN17 is a 45-kDa eukaryotic DNA- and RNA-binding protein that plays an important role in nuclear metabolism and in particular in the general response to genotoxics. Its amino acids sequence contains a zinc finger motif (residues 28-50) within a 30-kDa N-terminal region conserved from yeast to human, and a 15-kDa C-terminal tandem of SH3-like subdomains (residues 268-393) only found in higher eukaryotes. Here we report the solution structure of the region 51-160 of human KIN17. We show that this fragment folds into a three-alpha-helix bundle packed against a three-stranded beta-sheet. It belongs to the winged helix (WH) family. Structural comparison with analogous WH domains reveals that KIN17 WH module presents an additional and highly conserved 3(10)-helix. Moreover, KIN17 WH helix H3 is not positively charged as in classical DNA-binding WH domains. Thus, human KIN17 region 51-160 might rather be involved in protein-protein interaction through its conserved surface centered on the 3(10)-helix.

Peptidomic analysis of skin secretions of the Mexican burrowing toad Rhinophrynus dorsalis (Rhinophrynidae): Insight into the origin of host-defense peptides within the Pipidae and characterization of a proline-arginine-rich peptide.

The Mexican burrowing toad Rhinophrynus dorsalis is the sole extant representative of the Rhinophrynidae. United in the superfamily Pipoidea, the Rhinophrynidae is considered to be the sister-group to the extant Pipidae which comprises Hymenochirus, Pipa, Pseudhymenochirus and Xenopus. Cationic, α-helical host-defense peptides of the type found in Hymenochirus, Pseudhymenochirus, and Xenopus species (hymenochirins, pseudhymenochirins, magainins, and peptides related to PGLa, XPF, and CPF) were not detected in norepinephrine-stimulated skin secretions of R. dorsalis. Skin secretions of representatives of the genus Pipa also do not contain cationic α-helical host-defense peptides which suggest, as the most parsimonious hypothesis, that the ability to produce such peptides by frogs within the Pipidae family arose in the common ancestor of (Hymenochirus+Pseudhymenochirus)+Xenopus after divergence from the line of evolution leading to extant Pipa species. Peptidomic analysis of the R. dorsalis secretions led to the isolation of rhinophrynin-27, a proline-arginine-rich peptide with the primary structure ELRLPEIARPVPEVLPARLPLPALPRN, together with rhinophrynin-33 containing the C-terminal extension KMAKNQ. Rhinophrynin-27 shows limited structural similarity to the porcine multifunctional peptide PR-39 but it lacks antimicrobial and cytotoxic activities. Like PR-39, the peptide adopts a poly-l-proline helix but some changes in the circular dichroism spectrum were observed in the presence of anionic sodium dodecylsulfate micelles consistent with the stabilization of turn structures.

Structure and functions of the novel hypothalamic RFamide neuropeptides R-RFa and 26RFa in vertebrates.

A number of RFamide peptides have been characterized in invertebrate species and these peptides have been found to exert a broad spectrum of biological activities. In contrast, in vertebrates, our knowledge on RFamide peptides is far more limited and only a few members of the RFamide peptide family have been identified in various vertebrate classes during the last years. The present review focuses on two novel RFamide peptides, Rana RFamide (R-RFa) and 26RFa, that have been recently isolated from the amphibian brain. R-RFa shares the C-terminal LPLRFamide motif with other RFamide peptides previously identified in mammals, birds and fish. The distribution of R-RFa in the frog brain exhibits strong similarities with those of other LPLRFamide peptides, notably in the periventricular region of the hypothalamus. There is also evidence that the physiological functions of R-RFa and other LPLRFamide peptides have been conserved from fish to mammals; in particular, all these peptides appear to be involved in the control of pituitary hormone secretion. 26RFa does not exhibit any significant structural identity with other RFamide peptides and this peptide is the only member of the family that possesses an FRFamide motif at its C-terminus. The strong conservation of the primary structure of 26RFa from amphibians to mammals suggests that this RFamide peptide is involved in important biological functions in vertebrates. As for several other RFamide peptides, 26RFa-containing neurons are present in the hypothalamus, notably in two nuclei involved in the control of feeding behavior. Indeed, 26RFa is a potent stimulator of appetite in mammals. Concurrently, recent data suggest that 26RFa exerts various neuroendocrine regulatory activities at the pituitary and adrenal level.