Peptides (v.53, #C)
IFC (editorial board) (CO2).
Gayle & Richard Olson prize pages (III-IV).
Introduction: Invertebrate Neuropeptides XIV by Ronald J. Nachman (1-2).
A novel sea anemone peptide that inhibits acid-sensing ion channels by Armando Alexei Rodríguez; Emilio Salceda; Anoland Georgina Garateix; André Junqueira Zaharenko; Steve Peigneur; Omar López; Tirso Pons; Michael Richardson; Maylín Díaz; Yasnay Hernández; Ludger Ständker; Jan Tytgat; Enrique Soto (3-12).
Sea anemones produce ion channels peptide toxins of pharmacological and biomedical interest. However, peptides acting on ligand-gated ion channels, including acid-sensing ion channel (ASIC) toxins, remain poorly explored. PhcrTx1 is the first compound characterized from the sea anemone Phymanthus crucifer, and it constitutes a novel ASIC inhibitor. This peptide was purified by gel filtration, ion-exchange and reversed-phase chromatography followed by biological evaluation on ion channels of isolated rat dorsal root ganglia (DRG) neurons using patch clamp techniques. PhcrTx1 partially inhibited ASIC currents (IC50 ∼ 100 nM), and also voltage-gated K+ currents but the effects on the peak and on the steady state currents were lower than 20% in DRG neurons, at concentrations in the micromolar range. No significant effect was observed on Na+ voltage-gated currents in DRG neurons. The N-terminal sequencing yielded 32 amino acid residues, with a molecular mass of 3477 Da by mass spectrometry. No sequence identity to other sea anemone peptides was found. Interestingly, the bioinformatic analysis of Cys-pattern and secondary structure arrangement suggested that this peptide presents an Inhibitor Cystine Knot (ICK) scaffold, which has been found in other venomous organisms such as spider, scorpions and cone snails. Our results show that PhcrTx1 represents the first member of a new structural group of sea anemones toxins acting on ASIC and, with much lower potency, on Kv channels. Moreover, this is the first report of an ICK peptide in cnidarians, suggesting that the occurrence of this motif in venomous animals is more ancient than expected.
Keywords: ASIC; Ion channel; Sea anemone; Animal venom; Toxin; Inhibitor Cystine Knot;
Echinometrin: A novel mast cell degranulating peptide from the coelomic liquid of Echinometra lucunter sea urchin by Juliana Mozer Sciani; Marlos Cortez Sampaio; Bianca Cestari Zychar; Luis Roberto de Camargo Gonçalves; Renata Giorgi; Thiago de Oliveira Nogueira; Robson Lopes de Melo; Catarina de Fátima Pereira Teixeira; Daniel Carvalho Pimenta (13-21).
Echinometra lucunter is an abundant sea urchin found in Brazilian waters. Accidents caused by this animal are common and are characterized by the penetration of the spines in the skin, which raises an inflammatory reaction through mechanical trauma as well as by the presumable action of toxins. Additionally, there have been reports of inflammatory reaction after the consumption of raw sea urchin eggs. In this work, we have isolated a peptide from E. lucunter coelomic fluid that could elicit inflammatory reactions, such as paw edema, leukocyte recruitment and diminishment of the pain threshold. This peptide was termed Echinometrin. Moreover, the peptide administration was able to produce in vivo degranulation of mouse mast cells, in a dose–response manner. The peptide was ‘de novo’ sequenced by mass spectrometry and its synthetic analog could reproduce all the observed effects. Sequence alignment indicates that this peptide is comprised in vitellogenin, an abundant nutrient protein present in the gametogenic cells of sea urchins, making it possible that echinometrin would be a cryptide with pro-inflammatory effects.
Keywords: Sea urchin; Echinometra lucunter; Coelomic fluid; Inflammation; Peptide; Cryptide; Mast cell;
Identification, by RT-PCR, of eight novel I2-conotoxins from the worm-hunting cone snails Conus brunneus, Conus nux, and Conus princeps from the eastern Pacific (Mexico) by R. Zamora-Bustillos; R. Rivera-Reyes; M.B. Aguilar; E. Michel-Morfín; V. Landa-Jaime; A. Falcón; E.P. Heimer (22-29).
Marine snails of the genus Conus (∼500 species) are tropical predators that produce venoms for capturing prey, defense and competitive interactions. These venoms contain 50–200 different peptides (“conotoxins”) that generally comprise 7–40 amino acid residues (including 0–5 disulfide bridges), and that frequently contain diverse posttranslational modifications, some of which have been demonstrated to be important for folding, stability, and biological activity. Most conotoxins affect voltage- and ligand-gated ion channels, G protein-coupled receptors, and neurotransmitter transporters, generally with high affinity and specificity. Due to these features, several conotoxins are used as molecular tools, diagnostic agents, medicines, and models for drug design. Based on the signal sequence of their precursors, conotoxins have been classified into genetic superfamilies, whereas their molecular targets allow them to be classified into pharmacological families. The objective of this work was to identify and analyze partial cDNAs encoding precursors of conotoxins belonging to I superfamily from three vermivorous species of the Mexican Pacific coast: C. brunneus, C. nux and C. princeps. The precursors identified contain diverse numbers of amino acid residues (C. brunneus, 65 or 71; C. nux, 70; C. princeps, 72 or 73), and all include a highly conserved signal peptide, a C-terminal propeptide, and a mature toxin. All the latter have one of the typical Cys frameworks of the I-conotoxins (C-C-CC-CC-C-C). The prepropeptides belong to the I2-superfamily, and encode eight different hydrophilic and acidic mature toxins, rather similar among them, and some of which have similarity with I2-conotoxins targeting voltage- and voltage-and-calcium-gated potassium channels.
Keywords: Conus brunneus; Conus nux; Conus princeps; I-conotoxin; cDNA cloning; Conotoxin precursor;
Genomic and functional characterization of three new venom peptides from the scorpion Heterometrus spinifer by Shifen Wu; Yao Nie; Xian-Chun Zeng; Hanjun Cao; Lei Zhang; Lingli Zhou; Ye Yang; Xuesong Luo; Yichen Liu (30-41).
Three new cysteine-free venom peptides, which are referred to as Heterin-1, Heterin-2 and Spiniferin, respectively, were identified from the scorpion Heterometrus spinifer. Heterin-1, Heterin-2 and Spiniferin contain 43, 24 and 13 amino acid residues, respectively. Genomic analysis showed that the genomic organizations of the three peptides are consistent with those of the known Na+, K+ or Cl−-channel specific toxins from scorpions; this suggests that the genes of the cysteine-free and cysteine-rich peptides from scorpions were derived from a common ancestor. Antimicrobial assay demonstrated that Heterin-1 possesses potent activities against both Gram-positive and Gram-negative bacteria. Among the tested bacterial species, Heterin-1 is the most active against Bacillus megaterium and Micrococcus luteus with MICs of 4.0 μM and 4.0 μM, respectively. Heterin-2 is able to potently inhibit the growth of Gram-positive bacteria with MICs from 5.6 μM to 30.0 μM; however, it has weaker activities against the tested Gram-negative bacteria. It is interesting to see that deletion of the C-terminal random coiled tail (KKD) in Heterin-2 markedly changed the antimicrobial specificity and activity of the peptide. Spiniferin has very weak antimicrobial activities against both Gram-positive and Gram-negative bacteria. We found that introducing three net charges into the polar face of Spiniferin significantly increased its antimicrobial activity against the majority of the tested bacteria; however, in some instances, net charge on the polar face is not important for the antimicrobial activity of the peptide. These studies have expanded our understanding of the diversity, evolution and structure/function relationships of the cysteine-free peptides from scorpions.
Keywords: Antimicrobial peptide; Cysteine-free peptide; Genomic organization; Heterometrus spinifer; Scorpion; Structure/function relationship;
A K+ channel blocking peptide from the Cuban scorpion Rhopalurus garridoi by Rodolfo Rodríguez-Ravelo; Rita Restano-Cassulini; Fernando Z. Zamudio; Fredy I.V. Coronas; Georgina Espinosa-López; Lourival D. Possani (42-47).
A proteomic analysis of the venom obtained from the Cuban scorpion Rhopalurus garridoi was performed. Venom was obtained by electrical stimulation, separated by high performance liquid chromatography, and the molecular masses of their 50 protein components were identified by mass spectrometry. A peptide of 3940 Da molecular mass was obtained in pure form and its primary structure determined. It contains 37 amino acid residues, including three disulfide bridges. Electrophysiological experiments showed that this peptide is capable of blocking reversibly K+-channels hKv1.1 with a K d close to 1 μM, but is not effective against hKv1.4, hERG1 and EAG currents, at the same concentration. This is the first protein component ever isolated from this species of scorpion and was assigned the systematic number α-KTx 2.14.
Keywords: Amino acid sequence; K+-channel; Proteome; Rhopalurus garridoi; Scorpion venom;
Purification, structural elucidation and bioactivity of tryptophan containing diketopiperazines, from Comamonas testosteroni associated with a rhabditid entomopathogenic nematode against major human-pathogenic bacteria by S. Nishanth Kumar; C. Mohandas; Bala Nambisan (48-58).
The cell free culture filtrate of a Comamonas testosteroni associated with an Entomopathogenic nematode (EPN), Rhabditis (Oscheius) sp. exhibited promising antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain five diketopiperazines or cyclic dipeptides (DKP 1–5). The structure and absolute stereochemistry of the compounds were determined based on extensive spectroscopic analyses (HR-MS, 1HNMR, 13CNMR, 1H–1H COSY, 1H–13C HMBC) and Marfey's method. Based on the spectral data the compounds were identified as Cyclo-(L-Trp-L-Pro) (1), Cyclo-(L-Trp-L-Tyr) (2), Cyclo-(L-Trp-L-Ile) (3), Cyclo-(L-Trp-L-Leu) (4) and Cyclo-(L-Trp-L-Phe) (5), respectively. Three diketopiperazines (DKP 2, 3 and 5) were active against all the ten bacteria tested. The highest activity of 0.5 μg/ml by Cyclo-(L-Trp-L-Phe) was recorded against Vibrio cholerae followed by Salmonella typhi (1 μg/ml) a human pathogen responsible for life threatening diseases like profuse watery diarrhea and typhoid or enteric fever. The activity of this compound against V. cholerae and S. typhi is more effective than ciprofloxacin and ampicillin, the standard antibiotics. Cyclo-(L-Trp-L-Phe) recorded significant antibacterial activity against all the test bacteria when compared to other compounds. Five diketopiperazines were active against all the test fungi and are more effective than bavistin the standard fungicide. Diketopiperazines recorded no cytotoxicity to FS normal fibroblast and VERO cells (African green monkey kidney) except DKP 3 and 4. To our best knowledge this is the first report of antimicrobial activity of the tryptophan containing diketopiperazines against the human pathogenic microbes. The production of cyclic dipeptides by C. testosteroni is also reported here for the first time. We conclude that the C. testosteroni is promising sources of natural bioactive secondary metabolites against human pathogenic bacteria which may receive great benefit in the field of human medicine in near future.
Keywords: Comamonas testosteroni; Tryptophan; Cyclic dipeptide; Antibacterial; Vibrio cholerae;
Three bioactive cyclic dipeptides from the Bacillus sp. N strain associated with entomopathogenic nematode by Sasidharan Kumar Nishanth; Bala Nambisan; C. Dileep (59-69).
In continuation of our search for new bioactive secondary metabolites from Bacillus cereus associated with entomopathogenic nematode (EPN), three cyclic dipeptides (CDPs), cyclo(l-Leu-d-Arg) (1), cyclo(2-hydroxy-Pro-l-Leu) (2), and cyclo(l-Val-l-Pro) (3) were purified from the ethyl acetate extract of B. cereus. The chemical structure of the compounds was identified by 1D, 2D NMR and HR-ESI-MS. Cyclo(l-Leu-d-Arg) recorded best antifungal activity and the highest activity was recorded against Cryptococcus neoformans (1 μg/mL), which is better than the standard antifungal agent amphotericin B. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used for finding cell proliferation inhibition and cyclo(l-Leu-d-Arg) recorded significant activity against breast cancer cell line (MDAM-B231) (IC50 value: 25 μM) and the three cyclic dipeptides recorded no toxicity against normal human cell (fore skin (FS) normal fibroblast) up to 50 μM except cyclo(l-Val-l-Pro). Cyclo(l-Leu-d-Arg) induced significant morphological changes and DNA fragmentation associated with apoptosis in MDAM-B231 cells by acridine orange/ethidium bromide staining and flow cytometry analysis. Out of three cyclic dipeptides tested only cyclo(2-hydroxy-Pro-l-Leu) recorded significant antioxidant activity. The hydroxyl radical scavenging activity of cyclo(2-hydroxy-Pro-l-Leu) is greater than BHA, the standard antioxidant agent. Cyclo(l-Leu-d-Arg) was isolated for the first time from a natural source with a d-arginine residue. To the best of our knowledge, this is the first time that the bioactivity of the isolated cyclic dipeptides is reported against medically important fungi and cancer cells. This study is a significant contribution to the knowledge of cyclo(l-Leu-d-Arg) from B. cereus as potential sources of new drugs in the pharmacological industry, especially as potent antifungal and anticancer agent.
Keywords: Cryptococcus neoformans; Cyclo(l-Leu-d-Arg); Cytotoxicity; Apoptosis; Antioxidant;
A novel peptide inhibitor of platelet aggregation from stiff silkworm, Bombyx batryticatus by Yi Kong; Cheng Xu; Zhi-Long He; Qiu-Mei Zhou; Jin-Bin Wang; Zhi-Yu Li; Xin Ming (70-78).
A novel platelet aggregation inhibitory peptide, named BB octapeptide, was isolated from stiff silkworm (Bombyx batryticatus) by gel filtration, anion-exchange, and reverse-phase high performance liquid chromatography. The molecular mass of the peptide was determined to be 885 Da using electrospray ionization mass spectrometry, and the sequence was identified as Asp-Pro-Asp-Ala-Asp-IIe-Leu-Gln using the Edman degradation method. To test its biological activity, the peptide was chemically synthesized using Fmoc solid-phase synthesis method. BB octapeptide inhibited rabbit platelet aggregation that was induced by collagen and epinephrine, with the IC50 values of 91.14 μM and 104.50 μM, respectively. After intravenous administrated in mice (30 mg/kg, 4 days), BB octapeptide showed similar ex vivo efficacy of inhibiting platelet aggregation as aspirin (10 mg/kg). In addition, this peptide prevented paralysis and death in pulmonary thromboembolism model and significantly reduced ferric chloride-induced thrombus formation in rats. Moreover, it exhibited low cytotoxicity in a cellular model. In conclusion, this is the first report that a novel platelet aggregation inhibitory peptide was isolated from stiff silkworm (B. batryticatus). Due to the excellent efficacy in reducing platelet aggregation and low toxicity, it can be a valuable lead compound for new drug design and development.
Keywords: Bombyx batryticatus; BB octapeptide; Platelet aggregation; Pulmonary thromboembolism; Ferric chloride-induced thrombosis; Bleeding time;
A novel single-domain peptide, anti-LPS factor from prawn: Synthesis of peptide, antimicrobial properties and complete molecular characterization by Jesu Arockiaraj; Venkatesh Kumaresan; Prasanth Bhatt; Rajesh Palanisamy; Annie J. Gnanam; Mukesh Pasupuleti; Marimuthu Kasi; Mukesh Kumar Chaurasia (79-88).
In this study, we reported a complete molecular characterization including bioinformatics features, gene expression, peptide synthesis and its antimicrobial activities of an anti-lipopolysaccharide (LPS) factor (ALF) cDNA identified from the established cDNA library of freshwater prawn Macrobrachium rosenbergii (named as MrALF). The mature protein has an estimated molecular weight of 11.240 kDa with an isoelectric point of 9.46. The bioinformatics analysis showed that the MrALF contains an antimicrobial peptide (AMP) region between T54 and P77 with two conserved cysteine residues (Cys55 and Cys76) which have an anti-parallel β-sheet confirmation. The β-sheet is predicted as cationic with hydrophobic nature containing a net charge of +5. The depicted AMP region is determined to be amphipathic with a predicted hydrophobic face ‘FPVFI’. A highest MrALF gene expression was observed in hemocytes and is up-regulated with virus [white spot syndrome baculovirus (WSBV)], bacteria (Aeromonas hydrophila) and Escherichia coli LPS at various time points. The LPS binding region of MrALF peptide was synthesized to study the antimicrobial property, bactericidal efficiency and hemolytic capacity. The peptide showed antimicrobial activity against both the Gram-negative and Gram-positive bacteria. The bactericidal assay showed that the peptide recognized the LPS of bacterial cell walls and binding on its substrate and thereby efficiently distinguishing the pathogens. The hemolytic activity of MrALF peptide is functioning in a concentration dependant manner. In summary, the comprehensive analysis of MrALF showed it to be an effective antimicrobial peptide and thus it plays a crucial role in the defense mechanism of M. rosenbergii.
Keywords: Prawn; Anti-lipopolysaccharide; Antimicrobial peptide; Hemolysis; Pathogen;
Purification, characterization, and bioactivity of a new analgesic-antitumor peptide from Chinese scorpion Buthus martensii Karsch by Jian-Hua Shao; Yong Cui; Ming-Yi Zhao; Chun-Fu Wu; Yan-Feng Liu; Jing-Hai Zhang (89-96).
Scorpion venoms are complex mixtures of dozens or even hundreds of distinct proteins, many of which have diverse bioactivities. In this study, after bioassay-driven chromatographic purification, a new dual-function peptide with analgesic and antitumor activities was isolated and designated BmK AGAP-SYPU2. The first 12 amino acid residues were sequenced with Edman degradation. The cDNA was cloned by using rapid amplification of cDNA ends from the cDNA pool from scorpion glands. The amino acid sequence of BmK AGAP-SYPU2 was then deduced, and is consistent with the molecular mass measured with MALDI-TOF-MS. A preliminary pharmacological analysis revealed the following: in the dose–effect curve plotted with the mouse-twisting test, BmK AGAP-SYPU2 showed analgesic activity with an ED50 value of 1.42 mg/kg; in the time–effect curves plotted with a hot-plate procedure, BmK AGAP-SYPU2 had similar effects to those of the painkiller morphine, except for its longer duration. BmK AGAP-SYPU2 also showed antitumor activity against Ehrlich ascites tumor and S-180 fibrosarcoma models in vivo. Sequence alignment and homology modeling showed that BmK AGAP-SYPU2 is highly conserved relative to other scorpion α-toxins. However, a few different amino acids endow it with unique molecular properties, which may be responsible for its specific bioactivities. BmK AGAP-SYPU2, a new scorpion neurotoxin with dual functions, is a potential candidate drug amenable to exploitation and modification.
Keywords: Buthus martensii Karsch; BmK AGAP-SYPU2; Purification; cDNA clone; Analgesic activity; Antitumor activity;
cDNA cloning and transcript distribution of two novel members of the neuroparsin peptide family in a hemipteran insect (Nezara viridula) and a decapod crustacean (Jasus lalandii) by Heather G. Marco; Lance Anders; Gerd Gäde (97-105).
Two novel neuroparsin (NP) precursor cDNAs were cloned: one from the corpora cardiaca of an insect, the green stink bug Nezara viridula, and the other from the X-organ of a decapod crustacean, the spiny lobster Jasus lalandii. The translated NP precursor consists of 106 amino acid residues in N. viridula and 103 amino acid residues in J. lalandii, with 14 and 12 cysteine residues, respectively, in conserved positions when aligned with known NPs. Reverse transcriptase PCR shows that in both arthropods, NP is expressed in some neural tissues: corpora cardiaca, sub-esophageal ganglion and brain of N. viridula; X-organ, brain, sub-esophageal and thoracic ganglion in J. lalandii. Additionally, NP is also expressed in non-neural tissues, such as fat body, leg muscle, flight muscle, reproductive organs and antennae in N. viridula, and heart and ovary in J. lalandii. There are no major differences in the NP transcript expression in mature and immature stink bugs, and also no difference between male and female stink bugs.
Keywords: Insecta; Crustacea; Neuropeptide; Neuroparsin; Molecular cloning;
Genome-wide search and comparative genomic analysis of the trypsin inhibitor-like cysteine-rich domain-containing peptides by Xian-Chun Zeng; Yichen Liu; Wanxia Shi; Lei Zhang; Xuesong Luo; Yao Nie; Ye Yang (106-114).
It was shown that peptides containing trypsin inhibitor-like cysteine-rich (TIL) domain are able to inhibit proteinase activities, and thus play important roles in various biological processes, such as immune response and anticoagulation. However, only a limited number of the TIL peptides have been identified and characterized so far; and little has been known about the evolutionary relationships of the genes encoding the TIL peptides. BmKAPi is a TIL domain-containing peptide that was identified from Mesobuthus martensii Karsch. Here, we conducted genome-wide searches for new peptides that are homologous to BmKAPi or possess a cysteine pattern similar to that of BmKAPi. As a result, we identified a total of 80 different TIL peptides from 34 species of arthropods. We found that these peptides can be classified into seven evolutionarily distinct groups. Furthermore, we cloned the genomic sequence of BmKAPi; the genomic sequences of the majority of other TIL peptides were also identified from the GenBank database using bioinformatical approaches. Through phylogenetic and comparative genomic analysis, we found 26 cases of intron gain events occurred in the genes of the TIL peptides; however, no instances of intron loss were observed. Moreover, we found that alternative splicing contributes to the diversification of the TIL peptides. It is interesting to see that four genes of the TIL domain-containing peptides overlap in a DNA region located on the chromosome LG B15 of Bombus terretris. These data suggest that the evolution of the TIL peptide genes are dynamic, which was dominated by intron gain.
Keywords: TIL domain; Intron gain; Genomic organization; Alternative splicing; Overlapping gene; Scorpion Mesobuthus martensii Karsch; Protease inhibitor;
Molecular cloning, characterization and recombinant expression of crustacean hyperglycemic hormone in white shrimp Litopenaeus vannamei by Maoqi Liu; Luqing Pan; Li Li; Debin Zheng (115-124).
Keywords: Crustacean hyperglycemic hormone; cDNA cloning; mRNA expression; Recombinant expression, Salinity challenge, Litopenaeus vannamei;
Transcriptome analysis of neuropeptides and G-protein coupled receptors (GPCRs) for neuropeptides in the brown planthopper Nilaparvata lugens by Yoshiaki Tanaka; Yoshitaka Suetsugu; Kimiko Yamamoto; Hiroaki Noda; Tetsuro Shinoda (125-133).
The genes encoding neuropeptides, neurohormones and their putative G-protein coupled receptors were identified in the brown planthopper (BPH), Nilaparvata lugens (Stål) by transcriptome analysis (RNA-seq). Forty-eight candidate genes were found to encode neuropeptides or peptide hormones. These include all known insect neuropeptides and neurohormones, with the exception of neuropeptide-like precursor 2 (NPLP2) and trissin. The gene coding for prothoracicotropic hormone (PTTH) was first identified from hemimetabolous insect. A total of 57 putative neuropeptide GPCR genes were identified and phylogenetic analysis showed most of them to be closely related to insect GPCRs. A notable finding was the occurrence of vertebrate hormone receptors, thyrotropin-releasing hormone receptor (TRHR)-like GPCR and parathyroid hormone receptor (PTHR)-like GPCRs. These results suggest that N. lugens possesses the most comprehensive neuropeptide system yet found in insects. Moreover, our findings demonstrate the power of RNA-seq as a tool for analyzing the neuropeptide-related genes in the absence of whole genome sequence information.
Keywords: Neuropeptide; G-protein coupled receptor (GPCR); Nilaparvata lugens; Prothoracicotropic hormone (PTTH); Thyrotropin-releasing hormone receptor (TRHR); Parathyroid hormone receptor (PTHR);
Identification of the short neuropeptide F precursor in the desert locust: Evidence for an inhibitory role of sNPF in the control of feeding by Senne Dillen; Rik Verdonck; Sven Zels; Pieter Van Wielendaele; Jozef Vanden Broeck (134-139).
Peptides of the short neuropeptide F (sNPF) family have been shown to modulate feeding behavior in a wide variety of insect species. While these peptides stimulate feeding and food-searching behavior in Drosophila melanogaster and Apis mellifera, an opposite effect has recently been demonstrated in the desert locust, Schistocerca gregaria. In this study, we elaborate on these observations with the identification of the nucleotide sequence encoding the Schgr-sNPF precursor and the study of its role in the regulation of locust feeding behavior. We confirm that both Schgr-sNPF-like peptides, previously identified in mass spectrometric studies, are genuine precursor-encoded peptides. RNA interference mediated silencing of the Schgr-sNPF precursor transcript generates novel evidence for an inhibitory role of Schgr-sNPF in the regulation of feeding in S. gregaria. Furthermore, we show that starvation reduces the Schgr-sNPF precursor transcript level in the optic lobes, the primary visual centers of the locust brain. Our data indicate that Schgr-sNPF exerts an inhibitory effect on food uptake in the desert locust, which contrasts with effects of sNPF reported for several other insect species.
Keywords: sNPF; Short neuropeptide F; Precursor; Feeding; Locust; Neuropeptide; Insect; Satiety; Schistocerca;
The female reproductive system of the kissing bug, Rhodnius prolixus: Arrangements of muscles, distribution and myoactivity of two endogenous FMRFamide-like peptides by Laura Sedra; Angela B. Lange (140-147).
Phalloidin staining F-actin was used to image muscle fiber arrangements present in the reproductive system of the adult female Rhodnius prolixus. A mesh of muscle fibers encircles the ovaries whereas a criss-cross pattern of finer muscle fibers covers each ovariole. Two layers of muscle fibers (arranged longitudinally and circularly) form the lateral oviducts. The circular layer of muscle fibers extends throughout the common oviduct and spermathecae. A chevron pattern of thicker muscle fibers makes up the bursa. All of these structures show spontaneous contractions that are stimulated in a dose-dependent manner by the endogenous peptides, GNDNFMRFamide and AKDNFIRFamide which belong to the family of the FMRFamide-like peptides (FLP). Immunohistochemistry shows that these peptides could be supplied via nerves to the oviducts, spermathecae and bursa. Although no FMRF-like immunoreactivity was observed on the ovarioles/ovary they still exhibited a stimulatory response to the peptides indicating that they may be under the influence of FLPs as neurohormones. This work implicates FLPs in the control of ovulation, egg movement and oviposition in this insect.
Keywords: Insect; Neuropeptides; Immunohistochemistry; Reproductive system; Muscle contraction; F-actin;
The molecular characterization of the kinin transcript and the physiological effects of kinins in the blood-gorging insect, Rhodnius prolixus by Garima Bhatt; Rosa da Silva; Ronald J. Nachman; Ian Orchard (148-158).
The dramatic feeding-related activities of the Chagas’ disease vector, Rhodnius prolixus are under the neurohormonal regulation of serotonin and various neuropeptides. One such family of neuropeptides, the insect kinins, possess diuretic, digestive and myotropic activities in many insects. In this study, we have cloned and examined the spatial expression of the R. prolixus kinin (Rhopr-kinin) transcript. In addition, in situ hybridization has been used to map the distribution of neurons expressing the kinin transcript. Physiological bioassays demonstrate the myostimulatory effects of selected Rhopr-kinin peptides and also illustrate the augmented responses of hindgut contractions to co-application of Rhopr-kinin and a R. prolixus diuretic hormone. Two synthetic kinin analogs have also been examined on the hindgut. These reveal interesting properties including a relatively irreversible effect on hindgut contractions and activity at very low concentrations.
Keywords: Insect; Neuropeptide; In situ hybridization; Kinin analogs; Hindgut;
Molecular characterization and possible biological roles of allatotropin in Rhodnius prolixus by Maryam Masood; Ian Orchard (159-171).
Allatotropins (ATs) are a family of neuropeptides that have been shown to stimulate the biosynthesis of juvenile hormone in certain insect species, and to have stimulatory activity on some visceral muscles. Here, we have examined the AT in Rhodnius prolixus. Molecular analysis revealed a cDNA fragment of 973 bp encoding one mature amidated AT (Rhopr-AT) with transcript levels observed in the central nervous system (CNS) and pool of fat body, trachea and associated peripheral nerves. AT-like immunoreactive neurons were found throughout the CNS and AT-like immunoreactive processes were present on some peripheral tissues. Bioassays based upon changes in hindgut and dorsal vessel contractions failed to demonstrate any myotropic effects of Rhopr-AT on these tissues; however Rhopr-AT stimulated contractions of muscles surrounding the salivary glands and secretion of saliva, as judged by the reduction in content of the cherry red saliva from the salivary glands. Serotonin stimulated an increase in peristaltic contractions of the gland though no secretion was observed. Co-application of Rhopr-AT and serotonin resulted in a more rapid secretion than either chemical alone.
Keywords: Insect; Immunohistochemistry; Gene transcript; Dorsal vessel; Hindgut; Salivary gland; Serotonin;
Functional activity of allatotropin and allatostatin in the pupal stage of a holometablous insect, Tribolium castaneum (Coleoptera, Tenebrionidae) by Mohatmed Abdel-latief; Klaus H. Hoffmann (172-184).
Allatotropin (AT) and allatostatin (AS) neuropeptides are known to regulate the biosynthesis of juvenile hormones (JH) in insects. Furthermore, they possess myoregulatory and other activities in a wide range of insect species. The genome of Tribolium castaneum encodes two AS and one AT precursors. Here we cloned the cDNAs of the precursors, followed their expression patterns during the pupal stage, and established their putative roles in adult development and oviposition of the females using RNA interference (RNAi). Cloning of the cDNA and gene structure analyses of the Tc-AT gene confirmed that the gene is expressed in three mRNA isoforms. Real-time PCR data demonstrate that the Tc-AT isoforms and the AS genes, Tc-AS C and Tc-AS B, are expressed in discerning developmental and tissue-specific patterns. Single injections of dsRNAi (targeted against the Tc-AT, Tc-AS C, and Tc-AS B, respectively), into young pupae resulted in abnormal adult phenotypes, whereby about half of the animals (P1 phenotype) looked relatively normal, but the females laid low numbers of eggs. The other halves (P2) exhibited strong developmental defects with abnormal duration of the pupal stage, abnormal head and body sizes, short elytra, and incomplete sclerotization. Moreover, these females deposited no eggs and died within one week after emergence. Individual silencing of the Tc-AT mRNA isoforms showed that Tc-AT3 had the most disruptive influence on adult development and fecundity of the females. Our findings clearly indicate a significant role of AT and AS neuropeptides in the pupa. The distinct mechanisms of action, however, remain to be determined.
Keywords: Coleoptera; Neuropeptides; Pupal stage; Metamorphosis; Oviposition; RNA interference;
Functional characterization of naturally occurring melittin peptide isoforms in two honey bee species, Apis mellifera and Apis cerana by Doori Park; Je Won Jung; Mi Ok Lee; Si Young Lee; Boyun Kim; Hye Jun Jin; Jiyoung Kim; Young-Joon Ahn; Ki Won Lee; Yong Sang Song; Seunghun Hong; James E. Womack; Hyung Wook Kwon (185-193).
Insect-derived antimicrobial peptides (AMPs) have diverse effects on antimicrobial properties and pharmacological activities such as anti-inflammation and anticancer properties. Naturally occurring genetic polymorphism have a direct and/or indirect influence on pharmacological effect of AMPs, therefore information on single nucleotide polymorphism (SNP) occurring in natural AMPs provides an important clue to therapeutic applications. Here we identified nucleotide polymorphisms in melittin gene of honey bee populations, which is one of the potent AMP in bee venoms. We found that the novel SNP of melittin gene exists in these two honey bee species, Apis mellifera and Apis cerana. Nine polymorphisms were identified within the coding region of the melittin gene, of which one polymorphism that resulted in serine (Ser) to asparagine (Asp) substitution that can potentially effect on biological activities of melittin peptide. Serine-substituted melittin (Mel-S) showed more cytotoxic effect than asparagine-substituted melittin (Mel-N) against E. coli. Also, Mel-N and Mel-S had different inhibitory effects on the production of inflammatory factors such as IL-6 and TNF-α in BV-2 cells. Moreover, Mel-S showed stronger cytotoxic activities than Mel-N peptide against two human ovarian cancer cell lines. Using carbon nanotube-based transistor, we here characterized that Mel-S interacted with small unilamellar liposomes more strongly than Mel-N. Taken together, our present study demonstrates that there exist different characteristics of the gene frequency and the biological activities of the melittin peptide in two honey bee species, Apis mellifera and A. cerana.
Keywords: Antimicrobial peptide (AMP); Honey bee; Single nucleotide polymorphism (SNP); Melittin; Anti-inflammatory effect; Anti-cancer activity;
Studies on the role of insect hemolymph polypeptides: Galleria mellonella anionic peptide 2 and lysozyme by Aneta Sowa-Jasiłek; Agnieszka Zdybicka-Barabas; Sylwia Stączek; Jerzy Wydrych; Paweł Mak; Teresa Jakubowicz; Małgorzata Cytryńska (194-201).
The lysozymes are well known antimicrobial polypeptides exhibiting antibacterial and antifungal activities. Their antibacterial potential is related to muramidase activity and non-enzymatic activity resembling the mode of action of cationic defense peptides. However, the mechanisms responsible for fungistatic and/or fungicidal activity of lysozyme are still not clear. In the present study, the anti-Candida albicans activity of Galleria mellonella lysozyme and anionic peptide 2 (AP2), defense factors constitutively present in the hemolymph, was examined. The lysozyme inhibited C. albicans growth in a dose-dependent manner. The decrease in the C. albicans survival rate caused by the lysozyme was accompanied by a considerable reduction of the fungus metabolic activity, as revealed by LIVE/DEAD staining. In contrast, although AP2 reduced C. albicans metabolic activity, it did not influence its survival rate. Our results suggest fungicidal action of G. mellonella lysozyme and fungistatic activity of AP2 toward C. albicans cells. In the presence of AP2, the anti-C. albicans activity of G. mellonella lysozyme increased. Moreover, when the fungus was incubated with both defense factors, true hyphae were observed besides pseudohyphae and yeast-like C. albicans cells. Atomic force microscopy analysis of the cells exposed to the lysozyme and/or AP2 revealed alterations in the cell surface topography and properties in comparison with the control cells. The results indicate synergistic action of G. mellonella AP2 and lysozyme toward C. albicans. The presence of both factors in the hemolymph of naive larvae suggests their important role in the early stages of immune response against fungi in G. mellonella.
Keywords: Anionic peptide 2; Lysozyme; Galleria mellonella; Candida albicans; Atomic force microscopy; Synergy;
Mitochondrial inactivation by Anopheles albimanus cecropin 3: Molecular mechanisms by Natalia Pavón; Mabel Buelna-Chontal; Luz Hernández-Esquivel; Sauri Hernández; Edmundo Chávez; Renaud Condé; Humberto Lanz-Mendoza (202-209).
Cecropin 3 (Ccrp3) is an antimicrobial peptide from Anopheles albimanus, which is expressed during Plasmodium berghei infection. Here, we report that synthetic Ccrp3, aside from antibacterial activity, also shows cardio regulatory functions. In rats, Ccrp3 significantly diminishes blood pressure as well as the heartbeat frequency at nanomolar concentration. Ccrp3 affect the rat cardiac muscle mitochondria, inducing uncoupling of oxidative phosphorylation, oxygen consumption and transport of Ca2. Ccrp3 treatment of the mitochondria causes mitochondrial damage promoting oxidative stress, causing overproduction of reactive oxygen species (ROS) and inhibition of superoxide dismutase. At nM concentration, Ccrp3 inhibits superoxide dismutase activity through direct interaction, diminishing by its enzymatic activity. Ccrp3 induces the release of the pro-apoptotic marker Bax from the mitochondria. Altogether, these results suggest that Ccrp3 pro-oxidative activity on cardiac muscle mitochondria could be responsible for triggering the heartbeat frequency and blood pressure lowering observed the Ccrp3 injected rats.
Keywords: Cecropin 3; Anopheles albimanus; Mitochondria; Oxidative stress; Blood pressure; Cardiomyocyte;
Plasmodium falciparum rhoptry neck protein 5 peptides bind to human red blood cells and inhibit parasite invasion by Hernando Curtidor; Liliana C. Patiño; Gabriela Arévalo-Pinzón; Magnolia Vanegas; Manuel E. Patarroyo; Manuel A. Patarroyo (210-217).
Plasmodium falciparum malaria parasite invasion of erythrocytes is an essential step in host infection and the proteins involved in such invasion are the main target in developing an antimalarial vaccine. Secretory organelle-derived proteins (micronemal AMA1 protein and the RON2, 4, and 5 rhoptry neck proteins) have been recently described as components of moving junction complex formation allowing merozoites to move into a newly created parasitophorous vacuole. This study led to identifying RON5 regions involved in binding to human erythrocytes by using a highly robust, sensitive and specific receptor–ligand interaction assay; it is further shown that the RON5 protein remains highly conserved throughout different parasite strains. It is shown that the binding peptide–erythrocyte interaction is saturable and sensitive to chymotrypsin and trypsin. Invasion inhibition assays using erythrocyte binding peptides showed that the RON5–erythrocyte interaction could be critical for merozoite invasion of erythrocytes. This work provides evidence (for the first time) suggesting a fundamental role for RON5 in erythrocyte invasion.
Keywords: Malaria; Merozoite; Rhoptry neck; Synthetic peptide; Binding;
The D. melanogaster capa-1 neuropeptide activates renal NF-kB signaling by Selim Terhzaz; Gayle Overend; Sujith Sebastian; Julian A.T. Dow; Shireen-A. Davies (218-224).
The capa peptide family exists in a very wide range of insects including species of medical, veterinary and agricultural importance. Capa peptides act via a cognate G-protein coupled receptor (capaR) and have a diuretic action on the Malpighian tubules of Dipteran and Lepidopteran species. Capa signaling is critical for fluid homeostasis and has been associated with desiccation tolerance in the fly, Drosophila melanogaster. The mode of capa signaling is highly complex, affecting calcium, nitric oxide and cyclic GMP pathways. Such complex physiological regulation by cell signaling pathways may occur ultimately for optimal organismal stress tolerance to multiple stressors. Here we show that D. melanogaster capa-1 (Drome-capa-1) acts via the Nuclear Factor kappa B (NF-kB) stress signaling network. Human PCR gene arrays of capaR-transfected Human Embryonic Kidney (HEK) 293 cells showed that Drome-capa-1 increases expression of NF-kB, NF-kB regulated genes including IL8, TNF and PTGS2, and NF-kB pathway-associated transcription factors i.e. EGR1, FOS, cJUN. Furthermore, desiccated HEK293 cells show increased EGR1, EGR3 and PTGS2 – but not IL8, expression. CapaR-transfected NF-kB reporter cells showed that Drome-capa-1 increased NF-kB promoter activity via increased calcium. In Malpighian tubules, both Drome-capa-1 stimulation and desiccation result in increased gene expression of the D. melanogaster NF-kB orthologue, Relish; as well as EGR-like stripe and klumpfuss. Drome-capa-1 also induces Relish translocation in tubule principal cells. Targeted knockdown of Relish in only tubule principal cells reduces desiccation stress tolerance of adult flies. Together, these data suggest that Drome-capa-1 acts in desiccation stress tolerance, by activating NF-kB signaling.
Keywords: Neuroendocrine; Capa; Malpighian tubules; D. melanogaster; NF-kB; Relish; Desiccation; Renal;
Analysis of interaction of phenolic compounds with the cholecystokinin signaling pathway to explain effects on reducing food intake by Nadin Al Shukor; Katleen Raes; John Van Camp; Guy Smagghe (225-231).
Previous animal experiments demonstrated that phenolic compounds can reduce weight and food intake, but the exact mechanism(s) behind these effects remain unknown. For regulation of food intake, the cholecystokinin (CCK) hormone signaling pathway plays an important role as it induces satiety by binding on its specific receptor (CCK1R), hereby reducing food intake. In this study, we investigated the possible interactions of eight phenolic compounds of different classes (tannic acid, gallic acid, benzoic acid, hydroxybenzoic acid, protocatechuic acid, quercetin, kaempferol and resveratrol) with the CCK1R signaling pathway. As major results, the tested phenolic compounds could not activate the CCK1R in a specific cell-based bioassay. In contrast, we observed an anti-CCK1R activity. This antagonistic action might be explained by blocking of the functioning of the CCK1R receptor, although the exact mechanism of interaction remains unknown. For tannic acid, we also measured a sequestration activity of the CCK hormone in vitro. In conclusion, the reported activity of phenolic compounds against food intake and weight is not based on an activation of the CCK1R. Taking into account the complex regulation of food intake, further work is necessary to unravel other essential mechanisms involved to explain the reported effects of phenolic compounds against food intake.
Keywords: Cholecystokinin; Cholecystokinin type 1 receptor; Phenolic compounds;
Characterization of sulfakinin receptor 2 and its role in food intake in the red flour beetle, Tribolium castaneum by Na Yu; Guy Smagghe (232-237).
Insects acquire essential nutrients from their food to support a diverse range of biological processes such as development and reproduction. An important role in the food intake regulation is attributed to the neurohormone sulfakinin signaling pathway. Sulfakinins (SKs) elicit satiety together with sulfakinin receptors (SKRs). In this project, the gene coding a second putative SKR, namely skr2, was cloned, characterized and functionally studied in the red flour beetle Tribolium castaneum, with the purpose to understand its role in food intake regulation. The gene skr2 encoded a seven-transmembrane SKR2 protein with 420 amino acids and is evolutionarily close to the two SKRs in Drosophila. A distribution analysis by means of quantitative real-time PCR revealed a tissue- and stage-specific expression pattern with skr2 being dominantly expressed in head, and in the pupal and adult stages. Besides, its expression in gut was notable. With use of dsRNA of skr2, larval food intake was stimulated in an injection RNAi assay. Based on the data obtained, skr2 is considered as an indispensable component in the regulation of food intake in T. castaneum.
Keywords: Sulfakinin; Sulfakinin receptor; Tribolium castaneum; Food intake; RNA interference;
Development of cell-based bioassay with Sf9 cells expressing TcSKR1 and TcSKR2 and differential activation by sulfated and non-sulfated SK peptides by Na Yu; Luc Swevers; Ronald J. Nachman; Guy Smagghe (238-242).
Insect sulfakinin receptors (SKRs) are G-protein-coupled receptors (GPCRs) that interact with sulfakinins (SKs) to modulate diverse biological processes. One of the indispensable roles of SKs is in the regulation of food intake in insects. In this project we report on the development of a cell-based receptor assay system with insect Sf9 cells, expressing TcSKR1 and TcSKR2 from the red flour beetle Tribolium castaneum, a model and important pest insect in agriculture. In this system, a stable presence of the two TcSKRs was supported by Western blotting. The expressed TcSKRs were coupled to Gαs-protein upon activation and stimulated cAMP accumulation in Sf9 cells. Exposure of the transfected cell lines to sulfated SK (sSK) activated TcSKR1 at 1 nM; the EC50 of sSK to obtain 50% of receptor activation was similar for both receptors. In contrast, μM concentrations of non-sulfated SK were necessary to activate both TcSKRs. In conclusion, this cell-based TcSKR assay system is useful to screen SK-related peptides and mimetics and to better document ligand-receptor structure-activity relationships. Given the importance of SK signaling system in insects, the present study may provide new insights on the development of new methods to control pest insects.
Keywords: Sulfakinin; Sulfakinin receptor; G-protein-coupled receptor; Cell-based assay; Gαs-protein;
Molecular cloning and functional characterization of the diapause hormone receptor in the corn earworm Helicoverpa zea by Hongbo Jiang; Zhaojun Wei; Ronald J. Nachman; Yoonseong Park (243-249).
The diapause hormone (DH) in the heliothine moth has shown its activity in termination of pupal diapause, while the orthology in the silkworm is known to induce embryonic diapause. In the current study, we cloned the diapause hormone receptor from the corn earworm Helicoverpa zea (HzDHr) and tested its ligand specificities in a heterologous reporter system. HzDHr was expressed in Chinese Hamster Ovary (CHO) cells, which were co-transfected with the aequorin reporter, and was used to measure the ligand activities. A total of 68 chemicals, including natural DH analogs and structurally similar peptide mimetics, were tested for agonistic and antagonistic activities. Several peptide mimetics with a 2-amino-7-bromofluorene-succinoyl (2Abf-Suc) N-terminal modification showed strong agonistic activities; these mimetics included 2Abf-Suc-F[dA]PRLamide, 2Abf-Suc-F[dR]PRLamide, 2Abf-Suc-FKPRLamide and 2Abf-Suc-FGPRLamide. Antagonistic activity was found in the ecdysis triggering hormone in Drosophila melanogaster (FFLKITKNVPRLamide). Interestingly, HzDHr does not discriminate between DH (WFGPRLamide C-terminal motif) and another closely related endogenous peptide, pyrokinin 1 (FXPRXamide; a C-terminal motif that is separate from WFGPRLamide). We provide large-scale in vitro data that serve as a reference for the development of agonists and antagonists to disrupt the DH signaling pathway.
Keywords: GPCR; Neuropeptide; Insect; Peptidomimetic;
Molecular identification and expression analysis of a diapause hormone receptor in the corn earworm, Helicoverpa zea by Qirui Zhang; Peter M. Piermarini; Ronald J. Nachman; David L. Denlinger (250-257).
Diapause hormone (DH) is an insect neuropeptide that is highly effective in terminating the overwintering pupal diapause in members of the Helicoverpa/Heliothis complex of agricultural pests, thus DH and related compounds have promise as tools for pest management. To augment our development of effective DH analogs and antagonists that could be used as diapause disruptors this study focuses on the cloning and identification of the DH receptor (DHR) in the corn earworm, Helicoverpa zea. The full-length dhr cDNA contains 2153 nucleotides encoding 511 amino acids. Our results suggest there are at least two splicing variants of Hezea-DHR. Hydrophobicity analysis and sequence alignment indicate that Hezea-DHR has 7 transmembrane regions and a highly conserved C-terminal region that is also present in related receptors. Hezea-DHR has 95%, 82% and 79% identity to a partial DHR sequence from Heliothis virescens, a full-length DHR in Orgyia thyellina, and DHR-1 in Bombyx mori, but only 45–49% identity to pheromone biosynthesis activating neuropeptide receptor (PBANR). Expression of dhr mRNA remained low in whole body extracts throughout diapause and in young nondiapausing pupae, but was distinctly elevated as development ensued in pharate adults 7 days after pupation. The highest expression of dhr mRNA we noted was in the ovary. A DHR fusion protein with enhanced-green fluorescent protein was successfully expressed heterologously in X. laevis oocytes, as verified by fluorescent imaging and Western blots, but an electrophysiological assay failed to detect receptor–ligand binding activity, which suggests that an essential cofactor and/or accessory protein is required for functional activity of the DHR.
Keywords: Diapause hormone; Receptor; Xenopus oocytes; Analogs; Diapause termination;
The degradome and the evolution of Drosophila sex peptide as a ligand for the MIP receptor by R. Elwyn Isaac; Young-Joon Kim; Neil Audsley (258-264).
The male sex peptide (SP) of Drosophila melanogaster has wide ranging effects on females, including rejection of courting males, increased egg production, changes to the feeding habit, increased synthesis of antimicrobial peptides and elevated locomotor activity during day-time. The peptide activates receptors in sensory neurons of the female reproductive tract and can also traverse into the hemolymph and reach the central nervous system. The SP receptor involved in rejection and egg-laying responses has been shown to be identical to the receptor for the evolutionary conserved myoinhibitory peptides (MIPs) that function as neuropeptides in both males and females. Intriguingly, MIPs cannot substitute for SP when either expressed in the male accessory glands or injected into virgin females. MIPs are linear peptides with an amidated C-terminus which protects them from cleavage by carboxypeptidases, but leaves them exposed to potential attack from aminopeptidase and endopeptidase activities. In contrast, the SP region responsible for eliciting the post-mating response is cyclic and has several hydroxyproline residues N-terminal to the disulfide bridge which is expected to protect the biological activity of SP from peptidases of the male accessory gland and seminal fluid. We now present in vitro data showing that SP is metabolically stable, whereas MIPs are much more susceptible to degradation by peptidases of the male accessory gland and the hemolymph of virgin female D. melanogaster. SP has evolved relatively recently as a MIP receptor ligand that is particularly well adapted to surviving in the hostile degradome of the male accessory gland and seminal fluid.
Keywords: Sex peptide; Myoinhibitory peptide; Drosophila melanogaster; Peptidase; Male accessory glands; Seminal fluid;
Human β-defensin HBD3 binds to immobilized Bla g2 from the German cockroach (Blattella germanica) by Deborah E. Dietrich; Aaron D. Martin; Kim A. Brogden (265-269).
Human β-defensin 3 (HBD3) is a small, well-characterized peptide in mucosal secretions with broad antimicrobial activities and diverse innate immune functions. Among these functions is the ability of HBD3 to bind to antigens. In this study, we hypothesize that HBD3 binds to the allergen Bla g2 from the German cockroach (Blattella germanica). The ability of HBD1 (used as a control β-defensin) and HBD3 to bind to Bla g2 and human serum albumin (HSA, used as a control ligand) was assessed using the SensíQ Pioneer surface plasmon resonance (SPR) spectroscopy biosensor system. HBD1 was observed to bind weakly to Bla g2, while HBD3 demonstrated a stronger affinity for the allergen. HBD3 was assessed under two buffer conditions using 0.15 M and 0.3 M NaCl to control the electrostatic attraction of the peptide to the chip surface. The apparent K D of HBD3 binding Bla g2 was 5.9 ± 2.1 μM and for binding HSA was 4.2 ± 0.7 μM, respectively. Thus, HBD3, found in mucosal secretions has the ability to bind to allergens like Bla g2 possibly by electrostatic interaction, and may alter the ability of Bla g2 to induce localized allergic and/or inflammatory mucosal responses.
Keywords: Human β-defensin; HBD3; Allergen; Bla g2; Surface plasmon resonance spectroscopy;
Structural studies of adipokinetic hormones in water and DPC micelle solution using NMR distance restrained molecular dynamics by Graham E. Jackson; Riedaa Gamieldien; Grace Mugumbate; Gerd Gäde (270-277).
Melme-CC (pGlu-Leu-Asn-Tyr-Ser-Pro-Asp-Trp amide) and Declu-CC (pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp-Gly-Asn amide) are members of the insect adipokinetic hormone family with very different activities in the locust bioassay. The conformations of both peptides were determined in water and in a phospholipid (DPC) micelle solution using nuclear magnetic resonance (NMR) restrained molecular dynamics simulations.In water, Melme-CC has one dominant conformation while in DPC solution it has two preferred conformation. In water, Declu-CC has two conformations but in DPC solution it has one preferred conformation, which is similar to one of the water conformations. All the conformations have type IV β-turn between residues 4 and 7.The binding of the two peptides to the DPC micelle is different. Melme-CC does not bind strongly to the surface and is oriented with the β-turn facing the surface. Declu-CC interacts more strongly with the β-turn facing away from the surface. Both termini having hydrophobic interactions with the surface. In Declu-CC the side chain of Asn7 projects away from the chain while in Melme-CC the Asp7 side chain is folded inside the chain. The different orientation of these side chains may account for the much higher biological activity of Declu-CC in mobilizing lipids in the locust compared to the poor biological effect of Melme-CC in this bioassay. Receptor binding of Declu-CC was tested using a model AKH receptor from Anopheles gambiae. A free energy of binding of −38.5 kJ mol−1 was found.
Keywords: Adipokinetic hormones (AKHs); Melme-CC; Declu-CC; Molecular dynamics simulations and receptor docking;
Conserved residues in RF-NH2 receptor models identify predicted contact sites in ligand–receptor binding by C. Bass; C. Katanski; B. Maynard; I. Zurro; E. Mariane; M. Matta; M. Loi; V. Melis; V. Capponi; P. Muroni; M. Setzu; R. Nichols (278-285).
Peptides in the RF-NH2 family are grouped together based on an amidated dipeptide C terminus and signal through G-protein coupled receptors (GPCRs) to influence diverse physiological functions. By determining the mechanisms underlying RF-NH2 signaling targets can be identified to modulate physiological activity; yet, how RF-NH2 peptides interact with GPCRs is relatively unexplored. We predicted conserved residues played a role in Drosophila melanogaster RF-NH2 ligand–receptor interactions. In this study D. melanogaster rhodopsin-like family A peptide GPCRs alignments identified eight conserved residues unique to RF-NH2 receptors. Three of these residues were in extra-cellular loops of modeled RF-NH2 receptors and four in transmembrane helices oriented into a ligand binding pocket to allow contact with a peptide. The eighth residue was unavailable for interaction; yet its conservation suggested it played another role. A novel hydrophobic region representative of RF-NH2 receptors was also discovered. The presence of rhodopsin-like family A GPCR structural motifs including a toggle switch indicated RF-NH2s signal classically; however, some features of the DMS receptors were distinct from other RF-NH2 GPCRs. Additionally, differences in RF-NH2 receptor structures which bind the same peptide explained ligand specificity. Our novel results predicted conserved residues as RF-NH2 ligand–receptor contact sites and identified unique and classic structural features. These discoveries will aid antagonist design to modulate RF-NH2 signaling.
Keywords: Dromyosuppressin (DMS); Drosulfakinin (DSK); FMRF-NH2; FMRF-NH2-related peptide (FaRP); Neuropeptide F (NPF); Short NPF (sNPF);
Membrane binding and oligomer membrane insertion are necessary but insufficient for Bacillus thuringiensis Cyt1Aa toxicity by Pablo Emiliano Cantón; Jazmin A. López-Díaz; Sarjeet S. Gill; Alejandra Bravo; Mario Soberón (286-291).
Bacillus thuringiensis Cyt proteins are pore-forming toxins that have insecticidal activity mainly against dipteran insects. However, certain Cyt proteins have toxicity to some insect orders, but not toxicity of Cyt1Aa against lepidopteran larvae has been found. Insect specificity has been proposed to rely in specific binding to certain lipids on the brush border membrane of midgut cells since no protein receptors have been described so far. To determine the molecular basis of Cyt1Aa insect specificity we compared different steps of Cyt1Aa mode of action in a susceptible insect as the dipteran Aedes aegypti and also in the non-susceptible lepidopteran Manduca sexta. Our data shows that the lack toxicity of Cyt1Aa to M. sexta larvae does not rely on protoxin processing, membrane binding interaction, and oligomerization of Cyt1Aa since these steps were similar in the two insect species analyzed.
Keywords: Cyt toxins; Bacillus thuringiensis; Mode of action; Membrane binding; Oligomerization;
Toxicity and mode of action of insecticidal Cry1A proteins from Bacillus thuringiensis in an insect cell line, CF-1 by Leivi Portugal; J. Lawrence Gringorten; Guido F. Caputo; Mario Soberón; Carlos Muñoz-Garay; Alejandra Bravo (292-299).
Bacillus thuringiensis Cry toxins are insecticidal proteins used to control insect pests. The interaction of Cry toxins with the midgut of susceptible insects is a dynamic process involving activation of the toxin, binding to midgut receptors in the apical epithelium and conformational changes in the toxin molecule, leading to pore formation and cell lysis. An understanding of the molecular events underlying toxin mode of action is essential for the continued use of Cry toxins. In this work, we examined the mechanism of action of Cry1A toxins in the lepidopteran cell line CF-1, using native Cry1Ab and mutant forms of this protein that interfer with different steps in the mechanism of action, specifically, receptor binding, oligomerization or pore formation. These mutants lost activity against both Manduca sexta larvae and CF-1 cells. We also analyzed a mutation created in domain I of Cry1Ab, in which helix α-1 and part of helix α-2 were deleted (Cry1AbMod). Cry1AbMod is able to oligomerize in the absence of toxin receptors, and although it shows reduced activity against some susceptible insects, it kills insect pests that have developed resistance to native Cry1Ab. Cry1AbMod showed enhanced toxicity to CF-1, suggesting that oligomerization of native Cry1Ab may be a limiting step in its activity against CF-1 cells. The toxicity of Cry1Ac and Cry1AcMod were also analyzed. Our results suggest that some of the steps in the mode of action of Cry1A toxins are conserved in vivo in insect midgut cells and in vitro in an established cell line, CF-1.
Keywords: Bacillus thuringiensis; Cry proteins; Toxicity; Manduca sexta; CF-1 cells; Mode of action; Oligomerization;
Ala-7, His-10 and Arg-12 are crucial amino acids for activity of a synthetically engineered μ-conotoxin by Eline K.M. Lebbe; Steve Peigneur; Ward Brullot; Thierry Verbiest; Jan Tytgat (300-306).
Cone snail toxins or conotoxins are often small cysteine-rich peptides which have shown to be highly selective ligands for a wide range of ion channels such as voltage-gated sodium channels (NaVs). NaVs participate in a wide range of electrophysiological processes. Consequently, their malfunction has been associated with numerous diseases. The development of subtype-selective modulators of NaVs remains highly important in the treatment of such disorders. In order to expand our knowledge in the search for novel therapeutics to treat NaV-related diseases, we explored the field of peptide engineering. In the current study, the impact of well considered point mutations into a bioactive peptide that was found to be a very potent and selective inhibitor of NaVs (i.e. Midi R2) was examined. We designed two peptides, named Midi R2[A7G] and Midi R2[H10A, R12A] which have mutations at position 7, and both 10 and 12, respectively. Electrophysiological recordings indicated that an Ala to Gly mutation at position 7 increased IC50-values from the nanomolar range to the micromolar range. For Midi R2[H10A, R12A] at a concentration of 10 μM, activity is even reduced to 0–10% for all of the tested NaV-channels. Circular dichroism measurements proved that overall structural conformations did not change. These findings suggest that the minimal space between the second and the third intercysteine loop of Midi R2 is the sequence RRWARDHSR and that His at position 10 and Arg at position 12 are crucial amino acids for the potency and specificity of Midi R2. In this way, new insights into the structure–activity relationships of μ-conotoxins were found.
Keywords: (μ-)Conotoxin; Voltage-gated sodium channel; Midi R2; Circular dichroism; Cone snail; Peptidomimetics;
DsRNA degradation in the pea aphid (Acyrthosiphon pisum) associated with lack of response in RNAi feeding and injection assay by Olivier Christiaens; Luc Swevers; Guy Smagghe (307-314).
Over the past decade, RNA interference (RNAi), the sequence-specific suppression of gene expression, has proven very promising for molecular research in many species, including model insects as Tribolium castaneum and Apis mellifera. It showed its usefulness to analyze gene function and its potential to manage pest populations and reduce disease pathogens. However, in several insects, the efficiency of RNAi is low or very variable at best. One of the factors that could influence RNAi efficiency in insects is degradation of dsRNA after administration to the insect. In this paper, we report on the importance of dsRNA breakdown in the pea aphid (Acyrthosiphon pisum) associated with the absence of an RNAi response upon oral feeding and injection with dsRNA targeting different genes such as the ecdysone hormone receptor and ultraspiracle. In essence, we discovered that both the salivary secretions of aphids and the hemolymph were able to degrade the dsRNA. In parallel, introduction of dsRNA in the aphid body was not able to provoke a response in the expression of the siRNA core machinery genes.
Keywords: Insects; Hemiptera; Acyrthosiphon pisum; Aphids; RNAi; dsRNA degradation;
Potent and rapid antigonococcal activity of the venom peptide BmKn2 and its derivatives against different Maldi biotype of multidrug-resistant Neisseria gonorrhoeae by Teerakul Arpornsuwan; Brisana Buasakul; Janthima Jaresitthikunchai; Sittiruk Roytrakul (315-320).
The emergence of multidrug-resistant strains of Neisseria gonorrhoeae constitutes a serious threat to public health and necessitates the discovery of new types of antimicrobial agents. Among the 18 clinical isolates of N. gonorrhoeae with susceptible to spectinomycin, ceftriaxone and cefixime, 14 isolates were resistance to penicillin, tetracycline and ciprofloxacin, while 2 isolates were susceptible to tetracycline and another was penicillin intermediate isolate. Significant differences between laboratory strain and multidrug resistant strains were revealed by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling and bioinformatics examination using the MALDI BioTyper software. However, Maldi Biotyper was not successfully separated ciprofloxacin-penicillin resistance and ciprofloxacin-tetracycline resistance from ciprofloxacin-penicillin-tetracycline resistant N. gonorrhoeae isolates. BmKn2 is a basic, alpha-helical peptide with no disulfide-bridge venom peptides that was first isolated from Buthus martensii Kasch. A panel of BmKn2 scorpion venom peptide and its derivatives of varying length and characteristics were synthesized chemically and evaluated for their ability to inhibit the growth of clinical N. gonorrhoeae isolates. Synthetic BmKn2 displayed potent activity against 18 clinical isolates of N. gonorrhoeae with MIC50 values of 6.9–27.6 μM. BmKn2 exerted its antibacterial activity via a bactericidal mechanism. Cyclic BmKn1 did not show antigonococcal activity. Decreasing the cationicity and helix percentage at the C-terminus of BmKn2 reduced the potency against N. gonorrhoeae. Taken together, the BmKn1 peptide can be developed as a topical therapeutic agent for treating multidrug-resistant strains of N. gonorrhoeae infections.
Keywords: Antimicrobial peptide; Antibiotic-resistant N. gonorrhoeae; MALDI Biotyper;