Probing the production of amidated peptides following genetic and dietary copper manipulations

Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM(+/-)) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM(+/-) mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM(+/-) mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.     

Functional analysis of four neuropeptides, EH, ETH, CCAP and bursicon, and their receptors in adult ecdysis behavior of the red flour beetle, Tribolium castaneum

Ecdysis behavior in arthropods is driven by complex interactions among multiple neuropeptide signaling systems. To understand the roles of neuropeptides and their receptors in the red flour beetle, Tribolium castaneum, we performed systemic RNA interference (RNAi) experiments utilizing post-embryonic injections of double-stranded (ds) RNAs corresponding to ten gene products representing four different peptide signaling pathways: eclosion hormone (EH), ecdysis triggering hormone (ETH), crustacean cardioactive peptide (CCAP) and bursicon. Behavioral deficiencies and developmental arrests occurred as follows: RNAi of (1) eh or eth disrupted preecdysis behavior and prevented subsequent ecdysis behavior; (2) ccap interrupted ecdysis behavior; and (3) bursicon subunits resulted in wrinkled elytra due to incomplete wing expansion, but there was no effect on cuticle tanning or viability. RNAi of genes encoding receptors for those peptides produced phenocopies comparable to those of their respective cognate neuropeptides, except in those cases where more than one receptor was identified. The phenotypes resulting from neuropeptide RNAi in Tribolium differ substantially from phenotypes of the respective Drosophila mutants. Results from this study suggest that the functions of neuropeptidergic systems that drive innate ecdysis behavior have undergone significant changes during the evolution of arthropods.     

Isolation and characterization of a Drosophila neuropeptide gene

We have purified a 9 amino acid amidated neuropeptide, DPKQDFMRFamide, from whole adult D. melanogaster. This peptide exhibits sequence homology to the molluscan bioactive tetrapeptide FMRFamide and is a novel member of the FMRFamide peptide family. The gene encoding DPKQDFMRFamide has been cloned and characterized. It is present in a single copy per haploid genome, is expressed as a unique 1.7 kb mRNA species, and cytologically maps to 46C on the right arm of chromosome 2. Characterization of a cDNA clone indicates that the precursor protein is 347 amino acids in length and contains 5 copies of DPKQDFMRFamide, as well as 10 additional amidated peptides exhibiting varying degrees of structural relatedness. The Drosophila DPKQDFMRFamide gene and the Aplysia FMRFamide gene are ancestrally related; however, peptides display a higher degree of homology within a species than between species, suggesting intragenic concerted evolution of these neuropeptides.     

In silico identification of new secretory peptide genes in Drosophila melanogaster

Bioactive peptides play critical roles in regulating most biological processes in animals. The elucidation of the amino acid sequence of these regulatory peptides is crucial for our understanding of animal physiology. Most of the (neuro)peptides currently known were identified by purification and subsequent amino acid sequencing. With the entire genome sequence of some animals now available, it has become possible to predict novel putative peptides. In this way, BLAST (Basic Local Alignment Searching Tool) analysis of the Drosophila melanogaster genome has allowed annotation of 36 secretory peptide genes so far. Peptide precursor genes are, however, poorly predicted by this algorithm, thus prompting an alternative approach described here. With the described searching program we scanned the Drosophila genome for predicted proteins with the structural hallmarks of neuropeptide precursors. As a result, 76 additional putative secretory peptide genes were predicted in addition to the 43 annotated ones. These putative (neuro)peptide genes contain conserved motifs reminiscent of known neuropeptides from other animal species. Peptides that display sequence similarities to the mammalian vasopressin, atrial natriuretic peptide, and prolactin precursors and the invertebrate peptides orcokinin, prothoracicotropic hormones, trypsin modulating oostatic factor, and Drosophila immune induced peptides (DIMs) among others were discovered. Our data hence provide further evidence that many neuropeptide genes were already present in the ancestor of Protostomia and Deuterostomia prior to their divergence. This bioinformatic study opens perspectives for the genome-wide analysis of peptide genes in other eukaryotic model organisms.     

cDNA cloning and sequence determination of the pheromone biosynthesis activating neuropeptide of the silkworm, Bombyx mori.

We have identified the cDNAs encoding pheromone biosynthesis activating neuropeptide (PBAN) using PCR technique. The nucleotide sequence showed that the PBAN gene encodes, besides PBAN, diapause hormone and three putative amidated peptides. These four peptides share with PBAN the C-terminal pentapeptide amide which is corresponding to the shortest fragment with pheromonotropic activity. The organization of the PBAN gene is characteristic of several short neuropeptides and has some degree of similarity to that of the gene for the insect neuropeptide FMRFamide. Thus, the PBAN gene products construct a family of structurally related peptides and have various biological functions.     

Ap-let neurons--a peptidergic circuit potentially controlling ecdysial behavior in Drosophila

Here we describe a novel set of peptidergic neurons conserved throughout all developmental stages in the Drosophila central nervous system (CNS). We show that a small complement of 28 apterous-expressing cells (Ap-let neurons) in the ventral nerve cord (VNC) of Drosophila larvae co-express numerous gene products. The products include the neuroendocrine-specific bHLH regulator called Dimmed (Dimm), four neuropeptide biosynthetic enzymes (PC2, Fur1, PAL2, and PHM), and a specific dopamine receptor subtype (dDA1). For the PC2, Fur1, and PAL2 enzymes, and for the dDA1 receptor, this neuronal pattern represents the vast majority of their total expression in the VNC. In addition, while Dimm and PHM are present in the peritracheal Inka cells in larvae, pupae, and adults, Ap, PC2, Fur1, PAL2, and dDA1 are not. PC2, PAL2, and DA1 receptor expression were all controlled by both dimm and ap. Previous genetic analysis of animals deficient in PC2 revealed an abnormal larval ecdysis phenotype. Together, these data support the hypothesis that the small cohort of Ap-let interneurons regulates larval ecdysis behavior by secretion of an unidentified amidated peptide(s). This hypothesis further predicts that the production of the Ap-let neuropeptide(s) is dependent on each of four specific enzymes, and that a certain aspect(s) of its production and/or release is regulated by dopamine input.     

Deficiency of prohormone convertase dPC2 (AMONTILLADO) results in impaired production of bioactive neuropeptide hormones in Drosophila

Peptide hormones synthesized by secretory neurons in the CNS are important regulators of physiology, behavior, and development. Like other neuropeptides, they are synthesized from larger precursor molecules by a specific set of enzymes. Using a combination of neurogenetics, immunostainings, and direct mass spectrometric profiling, we show that the presence of Drosophila prohormone convertase 2 encoded by the gene amontillado (amon) is a prerequisite for the proper processing of neuropeptide hormones from the major neurohemal organs of the CNS. A loss of amon correlates with a loss of neuropeptide hormone signals from the larval ring gland and perisympathetic organs. Neuropeptide hormone signals were still detectable in the adult corpora cardiaca of older amon-deficient flies which were amon heat-shock-rescued until eclosion. A semiquantification by direct peptide profiling using stable isotopic standards showed, however, that their neuropeptide hormone levels are strongly reduced. Targeted expression of GFP under the control of amon regulatory regions revealed a co-localization with the investigated peptide hormones in secretory neurons of the brain and ventral nerve cord. The lack of AMON activity resulted in a deficiency of L3 larva to enter the wandering phase. In conclusion, our findings provide the first direct evidence that AMON is a key enzyme in the production of neuropeptides in the fruitfly.     

Discovery of multiple neuropeptide families in the phylum Platyhelminthes

Available evidence shows that short amidated neuropeptides are widespread and have important functions within the nervous systems of all flatworms (phylum Platyhelminthes) examined, and could therefore represent a starting point for new lead drug compounds with which to combat parasitic helminth infections. However, only a handful of these peptides have been characterised, the rigorous exploration of the flatworm peptide signalling repertoire having been hindered by the dearth of flatworm genomic data. Through searches of both expressed sequence tags and genomic resources using the basic local alignment search tool (BLAST), we describe 96 neuropeptides on 60 precursors from 10 flatworm species. Most of these (51 predicted peptides on 14 precursors) are novel and are apparently restricted to flatworms; the remainder comprise nine recognised peptide families including FMRFamide-like (FLPs), neuropeptide F (NPF)-like, myomodulin-like, buccalin-like and neuropeptide FF (NPFF)-like peptides; notably, the latter have only previously been reported in vertebrates. Selected peptides were localised immunocytochemically to the Schistosoma mansoni nervous system. We also describe several novel flatworm NPFs with structural features characteristic of the vertebrate neuropeptide Y (NPY) superfamily, previously unreported characteristics which support the common ancestry of flatworm NPFs with the NPY-superfamily. Our dataset provides a springboard for investigation of the functional biology and therapeutic potential of neuropeptides in flatworms, simultaneously launching flatworm neurobiology into the post-genomic era.     

Physiological differentiation of DH-PBAN-producing neurosecretory cells in the silkworm embryo

Embryonic diapause of the silkworm, Bombyx mori, is induced by a neuropeptide hormone, the diapause hormone (DH), which is secreted from a limited number of neurosecretory cells in the subesophageal ganglion (SG) at the maternal generation. We examined the developmental fate of the hormone-producing cell (DH-pheromone biosynthesis activating neuropeptide [PBAN]-producing cell) in the embryonic stage at the level of gene expression and cell biology. The DH-PBAN gene expression started at the histogenesis stage and gradually increased toward hatching. DH is an amidated peptide belonging to FXPRLamide family. The immunoreactive somata against anti FXPRLamide antiserum were found in the SG from blastokinesis. Immunoreactive neural processes with varicosites were also found on the corpus cardiacum and the corpus allatum. The implantation of a part of a developing embryo including the SG into the pupae with the SG removed induced diapause eggs in the progeny. These results were obtained from eggs incubated under diapause-averting conditions as well as diapause-inducing conditions. Thus, a neurosecretory system responsible for biosynthesis of FXPRLamide neuropeptides is established as early as histogenesis, although the system to regulate the secretion of neuropeptide hormones has not been fully formed by that time.     

Role of neuropeptides in sex pheromone production in moths

Sex pheromone biosynthesis in many moth species is controlled by a cerebral neuropeptide, termed pheromone biosynthesis activating neuropeptide (PBAN). PBAN is a 33 amino acid C-terminally amidated neuropeptide that is produced by neuroendocrine cells of the subesophageal ganglion (SEG). Studies of the regulation of sex pheromone biosynthesis in moths have revealed that this function can be elicited by additional neuropeptides all of which share the common C-terminal pentapeptide FXPRL-amide (X = S, T, G, V). In the past two decades extensive studies were carried out on the chemical, cellular and molecular aspects of PBAN and the other peptides (termed the pyrokinin (PK)/PBAN family) aiming to understand the mode of their action on sex pheromone biosynthesis. In the present review we focus on a few of these aspects, specifically on the: (i) structure-activity relationship (SAR) of the PK/PBAN family, (ii) characterization of the PK/PBAN receptor and (iii) development of a novel strategy for the generation of PK/PBAN antagonists and their employment in studying the mode of action of the PK/PBAN peptides.     

Functional significance of the copper transporter ATP7 in peptidergic neurons and endocrine cells in Drosophila melanogaster

The Drosophila ATP7 copper transporter has sequence homology to the human copper transporters ATP7A and ATP7B, which are defective in Menkes and Wilson disease, respectively. We show here that in Drosophila ATP7 is expressed by many peptidergic neurons. As C-terminal amidation of neuropeptides depends on the copper-containing enzyme PHM, it seemed likely that in the absence of ATP7 the activity of PHM might be compromised. Indeed, inhibition of ATP7 expression by RNAi led to a decrease in mature amidated neuropeptides and the appearance of C-terminally Gly-extended neuropeptides. The strength of this effect differed from one cell type to another; it was very pronounced for AKH and corazonin, but much less so for SIFamide and myosuppressin. Nevertheless, down-regulation of ATP7 specifically in the SIFamide-expressing neurons resulted in male-male courtship behavior.     

Drosophila uses two distinct neuropeptide amidating enzymes, dPAL1 and dPAL2

Neuropeptide alpha-amidation is a common C-terminal modification of secretory peptides, frequently required for biological activity. In mammals, amidation is catalyzed by the sequential actions of two enzymes [peptidylglycine-alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL)] that are co-synthesized within a single bifunctional precursor. The Drosophila genome predicts expression of one monofunctional PHM gene and two monofunctional PAL genes. Drosophila PHM encodes an active enzyme that is required for peptide amidation in vivo. Here we initiate studies of the two Drosophila PAL genes. dPAL1 has two predicted transmembrane domains, whereas dPAL2 is predicted to be soluble and secreted. dPAL2 expressed in heterologous cells is secreted readily and co-localized with hormone. In contrast, dPAL1 is secreted poorly, even when expressed with a cleaved signal replacing the predicted transmembrane domains; the majority of dPAL1 stays in the endoplasmic reticulum. Both proteins display PAL enzymatic activity. Compared to the catalytic core of rat PAL, the two Drosophila lyases have higher K(m) values, higher pH optima and similarly broad divalent metal ion requirements. Antibodies to dPAL1 and dPAL2 reveal co-expression in many identified neuroendocrine neurons. Although dPAL1 is broadly expressed, dPAL2 is found in only a limited subset of neurons. dPAL1 expression is highly correlated with the non-amidated peptide proctolin. Tissue immunostaining demonstrates that dPAL1 is largely localized to the cell soma, whereas dPAL2 is distributed throughout neuronal processes.     

amontillado,the Drosophila homolog of the prohormone processing protease PC2, is required during embryogenesis and early larval development

Biosynthesis of most peptide hormones and neuropeptides requires proteolytic excision of the active peptide from inactive proprotein precursors, an activity carried out by subtilisin-like proprotein convertases (SPCs) in constitutive or regulated secretory pathways. The Drosophila amontillado (amon) gene encodes a homolog of the mammalian PC2 protein, an SPC that functions in the regulated secretory pathway in neuroendocrine tissues. We have identified amon mutants by isolating ethylmethanesulfonate (EMS)-induced lethal and visible mutations that define two complementation groups in the amon interval at 97D1 of the third chromosome. DNA sequencing identified the amon complementation group and the DNA sequence change for each of the nine amon alleles isolated. amon mutants display partial embryonic lethality, are defective in larval growth, and arrest during the first to second instar larval molt. Mutant larvae can be rescued by heat-shock-induced expression of the amon protein. Rescued larvae arrest at the subsequent larval molt, suggesting that amon is also required for the second to third instar larval molt. Our data indicate that the amon proprotein convertase is required during embryogenesis and larval development in Drosophila and support the hypothesis that AMON acts to proteolytically process peptide hormones that regulate hatching, larval growth, and larval ecdysis.     

Peptidergic innervation of the human clitoris.

In the present study, the distributions of neuropeptides in the normal human clitoris and in a clitoris from an adrenogenital syndrome (AGS) was demonstrated by immunohistochemistry (IHC). Immunohistochemical screening detected a complex network of nerve fibers containing vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), neuropeptide tyrosine (neuropeptide Y), C-flanking peptide of neuropeptide Y (CPON), calcitonin gene-related peptide (CGRP) and substance P immunoreactivities. Special attention was given to the VIP-related peptide helospectin, that has been detected in neuronal elements in the clitoris. No visible differences between the localization and distribution of peptidergic nerve fibers of normal and hypertrophic clitoris from AGS have been observed. Co-localization studies showed the co-existence of VIP, PHM and partly helospectin and neuropeptide Y with CPON within nerve fibers in the cavernous tissue and substance P and CGRP co-expression in nerve fibers especially underneath and within the glans clitoris.     

Drosophila short neuropeptide F regulates food intake and body size

Neuropeptides regulate a wide range of animal behavior including food consumption, circadian rhythms, and anxiety. Recently, Drosophila neuropeptide F, which is the homolog of the vertebrate neuropeptide Y, was cloned, and the function of Drosophila neuropeptide F in feeding behaviors was well characterized. However, the function of the structurally related short neuropeptide F (sNPF) was unknown. Here, we report the cloning, RNA, and peptide localizations, and functional characterizations of the Drosophila sNPF gene. The sNPF gene encodes the preprotein containing putative RLRF amide peptides and was expressed in the nervous system of late stage embryos and larvae. The embryonic and larval localization of the sNPF peptide in the nervous systems revealed the larval central nervous system neural circuit from the neurons in the brain to thoracic axons and to connective axons in the ventral ganglion. In the adult brain, the sNPF peptide was localized in the medulla and the mushroom body. However, the sNPF peptide was not detected in the gut. The sNPF mRNA and the peptide were expressed during all developmental stages from embryo to adult. From the feeding assay, the gain-of-function sNPF mutants expressed in nervous systems promoted food intake, whereas the loss-of-function mutants suppressed food intake. Also, sNPF overexpression in nervous systems produced bigger and heavier flies. These findings indicate that the sNPF is expressed in the nervous systems to control food intake and regulate body size in Drosophila melanogaster.     

Secretory vesicle aminopeptidase B related to neuropeptide processing: molecular identification and subcellular localization to enkephalin- and NPY-containing chromaffin granules

Biosynthesis of peptide hormones and neurotransmittters involves proteolysis of proprotein precursors by secretory vesicle cathepsin L. Cathepsin L generates peptide intermediates with basic residues at their NH(2)-termini, indicating that Arg/Lys aminopeptidase is needed to generate the smaller biologically active peptide. Therefore, this study identified the Arg/Lys aminopeptidase that is present in secretory vesicles of adrenal medulla and neuroendocrine tissues, achieved by molecular cloning and localization in 'model' neuropeptide-containing secretory vesicles (bovine). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA defined its primary sequence that allowed selection of antisera for immunolocalization studies. AP-B was present in secretory vesicles that contain cathepsin L with the neuropeptides enkephalin and neuropeptide Y. The AP-B in several neuroendocrine tissues was detected by western blots. Recombinant bovine AP-B showed preference for Arg-methylcoumarinamide substrate. AP-B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP-B showed similar activities for Arg-(Met)enkephalin (ME) and Lys-ME neuropeptide substrates to generate ME, while rat AP-B preferred Arg-ME. Furthermore, AP-B possesses an acidic pH optimum of 5.5-6.5 that is similar to the internal pH of secretory vesicles. The significant finding of the secretory vesicle localization of AP-B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides.     

Peptidomics of the Agriculturally Damaging Larval Stage of the Cabbage Root Fly Delia radicum (Diptera: Anthomyiidae)

The larvae of the cabbage root fly induce serious damage to cultivated crops of the family Brassicaceae. We here report the biochemical characterisation of neuropeptides from the central nervous system and neurohemal organs, as well as regulatory peptides from enteroendocrine midgut cells of the cabbage maggot. By LC-MALDI-TOF/TOF and chemical labelling with 4-sulfophenyl isothiocyanate, 38 peptides could be identified, representing major insect peptide families: allatostatin A, allatostatin C, FMRFamide-like peptides, kinin, CAPA peptides, pyrokinins, sNPF, myosuppressin, corazonin, SIFamide, sulfakinins, tachykinins, NPLP1-peptides, adipokinetic hormone and CCHamide 1. We also report a new peptide (Yamide) which appears to be homolog to an amidated eclosion hormone-associated peptide in several Drosophila species. Immunocytochemical characterisation of the distribution of several classes of peptide-immunoreactive neurons and enteroendocrine cells shows a very similar but not identical peptide distribution to Drosophila. Since peptides regulate many vital physiological and behavioural processes such as moulting or feeding, our data may initiate the pharmacological testing and development of new specific peptide-based protection methods against the cabbage root fly and its larva.     

Cathepsin L expression is directed to secretory vesicles for enkephalin neuropeptide biosynthesis and secretion

Proteases within secretory vesicles are required for conversion of neuropeptide precursors into active peptide neurotransmitters and hormones. This study demonstrates the novel cellular role of the cysteine protease cathepsin L for producing the (Met)enkephalin peptide neurotransmitter from proenkephalin (PE) in the regulated secretory pathway of neuroendocrine PC12 cells. These findings were achieved by coexpression of PE and cathepsin L cDNAs in PC12 cells with analyses of PE-derived peptide products. Expression of cathepsin L resulted in highly increased cellular levels of (Met)enkephalin, resulting from the conversion of PE to enkephalin-containing intermediates of 23, 18-19, 8-9, and 4.5 kDa that were similar to those present in vivo. Furthermore, expression of cathepsin L with PE resulted in increased amounts of nicotine-induced secretion of (Met)enkephalin. These results indicate increased levels of (Met)enkephalin within secretory vesicles of the regulated secretory pathway. Importantly, cathespin L expression was directed to secretory vesicles, demonstrated by colocalization of cathepsin L-DsRed fusion protein with enkephalin and chromogranin A neuropeptides that are present in secretory vesicles. In vivo studies also showed that cathepsin L in vivo was colocalized with enkephalin. The newly defined secretory vesicle function of cathepsin L for biosynthesis of active enkephalin opioid peptide contrasts with its function in lysosomes for protein degradation. These findings demonstrate cathepsin L as a distinct cysteine protease pathway for producing the enkephalin member of neuropeptides.     

An NPY-like peptide may function as MSH-release inhibiting factor in Xenopus laevis

This study demonstrates the presence of a rich plexus of neuropeptide Y (NPY) immunoreactive fibers in the hypothalamus and in the intermediate lobe of the pituitary of Xenopus laevis. During superfusion of neurointermediate lobe tissue, synthetic NPY induces a rapid, powerful and dose-dependent inhibition of in vitro release of MSH, endorphin and other proopiomelanocortin (POMC) derived peptides. Therefore, NPY undoubtedly is one of the growing number of neuropeptides that are likely involved in control of the amphibian MSH cells. Although a number of stimulatory neuropeptides have been found, this is the first neuropeptide to apparently function through an inhibitory mechanism. In that a 2-hr treatment with NPY did not influence POMC biosynthesis, nor processing of this prohormone to smaller peptides, we conclude that the primary action of NPY is a direct effect on the secretory process of the MSH cell.