Toward a consensus nomenclature for insect neuropeptides and peptide hormones

The nomenclature currently in use for insect neuropeptide and peptide hormone families is reviewed and suggestions are made as to how it can be rationalized. Based upon this review, a number of conventions are advanced as a guide to a more rationale nomenclature. The scheme that is put forward builds upon the binomial nomenclature scheme proposed by Raina and Gäde in 1988 [100], when just over 20 insect neuropeptides had been identified. Known neuropeptides and peptide hormones are assigned to 32 structurally distinct families, frequently with overlapping functions. The names given to these families are those that are currently in use, and describe a biological function, homology to known invertebrate/vertebrate peptides, or a conserved structural motif. Interspecific isoforms are identified using a five-letter code to indicate genus and species names, and intraspecific isoforms are identified by Roman or Arabic numerals, with the latter used to signify the order in which sequences are encoded on a prepropeptide. The proposed scheme is sufficiently flexible to allow the incorporation of novel peptides, and could be extended to other arthropods and non-arthropod invertebrates.     

Conformational and dynamic considerations in the design of peptide hormone analogs

Efforts to understand the chemical-physical basis for peptide hormone and neurotransmitter action requires integration of conformational parameters and biological properties. Since most peptide hormones are conformationally flexible, the question arises as to which of the manifold of conformations is of biological significance. In molecular terms, it is necessary to carefully distinguish chemical-physical features important to binding (the binding message) from those involved in transduction (the biological activity message). One approach to this involves the design, synthesis, and conformational analysis of semirigid hormone analogs. The distinction between binding and transduction can best be examined by evaluation of full biological profiles of partial agonists, antagonists, and analogs with prolonged biological activity. Using this multidisciplinary approach, we have prepared several semirigid [Pen¹]-oxytocin antagonist analogs and evaluated their conformational properties and biological activities. Specific conformational features can be related to inhibitory activities in several cases. On the basis of structure–activity relationships and conformational considerations, we have designed a series of conformationally restricted cyclic and acyclic analogs of the linear peptide α-melanotropin. Some of these peptides have exceptionally prolonged in vivo activity (weeks), and others exhibit superagonist potency (10,000 times the native hormone). We have evidence that potency and prolonged activity have different structural and conformational requirements. It is suggested that potency is primarily a function of receptor recognition (the binding message), whereas prolonged activity is related to transduction (the biological activity message).     

Site-directed mutagenesis and PBAN activation of the Helicoverpa zea PBAN-receptor

Pheromone biosynthesis-activating neuropeptide (PBAN) and pyrokinins belong to a family of insect peptide hormones that have a common FXPRLamide C-terminal ending. The G-protein-coupled receptors (GPCRs) for this peptide family were first identified from a moth and Drosophila with sequence similarity to neuromedin U receptors from vertebrates. We have characterized the PBAN-receptor (PBAN-R or PR) active binding domains using chimeric GPCRs and proposed that extracellular loop 3 is critical for ligand selection. Here, we characterized the 3rd extracellular domain of PBAN-R through site-directed point mutations. Results are discussed in context of the structural features required for receptor activation using receptor activation experiments and in silico computational modeling. This research will help in characterizing these receptors towards a goal of finding agonists and/or antagonists for PBAN/pyrokinin receptors.     

New field of insect science: Research on the use of insect properties

In recent years, research on “insect properties” has been attracting much attention for industrial applications of insect technology. This is a new field of research that attempts to analyze specific physiological properties of insects to develop technology for helping humankind. The term “insect properties” has been used to refer to “specific biological functions of insects” since around 1986, and it is now widely accepted in Japan. From 1996, the National Institute of Sericultural and Entomological Science (NISES) promoted “Research for utilization of insect properties” as a “Center of Excellence (COE)” project funded by the Science and Technology Agency. At this point, a new research field called “Research for utilization of insect properties” was initiated, and this led to the recognition of this field by the academic community. In the 21st century, remarkable results, including the development of transgenic silkworms and the full decoding of the silkworm genome, have been achieved. It is expected that this advanced technology will be a powerful tool for progress of research on the use of insect properties. This review presents an overview of the current state of research on use of insect properties as a new technology.     

Design of biologically active peptides with non-peptidic structural elements. Biological and physical properties of a synthetic analogue of beta-endorphin with unnatural amino acids in the region 6-12

Modelling studies with beta-endorphin have clearly demonstrated that an amphiphilic secondary structural segment is a salient feature of the biologically active conformation of this 31-residue opioid peptide hormone. Here, we have initiated the synthesis of peptide models using unnatural building blocks by designing a beta-endorphin analogue (peptide 6) in which the hydrophilic linker region between the NH2-terminal enkephalin (residues 1-5) and the COOH-terminal helix (residues 10-28, sequence identical to that of peptide 3 in region 13-31, Fig. 1) consists of four units of gamma-amino-gamma-hydroxymethylbutyric acid connected by isopeptidic linkages. Peptide 6 has physical properties similar to that of peptide 3, as shown by surface monolayer and circular dichroism studies. The binding affinities of the two peptides to delta- and mu-receptors are also similar. In rat vas deferens assays, the present model is equipotent to peptide 3. The most striking result of all is the potent analgesic activity displayed by peptide 6 when injected intracerebroventricularly into mice. The potencies of peptides 6 and 3 are comparable in these assays. These studies clearly illustrate that one can use unusual building blocks to construct structural regions of synthetic analogues and still preserve the biological activity of peptide hormones.     

Locustakinin, a novel myotropic peptide from Locusta migratoria, isolation, primary structure and synthesis.

The isolated hindgut of the cockroach, Leucophaea maderae is a very efficient bioassay tool for the monitoring of certain structural types of insect myotropic peptides during HPLC purification. Using this detection system, a six residue peptide has been isolated from an extract of 9000 brain corpora cardiaca-corpora allata suboesophageal ganglion complexes of Locusta migratoria. Amino acid composition and sequence analysis combined with enzymatic digestion data established the structure of the novel peptide as Ala-Phe-Ser-Ser-Trp-Gly-amide. The chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis. The carboxy-terminal pentamer sequence is the active core of leucokinins II, V and VII and of achetakinin III (myotropic neuropeptides isolated from Leucophaea m. and from Acheta domesticus; Holman et al., 1990). Furthermore, the octapeptide leucokinin VII contains the novel sequence as its carboxy-terminal hexamer and Achetakinin V (AFHSWGamide) differs from it by one residue. This new peptide designated as locustakinin I (locusts) may therefore represent an evolutionary molecular link between leucokinin VII (cockroaches) and achetakinin V (crickets). Using synthetic locustakinin, physiological studies will be performed in the locust. In view of the known effects of leucokinins, locustakinin may be important in the stimulation of ion transport and inhibition of diuretic activity in Malpighian tubules. This study indicates that the AFXSWGamide sequence appears to have been well conserved and that members of this peptide family may be widely distributed among insects and posses a number of functions.     

The periviscerokinin (PVK) peptide family in insects: evidence for the inclusion of CAP(2b) as a PVK family member

Periviscerokinins (PVKs) are a distinct insect peptide family with unusual distribution in the central nervous system and neurohemal release sites. PVKs were first isolated from the abdominal perisympathetic organs of Periplaneta americana, but can be found in other insect species. Peptides with structural similarity to PVKs have been identified through searches of the Drosophila genome. The cardioacceleratory peptide CAP(2b) of the hawkmoth Manduca sexta shares close amino acid identity with the PVKs and may thus be included as a structural member of the PVK peptide family. In this review, we provide support for grouping CAP(2b) as a PVK family member based on published sequences, and new immunocytochemical findings and mass spectrometric data.     

Evolutionary Sequence Modeling for Discovery of Peptide Hormones

There are currently a large number of “orphan” G-protein-coupled receptors (GPCRs) whose endogenous ligands (peptide hormones) are unknown. Identification of these peptide hormones is a difficult and important problem. We describe a computational framework that models spatial structure along the genomic sequence simultaneously with the temporal evolutionary path structure across species and show how such models can be used to discover new functional molecules, in particular peptide hormones, via cross-genomic sequence comparisons. The computational framework incorporates a priori high-level knowledge of structural and evolutionary constraints into a hierarchical grammar of evolutionary probabilistic models. This computational method was used for identifying novel prohormones and the processed peptide sites by producing sequence alignments across many species at the functional-element level. Experimental results with an initial implementation of the algorithm were used to identify potential prohormones by comparing the human and non-human proteins in the Swiss-Prot database of known annotated proteins. In this proof of concept, we identified 45 out of 54 prohormones with only 44 false positives. The comparison of known and hypothetical human and mouse proteins resulted in the identification of a novel putative prohormone with at least four potential neuropeptides. Finally, in order to validate the computational methodology, we present the basic molecular biological characterization of the novel putative peptide hormone, including its identification and regional localization in the brain. This species comparison, HMM-based computational approach succeeded in identifying a previously undiscovered neuropeptide from whole genome protein sequences. This novel putative peptide hormone is found in discreet brain regions as well as other organs. The success of this approach will have a great impact on our understanding of GPCRs and associated pathways and help to identify new targets for drug development.     

Roles of aromatic residues in the structure and biological activity of the small cytokine,growth-blocking peptide (GBP)

Growth-blocking peptide (GBP) is a small (25 amino acids) insect cytokine with a variety of functions: controlling the larval development of lepidopteran insects, acting as a mitogen for various types of cultured cells, and stimulating insect blood cells. The aromatic residues of GBP (Phe-3, Tyr-11, and Phe-23) are highly conserved in the ENF peptide family found in lepidopteran insects. We investigated the relationship between the biological activities and structural properties of a series of GBP mutants, in which each of the three aromatic residues is replaced by a different residue. The results of the hemocytes-stimulating assays of GBP mutants indicated that Phe-3 is the key residue in this activity: Ala or Tyr replacement resulted in significant loss of the activity, but Leu replacement did not. The replacements of other aromatic residues hardly affected the activity. On the other hand, NMR analysis of the mutants suggested that Tyr-11 is a key residue for maintaining the core structure of GBP. Surprisingly, the Y11A mutant maintained its biological activity, although its native-like secondary structure was disordered. Detailed analyses of the (15)N-labeled Y11A mutant by heteronuclear NMR spectroscopy showed that the native-like beta-sheet structure of Y11A was induced by the addition of 2,2,2-trifluoroethanol. The results suggest that Y11A has a tendency to form a native-like structure, and this property may give the Y11A mutant native-like activity.     

Structural determinants of natriuretic peptide receptor specificity and degeneracy

Cardiovascular homeostasis and blood pressure regulation are reliant, in part, on interactions between natriuretic peptide (NP) hormones and natriuretic peptide receptors (NPR). The C-type NPR (NPR-C) is responsible for clearance of NP hormones from the circulation, and displays a cross-reactivity for all NP hormones (ANP, BNP, and CNP), in contrast to other NPRs, which are more restricted in their specificity. In order to elucidate the structural determinants for the binding specificity and cross-reactivity of NPR-C with NP hormones, we have determined the crystal structures of the complexes of NPR-C with atrial natriuretic peptide (ANP), and with brain natriuretic peptide (BNP). A structural comparison of these complexes, with the previous structure of the NPR-C/CNP complex, reveals that NPR-C uses a conformationally inflexible surface to bind three different, highly flexible, NP ligands. The complex structures support a mechanism of rigid promiscuity rather than conformational plasticity by the receptor. While ANP and BNP appear to adopt similar receptor-bound conformations, the CNP structure diverges, yet shares sets of common receptor contacts with the other ligands. The degenerate versus selective hormone recognition properties of different NPRs appears to derive largely from two cavities on the receptor surfaces, pocket I and pocket II, that serve as anchoring sites for hormone side-chains and modulate receptor selectivity.     

Invertebrate neuropeptide hormones

The development of a long-term research program on the neurosecretory hormones of arthropods is described. The purification and full characterization of the first invertebrate neurohormones, the red pigment-concentrating hormone (RPCH) and the distal retinal pigment hormone (DRPH) demonstrated that they are peptides, an octapeptide and an octadecapeptide, respectively. Physiological function studies with the pure hormones and their synthetic preparations showed that the RPCH acts as a general pigment-concentrating hormone (PCH), and that the DRPH, in addition to its light-adaptive function, also constitutes a general pigment-dispersing hormone (PDH). In the regulation of the color-adaptation of the animals, the two hormones act as antagonists. The chromatophorotropic activities are widely distributed within the arthropod neuroendocrine systems. Purification of the pigment-concentrating activities from the locust corpora cardiaca lead to the isolation and characterization of the first insect neurohormones, the adipokinetic hormones (AKH I and AKH II). These two hormones, AKH I being a decapeptide and AKH II being an octapeptide, are close structural analogs to the crustacean PCH, demonstrating a common evolution of arthropod neurohormones. The hormones of this PCH-family all cross-react, but structure-function studies of the hormones show that quite different parts of their structure are involved in their binding to the various receptors.     

Insect oostatic activity of GnRH and its fragments

Mammal, ¹²⁵I-mammal, salmon, chicken I and II GnRHs and three fragments of mammal GnRH were synthesized and their effect on oogenesis in the flesh fly Neobellieria(formerly Sarcophaga) bullata (Diptera) was investigated. The peptides were prepared by the Merrifield solid phase synthesis on polystyrene/divinylbenzene polymer using the N-Boc strategy in DMF and were purified by preparative RP-HPLC in a gradient of water-MeOH. From the peptides assayed, only mammal GnRH and two of its carboxy-terminus truncated analogs remarkably affected the processes of egg development in ovarioles, causing changes in the follicular epithelium, proliferation of its nuclei and cell division towards the inner part of the egg chamber. The process led to the occurrence of multinuclear follicular epithelium which finally filled up almost the whole egg chamber and then it degenerated. The inability of GnRH of other animal species to evoke the changes in the egg development establishes the question of primary structures of GnRH responsible for these biological effects. The identityof sequences of GnRHs from position 1 up to 6 (with the exception of chicken GnRH II) points to functionality of amino acids located in positions 7 and 8 of the peptide chain. The radioactivity of the ¹²⁵I-labelled mammal GnRH with maintained oostatic activity and its receptor competition with the non-labelled mammal GnRH were measured in selected insect organs and exhibited different residual values according to the organ and the time after applicationof the peptide. A transfer of the radioactivity into the next (F₁) generation was also observed.     

Prosequence-mediated disulfide coupled folding of the peptide hormones guanylin and uroguanylin

In contrast to their prohormones the mature peptide hormones guanylin and uroguanylin are not able to fold to their native disulfide connectivities upon oxidative folding. Structural properties of both peptide hormones and their precursor proteins as well as the role of their prosequences in proper disulfide coupled folding are reviewed. In addition, the structural behavior of a proguanylin mutant that closely resembles prouroguanylin has been investigated to gain further insight into structural properties of this homologous precursor protein.     

Post-translational modifications in secreted peptide hormones in plants

More than a dozen secreted peptides are now recognized as important hormones that coordinate and specify cellular functions in plants. Recent evidence has shown that secreted peptide hormones often undergo post-translational modification and proteolytic processing, which are critical for their function. Such 'small post-translationally modified peptide hormones' constitute one of the largest groups of peptide hormones in plants. This short review highlights recent progress in research on post-translationally modified peptide hormones, with particular emphasis on their structural characteristics and modification mechanisms.     

Xenorhabdus nematophila (enterobacteriacea) secretes a cation-selective calcium-independent porin which causes vacuolation of the rough endoplasmic reticulum and cell lysis

Xenorhabdus nematophila and Photorhabdus luminescens are two related enterobacteriaceae studied for their use in biological control and for synthesis of original virulence factors and new kinds of antibiotics. X. nematophila broth growth exhibits different cytotoxic activities on insect (Spodoptera littoralis, lepidoptera) immunocytes (hemocytes). Here we report the purification of the flhDC-dependent cytotoxin, a 10,790-Da peptide we have called alpha-Xenorhabdolysin (alpha X). We show that plasma membrane of insect hemocytes and of mammal red blood cells is the first target of this toxin. Electrophysiological and pharmacological approaches indicate that the initial effect of alpha X on macrophage plasma membrane is an increase of monovalent cation permeability, sensitive to potassium channel blockers. As a consequence, several events can occur intracellularly, such as selective vacuolation of the endoplasmic reticulum, cell swelling, and cell death by colloid-osmotic lysis. These effects, inhibited by potassium channel blockers, are totally independent of Ca(2+). However, the size of the pores created by alpha X on macrophage or red blood cell plasma membrane increases with toxin concentration, which leads to a rapid cell lysis.     

The folliculostatins of two dipteran insect species, their relation to matrix proteins and prospects for practical applications

The first insect folliculostatic peptide was isolated from vitellogenic ovaries of the mosquitoAedes aegypti. This decapeptide directly inhibits trypsin biosynthesis in the gut, and indirectly ovarian development. The factor was named Trypsin Modulating Oostatic Factor or TMOF by its discoverers. From the fleshfly Neobellieria bullata 2 folliculostatins have been isolated, the hexapeptide Neb-TMOF and the 19-mer Neb-colloostatin. The available data suggest that at least 2 of the 3 folliculostatins originate from matrix (like) proteins present in the ovary, a hitherto unknown source of hormones. Furthermore, one of the folliculostatins (Neb-TMOF) is a potent inhibitor of ecdysone biosynthesis by larval ring glands of fleshflies. The discovery of the dipteran folliculostatins, which do not show any resemblance to inhibins of vertebrates, may significantly contribute to a better understanding of the hormonal control of growth in insects and perhaps, in other animals as well. None of the 3 folliculostatins is blocked at its N- or C-terminus. This, in combination with the pleiotropy of their effects and the narrow species specificity make such peptides prime candidates for, testing their potential in insect pest control by means of molecular biological methods.     

Gut hormones with activity to modulate cellular growth

Recent progress in cancer research revealed that gut hormones have the activity to regulate the cellular growth of cancer cells. Gastrin, cholecystokinin and vasoactive intestinal peptide were demonstrated to stimulate the growth of gastric cancer cells, pancreatic cancer cells and colon cancer cells, respectively. Accordingly, it is possible to assume that these gut hormones may play an important role in the progression of these cancers. Further studies will be required to clarify the role of gut hormones as physiological growth factors in gastrointestinal tissues. The other aspect of gut hormones related with cellular growth is their role as autocrine growth factors. Gastrin-releasing peptide (GRP) is classified as a gut hormone with the structural similarity with amphibian bombesin. Several reported findings indicate that GRP functions as an autocrine growth factor for human small cell lung carcinoma; a monoclonal antibody for GRP is now applied for the therapy of this cancer. It is important to find out other gut hormones functioning as autocrine growth factors.     

Insect gonadotropic peptide hormones: some recent developments.

Gonadotropic peptides are a new generation of peptide hormone regulators of insect reproduction. They have been isolated from ovaries, oviducts, or brains of insects. The subject of this paper is insect peptides that exert stimulatory or inhibitory effects on ovarian development and oocyte maturation. On the basis of the literature data and the results of our investigations, the structure and biological properties of different groups of peptides are presented.     

Prolactin-releasing activity of porcine intestinal peptide (PHI-27)

Porcine intestinal peptide (PHI), a twenty-seven amino acid peptide isolated from porcine gut extracts, is a close structural homolog of the secretin family hormones. The structural and biological similarities of PHI to vasoactive intestinal peptide (VIP) together with its presence in the rat hypothalamus suggested a possible role for the peptide in the control of prolactin (PRL) secretion. PHI induced significant, dose-related stimulations of PRL release from cultured, dispersed rat pituitary cells in vitro. The minimum effective dose is 10⁷ molar, compared to 10⁹ molar for VIP. No interactive effect with thyrotropin-releasing hormone was observed; however, PHI partially overcame the dopamine inhibition of PRL release.