Hydra Peptide Project 1993–2007

A systematic screening of peptide signaling molecules (<5000 da) in Hydra magnipapillata (the Hydra Peptide Project) was launched in 1993 and at least the first phase of the project ended in 2007. From the project a number of interesting suggestions and results have been obtained. First, a simple metazoan-like Hydra appears to contain a few hundred peptide signaling molecules: half of them neuropeptides and the rest epitheliopeptides that are produced by epithelial cells. Second, epitheliopeptides were identified for the first time in Hydra . Some exhibit morphogen-like activities, which accord with the notion that epithelial cells are primarily responsible for patterning in Hydra . A family of epitheliopeptides was involved in regulating neuron differentiation possibly through neuron–epithelial cell interaction. Third, many novel neuropeptides were identified. Most of them act directly on muscle cells inducing contraction or relaxation. Some were involved in cell differentiation and morphogenesis. During the course of this study, a number of important technical innovations (e.g. genetic manipulations in transgenic Hydra , high-throughput purification techniques, etc.) and expressed sequence tag (EST) and genome databases were introduced in Hydra research. They have already helped to identify and characterize novel peptides and will contribute even more to the Hydra Peptide Project in the near future.     

Important roles for epithelial cell peptides in hydra development

It has been convincingly shown that peptides play important roles in the regulation and maintenance of a variety of tissues and organs in living animals. However, little is known concerning the potential role of peptides as signaling molecules in developmental processes. In Hydra, there is circumstantial evidence that small diffusible molecules act as morphogens in the regulation of patterning processes. In order to view the entire spectrum of peptide signaling molecules, we initiated a project aiming at the systematic identification of peptide signaling molecules in Hydra. In this review, we describe three peptide signaling molecules and one family of peptides that function as signaling molecules in the processes of axial pattern formation and neuron differentiation in Hydra. These peptides are produced by epithelial cells and are therefore termed “epitheliopeptides”. We discuss the importance of epitheliopeptides in developmental processes within a subset of hydrozoans.     

Developmental signaling in Hydra: what does it take to build a "simple" animal?

Developmental processes in multicellular animals depend on an array of signal transduction pathways. Studies of model organisms have identified a number of such pathways and dissected them in detail. However, these model organisms are all bilaterians. Investigations of the roles of signal transduction pathways in the early-diverging metazoan Hydra have revealed that a number of the well-known developmental signaling pathways were already in place in the last common ancestor of Hydra and bilaterians. In addition to these shared pathways, it appears that developmental processes in Hydra make use of pathways involving a variety of peptides. Such pathways have not yet been identified as developmental regulators in more recently diverged animals. In this review I will summarize work to date on developmental signaling pathways in Hydra and discuss the future directions in which such work will need to proceed to realize the potential that lies in this simple animal.     

Polyps, peptides and patterning

Peptides serve as important signalling molecules in development and differentiation in the simple metazoan Hydra. A systematic approach (The Hydra Peptide Project) has revealed that Hydra contains several hundreds of peptide signalling molecules, some of which are neuropeptides and others emanate from epithelial cells. These peptides control biological processes as diverse as muscle contraction, neuron differentiation, and the positional value gradient. Signal peptides cause changes in cell behaviour by controlling target genes such as matrix metalloproteases. The abundance of peptides in Hydra raises the question of whether, in early metazoan evolution, cell-cell communication was based mainly on these small molecules rather than on the growth-factor-like cytokines that control differentiation and development in higher animals.     

A novel neuropeptide, Hym-355, positively regulates neuron differentiation in Hydra

During the course of a systematic screening of peptide signaling molecules in Hydra a novel peptide, Hym-355 (FPQSFLPRG-NH(2)), was identified. A cDNA encoding the peptide was isolated and characterized. Using both in situ hybridization and immunohistochemistry, Hym-355 was shown to be expressed in neurons and hence is a neuropeptide. The peptide was shown to specifically enhance neuron differentiation throughout the animal by inducing interstitial cells to enter the neuron pathway. Further, co-treatment with a PW peptide, which inhibits neuron differentiation, nullified the effects of both peptides, suggesting that they act in an antagonistic manner. This effect is discussed in terms of a feedback mechanism for maintaining the steady state neuron population in Hydra.     

Further characterization of the PW peptide family that inhibits neuron differentiation in <Emphasis Type="Italic">Hydra</Emphasis>

From an evolutionary point of view, Hydra has one of the most primitive nervous systems among metazoans. Two different groups of peptides that affect neuron differentiation were identified in a systematic screening of peptide signaling molecules in Hydra. Within the first group of peptides, a neuropeptide, Hym-355, was previously shown to positively regulate neuron differentiation. The second group of peptides encompasses the PW family of peptides that negatively regulate neuron differentiation. In this study, we identified the gene encoding PW peptide preprohormone. Moreover, we made the antibody that specifically recognizes LPW. In situ hybridization and immunohistochemical analyses showed that the PW peptides and the gene encoding them were expressed in ectodermal epithelial cells throughout the body except for the basal disk. The PW peptides are produced by epithelial cells and are therefore termed “epitheliopeptides.” Together with Hym-355, the PW family peptides mediate communication between neurons and epithelial cells and thereby maintain a specific density of neurons in Hydra. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Toshio Takahashi, Osamu Koizumi equally contributed to this study.     

A new case of neuropeptide coexpression (RGamide and LWamides) in Hydra,found by whole-mount,two-color double-labeling in situ hybridization

The freshwater polyp Hydra has a primitive nervous system that expresses at least six different neuropeptide genes: (1) three genes, coding for the preprohormones-A, -B, and -C that each gives rise to a variety of peptides with the C-terminal sequence Arg-Phe-NH(2) (the Hydra-RFamides); (2) one gene, coding for a preprohormone that gives rise to five peptides with the C-terminal sequence Leu-Trp-NH(2) (the Hydra-LWamides); (3) one gene, coding for a preprohormone that produces a peptide with the C-terminal sequence Lys-Val-NH(2) (Hydra-KVamide, also called Hym-176); and (4) one gene, coding for a preprohormone that gives rise to a peptide with the C-terminal sequence Arg-Gly NH(2) (Hydra-RGamide, also called Hym-355). In a previous paper, we described that a population of neurons in the peduncle (a region just above the foot) of Hydra coexpresses the preprohormone-A and KVamide genes, whereas neurons in the other regions only express either the preprohormone-A, -B, -C, LWamide, or the KVamide genes. Here, we investigated the RGamide gene expression, using whole-mount, two-color double-labeling in situ hybridization, and found that neurons in the basal disk (foot), gastric region, hypostome (a region around the mouth), and tentacles coexpress this gene together with the LWamide gene. A small population of neurons in the hypostome and upper gastric region expresses only the LWamide gene. No coexpression of the RGamide gene with any of the other neuropeptide genes was observed. This is the second example of coexpression of two neuropeptide genes in cnidarians. It demonstrates that many neurons in the primitive nervous systems of cnidarians use combinations ("cocktails") of neuropeptides for their signaling. It also shows that Hydra has at least seven neurochemically different populations of neurons.     

Enhancement of foot formation in Hydra by a novel epitheliopeptide, Hym-323

During the course of a systematic screening of peptide signaling molecules in Hydra magnipapillata, a novel peptide, Hym-323, which enhances foot regeneration was identified. The peptide is 16 amino acids long, and is encoded in the precursor protein as a single copy. Northern blot analysis, in situ hybridization analysis and immunohistochemistry showed that it was expressed in both ectodermal and endodermal epithelial cells throughout the body, except for the basal disk and the head region. The peptide enhanced foot regeneration by acting on epithelial cells. Lateral transplantation experiments indicated that the foot activation potential was increased in the peptide-treated tissue. These results suggest that Hym-323 is a peptide involved in a foot-patterning process in Hydra.     

Investigating endogenous peptides and peptidases using peptidomics

Rather than simply being protein degradation products, peptides have proven to be important bioactive molecules. Bioactive peptides act as hormones, neurotransmitters, and antimicrobial agents in vivo. The dysregulation of bioactive peptide signaling is also known to be involved in disease, and targeting peptide hormone pathways has been a successful strategy in the development of novel therapeutics. The importance of bioactive peptides in biology has spurred research to elucidate the function and regulation of these molecules. Classical methods for peptide analysis have relied on targeted immunoassays, but certain scientific questions necessitated a broader and more detailed view of the peptidome--all the peptides in a cell, tissue, or organism. In this review we discuss how peptidomics has emerged to fill this need through the application of advanced liquid chromatography--tandem mass spectrometry (LC-MS/MS) methods that provide unique insights into peptide activity and regulation.     

Suppression of S-methylglutathione-induced tentacle ball formation by peptides and nullification of the suppression by TGF-beta in Hydra

Tentacle ball formation (TBF) in Hydra elicited by S-methylglutathione (GSM) was modulated by a number of biologically active peptides. Hydra fed on Artemia, which had been hatched in a common salt solution supplemented with LiCl and ZnCl(2), easily induced TBF in response to GSM after pretreatment with trypsin. After Hydra were treated with 100 pg/ml trypsin for 10 min, the response to GSM (TBF) was sensitively suppressed by acidic fibroblast growth factor and other biologically active peptides for >10 h. Various peptides, but not transforming growth factor beta (TGF-beta), suppressed GSM-induced TBF in a specific pattern for each peptide. However, TGF-beta was unique in that it did not suppress the response to GSM, but nullified the suppressive effect of other peptides. Only active TGF-beta nullified the suppressive effect of the peptides, and the latent form of TGF-beta neither suppressed GSM-induced TBF nor nullified the suppressive effect of other peptides. Members of the TGF-beta family suppressed GSM-induced TBF. These results indicate that all peptides examined, except for TGF-beta suppressed the response to GSM in a manner specific to each peptide. This assay system would be useful in identification of biologically active peptides.     

Tapasin is a facilitator, not an editor, of class I MHC peptide binding

Tapasin has been proposed to function as a peptide editor to displace lower affinity peptides and/or to favor the binding of high affinity peptides. Consistent with this, cell surface HLA-B8 molecules in tapasin-deficient cells were less stable and the peptide repertoire was substantially altered. However, the binding affinities of peptides expressed in the absence of tapasin were unexpectedly higher, not lower. The peptide repertoire from cells expressing soluble tapasin was similar in both appearance and affinity to that presented in the presence of full-length tapasin, but the HLA-B8 molecules showed altered cell surface stability characteristics. Similarly, the binding affinities of HLA-A*0201-associated peptides from tapasin(+) and tapasin(-) cells were equivalent, although steady state HLA-A*0201 cell surface expression was decreased and the molecules demonstrated reduced cell surface stability on tapasin(-) cells. These data are inconsistent with a role for tapasin as a peptide editor. Instead, we propose that tapasin acts as a peptide facilitator. In this role, it stabilizes the peptide-free conformation of class I MHC molecules in the endoplasmic reticulum and thus increases the number and variety of peptides bound to class I MHC. Full-length tapasin then confers additional stability on class I MHC molecules that are already associated with peptides.     

Separation and identification of mouse brain tissue microproteins using top‐down method with high resolution nanocapillary liquid chromatography mass spectrometry

Microproteins and endogenous peptides in the brain contain important substances that have critical roles in diverse biological processes, contributing to signal transduction and intercellular signaling. However, variability in their physical or chemical characteristics, such as molecule size, hydrophobicity, and charge states, complicate the simultaneous analysis of these compounds, although this would be highly beneficial for the field of neuroscience research. Here, we present a top‐down analytical method for simultaneous analysis of microproteins and endogenous peptides using high‐resolution nanocapillary LC‐MS/MS. This method is detergent‐free and digestion‐free, which allows for extracting and preserving intact microproteins and peptides for direct LC‐MS analysis. Both higher energy collision dissociation and electron‐transfer dissociation fragmentations were used in the LC‐MS analysis to increase the identification rate, and bioinformatics tools ProteinGoggle and PEAKS Studio software were utilized for database search. In total, we identified 471 microproteins containing 736 proteoforms, including brain‐derived neurotrophic factor and a number of fibroblast growth factors. In addition, we identified 599 peptides containing 151 known or potential neuropeptides such as somatostatin‐28 and neuropeptide Y. Our approach bridges the gap for the characterization of brain microproteins and peptides, which permits quantification of a diversity of signaling molecules for biomarker discovery or therapy diagnosis in the future.     

Intracellullar peptides as putative natural regulators of protein interactions

Extralysosomal proteolysis by multicatalytic complexes such as the 26S proteasome produces large amounts of peptides in the cytosol, mitochondria and nuclei of eukaryotic cells, and there is increasing evidence that the resulting free intracellular peptides can modulate specific protein interactions. The demonstration that free peptides added to the intracellular milieu can regulate cellular functions mediated by protein interactions suggests new putative roles for these molecules in gene regulation, metabolism, cell signaling and protein targeting. Such interactions frequently involve specific consensus amino acid sequences that can be predicted based on similarities in domain composition. We have recently developed a new strategy for identifying novel natural peptides, the sequences of which correspond to fragments of intracellular proteins and contain putative post-translational modification sites. In this review, we examine the evidence that intracellular peptides released by proteasomes may be involved in regulating protein interactions. In particular, the role of endopeptidase 24.15 (thimet oligopeptidase; EC 3.4.24.15) is discussed in detail as this enzyme has been implicated in intracellular peptide metabolism in vivo in concert with the 26S proteasome.     

Identification of Peptide lv, a novel putative neuropeptide that regulates the expression of L-type voltage-gated calcium channels in photoreceptors

Neuropeptides are small protein-like signaling molecules with diverse roles in regulating neural functions such as sleep/wake cycles, pain modulation, synaptic plasticity, and learning and memory. Numerous drugs designed to target neuropeptides, their receptors, or relevant pathways have been developed in the past few decades. Hence, the discovery and characterization of new neuropeptides and their functions have received considerable attention from scientific research. Computational bioinformatics coupled with functional assays are powerful tools to address the difficulties in discovering new bioactive peptides. In this study, a new bioinformatic strategy was designed to screen full length human and mouse cDNA databases to search for novel peptides. One was discovered and named peptide Lv because of its ability to enhance L-type voltage-gated calcium channel (L-VGCC) currents in retinal photoreceptors. Using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), peptide Lv was detected in the culture media, which indicated that it was secreted from 661W cells transfected with the gene. In vitro treatments with either glutathione S-transferase (GST) fusion peptide Lv or synthesized peptide Lv enhanced L-VGCC channel activities in cone photoreceptors. At the molecular level, peptide Lv stimulated cAMP production, enhanced phosphorylation of extracellular signal-regulated kinase (ERK), and increased the protein expression of L-VGCCα1 subunits in cone photoreceptors. Therefore, the biological activities of peptide Lv may be very important in the modulation of L-VGCC dependent neural plasticity.     

A human CTL recognizes a caspase-8-derived peptide on autologous HLA-B*3503 molecules and two unrelated peptides on allogeneic HLA-B*3501 molecules

A CTL clone that recognizes autologous tumor cells was previously isolated from the blood of a head-and-neck cancer patient. The Ag was identified as peptide FPSDSWCYF presented by autologous HLA-B*3503 molecules. This peptide was encoded by a mutated CASP-8 gene, which is implicated in the triggering of apoptosis. Here, we show that this CTL clone, which expresses a single TCR, also recognizes two unrelated peptides on allogeneic HLA-B*3501 molecules. One peptide, HIPDVITY, is encoded by squalene synthase, and the other one, QFADVIVLF, is encoded by 2-hydroxyphytanoyl-CoA lyase. Both genes are expressed ubiquitously. These antigenic peptides are processed and presented by HLA-B*3501 cells. The two HLA-B35 alleles are closely related. Our results might reinforce the notion that the recognition of allogeneic HLA molecules depends on the presence in their groove of a limited number of peptides processed from ubiquitous proteins.     

A novel neuropeptide (FRamide) family identified by a peptidomic approach in Hydra magnipapillata

In the course of systematic identification of peptide signaling molecules combined with the expressed sequence tag database from Hydra, we have identified a novel neuropeptide family that consists of two members with FRamide at the C-terminus; FRamide-1 (IPTGTLIFRamide) and FRamide-2 (APGSLLFRamide). The precursor sequence deduced from cDNA contained a single copy each of FRamide-1 and FRamide-2 precursor sequences. Expression analysis by whole-mount in situ hybridization showed that the gene was expressed in a subpopulation of neurons that were distributed throughout the body from tentacles to basal disk. Double in situ hybridization analysis showed that the expressing cell population was further subdivided into one population consisting of neurons expressing both the FRamide and Hym176 (neuropeptide) genes and the other consisting of neurons expressing only the FRamide gene. FRamide-1 evoked elongation of the body column of 'epithelial' Hydra that was composed of epithelial cells and gland cells but lacked all the cells in the interstitial stem cell lineage, including neurons. In contrast, FRamide-2 evoked body column contraction. These results suggest that both of the neuropeptides directly act on epithelial cells as neurotransmitters and regulate body movement in an axial direction.     

Subcellular localization of the epitheliopeptide, Hym-301, in hydra

Peptides, as signaling molecules, play a number of roles in cell activities. An epitheliopeptide, Hym-301, has been described as a peptide involved in morphogenesis in hydra. However, little is known about the intracellular location of the peptide or its specific functions. To investigate the mechanism of morphogenesis that involves peptidic molecules, we have examined the intracellular localization of Hym-301 in hydra by using immunohistochemical and immunogold electron-microscopic analyses. We have found that the pattern of distribution of mature peptide is slightly different from that of its mRNA, and that the peptide is stored in vesicles located adjacent to the cell membrane. We have also found that the peptide is released both extracellularly and internally to the cytoplasm of the cells. Based upon these observations, we have constructed a possible model mechanism of homeostatic regulation of the distribution of the Hym-301 peptide in a dynamic tissue context.     

Synthetic peptides and non-peptidic molecules as probes of structure and function of Bcl-2 family proteins and modulators of apoptosis

The Bcl-2 family includes a growing number of proteins that play an essential role in regulating apoptosis or programmed cell death. Members of this family display diverse biological functions and can either inhibit or promote cell death signals. Abnormal gene expression of some Bcl-2 family members such as Bcl-2 that inhibits apoptosis is found in a wide variety of human cancers and contributes to the resistance of tumor cells to conventional therapies through interfering with the cell death signals triggered by chemotherapeutic agents. As such, elucidating the structure-function and mechanism of the Bcl-2 family is important for understanding some of the fundamental principles underling the death and survival of cells and of practical value for developing potential therapeutics to control apoptosis in pathological processes. Synthetic peptides derived from homologous or heterogeneous domains in Bcl-2 family proteins that might mediate different biological activities provide simplified and experimentally more tractable models as compared to their full-length counterparts to dissect and analyze the complex functional roles of these proteins. Non-peptidic molecules identified from random screening of natural products or designed by rational structure-based techniques can mimic the effect of synthetic peptides by targeting similar active sites on a Bcl-2 family member protein. In this article, we review recent progress in using these synthetic peptides and non-peptidic mimic molecules to obtain information about the structure and function of Bcl-2 family proteins and discuss their application in modulating and studying intracellular apoptotic signaling.