Is Proteolytic Cleavage Essential for the Enhanced Activity of Hydra Pore-Forming Toxin, HALT-4?

Hydra actinoporin-like toxin 4 (HALT-4) differs from other actinoporins due to its N-terminal propart that contains approximately 103 additional residues. Within this region, we identified five dibasic residues and assumed that, when cleaved, they could potentially exhibit HALT-4's cytolytic activity. We created five truncated versions of HALT-4 (tKK1, tKK2, tRK3, tKK4 and tKK5) to investigate the role of the N-terminal region and potential cleavage sites on the cytolytic activity of HALT-4. However, our results demonstrated that the propart-containing HALT-4 (proHALT-4), as well as the truncated versions tKK1 and tKK2, exhibited similar cytolytic activity against HeLa cells. In contrast, tRK3, tKK4 and tKK5 failed to kill HeLa cells, indicating that cleavage at the KK1 or KK2 sites did not enhance cytolytic activity but may instead facilitate the sorting of tKK1 and tKK2 to the regulated secretory pathway for eventual deposition in nematocysts. Moreover, RK3, KK4 and KK5 were unlikely to serve as proteolytic cleavage sites, as the amino acids between KK2 and RK3 are also crucial for pore formation.

The structural basis of Bcl-2 mediated cell death regulation in hydra.

Apoptosis is regulated by evolutionarily conserved signaling pathways to remove damaged, diseased or unwanted cells. Proteins homologous to the B-cell lymphoma 2 (Bcl-2) family of proteins, the primary arbiters of mitochondrially mediated apoptosis, are encoded by the cnidarian Hydra vulgaris. We mapped interactions between pro-survival and pro-apoptotic Bcl-2 proteins of H. vulgaris by affinity measurements between Hy-Bcl-2-4, the sole confirmed pro-survival Bcl-2 protein, with BH3 motif peptides of two Bcl-2 proteins from hydra that displayed pro-apoptotic activity, Hy-Bak1 and Hy-BH3-only-2, and the BH3 motif peptide of the predicted pro-apoptotic protein Hy-Bax. In addition to peptides from hydra encoded pro-apoptotic proteins, Hy-Bcl-2-4 also engaged BH3 motif peptides from multiple human pro-apoptotic Bcl-2 proteins. Reciprocally, human pro-survival Bcl-2 proteins Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1/Bfl-1 bound to BH3 spanning peptides from hydra encoded pro-apoptotic Hy-Bak1, Hy-BH3-only and Hy-Bax. The molecular details of the interactions were determined from crystal structures of Hy-Bcl-2-4 complexes with BH3 motif peptides of Hy-Bak1 and Hy-Bax. Our findings suggest that the Bcl-2 family in hydra may function in a manner analogous to the Bcl-2 family in humans, and less like the worm Caenorhabditis elegans where evolutionary gene deletion has simplified the apoptotic program. Combined, our results demonstrate the powerful conservation of the interaction pattern between hydra and human Bcl-2 family members. Furthermore, our data reveal mechanistic differences in the mode of binding between hydra and sponges such as Geodia cydonium, with hydra encoded Bcl-2 resembling the more promiscuous pro-apoptotic Bcl-2 members found in mammals compared with its sponge counterpart.

Antifibrotic strategies for medical devices.

A broad range of medical devices initiate an immune reaction known as the foreign body response (FBR) upon implantation. Here, collagen deposition at the surface of the implant occurs as a result of the FBR, ultimately leading to fibrous encapsulation and, in many cases, reduced function or failure of the device. Despite significant efforts, the prevention of fibrotic encapsulation has not been realized at this point in time. However, many next-generation medical technologies including cellular therapies, sensors and devices depend on the ability to modulate and control the FBR. For these technologies to become viable, significant advances must be made in understanding the underlying mechanism of this response as well as in the methods modulating this response. In this review, we highlight recent advances in the development of materials and coatings providing a reduced FBR and emphasize key characteristics of high-performing approaches. We also provide a detailed overview of the state-of-the-art in strategies relying on controlled drug release, the surface display of bioactive signals, materials-based approaches, and combinations of these approaches. Finally, we offer perspectives on future directions in this field.

New Class of Crosslinker-Free Nanofiber Biomaterials from Hydra Nematocyst Proteins.

Nematocysts, the stinging organelles of cnidarians, have remarkable mechanical properties. Hydra nematocyst capsules undergo volume changes of 50% during their explosive exocytosis and withstand osmotic pressures of beyond 100 bar. Recently, two novel protein components building up the nematocyst capsule wall in Hydra were identified. The cnidarian proline-rich protein 1 (CPP-1) characterized by a "rigid" polyproline motif and the elastic Cnidoin possessing a silk-like domain were shown to be part of the capsule structure via short cysteine-rich domains that spontaneously crosslink the proteins via disulfide bonds. In this study, recombinant Cnidoin and CPP-1 are expressed in E. coli and the elastic modulus of spontaneously crosslinked bulk proteins is compared with that of isolated nematocysts. For the fabrication of uniform protein nanofibers by electrospinning, the preparative conditions are systematically optimized. Both fibers remain stable even after rigorous washing and immersion into bulk water owing to the simultaneous crosslinking of cysteine-rich domains. This makes our nanofibers clearly different from other protein nanofibers that are not stable without chemical crosslinkers. Following the quantitative assessment of mechanical properties, the potential of Cnidoin and CPP-1 nanofibers is examined towards the maintenance of human mesenchymal stem cells.

Expansion of Hydra actinoporin-like toxin (HALT) gene family: Expression divergence and functional convergence evolved through gene duplication.

Hydra actinoporin-like toxin 1 (HALT-1) was previously shown to cause cytolysis and haemolysis in a number of human cells and has similar functional properties to the actinoporins equinatoxin and sticholysin. In addition to HALT-1, five other HALTs (HALTs 2, 3, 4, 6 and 7) were also isolated from Hydra magnipapillata and expressed as recombinant proteins in this study. We demonstrated that recombinant HALTs have cytolytic activity on HeLa cells but each exhibited a different range of toxicity. All six recombinant HALTs bound to sulfatide, while rHALT-1 and rHALT-3 bound to two additional sphingolipids, lysophosphatidic acid and sphingosine-1-phosphate as indicated by the protein-lipid overlay assay. When either tryptophan133 or tyrosine129 of HALT-1 was mutated, the mutant protein lost binding to sulfatide, lysophosphatidic acid and sphingosine-1-phosphate. As further verification of HALTs' binding to sulfatide, we performed ELISA for each HALT. To determine the cell-type specific gene expression of seven HALTs in Hydra, we searched for individual HALT expression in the single-cell RNA-seq data set of Single Cell Portal. The results showed that HALT-1, 4 and 7 were expressed in differentiating stenoteles. HALT-1 and HALT-6 were expressed in the female germline during oogenesis. HALT-2 was strongly expressed in the gland and mucous cells in the endoderm. Information on HALT-3 and HALT-5 could not be found in the single-cell data set. Our findings show that subfunctionalisation of gene expression following duplication enabled HALTs to become specialized in various cell types of the interstitial cell lineage.

The Sialic Acid-Dependent Nematocyst Discharge Process in Relation to Its Physical-Chemical Properties Is A Role Model for Nanomedical Diagnostic and Therapeutic Tools.

Formulas derived from theoretical physics provide important insights about the nematocyst discharge process of Cnidaria (Hydra, jellyfishes, box-jellyfishes and sea-anemones). Our model description of the fastest process in living nature raises and answers questions related to the material properties of the cell- and tubule-walls of nematocysts including their polysialic acid (polySia) dependent target function. Since a number of tumor-cells, especially brain-tumor cells such as neuroblastoma tissues carry the polysaccharide chain polySia in similar concentration as fish eggs or fish skin, it makes sense to use these findings for new diagnostic and therapeutic approaches in the field of nanomedicine. Therefore, the nematocyst discharge process can be considered as a bionic blue-print for future nanomedical devices in cancer diagnostics and therapies. This approach is promising because the physical background of this process can be described in a sufficient way with formulas presented here. Additionally, we discuss biophysical and biochemical experiments which will allow us to define proper boundary conditions in order to support our theoretical model approach. PolySia glycans occur in a similar density on malignant tumor cells than on the cell surfaces of Cnidarian predators and preys. The knowledge of the polySia-dependent initiation of the nematocyst discharge process in an intact nematocyte is an essential prerequisite regarding the further development of target-directed nanomedical devices for diagnostic and therapeutic purposes. The theoretical description as well as the computationally and experimentally derived results about the biophysical and biochemical parameters can contribute to a proper design of anti-tumor drug ejecting vessels which use a stylet-tubule system. Especially, the role of nematogalectins is of interest because these bridging proteins contribute as well as special collagen fibers to the elastic band properties. The basic concepts of the nematocyst discharge process inside the tubule cell walls of nematocysts were studied in jellyfishes and in Hydra which are ideal model organisms. Hydra has already been chosen by Alan Turing in order to figure out how the chemical basis of morphogenesis can be described in a fundamental way. This encouraged us to discuss the action of nematocysts in relation to morphological aspects and material requirements. Using these insights, it is now possible to discuss natural and artificial nematocyst-like vessels with optimized properties for a diagnostic and therapeutic use, e.g., in neurooncology. We show here that crucial physical parameters such as pressure thresholds and elasticity properties during the nematocyst discharge process can be described in a consistent and satisfactory way with an impact on the construction of new nanomedical devices.

Arminin 1a-C, a novel antimicrobial peptide from ancient metazoan Hydra, shows potent antileukemia activity against drug-sensitive and drug-resistant leukemia cells.

PURPOSE: Due to the emergence of multidrug resistance (MDR), traditional antileukemia drugs no longer meet the treatment needs. Therefore, new antileukemia drugs with different action mechanisms are urgently needed to cope with this situation. MATERIALS AND METHODS: Arminin 1a-C is an antimicrobial peptide (AMP) developed from the ancient metazoan marine Hydra. In this study, we first explored its antileukemia activity. RESULTS: Our results showed that Arminin 1a-C formed an α-helical structure and efficaciously suppressed the viability of leukemia cell lines whether or not they were multidrug resistant or sensitive, and there were no obvious differences between these cell lines. Arminin 1a-C exhibited distinct selectivity between noncancerous and cancerous cell lines. Arminin 1a-C interfered with K562/adriamycin (ADM) cell (a kind of multidrug-resistant leukemia cell line) proliferation in a very rapid manner and formed pores in its cell membrane, making it difficult to develop resistance against Arminin 1a-C. CONCLUSION: Our data show that Arminin 1a-C possesses great potential as a therapeutic candidate for the treatment of multidrug-resistant leukemia.

Mannose 6-phosphate-dependent lysosomal enzyme targeting in hydra: a biochemical, immunological and structural elucidation.

Mannose 6-phosphate (M6P)-dependent lysosomal enzyme targeting to endosome/lysosome complex is poorly understood among lower invertebrates. So far, only a M6P-independent lysosomal enzyme sorting protein, named LERP, has been described in Drosophila. Here, we have identified mannose 6-phosphate receptor (MPR) homologues in Hydra vulgaris, a basal Cnidarian, at genome level and further purified a cation-dependent MPR-like protein from hydra using affinity chromatography. Structural comparisons of hydra MPRs with mammalian MPRs confirm that the residues important for interacting with the M6P ligand are conserved. Based on our results, we report for the first time the occurrence of MPR-related proteins and M6P-dependent lysosomal enzyme targeting in H. vulgaris.

Evolution of miRNA Tailing by 3' Terminal Uridylyl Transferases in Metazoa.

In bilaterian animals the 3' ends of microRNAs (miRNAs) are frequently modified by tailing and trimming. These modifications affect miRNA-mediated gene regulation by modulating miRNA stability. Here, we analyzed data from three nonbilaterian animals: two cnidarians (Nematostella vectensis and Hydra magnipapillata) and one poriferan (Amphimedon queenslandica). Our analysis revealed that nonbilaterian miRNAs frequently undergo modifications like the bilaterian counterparts: the majority are expressed as different length isoforms and frequent modifications of the 3' end by mono U or mono A tailing are observed. Moreover, as the factors regulating miRNA modifications are largely uncharacterized in nonbilaterian animal phyla, in present study, we investigated the evolution of 3' terminal uridylyl transferases (TUTases) that are known to involved in miRNA 3' nontemplated modifications in Bilateria. Phylogenetic analysis on TUTases showed that TUTase1 and TUTase6 are a result of duplication in bilaterians and that TUTase7 and TUTase4 are the result of a vertebrate-specific duplication. We also find an unexpected number of Drosophila-specific gene duplications and domain losses in most of the investigated gene families. Overall, our findings shed new light on the evolutionary history of TUTases in Metazoa, as they reveal that this core set of enzymes already existed in the last common ancestor of all animals and was probably involved in modifying small RNAs in a similar fashion to its present activity in bilaterians.

[MORPHOMETRIC AND HISTOCHEMICAL CHARACTERISTICS OF THE THYROID GLAND AFTER ADMINISTRATION OF HYDRA PEPTIDE MORPHOGEN].

The effect of Hydra peptide morphogen (HPM) on quantitative histochemical and morphometric parameters of the thyroid gland (TG) was studied. The experiments were conducted on 40 outbred albino male mice weighing 20-25 g, which were injected intraperitoneally with HPM at the dose of 100 µg/kg of body weight per day for 5 days. Relative volumes occupied by the epithelium (E), including its follicular (E(f)), interfollicular (E(i)) components, and colloid (C) were determined using stereological method on TG transverse sections. E(f)/E(i) and E/C ratios were calculated as the indices of follicular organization and TG activity, respectively. Mitotic activity of thyrocytes was also evaluated. The enzymes, characterizing the metabolic activity of thyrocytes: NADH-diaphorase, succinate- and lactate dehydrogenases were demonstrated on cryostat sections of material, frozen in liquid nitrogen and their activity was assessed cytophotometrically. The results demonstrated that HPM administration lead to a significant increase in relative volume of thyroid epithelium with a concomitant reduction of the volume of the colloid. E(f)/E(i) ratio was not significantly different from that in the control. HPM also induced a significant increase of thyrocyte proliferation rate and of the activity of enzymes studied. Collectively, the quantitative histoenzymological and morphometric data obtained indicate the stimulating effect of HPM on TG functional activity and thyrocyte proliferation.

One short cysteine-rich sequence pattern - two different disulfide-bonded structures - a molecular dynamics simulation study.

The nematocyst walls of Hydra are formed by proteins containing small cysteine-rich domains (CRDs) of ~25 amino acids. The first CRD of nematocyst outer all antigen (NW1) and the C-terminal CRD of minicollagen-1 (Mcol1C) contain six cysteines at identical sequence positions, however adopt different disulfide bonded structures. NW1 shows the disulfide connectivities C2-C14/C6-C19/C10-C18 and Mcol1C C2-C18/C6-C14/C10-C19. To analyze if both show structural preferences in the open, non-disulfide bonded form, which explain the formation of either disulfide connectivity pattern, molecular dynamics (MD) simulations at different temperatures were performed. NW1 maintained in the 100-ns MD simulations at 283 K a rather compact fold that is stabilized by specific hydrogen bonds. The Mcol1C structure fluctuated overall more, however stayed most of the time also rather compact. The analysis of the backbone Φ/ψ angles indicated different turn propensities for NW1 and Mcol1C, which mostly can be explained based on published data about the influence of different amino acid side chains on the local backbone conformation. Whereas a folded precursor mechanism may be considered for NW1, Mcol1C may fold according to the quasi-stochastic folding model involving disulfide bond reshuffling and conformational changes, locking the native disulfide conformations. The study further demonstrates the power of MD simulations to detect local structural preferences in rather dynamic systems such as the open, non-disulfide bonded forms of NW1 and Mcol1C, which complement published information from NMR backbone residual dipolar couplings. Because the backbone structural preferences encoded by the amino acid sequence embedding the cysteines influence which disulfide connectivities are formed, the data are generally interesting for a better understanding of oxidative folding and the design of disulfide stabilized therapeutics.

Hydra actinoporin-like toxin-1, an unusual hemolysin from the nematocyst venom of Hydra magnipapillata which belongs to an extended gene family.

Cnidarians rely on their nematocysts and the venom injected through these unique weaponry systems to catch prey and protect themselves from predators. The development and physiology of the nematocysts of Hydra magnipapillata, a classic model organism, have been intensively studied, yet the composition and biochemical activity of their venom components are mostly unknown. Here, we show that hydra actinoporin-like toxins (HALTs), which have previously been associated with Hydra nematocysts, belong to a multigene family comprising six genes, which have diverged from a single common ancestor. All six genes are expressed in a population of Hydra magnipapillata. When expressed recombinantly, HALT-1 (Δ-HYTX-Hma1a), an actinoporin-like protein found in the stenoteles (the main penetrating nematocysts used in prey capture), reveals hemolytic activity, albeit about two-thirds lower than that of the anemone actinoporin equinatoxin II (EqTII, Δ-AITX-Aeq1a). HALT-1 also differs from EqTII in the size of its pores, and likely does not utilize sphingomyelin as a membrane receptor. We describe features of the HALT-1 sequence which may contribute to this difference in activity, and speculate on the role of this unusual family of pore-forming toxins in the ecology of Hydra.

Neuropeptides and epitheliopeptides: structural and functional diversity in an ancestral metazoan Hydra.

Peptides are known to play important developmental and physiological roles in signaling. The rich diversity of peptides, with functions as diverse as intercellular communication, neurotransmission and signaling that spatially and temporally controls axis formation and cell differentiation, hints at the wealth of information passed between interacting cells. Little is known about peptides that control developmental processes such as cell differentiation and pattern formation in metazoans. The cnidarian Hydra is one of the most basic metazoans and is a key model system for study of the peptides involved in these processes. We developed a novel peptidomic approach for the isolation and identification of functional peptide signaling molecules from Hydra (the Hydra Peptide Project). Over the course of this project, a wide variety of novel neuropeptides were identified. Most of these peptides act directly on muscle cells and their functions include induction of contraction and relaxation. Some peptides are involved in cell differentiation and morphogenesis. Moreover, epitheliopeptides that are produced by epithelial cells were originally identified in Hydra. Some of these epitheliopeptides exhibit morphogen-like activities, whereas others are involved in regulating neuron differentiation, possibly through neuron-epithelial cell interactions. We also describe below our high-throughput reverse-phase nano-flow LCMALDI- TOF-MS/MS approach, which has proved a powerful tool for the discovery of novel peptide signaling molecules in Hydra.

Components, structure, biogenesis and function of the Hydra extracellular matrix in regeneration, pattern formation and cell differentiation.

The body wall of Hydra is organized as an epithelial bilayer (ectoderm and endoderm) with an intervening extracellular matrix (ECM), termed mesoglea by early biologists. Morphological studies have determined that Hydra ECM is composed of two basal lamina layers positioned at the base of each epithelial layer with an intervening interstitial matrix. Molecular and biochemical analyses of Hydra ECM have established that it contains components similar to those seen in more complicated vertebrate species. These components include such macromolecules as laminin, type IV collagen, and various fibrillar collagens. These components are synthesized in a complicated manner involving cross-talk between the epithelial bilayer. Any perturbation to ECM biogenesis leads to a blockage in Hydra morphogenesis. Blockage in ECM/cell interactions in the adult polyp also leads to problems in epithelial transdifferentiation processes. In terms of biophysical parameters, Hydra ECM is highly flexible; a property that facilitates continuous movements along the organism's longitudinal and radial axis. This is in contrast to the more rigid matrices often found in vertebrates. The flexible nature of Hydra ECM can in part now be explained by the unique structure of the organism's type IV collagen and fibrillar collagens. This review will focus on Hydra ECM in regard to: 1) its general structure, 2) its molecular composition, 3) the biophysical basis for the flexible nature of Hydra's ECM, 4) the relationship of the biogenesis of Hydra ECM to regeneration of body form, and 5) the functional role of Hydra ECM during pattern formation and cell differentiation.

In vivo imaging of basement membrane movement: ECM patterning shapes Hydra polyps.

Growth and morphogenesis during embryonic development, asexual reproduction and regeneration require extensive remodeling of the extracellular matrix (ECM). We used the simple metazoan Hydra to examine the fate of ECM during tissue morphogenesis and asexual budding. In growing Hydra, epithelial cells constantly move towards the extremities of the animal and into outgrowing buds. It is not known, whether these tissue movements involve epithelial migration relative to the underlying matrix or whether cells and ECM are displaced as a composite structure. Furthermore, it is unclear, how the ECM is remodeled to adapt to the shape of developing buds and tentacles. To address these questions, we used a new in vivo labeling technique for Hydra collagen-1 and laminin, and tracked the fate of ECM in all body regions of the animal. Our results reveal that Hydra 'tissue movements' are largely displacements of epithelial cells together with associated ECM. By contrast, during the evagination of buds and tentacles, extensive movement of epithelial cells relative to the matrix is observed, together with local ECM remodeling. These findings provide new insights into the nature of growth and morphogenesis in epithelial tissues.

Peptidomic approaches to the identification and characterization of functional peptides in Hydra.

Little is known about peptides that control developmental processes such as cell differentiation and pattern formation in metazoans. The cnidarian Hydra is one of the most basal metazoans and is a key model system for studying the peptides involved in these processes. We developed a novel peptidomic approach to the isolation and identification of functional signalling peptides from Hydra (the Hydra peptide project). First, peptides extracted from the tissue of Hydra magnipapillata are purified to homogeneity using high-performance liquid chromatography (HPLC). The isolated peptides are then tested for their ability to alter gene expression in Hydra using differential display-PCR (DD-PCR). If gene expression is altered, the peptide is considered as a putative signalling peptide and is subjected to amino acid sequencing. Following the sequencing, synthetic peptides are produced and compared to their native counterparts by HPLC and/or mass spectrometry (MS). The synthetic peptides, which are available in larger quantities than their native analogues, are then tested in a variety of biological assays in Hydra to determine their functions. Here we present our strategies and a systematic approach to the identification and characterization of novel signalling peptides in Hydra. We also describe our high-throughput reverse-phase nano-flow liquid chromatography matrix-assisted laser desorption ionization time-of-flight mass spectrometry (LC-MALDI-TOF-MS/MS) approach, which was proved to be a powerful tool in the discovery of novel signalling peptides.

Asymmetric total synthesis and stereochemical revision of gymnangiamide.

The asymmetric total synthesis of the originally proposed structure of gymnangiamide, a cytotoxic pentapeptide isolated from the marine hydroid Gymnangium regae Jaderholm, has been achieved. Key to the synthesis was the use of asymmetric hydrogenation of alpha-substituted beta-ketoesters through dynamic kinetic resolution for the preparation of nonproteinogenic chiral amino acids. The disparity of the NMR spectra between the synthetic material containing the L-serine residue and the natural product required a revision of the proposed structure.

A hydra with many heads: protein and polypeptide toxins from hydra and their biological roles.

Hydra have been classical model organisms for over 250 years, yet little is known about the toxins they produce, and how they utilize these toxins to catch prey, protect themselves from predators and fulfill other biological roles necessary for survival. Unlike typical venomous organisms the hydra allomonal system is complex and "holistic", produced by various stinging cells (in the hunting tentacles and body ectoderm) as well as by non-nematocystic tissue. Toxic proteins also fulfill novel, non-allomonal roles in hydra. This review described the toxins produced by hydra within the context of their biology and natural history. Hydra nematocyst venom contains a high-molecular weight (>100 kDa) hemolytic and paralytic protein and a protein of approximately 30 kDa which induces a long-lasting flaccid paralysis. No low-molecular weight toxicity is observed, suggesting the lack of "classical" 4-7 kDa neurotoxins. The occurrence of a potent phospholipase activity in the venom is supported by the detection of several venom-like phospholipase A2 genes expressed by hydra. Hydra also produce toxins which are not part of the nematocyst venom. In the green hydra, Hydralysins, a novel family of Pore-Forming Proteins, are secreted into the gastrovascular cavity during feeding, probably helping in disintegration of the prey. Other putative non-nematocystic "toxins" may be involved in immunity, development or regulation of behavior. As the first venomous organism for which modern molecular tools are available, hydra provide a useful model to answer many outstanding questions on the way venomous organisms utilize their toxins to survive.

Further characterization of the PW peptide family that inhibits neuron differentiation in Hydra.

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.

Hydramacin-1, structure and antibacterial activity of a protein from the basal metazoan Hydra.

Hydramacin-1 is a novel antimicrobial protein recently discovered during investigations of the epithelial defense of the ancient metazoan Hydra. The amino acid sequence of hydramacin-1 shows no sequence homology to any known antimicrobial proteins. Determination of the solution structure revealed that hydramacin-1 possesses a disulfide bridge-stabilized alphabeta motif. This motif is the common scaffold of the knottin protein fold. The structurally closest relatives are the scorpion oxin-like superfamily. Within this superfamily hydramacin-1 establishes a new family of proteins that all share antimicrobial activity. Hydramacin-1 is potently active against Gram-positive and Gram-negative bacteria including multi-resistant human pathogenic strains. It leads to aggregation of bacteria as an initial step of its bactericidal mechanism. Aggregated cells are connected via electron-dense contacts and adopt a thorn apple-like morphology. Analysis of the hydramacin-1 structure revealed an unusual distribution of amino acid side chains on the surface. A belt of positively charged residues is sandwiched by two hydrophobic areas. Based on this characteristic surface feature and on biophysical analysis of protein-membrane interactions, we propose a model that describes the aggregation effect exhibited by hydramacin-1.