A new invertebrate NPY-like polypeptide, ZoaNPY, from the Zoanthus sociatus, as a novel ligand of human NPY Y2 receptor rescues vascular insufficiency via PLC/PKC and Src- FAK-dependent signaling pathways.

Our recent multi-omics studies have revealed rich sources of novel bioactive proteins and polypeptides from marine organisms including cnidarians. In the present study, we initially conducted a transcriptomic analysis to review the composition profile of polypeptides from Zoanthus sociatus. Then, a newly discovered NPY-like polypeptide-ZoaNPY was selected for further in silico structural, binding and virtually pharmacological studies. To evaluate the pro-angiogenic effects of ZoaNPY, we employed an in vitro HUVECs model and an in vivo zebrafish model. Our results indicate that ZoaNPY, at 1-100 pmol, enhances cell survival, migration and tube formation in the endothelial cells. Besides, treatment with ZoaNPY could restore a chemically-induced vascular insufficiency in zebrafish embryos. Western blot results demonstrated the application of ZoaNPY could increase the phosphorylation of proteins related to angiogenesis signaling including PKC, PLC, FAK, Src, Akt, mTOR, MEK, and ERK1/2. Furthermore, through molecular docking and surface plasmon resonance (SPR) verification, ZoaNPY was shown to directly and physically interact with NPY Y2 receptor. In view of this, all evidence showed that the pro-angiogenic effects of ZoaNPY involve the activation of NPY Y2 receptor, thereby activating the Akt/mTOR, PLC/PKC, ERK/MEK and Src- FAK-dependent signaling pathways. Furthermore, in an excision wound model, the treatment with ZoaNPY was shown to accelerate the wound healing process in mice. Our findings provide new insights into the discovery and development of novel pro-angiogenic drugs derived from NPY-like polypeptides in the future.

TRPV1 Modulator Ameliorates Alzheimer-Like Amyloid-ß Neuropathology via Akt/Gsk3ß-Mediated Nrf2 Activation in the Neuro-2a/APP Cell Model.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder for which there is no effective therapeutic strategy. PcActx peptide from the transcriptome of zoantharian Palythoa caribaeorum has recently been identified and verified as a novel antagonist of transient receptor potential cation channel subfamily V member 1 (TRPV1). In the present study, we further investigated the neuroprotective potential of PcActx peptide and its underlying mechanism of action, in an N2a/APP cell model of AD. Both Western blot and RT-PCR analysis revealed that PcActx peptide markedly inhibited the production of amyloid-related proteins and the expression of BACE1, PSEN1, and PSEN2. Moreover, PcActx peptide notably attenuated the capsaicin-stimulated calcium response and prevented the phosphorylation of CaMKII and CaMKIV (calcium-mediated proteins) in N2a/APP cells. Further investigation indicated that PcActx peptide significantly suppressed ROS generation through Nrf2 activation, followed by enhanced NQO1 and HO-1 levels. In addition, PcActx peptide remarkably improved Akt phosphorylation at Ser 473 (active) and Gsk3ß phosphorylation at Ser 9 (inactive), while pharmacological inhibition of the Akt/Gsk3ß pathway significantly attenuated PcActx-induced Nrf2 activation and amyloid downregulation. In conclusion, PcActx peptide functions as a TRPV1 modulator of intercellular calcium homeostasis, prevents AD-like amyloid neuropathology via Akt/Gsk3ß-mediated Nrf2 activation, and shows promise as an alternative therapeutic agent for AD.

Anti-epileptic Kunitz-like peptides discovered in the branching coral Acropora digitifera through transcriptomic analysis.

Approximately 50 million people are suffering from epilepsy worldwide. Corals have been used for treating epilepsy in traditional Chinese medicine, but the mechanism of this treatment is unknown. In this study, we analyzed the transcriptome of the branching coral Acropora digitifera and obtained its Kyoto Encyclopedia of Genes and Genomes (KEGG), EuKaryotic Orthologous Groups (KOG) and Gene Ontology (GO) annotation. Combined with multiple sequence alignment and phylogenetic analysis, we discovered three polypeptides, we named them AdKuz1, AdKuz2 and AdKuz3, from A. digitifera that showed a close relationship to Kunitz-type peptides. Molecular docking and molecular dynamics simulation indicated that AdKuz1 to 3 could interact with GABAA receptor but AdKuz2-GABAA remained more stable than others. The biological experiments showed that AdKuz1 and AdKuz2 exhibited an anti-inflammatory effect by decreasing the aberrant level of nitric oxide (NO), IL-6, TNF-α and IL-1ß induced by LPS in BV-2 cells. In addition, the pentylenetetrazol (PTZ)-induced epileptic effect on zebrafish was remarkably suppressed by AdKuz1 and AdKuz2. AdKuz2 particularly showed superior anti-epileptic effects compared to the other two peptides. Furthermore, AdKuz2 significantly decreased the expression of c-fos and npas4a, which were up-regulated by PTZ treatment. In addition, AdKuz2 reduced the synthesis of glutamate and enhanced the biosynthesis of gamma-aminobutyric acid (GABA). In conclusion, the results indicated that AdKuz2 may affect the synthesis of glutamate and GABA and enhance the activity of the GABAA receptor to inhibit the symptoms of epilepsy. We believe, AdKuz2 could be a promising anti-epileptic agent and its mechanism of action should be further investigated.

Toxic Peptide From Palythoa caribaeorum Acting on the TRPV1 Channel Prevents Pentylenetetrazol-Induced Epilepsy in Zebrafish Larvae.

PcActx peptide, identified from the transcriptome of zoantharian Palythoa caribaeorum, was clustered into the phylogeny of analgesic polypeptides from sea anemone Heteractis crispa (known as APHC peptides). APHC peptides were considered as inhibitors of transient receptor potential cation channel subfamily V member 1 (TRPV1). TRPV1 is a calcium-permeable channel expressed in epileptic brain areas, serving as a potential target for preventing epileptic seizures. Through in silico and in vitro analysis, PcActx peptide was shown to be a potential TRPV1 channel blocker. In vivo studies showed that the linear and oxidized PcActx peptides caused concentration-dependent increases in mortality of zebrafish larvae. However, monotreatment with PcActx peptides below the maximum tolerated doses (MTD) did not affect locomotor behavior. Moreover, PcActx peptides (both linear and oxidized forms) could effectively reverse pentylenetetrazol (PTZ)-induced seizure-related behavior in zebrafish larvae and prevent overexpression of c-fos and npas4a at the mRNA level. The excessive production of ROS induced by PTZ was markedly attenuated by both linear and oxidized PcActx peptides. It was also verified that the oxidized PcActx peptide was more effective than the linear one. In particular, oxidized PcActx peptide notably modulated the mRNA expression of genes involved in calcium signaling and γ-aminobutyric acid (GABA)ergic-glutamatergic signaling, including calb1, calb2, gabra1, grm1, gria1b, grin2b, gat1, slc1a2b, gad1b, and glsa. Taken together, PcActx peptide, as a novel neuroactive peptide, exhibits prominent anti-epileptic activity, probably through modulating calcium signaling and GABAergic-glutamatergic signaling, and is a promising candidate for epilepsy management.

Combined transcriptomic and proteomic analysis reveals a diversity of venom-related and toxin-like peptides expressed in the mat anemone Zoanthus natalensis (Cnidaria, Hexacorallia).

Venoms from marine animals have been recognized as a new emerging source of peptide-based therapeutics. Several peptide toxins from sea anemone have been investigated as therapeutic leads or pharmacological tools. Venom complexity should be further highlighted using combined strategies of large-scale sequencing and data analysis which integrated transcriptomics and proteomics to elucidate new proteins or peptides to be compared among species. In this work, transcriptomic and proteomic analyses were combined to identify six groups of expressed peptide toxins in Zoanthus natalensis. These include neurotoxin, hemostatic and hemorrhagic toxin, protease inhibitor, mixed function enzymes, venom auxiliary proteins, allergen peptides, and peptides related to the innate immunity. Molecular docking analysis indicated that one expressed Zoanthus Kunitz-like peptide, ZoaKuz1, could be a voltage-gated potassium channels blocker and, hence, it was selected for functional studies. Functional bioassays revealed that ZoaKuz1 has an intrinsic neuroprotective activity in zebrafish model of Parkinson's disease. Since pharmacological blockade of KV channels is known to induce neuroprotective effects, ZoaKuz1 holds the potential to be developed in a therapeutic tool to control neural dysfunction by slowing or even halting neurodegeneration mediated by ion-channel hyperactivity.

Novel neurotoxic peptides from Protopalythoa variabilis virtually interact with voltage-gated sodium channel and display anti-epilepsy and neuroprotective activities in zebrafish.

We previously reported a novel toxic peptide identified from the anthozoan Protopalythoa variabilis transcriptome which is homologous to a novel structural type of sodium channel toxin isolated from a parental species (Palythoa caribaeorum). The peptide was named, according to its homologous, as Pp V-shape α-helical peptide (PpVα) in the present study. Through molecular docking and dynamics simulation, linear and hairpin folded PpVα peptides were shown to be potential voltage-gated sodium channel blockers. Nowadays, sodium channel blockers have been the mainstream of the pharmacological management of epileptic seizures. Also, sodium channel blockers could promote neuronal survival by reducing sodium influx and reducing the likelihood of calcium importation resulting in suppressing microglial activation and protecting dopaminergic neurons from degeneration. The folded PpVα peptide could decrease pentylenetetrazol (PTZ)-induced c-fos and npas4a expression level leading to reverse PTZ-induced locomotor hyperactivity in zebrafish model. In vitro, the folded PpVα peptide protected PC12 cells against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity via activating heme oxygenase-1 (HO-1) and attenuating inducible nitric oxide synthase (iNOS) expression. In vivo, PpVα peptide suppressed the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish and, importantly, prevented the 6-OHDA-induced excessive ROS generation and subsequent dopaminergic neurons loss. This study indicates that the single S-S bond folded PpVα peptide arises as a new structural template to develop sodium channel blockers and provides an insight on the peptide discovery from cnidarian transcriptome to potentially manage epilepsy and neurodegenerative disorders.

A Novel ShK-Like Toxic Peptide from the Transcriptome of the Cnidarian Palythoa caribaeorum Displays Neuroprotection and Cardioprotection in Zebrafish.

Palythoa caribaeorum (class Anthozoa) is a zoantharian which, together with other cnidarians, like jellyfishes, hydra, and sea anemones, possesses specialized structures in its tissues, the cnidocytes, which deliver an array of toxins in order to capture prey and deter predators. The whole transcriptome of P. caribaeroum was deep sequenced, and a diversity of toxin-related peptide sequences were identified, and some retrieved for functional analysis. In this work, a peptide precursor containing a ShK domain, named PcShK3, was analyzed by means of computational processing, comprising structural phylogenetic analysis, model prediction, and dynamics simulation of peptide-receptor interaction. The combined data indicated that PcShK3 is a distinct peptide which is homologous to a cluster of peptides belonging to the ShK toxin family. In vivo, PcShK3 distributed across the vitelline membrane and accumulated in the yolk sac stripe of zebrafish larvae. Notably, it displayed a significant cardio-protective effect in zebrafish in concentrations inferior to the IC50 (<43.53 ± 6.45 µM), while in high concentrations (>IC50), it accumulated in the blood and caused pericardial edema, being cardiotoxic to zebrafish larvae. Remarkably, PcShK3 suppressed the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish. The present results indicated that PcShK3 is a novel member of ShK toxin family, and has the intrinsic ability to induce neuro- and cardio-protective effects or cause cardiac toxicity, according to its effective concentration.

Novel Kunitz-like Peptides Discovered in the Zoanthid Palythoa caribaeorum through Transcriptome Sequencing.

Palythoa caribaeorum (class Anthozoa) is a zoanthid that together jellyfishes, hydra, and sea anemones, which are venomous and predatory, belongs to the Phyllum Cnidaria. The distinguished feature in these marine animals is the cnidocytes in the body tissues, responsible for toxin production and injection that are used majorly for prey capture and defense. With exception for other anthozoans, the toxin cocktails of zoanthids have been scarcely studied and are poorly known. Here, on the basis of the analysis of P. caribaeorum transcriptome, numerous predicted venom-featured polypeptides were identified including allergens, neurotoxins, membrane-active, and Kunitz-like peptides (PcKuz). The three predicted PcKuz isotoxins (1-3) were selected for functional studies. Through computational processing comprising structural phylogenetic analysis, molecular docking, and dynamics simulation, PcKuz3 was shown to be a potential voltage gated potassium-channel inhibitor. PcKuz3 fitted well as new functional Kunitz-type toxins with strong antilocomotor activity as in vivo assessed in zebrafish larvae, with weak inhibitory effect toward proteases, as evaluated in vitro. Notably, PcKuz3 can suppress, at low concentration, the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish, which indicated PcKuz3 may have a neuroprotective effect. Taken together, PcKuz3 figures as a novel neurotoxin structure, which differs from known homologous peptides expressed in sea anemone. Moreover, the novel PcKuz3 provides an insightful hint for biodrug development for prospective neurodegenerative disease treatment.

Discovery of a linear cyclotide from the bracelet subfamily and its disulfide mapping by top-down mass spectrometry.

Cyclotides are heat-stable macrocyclic peptides from plants that display a wide range of biological activities. They can be divided into two subfamilies: Möbius or bracelet, based on the presence or absence of a cis-proline residue in loop 5, respectively. Currently, over 150 cyclotides have been discovered, but only four linear variants of the Möbius subfamily have been hitherto isolated. In this study, we report the discovery of two novel cyclotides, hedyotide B1 and hedyotide B2, from the aerial parts of Hedyotis biflora. Hedyotide B1 has a cyclic cystine knot structure typical of cyclotides. Interestingly, hedyotide B2 possesses a linear backbone and is the first linear representative of the bracelet subfamily. Disulfide mapping of hedyotide B2 by a top-down MS/MS approach showed that it shares the same knotted disulfide arrangement as conventional cyclotides. Its unfolding pathway also showed that the penetrating disulfide bond Cys III-VI is the most stable disulfide linkage. Cloning of the gene encoding hedyotide B2 revealed a nonsense mutation that introduces a premature stop codon at the conserved Asn residue position, which is essential for an end-to-end backbone ligation. Biophysical characterization showed that hedyotide B2 was more susceptible to exopeptidase degradation as compared with hedyotide B1. Hedyotide B2 was also inactive against all four tested bacterial strains, whereas hedyotide B1 was bactericidal to Escherichia coli and Streptococcus salivarius at low micromolar concentration. Our results provide a deeper understanding of the structures, functions, and biosynthetic processing of cyclotides and uncyclotides in plants.