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.

The Place for Enzymes and Biologically Active Peptides from Marine Organisms for Application in Industrial and Pharmaceutical Biotechnology.

Since the beginning of written history, diverse texts have reported the use of enzymatic preparations in food processing and have described the medicinal properties of crude and fractionated venoms to treat various diseases and injuries. With the biochemical characterization of enzymes from distinct sources and bioactive polypeptides from animal venoms, the last sixty years have testified the advent of industrial enzymology and protein therapeutics, which are currently applicable in a wide variety of industrial processes, household products, and pharmaceuticals. Bioprospecting of novel biocatalysts and bioactive peptides is propelled by their unsurpassed properties that are applicable for current and future green industrial processes, biotechnology, and biomedicine. The demand for both novel enzymes with desired characteristics and novel peptides that lead to drug development, has experienced a steady increase in response to the expanding global market for industrial enzymes and peptidebased drugs. Moreover, although largely unexplored, oceans and marine realms, with their unique ecosystems inhabited by a large variety of species, including a considerable number of venomous animals, are recognized as untapped reservoirs of molecules and macromolecules (enzymes and bioactive venom-derived peptides) that can potentially be converted into highly valuable biopharmaceutical products. In this review, we have focused on enzymes and animal venom (poly)peptides that are presently in biotechnological use, and considering the state of prospection of marine resources, on the discovery of useful industrial biocatalysts and drug leads with novel structures exhibiting selectivity and improved performance.

The dinoponeratoxin peptides from the giant ant Dinoponera quadriceps display in vitro antitrypanosomal activity.

The crude venom of the giant ant Dinoponera quadriceps is a cocktail of polypeptides and organic compounds that shows antiparasitic effects against Trypanosoma cruzi, the causative agent of Chagas disease. In order to investigate the venom-derived components responsible for such antitrypanosomal activity, four dinoponeratoxins (DnTxs) were identified, namely M-PONTX-Dq3a, -Dq3b, -Dq3c and -Dq4e, that are diverse in size, net charge, hydrophobicity and propensity to interact with eukaryote cell membranes. These peptides were tested against epimastigote, trypomastigote and amastigote forms of benznidazole (Bz)-resistant Y strain of T. cruzi and in mammalian host cells. The M-PONTX-Dq3a and -Dq4e inhibited all developmental forms of T. cruzi, including amastigotes, the responsible form for the maintenance of infection on chronic phase of the disease. The M-PONTX-Dq3a showed the highest selectivity index (SI) (80) and caused morphological alterations in T. cruzi, as observed by scanning electron microscopy (SEM), and induced cell death through necrosis, as seen by multiparametric flow cytometry analysis with specific biochemical markers. Altogether, the D. quadriceps venom appears as a source for the prospection of trypanocidal peptides and the M-PONTX-Dq3a arises as a candidate among the dinoponeratoxin-related peptides in the development of compounds against Chagas disease.

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.

The holo-transcriptome of the zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa): a plentiful source of enzymes for potential application in green chemistry, industrial and pharmaceutical biotechnology

Marine invertebrates, such as sponges, tunicates and cnidarians (zoantharians and scleractinian corals), form functional assemblages, known as holobionts, with numerous microbes. This type of species-specific symbiotic association can be a repository of myriad valuable low molecular weight organic compounds, bioactive peptides and enzymes. The zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa) is one such example of a marine holobiont that inhabits the coastal reefs of the tropical Atlantic coast and is an interesting source of secondary metabolites and biologically active polypeptides. In the present study, we analyzed the entire holo-transcriptome of P. variabilis, looking for enzyme precursors expressed in the zoantharian-microbiota assemblage that are potentially useful as industrial biocatalysts and biopharmaceuticals. In addition to hundreds of predicted enzymes that fit into the classes of hydrolases, oxidoreductases and transferases that were found, novel enzyme precursors with multiple activities in single structures and enzymes with incomplete Enzyme Commission numbers were revealed. Our results indicated the predictive expression of thirteen multifunctional enzymes and 694 enzyme sequences with partially characterized activities, distributed in 23 sub-subclasses. These predicted enzyme structures and activities can prospectively be harnessed for applications in diverse areas of industrial and pharmaceutical biotechnology.

The holo-transcriptome of the zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa): a plentiful source of enzymes for potential application in green chemistry, industrial and pharmaceutical biotechnology

Marine invertebrates, such as sponges, tunicates and cnidarians (zoantharians and scleractinian corals), form functional assemblages, known as holobionts, with numerous microbes. This type of species-specific symbiotic association can be a repository of myriad valuable low molecular weight organic compounds, bioactive peptides and enzymes. The zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa) is one such example of a marine holobiont that inhabits the coastal reefs of the tropical Atlantic coast and is an interesting source of secondary metabolites and biologically active polypeptides. In the present study, we analyzed the entire holo-transcriptome of P. variabilis, looking for enzyme precursors expressed in the zoantharian-microbiota assemblage that are potentially useful as industrial biocatalysts and biopharmaceuticals. In addition to hundreds of predicted enzymes that fit into the classes of hydrolases, oxidoreductases and transferases that were found, novel enzyme precursors with multiple activities in single structures and enzymes with incomplete Enzyme Commission numbers were revealed. Our results indicated the predictive expression of thirteen multifunctional enzymes and 694 enzyme sequences with partially characterized activities, distributed in 23 sub-subclasses. These predicted enzyme structures and activities can prospectively be harnessed for applications in diverse areas of industrial and pharmaceutical biotechnology.

Identification of long non-coding RNAs in two anthozoan species and their possible implications for coral bleaching.

Long non-coding RNAs (lncRNAs) have been shown to play regulatory roles in a diverse range of biological processes and are associated with the outcomes of various diseases. The majority of studies about lncRNAs focus on model organisms, with lessened investigation in non-model organisms to date. Herein, we have undertaken an investigation on lncRNA in two zoanthids (cnidarian): Protolpalythoa varibilis and Palythoa caribaeorum. A total of 11,206 and 13,240 lncRNAs were detected in P. variabilis and P. caribaeorum transcriptome, respectively. Comparison using NONCODE database indicated that the majority of these lncRNAs is taxonomically species-restricted with no identifiable orthologs. Even so, we found cases in which short regions of P. caribaeorum's lncRNAs were similar to vertebrate species' lncRNAs, and could be associated with lncRNA conserved regulatory functions. Consequently, some high-confidence lncRNA-mRNA interactions were predicted based on such conserved regions, therefore revealing possible involvement of lncRNAs in posttranscriptional processing and regulation in anthozoans. Moreover, investigation of differentially expressed lncRNAs, in healthy colonies and colonial individuals undergoing natural bleaching, indicated that some up-regulated lncRNAs in P. caribaeorum could posttranscriptionally regulate the mRNAs encoding proteins of Ras-mediated signal transduction pathway and components of innate immune-system, which could contribute to the molecular response of coral bleaching.

Cell-penetrating peptides (CPPs): From delivery of nucleic acids and antigens to transduction of engineered nucleases for application in transgenesis.

Cell-penetrating peptides (CPPs) have been studied for their capacity to translocate across the lipid membrane of several cell types. In membrane translocation, these peptides can remarkably transport biologically active hydrophilic molecules, such as pharmaceuticals, nucleic acids (DNA and RNA) and even high-molecular-weight proteins, Fig. 3 into the cell cytoplasm and organelles. The development of CPPs as transduction agents includes the modification of gene and protein expression, the reprogramming and differentiation of induced pluripotent stem cells and the preparation of cellular vaccines. A relatively recent field of CPP application is the transduction of plasmid DNA vectors and CPP-fusion proteins to modify genomes and introduce new traits in cells and organisms. CPP-mediated transduction of components for genome editing is an advantageous alternative to viral DNA vectors. Engineered site-specific nucleases, such as Cre recombinase, ZFN, TALENs and CRISPR associated protein (Cas), have been coupled to CPPs, and the fused proteins have been used to permeate targeted cells and tissues. The functionally active fusion CPP-nucleases subsequently home to the nucleus, incise genomic DNA at specific sites and induce repair and recombination. This review has the objective of discussing CPPs and elucidating the prospective use of CPP-mediated transduction technology, particularly in genome modification and transgenesis.