RESUMEN
Sphingomyelin (SM) is a major sphingolipid in mammalian cells. SM is enriched in the extracellular leaflet of the plasma membrane (PM). Besides this localization, recent electron microscopic and biochemical studies suggest the presence of SM in the cytosolic leaflet of the PM. In the present study, we generated a non-toxic SM-binding variant (NT-EqtII) based on equinatoxin-II (EqtII) from the sea anemone Actinia equina, and examined the dynamics of SM in the cytosolic leaflet of living cell PMs. NT-EqtII with two point mutations (Leu26Ala and Pro81Ala) had essentially the same specificity and affinity to SM as wild-type EqtII. NT-EqtII expressed in the cytosol was recruited to the PM in various cell lines. Super-resolution microscopic observation revealed that NT-EqtII formed tiny domains that were significantly colocalized with cholesterol and N-terminal Lyn. Meanwhile, single molecule observation at high resolutions down to 1 ms revealed that all the examined lipid probes including NT-EqtII underwent apparent fast simple Brownian diffusion, exhibiting that SM and other lipids in the cytosolic leaflet rapidly moved in and out of domains. Thus, the novel SM-binding probe demonstrated the presence of the raft-like domain in the cytosolic leaflet of living cell PMs.
Asunto(s)
Membrana Celular , Venenos de Cnidarios , Citosol , Esfingomielinas , Esfingomielinas/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Animales , Venenos de Cnidarios/metabolismo , Venenos de Cnidarios/genética , Humanos , Anémonas de Mar/metabolismo , Anémonas de Mar/genética , Colesterol/metabolismoRESUMEN
Sea anemones are venomous animals that rely on their venom for prey capture, defense against predators, and intraspecific competition. Currently, comprehensive molecular and evolutionary analyses of the toxin repertoire for sea anemones are limited by a lack of proteomic data for most species. In this study, proteo-transcriptomic analysis was used to expand our knowledge of the proteinaceous components of sea anemone venom by determining the secreted venom proteome of Calliactis polypus. Electromechanical stimulation was used to obtain the secreted venom of C. polypus. We identified a low complexity proteome that was dominated by toxins with similarity to known neurotoxins, as well as six novel toxin candidates. The novel putative toxin candidates were found to be taxonomically restricted to species from the superfamily Metridioidea. Furthermore, the secreted venom of C. polypus had only three putative toxins in common with the venom of acontia from the same species and little similarity with the secreted venom of closely related species. Overall, this demonstrates that regionalized and lineage-specific variability in toxin abundance is common among sea anemone species. Moreover, the limited complexity of the toxin repertoire found in C. polypus supports the idea that peptide neurotoxins make up the dominant toxin arsenal found in the venom of sea anemones.
Asunto(s)
Proteoma , Anémonas de Mar , Animales , Anémonas de Mar/genética , Venenos de Cnidarios/genética , Venenos de Cnidarios/química , Venenos de Cnidarios/metabolismo , Neurotoxinas/genética , Filogenia , ProteómicaRESUMEN
Gene duplication is a major force driving evolutionary innovation. A classic example is generating new animal toxins via duplication of physiological protein-encoding genes and recruitment into venom. While this process drives the innovation of many animal venoms, reverse recruitment of toxins into nonvenomous cells remains unresolved. Using comparative genomics, we find members of the Membrane Attack Complex and Perforin Family (MAC) have been recruited into venom-injecting cells (cnidocytes), in soft and stony corals and sea anemones, suggesting that the ancestral MAC was a cnidocyte expressed toxin. Further investigation into the model sea anemone Nematostella vectensis reveals that three members have undergone Nematostella-specific duplications leading to their reverse recruitment into endomesodermal cells. Furthermore, simultaneous knockdown of all three endomesodermally expressed MACs leads to mis-development, supporting that these paralogs have nonvenomous function. By resolving the evolutionary history and function of MACs in Nematostella, we provide the first proof for reverse recruitment from venom to organismal development.
Asunto(s)
Evolución Molecular , Perforina , Anémonas de Mar , Animales , Anémonas de Mar/genética , Perforina/metabolismo , Perforina/genética , Duplicación de Gen , Venenos de Cnidarios/genética , Venenos de Cnidarios/metabolismo , Filogenia , Familia de MultigenesRESUMEN
Peptide toxins that adopt the ShK fold can inhibit the voltage-gated potassium channel KV1.3 with IC50 values in the pM range and are therefore potential leads for drugs targeting autoimmune and neuroinflammatory diseases. Nuclear magnetic resonance (NMR) relaxation measurements and pressure-dependent NMR have shown that, despite being cross-linked by disulfide bonds, ShK itself is flexible in solution. This flexibility affects the local structure around the pharmacophore for the KV1.3 channel blockade and, in particular, the relative orientation of the key Lys and Tyr side chains (Lys22 and Tyr23 in ShK) and has implications for the design of KV1.3 inhibitors. In this study, we have performed molecular dynamics (MD) simulations on ShK and a close homologue, HmK, to probe the conformational space occupied by the Lys and Tyr residues, and docked the different conformations with a recently determined cryo-EM structure of the KV1.3 channel. Although ShK and HmK have 60% sequence identity, their dynamic behaviors are quite different, with ShK sampling a broad range of conformations over the course of a 5 µs MD simulation, while HmK is relatively rigid. We also investigated the importance of conformational dynamics, in particular the distance between the side chains of the key dyad Lys22 and Tyr23, for binding to KV1.3. Although these peptides have quite different dynamics, the dyad in both adopts a similar configuration upon binding, revealing a conformational selection upon binding to KV1.3 in the case of ShK. Both peptides bind to KV1.3 with Lys22 occupying the pore of the channel. Intriguingly, the more flexible peptide, ShK, binds with significantly higher affinity than HmK.
Asunto(s)
Venenos de Cnidarios , Anémonas de Mar , Animales , Canal de Potasio Kv1.3/química , Canal de Potasio Kv1.3/metabolismo , Venenos de Cnidarios/química , Venenos de Cnidarios/metabolismo , Venenos de Cnidarios/farmacología , Anémonas de Mar/química , Anémonas de Mar/metabolismo , Péptidos/química , Conformación Molecular , Bloqueadores de los Canales de Potasio/farmacología , Bloqueadores de los Canales de Potasio/química , Canal de Potasio Kv.1.2/metabolismoRESUMEN
Hydractinia symbiolongicarpus is a colonial hydrozoan that displays a division of labor through morphologically distinct and functionally specialized polyp types. As with all cnidarians, their venoms are housed in nematocysts, which are scattered across an individual. Here, we investigate the spatial distribution of a specific protein family, jellyfish toxins, in which multiple paralogs are differentially expressed across the functionally specialized polyps. Jellyfish toxins (JFTs) are known pore-forming toxins in the venoms of medically relevant species such as box jellyfish (class Cubozoa), but their role in other medusozoan venoms is less clear. Utilizing a publicly available single-cell dataset, we confirmed that four distinct H. symbiolongicarpus JFT paralogs are expressed in nematocyst-associated clusters, supporting these as true venom components in H. symbiolongicarpus. In situ hybridization and immunohistochemistry were used to localize the expression of these JFTs across the colony. These expression patterns, in conjunction with known nematocyst type distributions, suggest that two of these JFTs, HsymJFT1c-I and HsymJFT1c-II, are localized to specific types of nematocysts. We further interpret JFT expression patterns in the context of known regions of nematogenesis and differential rates of nematocyst turnover. Overall, we show that JFT expression patterns in H. symbiolongicarpus are consistent with the subfunctionalization of JFT paralogs across a partitioned venom system within the colony, such that each JFT is expressed within a specific set of functionally distinct polyp types and, in some cases, specific nematocyst types.
Asunto(s)
Venenos de Cnidarios , Cubomedusas , Hidrozoos , Escifozoos , Toxinas Biológicas , Animales , Nematocisto , Hidrozoos/metabolismo , Venenos de Cnidarios/metabolismo , Escifozoos/metabolismo , Toxinas Biológicas/metabolismoRESUMEN
The dinoflagellate Ostreopsis cf. ovata produces several families of toxic polyketides. Despite only a few field measurements of these phycotoxins in seawater and aerosols, they are believed to be responsible for dermatitis and the toxic inhalations reported during blooms of this species. Therefore, the stability of these compounds in seawater is essential to understanding the causes of these symptoms, however, this has never been assessed. In the current study, the optimization of a solid phase extraction (SPE) procedure was first performed to ensure the most efficient extraction of all phycotoxins known to be produced by this strain, including the recently described liguriatoxins. The SPE cartridge SDBL® under non acidified conditions offered the best option. The stability of the ovatoxins and the liguriatoxins under biotic and abiotic stress was assessed by exposing the spent medium of a culture of Ostreopsis cf. ovata to its bacterial consortium and natural sunlight. A rapid biotic transformation was detected for both families of compounds. When exposed to bacteria, the half-lives of the ovatoxins were reached before 10 h and at 36 h, 97% of these toxins had been transformed. The half-lives of the liguriatoxins were 10 h under these conditions. Photolysis (abiotic degradation) of the ovatoxins (T1/2 < 36 h) was faster than for the liguriatoxins (T1/2 > 62 h). Although none of the catabolites of these phycotoxins were thoroughly identified, an untargeted metabolomics approach combined with molecular networking highlighted the presence of several compounds exhibiting structural similarities with the ovatoxins. Additional work should confirm the preliminary findings on these potential ovatoxins' catabolites and their biological properties. The rapid transformation of O. cf. ovata's phycotoxins introduces questions concerning their presence in seawater and their dispersion in the sea spray aerosols. The compounds involved in the toxic inhalations and dermatitis often experienced by beachgoers may stem from the catabolites of these toxins or even unrelated and as yet unidentified compounds.
Asunto(s)
Venenos de Cnidarios , Dermatitis , Dinoflagelados , Humanos , Toxinas Marinas/química , Dinoflagelados/química , Venenos de Cnidarios/metabolismo , Aerosoles , BacteriasRESUMEN
BACKGROUND: The application of multi-omics technologies provides a new perspective to solve three main problems including species identification, toxin screening and effective antagonist conformation in the studies of marine toxic jellyfish. METHODS: A series of transcriptome-proteome based analysis accompanied with toxicity evaluations were performed for the ornamental jellyfish Phacellophora camtschatica. RESULTS: Through combined morphological observation and Cytochrome c oxidase subunit â (CO1) molecular alignment, the sample jellyfish was identified as P. camtschatica. A total of 25,747 unigenes and 3058 proteins were obtained from the successfully constructed transcriptome and proteome, in which 6869 (26.68%) and 6618 (25.70%) unigenes, as well as 2536 (82.93%) and 2844 (93.00%) proteins were annotated against the databases of Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), respectively. The jellyfish displayed obvious in vivo lethal effects with significant increases of multi-organ functional indexes as well as in vitro activities. Total of 62 toxins from 120 toxin-related unigenes were screened including 16 metalloproteases, 11 phospholipases and others. Moreover, 11 toxins were further screened by using the erythrocyte model, where the zinc metalloproteinase nas-15-like (1) was the most abundant. Finally, Diltiazem greatly improved the survival rate while EDTA slightly prolonged the survival time in ICR mice. CONCLUSION: P. camtschatica is a poisonous jellyfish with diversified toxic components, in which metalloproteinase probably plays an important role in toxicities, and excessive Ca2+ entry may be the main mechanism of systemic lethal toxicity.
Asunto(s)
Venenos de Cnidarios , Proteoma , Animales , Venenos de Cnidarios/genética , Venenos de Cnidarios/metabolismo , Venenos de Cnidarios/toxicidad , Ratones , Ratones Endogámicos ICR , Proteoma/genética , Proteómica , TranscriptomaRESUMEN
Equinatoxin II (EqtII) and Fragaceatoxin C (FraC) are pore-forming toxins (PFTs) from the actinoporin family that have enhanced membrane affinity in the presence of sphingomyelin (SM) and phase coexistence in the membrane. However, little is known about the effect of these proteins on the nanoscopic properties of membrane domains. Here, we used combined confocal microscopy and force mapping by atomic force microscopy to study the effect of EqtII and FraC on the organization of phase-separated phosphatidylcholine/SM/cholesterol membranes. To this aim, we developed a fast, high-throughput processing tool to correlate structural and nano-mechanical information from force mapping. We found that both proteins changed the lipid domain shape. Strikingly, they induced a reduction in the domain area and circularity, suggesting a decrease in the line tension due to a lipid phase height mismatch, which correlated with proteins binding to the domain interfaces. Moreover, force mapping suggested that the proteins affected the mechanical properties at the edge, but not in the bulk, of the domains. This effect could not be revealed by ensemble force spectroscopy measurements supporting the suitability of force mapping to study local membrane topographical and mechanical alterations by membranotropic proteins.
Asunto(s)
Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Venenos de Cnidarios/metabolismo , Venenos de Cnidarios/toxicidad , Microdominios de Membrana/metabolismo , Anémonas de Mar/química , Anémonas de Mar/metabolismo , Esfingomielinas/metabolismo , Animales , Microdominios de Membrana/efectos de los fármacos , Microscopía de Fuerza Atómica , Microscopía ConfocalRESUMEN
Actinoporins (APs) are soluble pore-forming proteins secreted by sea anemones that experience conformational changes originating in pores in the membranes that can lead to cell death. The processes involved in the binding and pore-formation of members of this protein family have been deeply examined in recent years; however, the intracellular responses to APs are only beginning to be understood. Unlike pore formers of bacterial origin, whose intracellular impact has been studied in more detail, currently, we only have knowledge of a few poorly integrated elements of the APs' intracellular action. In this review, we present and discuss an updated landscape of the studies aimed at understanding the intracellular pathways triggered in response to APs attack with particular reference to sticholysin II, the most active isoform produced by the Caribbean Sea anemone Stichodactyla helianthus. To achieve this, we first describe the major alterations these cytolysins elicit on simpler cells, such as non-nucleated mammalian erythrocytes, and then onto more complex eukaryotic cells, including tumor cells. This understanding has provided the basis for the development of novel applications of sticholysins such as the construction of immunotoxins directed against undesirable cells, such as tumor cells, and the design of a cancer vaccine platform. These are among the most interesting potential uses for the members of this toxin family that have been carried out in our laboratory.
Asunto(s)
Muerte Celular/efectos de los fármacos , Venenos de Cnidarios/metabolismo , Venenos de Cnidarios/toxicidad , Inmunotoxinas/química , Inmunotoxinas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Anémonas de Mar/química , AnimalesRESUMEN
Many jellyfish species are known to cause a painful sting, but box jellyfish (class Cubozoa) are a well-known danger to humans due to exceptionally potent venoms. Cubozoan toxicity has been attributed to the presence and abundance of cnidarian-specific pore-forming toxins called jellyfish toxins (JFTs), which are highly hemolytic and cardiotoxic. However, JFTs have also been found in other cnidarians outside of Cubozoa, and no comprehensive analysis of their phylogenetic distribution has been conducted to date. Here, we present a thorough annotation of JFTs from 147 cnidarian transcriptomes and document 111 novel putative JFTs from over 20 species within Medusozoa. Phylogenetic analyses show that JFTs form two distinct clades, which we call JFT-1 and JFT-2. JFT-1 includes all known potent cubozoan toxins, as well as hydrozoan and scyphozoan representatives, some of which were derived from medically relevant species. JFT-2 contains primarily uncharacterized JFTs. Although our analyses detected broad purifying selection across JFTs, we found that a subset of cubozoan JFT-1 sequences are influenced by gene-wide episodic positive selection compared with homologous toxins from other taxonomic groups. This suggests that duplication followed by neofunctionalization or subfunctionalization as a potential mechanism for the highly potent venom in cubozoans. Additionally, published RNA-seq data from several medusozoan species indicate that JFTs are differentially expressed, spatially and temporally, between functionally distinct tissues. Overall, our findings suggest a complex evolutionary history of JFTs involving duplication and selection that may have led to functional diversification, including variability in toxin potency and specificity.
Asunto(s)
Cnidarios/genética , Venenos de Cnidarios/genética , Filogenia , Selección Genética , Transcriptoma , Animales , Cnidarios/metabolismo , Venenos de Cnidarios/metabolismo , Evolución MolecularRESUMEN
Jellyfish stings are a common issue globally, particularly in coastal areas in the summer. Victims can suffer pain, itching, swelling, shock, and even death. Usually, hot water, vinegar, or alumen is used to treat the normal symptoms of a jellyfish sting. However, a specific antivenom may be an effective treatment to deal with severe jellyfish stings. Cyanea nozakii often reach a diameter of 60 cm and are responsible for hundreds of thousands of stings per year in coastal Chinese waters. However, there has been no specific C. nozakii antivenom until now, and so the development of this antivenom is very important. Herein, we collected C. nozakii antisera from tentacle extract venom immunized rabbits and purified the immunoglobulin (IgG) fraction antivenom (AntiCnTXs). Subsequently, two complete procedures to produce a refined F(ab')2 type of antivenom (F(ab')2-AntiCnTXs) and Fab type of antivenom (Fab-AntiCnTXs) by multiple optimizations and purification were established. The neutralization efficacy of these three types of antivenoms was compared and analyzed in vitro and in vivo, and the results showed that all types of antibodies displayed some neutralization effect on the lethality of C. nozakii venom toxins, with the neutralization efficacy as follows: F(ab')2-AntiCnTXs ≥ AntiCnTXs > Fab-AntiCnTXs. This study describes the preparation of novel C. nozakii jellyfish antivenom preparations towards the goal of developing a new, effective treatment for jellyfish stings.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Antivenenos/farmacología , Mordeduras y Picaduras/tratamiento farmacológico , Venenos de Cnidarios/antagonistas & inhibidores , Fragmentos Fab de Inmunoglobulinas/farmacología , Inmunoglobulina G/farmacología , Escifozoos/metabolismo , Animales , Especificidad de Anticuerpos , Mordeduras y Picaduras/inmunología , Mordeduras y Picaduras/metabolismo , Venenos de Cnidarios/inmunología , Venenos de Cnidarios/metabolismo , ConejosRESUMEN
Sticholysins (Sts) I and II (StI and StII) are pore-forming proteins (PFPs), purified from the Caribbean Sea anemone Stichodactyla helianthus. StII encapsulated into liposomes induces a robust antigen-specific cytotoxic CD8+ T lymphocytes (CTL) response and in its free form the maturation of bone marrow-derived dendritic cells (BM-DCs). It is probable that the latter is partially supporting in part the immunomodulatory effect on the CTL response induced by StII-containing liposomes. In the present work, we demonstrate that the StII's ability of inducing maturation of BM-DCs is also shared by StI, an isoform of StII. Using heat-denatured Sts we observed a significant reduction in the up-regulation of maturation markers indicating that both PFP's ability to promote maturation of BM-DCs is dependent on their conformational characteristics. StII-mediated DC maturation was abrogated in BM-DCs from toll-like receptor (TLR) 4 and myeloid differentiation primary response gene 88 (MyD88)-knockout mice but not in cells from TLR2-knockout mice. Furthermore, the antigen-specific CTL response induced by StII-containing liposomes was reduced in TLR4-knockout mice. These results indicate that StII, and probably by extension StI, has the ability to induce maturation of DCs through a TLR4/MyD88-dependent pathway, and that this activation contributes to the CTL response generated by StII-containing liposomes.
Asunto(s)
Venenos de Cnidarios/metabolismo , Células Dendríticas/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/metabolismo , Compuestos Orgánicos/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The sea anemone Nematostella vectensis (Anthozoa, Cnidaria) is a powerful model for characterizing the evolution of genes functioning in venom and nervous systems. Although venom has evolved independently numerous times in animals, the evolutionary origin of many toxins remains unknown. In this work, we pinpoint an ancestral gene giving rise to a new toxin and functionally characterize both genes in the same species. Thus, we report a case of protein recruitment from the cnidarian nervous to venom system. The ShK-like1 peptide has a ShKT cysteine motif, is lethal for fish larvae and packaged into nematocysts, the cnidarian venom-producing stinging capsules. Thus, ShK-like1 is a toxic venom component. Its paralog, ShK-like2, is a neuropeptide localized to neurons and is involved in development. Both peptides exhibit similarities in their functional activities: They provoke contraction in Nematostella polyps and are toxic to fish. Because ShK-like2 but not ShK-like1 is conserved throughout sea anemone phylogeny, we conclude that the two paralogs originated due to a Nematostella-specific duplication of a ShK-like2 ancestor, a neuropeptide-encoding gene, followed by diversification and partial functional specialization. ShK-like2 is represented by two gene isoforms controlled by alternative promoters conferring regulatory flexibility throughout development. Additionally, we characterized the expression patterns of four other peptides with structural similarities to studied venom components and revealed their unexpected neuronal localization. Thus, we employed genomics, transcriptomics, and functional approaches to reveal one venom component, five neuropeptides with two different cysteine motifs, and an evolutionary pathway from nervous to venom system in Cnidaria.
Asunto(s)
Venenos de Cnidarios/genética , Duplicación de Gen , Sistema Nervioso/metabolismo , Neuropéptidos/genética , Anémonas de Mar/fisiología , Animales , Venenos de Cnidarios/metabolismo , Evolución Molecular , Neuropéptidos/metabolismo , FilogeniaRESUMEN
Cnidarians have been known since ancient times for the painful stings they induce to humans. The effects of the stings range from skin irritation to cardiotoxicity and can result in death of human beings. The noxious effects of cnidarian venoms have stimulated the definition of their composition and their activity. Despite this interest, only a limited number of compounds extracted from cnidarian venoms have been identified and defined in detail. Venoms extracted from Anthozoa are likely the most studied, while venoms from Cubozoa attract research interests due to their lethal effects on humans. The investigation of cnidarian venoms has benefited in very recent times by the application of omics approaches. In this review, we propose an updated synopsis of the toxins identified in the venoms of the main classes of Cnidaria (Hydrozoa, Scyphozoa, Cubozoa, Staurozoa and Anthozoa). We have attempted to consider most of the available information, including a summary of the most recent results from omics and biotechnological studies, with the aim to define the state of the art in the field and provide a background for future research.
Asunto(s)
Cnidarios/fisiología , Venenos de Cnidarios/química , Venenos de Cnidarios/metabolismo , Animales , Genómica , MetabolómicaRESUMEN
Tube anemones, or cerianthids, are a phylogenetically informative group of cnidarians with complex life histories, including a pelagic larval stage and tube-dwelling adult stage, both known to utilize venom in stinging-cell rich tentacles. Cnidarians are an entirely venomous group that utilize their proteinaceous-dominated toxins to capture prey and defend against predators, in addition to several other ecological functions, including intraspecific interactions. At present there are no studies describing the venom for any species within cerianthids. Given their unique development, ecology, and distinct phylogenetic-placement within Cnidaria, our objective is to evaluate the venom-like gene diversity of four species of cerianthids from newly collected transcriptomic data. We identified 525 venom-like genes between all four species. The venom-gene profile for each species was dominated by enzymatic protein and peptide families, which is consistent with previous findings in other cnidarian venoms. However, we found few toxins that are typical of sea anemones and corals, and furthermore, three of the four species express toxin-like genes closely related to potent pore-forming toxins in box jellyfish. Our study is the first to provide a survey of the putative venom composition of cerianthids and contributes to our general understanding of the diversity of cnidarian toxins.
Asunto(s)
Cnidarios/genética , Venenos de Cnidarios/genética , Perfilación de la Expresión Génica , Transcriptoma , Animales , Cnidarios/metabolismo , Venenos de Cnidarios/metabolismo , Venenos de Cnidarios/farmacología , Regulación de la Expresión Génica , Filogenia , Especificidad de la EspecieRESUMEN
Actinoporins are a family of pore-forming toxins produced by sea anemones as part of their venomous cocktail. These proteins remain soluble and stably folded in aqueous solution, but when interacting with sphingomyelin-containing lipid membranes, they become integral oligomeric membrane structures that form a pore permeable to cations, which leads to cell death by osmotic shock. Actinoporins appear as multigenic families within the genome of sea anemones: several genes encoding very similar actinoporins are detected within the same species. The Caribbean Sea anemone Stichodactyla helianthus produces three actinoporins (sticholysins I, II and III; StnI, StnII and StnIII) that differ in their toxic potency. For example, StnII is about four-fold more effective than StnI against sheep erythrocytes in causing hemolysis, and both show synergy. However, StnIII, recently discovered in the S. helianthus transcriptome, has not been characterized so far. Here we describe StnIII's spectroscopic and functional properties and show its potential to interact with the other Stns. StnIII seems to maintain the well-preserved fold of all actinoporins, characterized by a high content of ß-sheet, but it is significantly less thermostable. Its functional characterization shows that the critical concentration needed to form active pores is higher than for either StnI or StnII, suggesting differences in behavior when oligomerizing on membrane surfaces. Our results show that StnIII is an interesting and unexpected piece in the puzzle of how this Caribbean Sea anemone species modulates its venomous activity.
Asunto(s)
Venenos de Cnidarios/química , Proteínas Citotóxicas Formadoras de Poros/química , Anémonas de Mar/química , Secuencia de Aminoácidos , Animales , Venenos de Cnidarios/metabolismo , Hemólisis/efectos de los fármacos , Modelos Moleculares , Compuestos Orgánicos/química , Compuestos Orgánicos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Anémonas de Mar/metabolismo , Alineación de Secuencia , OvinosRESUMEN
Release of aqueous contents from model lipid vesicles has been a standard procedure to evaluate pore formation efficiency by actinoporins, such as sticholysin II (StnII), for the last few decades. However, regardless of the probe of choice, the results reported that StnII action was never able to empty the vesicles completely. This was hard to explain if StnII pores were to be stable and always leaky for the probes used. To address this question, we have used a variety of probes, including rhodamine 6G or Tb3+, to test the permeability of StnII's pores. Our results indicate that calcein was in fact too large to fit through StnII's pores, and that the standard method in the field is actually reporting StnII-induced transient permeation of the membrane rather than the passage of solutes through the stable assembled pores. In order to evaluate the permeability of these structures, we used a dithionite-based assay, which showed that the final pores were in fact open. Thus, our results indicate that the stable actinoporins' pores are open in spite of plateaued classic release curves. Besides the proper pore, the first stages of pore formation would inflict serious damage to living cells as well.
Asunto(s)
Venenos de Cnidarios/química , Membrana Dobles de Lípidos/química , Lípidos/química , Esfingomielinas/química , Animales , Venenos de Cnidarios/metabolismo , Membranas/efectos de los fármacos , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Rodaminas/química , Anémonas de Mar/químicaRESUMEN
Sticholysins are pore-forming toxins of biomedical interest and represent a prototype of proteins acting through the formation of protein-lipid or toroidal pores. Peptides spanning the N-terminus of sticholysins can mimic their permeabilizing activity and, together with the full-length toxins, have been used as a tool to understand the mechanism of pore formation in membranes. However, the lytic mechanism of these peptides and the lipid shape modulating their activity are not completely clear. In this article, we combine molecular dynamics simulations and experimental biophysical tools to dissect different aspects of the pore-forming mechanism of StII1-30, a peptide derived from the N-terminus of sticholysin II (StII). With this combined approach, membrane curvature induction and flip-flop movement of the lipids were identified as two important membrane remodeling steps mediated by StII1-30. Pore formation by this peptide was enhanced by the presence of the negatively curved lipid phosphatidylethanolamine in membranes. This lipid emerged not only as a facilitator of membrane interactions but also as a structural element of the StII1-30 pore that is recruited to the ring upon its assembly. Collectively, these, to our knowledge, new findings support a toroidal model for the architecture of the pore formed by StII1-30 and provide new molecular insight into the role of phosphatidylethanolamine as a membrane component that can easily integrate into the ring of toroidal pores, thus probably aiding in their stabilization. This study contributes to a better understanding of the molecular mechanism underlying the permeabilizing activity of StII1-30 and peptides or proteins acting via a toroidal pore mechanism and offers an informative framework for the optimization of the biomedical application of this and similar molecules.
Asunto(s)
Membrana Celular/metabolismo , Venenos de Cnidarios/metabolismo , Modelos Moleculares , Secuencia de Aminoácidos , Animales , Venenos de Cnidarios/química , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Permeabilidad , Fosfatidiletanolaminas/química , Soluciones , PorcinosRESUMEN
This paper provides a feasible model for molecular structure analysis and interaction mechanism of aptamer and micromolecule. In this study, modeling and dynamic simulation of ssDNA aptamer (P-18S2) and target (Palytoxin, PTX) were performed separately. Then, the complex structure between DNA and PTX was predicted, and docking results showed that PTX could combine steadily at the groove's top of DNA model by strong hydrogen-bonds and electrostatic interaction. Thus, we truncated and optimized P-18S2 by simulating. At the same time, we also confirmed the reliability of simulation results by experiments. With the experimental and computational results, the study provided a more reasonable interpretation for the high affinity and specific binding of P-18S2 and PTX, which laid the foundation for further optimization and development of aptamers in molecular diagnostics and therapeutic applications.
Asunto(s)
Acrilamidas/metabolismo , Aptámeros de Nucleótidos/metabolismo , Venenos de Cnidarios/metabolismo , Aptámeros de Nucleótidos/química , Sitios de Unión , Simulación del Acoplamiento Molecular , Conformación de Ácido NucleicoRESUMEN
Jellyfish is a common toxic zooplankton in ocean. We successfully captured a kind of jellyfish 200â¯m underwater in Antarctica, and identified it as a jellyfish Cyanea sp. through morphological examination and MT-CO1 phylogenetic analysis. A total of 40,468 unigenes were harvested through transcriptome sequencing. We also successfully annotated 12,955 (32.01%) unigenes with the NR database, 10,882 (26.89%) unigenes with the SWISSPROT database, 4951 (12.23%) unigenes with the GO database, and 4901 (12.11%) unigenes with the KEGG database. In the proteomic analysis, a total of 11,159 peptides and 2630 proteins were harvested using the constructed transcriptome as the database. A number of 771 (29.31%) and 841 (31.98%) proteins were annotated against the GO and KEGG database, respectively. Moreover, a number of 29 toxic proteins matched from the 145 toxin-related unigenes were successfully screened, including 6 metalloproteinases, 4 phospholipases, 2 serine proteases, 1 serine protease inhibitor, 7 toxin-related venom and 9 other toxins. Our study is the first to identify a polar jellyfish Cyanea sp. with transcriptomics and proteomics, and these data can further serve as a public database for the identification of potential polar jellyfish-derived lead compounds feasibly functioning in the cold environment. SIGNIFICANCE: With increasing discussions on marine biodiversity and global warming, polar species have gradually become a focus for research. To the best of our knowledge, there is only one paper in pubMed about the mitochondrial genome of the Antarctic stalked jellyfish Haliclystus antarcticus Pfeffer. In this study, we captured a type of jellyfish (named BD-4) from the Southern Ocean (60°29'57" S, 52°11'44"W) on the scientific expedition ship "Xue Long" at the end of 2016. Although the samples were stored and transported by the ship at only -20⯰C for more than two month, we successfully extracted the total RNA, and performed molecular species identification and combined analyses of de novo transcriptomics and proteomics. In addition to conventional bioinformatics techniques such as GO and KEGG annotation, we screened and listed toxic proteins, aligned the sequences, simulated three-dimensional structures and performed molecular phylogenetic analysis for typical components, including metalloproteinase and serine proteinase. Our study is the first to identify a polar jellyfish Cyanea sp. with de novo transcriptomics and proteomics, and these data can further serve as a public database for the identification of potential polar jellyfish-derived lead compounds.