Actinoporinas:un veneno letal

En la Naturaleza, tanto plantas como animales han desarrollado distintos mecanismos para sobrevivir. Los guepardos pueden alcanzar una velocidad de 120 km/h, lo que les permite cazar a sus presas con mayor facilidad; los leones y los tigres tienen grandes garras y potentes mandíbulas. Por otro lado, aquellos animales o plantas que no tienen libertad de movimiento recurren a los venenos, que les permiten atacar a sus predadores o presas sin necesidad de moverse

Structural and functional characterization of venom pore forming proteins

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Bioquímica y Biología Molecular, leída el 11-12-2020.Las proteínas formadoras de poros (PFP) son una familia de toxinas capaces de matar células por choque osmótico, precisamente porque forman poros en sus membranas. En disolución acuosa, estas proteínas permanecen plegadas y estables. Al interactuar con un receptor específico de la membrana (proteína, lípido o azúcar), se unen a ella, oligomerizan y forman un poro a través del núcleo hidrófobo de la membrana. Las actinoporinas son α-PFPs producidas por anémonas marinas como parte de su cóctel venenoso. Son pequeñas (≈ 20 kDa) y suelen tener un punto isoeléctrico básico ( ≈ 9). Comparten un motivo conformacional común, caracterizado por un sándwich β flanqueado por dos hélices α. Su receptor de membrana específico es la esfingomielina. Cuando las actinoporinas se unen a una membrana que contiene este esfingolípido, extienden su segmento helicoidal N-terminal, oligomerizan y forman un poro selectivo de cationes, insertando sus hélices α del extremo N-terminal a través del núcleo de la membrana. Sin embargo, el orden específico de las etapas que conducen a la formación del poro, así como la naturaleza de posibles estados intermedios, o su estequiometría final, todavía son objeto de debate. Estas actinoporinas constituyen además familias multigénicas: una sola especie produce una variedad de toxinas similares que no necesariamente muestran una actividad lítica o una especificidad idénticas. Debido a su simplicidad, son un modelo apropiado para estudiar la todavía no bien comprendida transición de una proteína soluble en agua a un estado en el que se integra en membrana...Pore forming proteins (PFPs) are a family of toxins that form pores in cell membranes leading to cell death by osmotic shock. These proteins remain stably folded and soluble in aqueous solution. Upon interaction with a specific receptor in the membrane (protein, lipid or sugar), they bind, oligomerize and form a pore through the membrane hydrophobic core.Actinoporins are α-PFPs produced by sea anemones as part of their venomous cocktail. They are small (≈ 20 kDa) and usually have a basic isoelectric point ( ≈ 9). They share a common fold characterized by a β-sandwich flanked by two α-helices. Their specific membrane receptor is sphingomyelin. When actinoporins bind to a membrane containing this sphingolipid, they extend their N-terminal α-helical segment, oligomerize and form a cation selective pore by inserting the α-helices through the membrane core. However, the specific step order leading to a final pore, the necessity of a pre-pore and the final stoichiometry are still debated. Actinoporins constitute multigene families: A single species produces a variety of similar toxins which not necessarily display identical lytic activity or specificity. Because of their simplicity, they are an appropriate model to study the biophysical challenging transition from a water-soluble protein to a membrane bound state...Fac. de Ciencias QuímicasTRUEunpu

Stichodactyla helianthus' de novo transcriptome assembly: Discovery of a new actinoporin isoform

Transcriptomic profiling of venom producing tissues from different animals is an effective approach for discovering new toxins useful in biotechnological and pharmaceutical applications, as well in evolutionary comparative studies of venomous animals. Stichodactyla helianthus is a Caribbean sea anemone which produces actinoporins as part of its toxic venom. This family of pore forming toxins is multigenic and at least two different isoforms, encoded by separate genes, are produced by S. helianthus. These isoforms, sticholysins I and II, share 93% amino acid identity but differ in their pore forming activity and act synergistically. This observation suggests that other actinoporin isoforms, if present in the venomous mixture, could offer an advantageous strategy to modulate whole venom activity. Using high-throughput sequencing we generated a de novo transcriptome of S. helianthus and determined the relative expression of assembled transcripts using RNA-Seq to better characterize components of this species' venom, focusing on actinoporin diversity. Applying this approach, we have discovered at least one new actinoporin variant from S. helianthus in addition to several other putative venom components

Synergistic action of actinoporin isoforms from the same sea anemone species assembled into functionally active heteropores

Among the toxic polypeptides secreted in the venom of sea anemones, actinoporins are pore forming toxins whose toxic activity relies on the formation of oligomeric pores within biological membranes. Intriguingly, actinoporins appear as multigene families which give rise to many protein isoforms in the same individual displaying high sequence identities but large functional differences. However, the evolutionary advantage of producing such similar isotoxins is not fully understood. Here, using sticholysins I and II (StnI and StnII) from the sea anemone Stichodactyla helianthus, it is shown that actinoporin isoforms can potentiate each other’s activity. Through hemolysis and calcein releasing assays, it is revealed that mixtures of StnI and StnII are more lytic than equivalent preparations of the corresponding isolated isoforms. It is then proposed that this synergy is due to the assembly of heteropores since (i) StnI and StnII can be chemically cross-linked at the membrane and (ii) the affinity of sticholysin mixtures for the membrane is increased with respect to any of them acting in isolation, as revealed by isothermal titration calorimetry experiments. These results help to understand the multigene nature of actinoporins and may be extended to other families of toxins that require oligomerization to exert toxicity

Synergistic Action of Actinoporin Isoforms from the Same Sea Anemone Species Assembled into Functionally Active Heteropores

Among the toxic polypeptides secreted in the venom of sea anemones, actinoporins are the pore-forming toxins whose toxic activity relies on the formation of oligomeric pores within biological membranes. Intriguingly, actinoporins appear as multigene families that give rise to many protein isoforms in the same individual displaying high sequence identities but large functional differences. However, the evolutionary advantage of producing such similar isotoxins is not fully understood. Here,using sticholysins I and II (StnI and StnII) from the sea anemone Stichodactyla helianthus, it is shown that actinoporin isoforms can potentiate each other’s activity. Through hemolysis and calcein releasing assays, it is revealed that mixtures of StnI and StnII are more lytic than equivalent preparations of the corresponding isolated isoforms. It is then proposed that this synergy is due to the assembly of heteropores because (i) StnI and StnII can be chemically cross-linked at the membrane and (ii) the affinity of sticholysin mixtures for the membrane is increased with respect to any of them acting in isolation, as revealed by isothermal titration calorimetry experiments. These results help us understand the multigene nature of actinoporins and may be extended to other families of toxins that require oligomerization to exert toxicity

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Fac. de Ciencias BiológicasFALSEsubmitte

Sticholysin, Sphingomyelin, and Cholesterol: A Closer Look at a Tripartite Interaction

Actinoporins are a group of soluble toxic proteins that bind to membranes containing sphingomyelin (SM) and oligomerize to form pores. Sticholysin II (StnII) is a member of the actinoporin family, produced by Stichodactyla helianthus. Cholesterol (Chol) is known to enhance the activity of StnII. However, the molecular mechanisms behind this activation have remained obscure, although the activation is not Chol specific but rather sterol specific. To further explore how bilayer lipids affect or are affected by StnII, we have used a multiprobe approach (fluorescent analogs of both Chol and SM) in combination with a series of StnII tryptophan (Trp)-mutants, to study StnII/bilayer interactions. First we compared StnII bilayer permeabilization in the presence of Chol or oleoyl-ceramide (OCer). The comparison was done since both Chol and OCer have a 1-hydroxyl which help to orient the molecule in the bilayer (although OCer have additional polar functional groups). Both Chol and OCer also have increased affinity for SM, which StnII may recognize. However, our results show that only Chol was able to activate StnII-induced bilayer permeabilization – OCer failed to active. To further examine possible Chol/StnII interactions, we measured Förster resonance energy transfer (FRET) between Trp in StnII and cholestatrienol (CTL), a fluorescent analog of Chol. We could show higher FRET efficiency between CTL and Trp:s in position 100 and 114 of StnII, when compared to three other Trp positions further away from the bilayer binding region of StnII. Taken together, our results suggest that StnII was able to attract Chol to its vicinity, maybe by showing affinity for Chol. SM interactions are known to be important for StnII binding to bilayers, and Chol is known to facilitate subsequent permeabilization of the bilayers by StnII. Our results help to better understand the role of these important membrane lipids for the bilayer properties of StnII

Pore-Forming Proteins from Cnidarians and Arachnids as Potential Biotechnological Tools

Animal venoms are complex mixtures of highly specialized toxic molecules. Cnidarians and arachnids produce pore-forming proteins (PFPs) directed against the plasma membrane of their target cells. Among PFPs from cnidarians, actinoporins stand out for their small size and molecular simplicity. While native actinoporins require only sphingomyelin for membrane binding, engineered chimeras containing a recognition antibody-derived domain fused to an actinoporin isoform can nonetheless serve as highly specific immunotoxins. Examples of such constructs targeted against malignant cells have been already reported. However, PFPs from arachnid venoms are less well-studied from a structural and functional point of view. Spiders from the Latrodectus genus are professional insect hunters that, as part of their toxic arsenal, produce large PFPs known as latrotoxins. Interestingly, some latrotoxins have been identified as potent and highly-specific insecticides. Given the proteinaceous nature of these toxins, their promising future use as efficient bioinsecticides is discussed throughout this Perspective. Protein engineering and large-scale recombinant production are critical steps for the use of these PFPs as tools to control agriculturally important insect pests. In summary, both families of PFPs, from Cnidaria and Arachnida, appear to be molecules with promising biotechnological applications

Química Inspirada por la Naturaleza: Lecciones en el Museo Nacional de Ciencias Naturales II

El proyecto Innova-Docencia 70 es una continuación del proyecto Innova-Docencia 30 que se desarrolló durante el curso académico 2016-2017 y su objetivo principal es implementar una nueva estrategia de enseñanza/aprendizaje que motive a los estudiantes hacia el estudio de Biología, favorezca la comprensión de los contenidos y les permita conectar los nuevos conocimientos adquiridos con el mundo real que les rodea. Motivación, comprensión e interdisciplinaridad son tres grandes retos a los que se enfrenta el profesorado de la asignatura de Biología del Grado en Química, que se imparte durante el primer cuatrimestre y que es de carácter obligatorio para los estudiantes de primero (6 ECTS), dado el escaso interés que despierta esta asignatura en los estudiantes de esta disciplina (además de otras, como física, arquitectura etc.), al considerarla una asignatura difícil de aprender y de poca utilidad. Esta estrategia docente tiene como eje metodológico principal la Biomímesis, pero a su vez se apoya en otras dos metodologías: (1) La enseñanza/aprendizaje basada en Proyectos y (2) Los Museos como espacios de enseñanza/aprendizaje. Todas ellas comparten un mismo principio: motivar al estudiante a aprender, implicándole en su propio proceso de aprendizaje. El estudiante ha de planificar, desarrollar y evaluar un proyecto (de carácter interdisciplinar) que le interesa, al ser elegido por él, y que le permite conectar lo académico con la vida

Structural and functional characterization of Sticholysin III: A newly discovered actinoporin within the venom of the sea anemone Stichodactyla helianthus.

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 β-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