Sea anemone actinoporins: The transition from a folded soluble state to a functionally active membrane-bound oligomeric pore

Actinoporins are a family of 20-kDa, basic proteins isolated from sea anemones, whose activity is inhibited by preincubation with sphingomyelin. They are produced in monomeric soluble form but, when binding to the plasma membrane, they oligomerize to produce functional pores which result in cell lysis. Equinatoxin II (EqtII) from Actinia equina and Sticholysin II (StnII) from Stichodactyla helianthus are the actinoporins that have been studied in more detail. Both proteins display a beta-sandwich fold composed of 10 beta-strands flanked on each side by two short alpha-helices. Twodimensional crystallization on lipid monolayers has allowed the determination of low-resolution models of tetrameric structures distinct from the pore. However, the actual structure of the pore is not known yet. Wild-type EqtII and StnII, as well as a nice collection of natural and artificially made variants of both proteins, have been produced in Escherichia coli and purified. Their characterization has allowed the proposal of a model for the mechanism of pore formation. Four regions of the actinoporins structure seem to play an important role. First, a phosphocholine-binding site and a cluster of exposed aromatic residues, together with a basic region, would be involved in the initial interaction with the membrane, whereas the amphipathic N-terminal region would be essential for oligomerization and pore formation. Accordingly, the model states that pore formation would proceed in at least four steps: Monomer binding to the membrane interface, assembly of four monomers, and at least two distinct conformational changes driving to the final formation of the functional pore

Calorimetric scrutiny of lipid binding by sticholysin II toxin mutants

The mechanisms by which pore-forming toxins are able to insert into lipid membranes are a subject of the highest interest in the field of lipid–protein interaction. Eight mutants affecting different regions of sticholysin II, a member of the pore-forming actinoporin family, have been produced, and their hemolytic and lipid-binding properties were compared to those of the wild-type protein. A thermodynamic approach to the mechanism of pore formation is also presented. Isothermal titration calorimetry experiments show that pore formation by sticholysin II is an enthalpy-driven process that occurs with a high affinity constant (1.7×108 M−1). Results suggest that conformational flexibility at the N-terminus of the protein does not provide higher affinity for the membrane, although it is necessary for correct pore formation. Membrane binding is achieved through two separate mechanisms, that is, recognition of the lipid–water interface by a cluster of aromatic residues and additional specific interactions that include a phosphocholinebinding site. Thermodynamic parameters derived from titration experiments are discussed in terms of a putative model for pore formation

Characterization of a new toxin from the entomopathogenic fungus Metarhizium anisopliae: the ribotoxin anisoplin

Metarhizium anisopliae is an entomopathogenic fungus relevant in biotechnology with applications like malaria vector control. Studies of its virulence factors are therefore of great interest. Fungal ribotoxins are toxic ribonucleases with extraordinary efficiency against target ribosomes and suggested as potential insecticides. Here, we describe this ribotoxin characteristic activity in M. anisopliae cultures. Anisoplin has been obtained as a recombinant protein and further characterized. It is structurally similar to hirsutellin A, the ribotoxin from the entomopathogen Hirsutella thompsonii. Moreover, anisoplin shows the ribonucleolytic activity typical of ribotoxins and cytotoxicity against insect cells. How Metarhizium uses this toxin and possible applications are on perspective

Differential toxicity of antifungal protein AFP against mutants of Fusarium oxysporum

Antifungal protein (AFP) from Aspergillus giganteus was assayed for toxicity against the Fusarium oxysporum wild-type strain and mutants in genes involved in cell signaling (ΔpacC, pacCc Δfmk1) or cell-wall biogenesis (ΔchsV, Δchs7, Δgas1). The mutants were classified into two groups according to their sensitivity to AFP: ΔpacC, Δgas1 and Δchs7, which were significantly more resistant to AFP than the wild-type, and pacCC, Δfmk1 and ΔchsV, which were more sensitive. Western blot analysis revealed increased binding of AFP to the three resistant mutants, ΔpacC, Δgas1 and Δchs7, but also to ΔchsV, indicating that differential binding may not be a key determinant for sensitivity. Addition of Ca2+ or K+ dramatically reduced antifungal activity and binding of AFP, suggesting that these cations compete for the same targets as AFP at the surface of the fungal cell. [Int Microbiol 2009; 12(2):115-121

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

Midiendo la entrada de talento en la Liga de Fútbol Profesional: un análisis de la década 2007-2017

El trabajo de fin de grado que hemos realizado intenta analizar la movilidad del talento en el deporte más popular del mundo. En particular nos hemos centrado en estudiar los últimos diez años de la Liga de Fútbol Profesional de España. A medida que pasan los años, la captación de nuevos jugadores para mejorar la productividad de las plantillas ha aumentado y esto ha llevado a invertir mayores cifras de dinero. El movimiento migratorio facilitado por la normativa favorable de la Unión Europea ha llevado a que se produzca una gran movilidad de jugadores de unos países a otros. Este trabajo se centra en poner en relieve la enorme cantidad de futbolistas extranjeros que se encuentran en la Liga de Fútbol Profesional, las consecuencias de este aumento y el estudio de algunas de las características de estos jugadores

The Effect of Cholesterol on the Long-Range Network of Interactions Established among Sea Anemone Sticholysin II Residues at the Water-Membrane Interface

Actinoporins are α-pore forming proteins with therapeutic potential, produced by sea anemones. Sticholysin II (StnII) from Stichodactyla helianthus is one of its most extensively characterized members. These proteins remain stably folded in water, but upon interaction with lipid bilayers, they oligomerize to form a pore. This event is triggered by the presence of sphingomyelin (SM), but cholesterol (Chol) facilitates pore formation. Membrane attachment and pore formation require changes involving long-distance rearrangements of residues located at the protein-membrane interface. The influence of Chol on membrane recognition, oligomerization, and/or pore formation is now studied using StnII variants, which are characterized in terms of their ability to interact with model membranes in the presence or absence of Chol. The results obtained frame Chol not only as an important partner for SM for functional membrane recognition but also as a molecule which significantly reduces the structural requirements for the mentioned conformational rearrangements to occur. However, given that the DOPC:SM:Chol vesicles employed display phase coexistence and have domain boundaries, the observed effects could be also due to the presence of these different phases on the membrane. In addition, it is also shown that the Arg51 guanidinium group is strictly required for membrane recognition, independently of the presence of Chol

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

Toxin-induced pore formation is hindered by intermolecular hydrogen bonding in sphingomyelin bilayers

Sticholysin I and II (StnI and StnII) are pore-forming toxins that use sphingomyelin (SM) for membrane binding. We examined how hydrogen bonding among membrane SMs affected the StnI- and StnII-induced pore formation process, resulting in bilayer permeabilization. We compared toxin-induced permeabilization in bilayers containing either SM or dihydro-SM (lacking the trans 4 double bond of the long-chain base), since their hydrogen-bonding properties are known to differ greatly. We observed that whereas both StnI and StnII formed pores in unilamellar vesicles containing palmitoyl-SM or oleoyl-SM, the toxins failed to similarly form pores in vesicles prepared from dihydro-PSM or dihydro-OSM. In supported bilayers containing OSM, StnII bound efficiently, as determined by surface plasmon resonance. However, StnII binding to supported bilayers prepared from dihydro-OSM was very low under similar experimental conditions. The association of the positively charged StnII (at pH 7.0) with unilamellar vesicles prepared from OSM led to a concentration-dependent increase in vesicle charge, as determined from zeta-potential measurements. With dihydro-OSM vesicles, a similar response was not observed. Benzyl alcohol, which is a small hydrogen-bonding compound with affinity to lipid bilayer interfaces, strongly facilitated StnII-induced pore formation in dihydro-OSM bilayers, suggesting that hydrogen bonding in the interfacial region originally prevented StnII from membrane binding and pore formation. We conclude that interfacial hydrogen bonding was able to affect the membrane association of StnI- and StnII, and hence their pore forming capacity. Our results suggest that other types of protein interactions in bilayers may also be affected by hydrogen-bonding origination from SMs

Minimized natural versions of fungal ribotoxins show improved active site plasticity

Fungal ribotoxins are highly specific extracellular RNases which cleave a single phosphodiester bond at the ribosomal sarcin-ricin loop, inhibiting protein biosynthesis by interfering with elongation factors. Most ribotoxins show high degree of conservation, with similar sizes and amino acid sequence identities above 85%. Only two exceptions are known: Hirsutellin A and anisoplin, produced by the entomopathogenic fungi Hirsutella thompsonii and Metarhizium anisopliae, respectively. Both proteins are similar but smaller than the other known ribotoxins (130 vs 150 amino acids), displaying only about 25% sequence identity with them. They can be considered minimized natural versions of their larger counterparts, best represented by α-sarcin. The conserved α-sarcin active site residue Tyr48 has been replaced by the geometrically equivalent Asp, present in the minimized ribotoxins, to produce and characterize the corresponding mutant. As a control, the inverse anisoplin mutant (D43Y) has been also studied. The results show how the smaller versions of ribotoxins represent an optimum compromise among conformational freedom, stability, specificity, and active-site plasticity which allow these toxic proteins to accommodate the characteristic abilities of ribotoxins into a shorter amino acid sequence and more stable structure of intermediate size between that of other nontoxic fungal RNases and previously known larger ribotoxins