Person: García Linares, Sara
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First Name
Sara
Last Name
García Linares
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Biológicas
Department
Bioquímica y Biología Molecular
Area
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7 results
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Item Sticholysin, Sphingomyelin, and Cholesterol: A Closer Look at a Tripartite Interaction(Biophysical Journal, 2019) Palacios Ortega, Juan; García Linares, Sara; Rivera de la Torre, Esperanza; Gavilanes, José G.; Martínez del Pozo, Álvaro; Slotte, J. PeterActinoporins 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.Item The metamorphic transformation of a water-soluble monomeric protein into an oligomeric transmembrane pore(Advances in Biomembranes and Lipid Self-Assembly, 2017) García Linares, Sara; Rivera de la Torre, Esperanza; Palacios Ortega, Juan; Gavilanes, José G.; Martínez del Pozo, ÁlvaroSea anemones produce venoms containing different toxic molecules. Among them, actinoporins are some of the best characterized ones. They constitute a family of toxic polypeptides that belong to the much larger group of pore-forming toxins. Actinoporins remain mostly monomeric and stably folded in aqueous solution but, upon interaction with lipid membranes of specific composition, they become oligomeric integral membrane structures to build a pore. They insert an α-helix stretch within biological membranes, forming cation-selective pores with a diameter of 1–2 nm, which result in a colloid osmotic shock that leads to cell death. They are believed to participate in functions like predation, defense, and digestion and have been shown to be lethal for small crustaceans, mollusks, and fish. The best-known actinoporins are equinatoxin II (from Actinia equina), fragaceatoxin C (from Actinia fragacea), and sticholysins I and II (from Stichodactyla helianthus). In order to fully understand the pore formation mechanism of these proteins, several approaches have been used: (i) characterization of natural and artificial variants of actinoporins to determine the role of specific residues, (ii) study of their water-soluble and transmembrane structures, and (iii) employment of different lipids to evaluate the influence of membrane properties and composition. Further research is still needed, however, in order to fully understand the complex mechanism underlying actinoporins’ functionality.Item Functional and Structural Variation among Sticholysins, Pore-Forming Proteins from the Sea Anemone Stichodactyla helianthus(International journal of molecular sciences, 2020) Rivera de la Torre, Esperanza; Palacios Ortega, Juan; Slotte, J. Peter; Gavilanes, José G.; Martínez del Pozo, Álvaro; García Linares, SaraVenoms constitute complex mixtures of many different molecules arising from evolution in processes driven by continuous prey–predator interactions. One of the most common compounds in these venomous cocktails are pore-forming proteins, a family of toxins whose activity relies on the disruption of the plasmatic membranes by forming pores. The venom of sea anemones, belonging to the oldest lineage of venomous animals, contains a large amount of a characteristic group of pore-forming proteins known as actinoporins. They bind specifically to sphingomyelin-containing membranes and suffer a conformational metamorphosis that drives them to make pores. This event usually leads cells to death by osmotic shock. Sticholysins are the actinoporins produced by Stichodactyla helianthus. Three different isotoxins are known: Sticholysins I, II, and III. They share very similar amino acid sequence and three-dimensional structure but display different behavior in terms of lytic activity and ability to interact with cholesterol, an important lipid component of vertebrate membranes. In addition, sticholysins can act in synergy when exerting their toxin action. The subtle, but important, molecular nuances that explain their different behavior are described and discussed throughout the text. Improving our knowledge about sticholysins behavior is important for eventually developing them into biotechnological tools.Item Synergistic Action of Actinoporin Isoforms from the Same Sea Anemone Species Assembled into Functionally Active Heteropores(Journal of Biological Chemistry, 2016) Rivera de la Torre, Esperanza; García Linares, Sara; Alegre Cebollada, Jorge; Lacadena, Javier; Gavilanes, José G.; Martínez del Pozo, AlvaroAmong 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.Item Role of the Tryptophan Residues in the Specific interaction of the Sea Anemone Stichodacty la helianthus’s Actinoporin Sticholysin II with Biological Membranes(Biochemistry, 2016) García Linares, Sara; Maula, Terhi; Rivera de la Torre, Esperanza; Gavilanes, José G.; Slotte, J.Peter; Martínez del Pozo, ÁlvaroActinoporins are pore-forming toxins from sea anemones. Upon interaction with sphingomyelin-containing bilayers, they become integral oligomeric membrane structures that form a pore. Sticholysin II from Stichodactyla helianthus contains five tryptophans located at strategic positions; its role has now been studied using different mutants. Results show that W43 and W115 play a eterminant role in maintaining the high thermostability of the protein, while W146 provides specific interactions for protomer−protomer assembly. W110 and W114 sustain the hydrophobic effect, which is one of the major driving forces for membrane binding in the presence of Chol. However, in its absence, additional interactions with sphingomyelin are required. These conclusions were confirmed with two sphingomyelin analogues, one of which had impaired hydrogen bonding properties. The results obtained support actinoporins’ Trp residues playing a major role in membrane recognition and binding, but their residues have an only minor influence on the diffusion and oligomerization steps needed to assemble a functional pore.Item Differential Effect of Bilayer Thickness on Sticholysin Activity(Langmuir, 2017) Palacios Ortega, Juan; García Linares, Sara; Rivera de la Torre, Esperanza; Gavilanes, José G.; Martínez del Pozo, Álvaro; Slotte, J. PeterIn this study, we examined the influence of bilayer thickness on the activity of the actinoporin toxins sticholysin I and II (StnI and StnII) at 25 °C. Bilayer thickness was varied using dimonounsaturated phosphatidylcholine (PC) analogues (with 14:1, 16:1, 18:1, 20:1, and 22:1 acyl chains). In addition, N-14:0-sphingomyelin (SM) was always included because StnI and StnII are SM specific. Cholesterol was also incorporated as indicated. In cholesterol-free large unilamellar vesicles (LUVs) the PC:SM molar ratio was 4:1, and when cholesterol was included, the complete molar ratio was 4:1:0.5 (PC:SM:cholesterol, respectively). Stn toxins promote bilayer leakage through pores formed by oligomerized toxin monomers. Initial calcein leakage was moderately dependent on bilayer PC acyl chain length (and thus bilayer thickness), with higher rates observed with di-16:1 and di-18:1 PC bilayers. In the presence of cholesterol, the maximum rates of calcein leakage were observed in di-14:1 and di-16:1 PC bilayers. Using isothermal titration calorimetry to study the Stn−LUV interaction, we observed that the bilayer affinity constant (Ka) peaked with LUVs containing di-18:1 PC, and was lower in shorter and longer PC acyl chain bilayers. The presence of cholesterol increased the binding affinity approximately 30-fold at the optimal bilayer thickness (di-18:1-PC). We conclude that bilayer thickness affects both functional and conformational aspects of Stn membrane binding and pore formation. Moreover, the length of the actinoporins’ N-terminal α-helix, which penetrates the membrane to form a functional pore, appears to be optimal for the membrane thickness represented by di-18:1 PC.Item Pore-Forming Proteins from Cnidarians and Arachnids as Potential Biotechnological Tools(Toxins, 2019) Rivera de la Torre, Esperanza; Palacios Ortega, Juan; Gavilanes, José G.; Martínez del Pozo, Álvaro; García Linares, SaraAnimal 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.