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
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17 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 Regulation of Sticholysin II-Induced Pore Formation by Lipid Bilayer Composition, Phase State, and Interfacial Properties(Langmuir, 2016) Palacios Ortega, Juan; García Linares, Sara; Astrand, Mia; Abdullah Al Sazzad, Md.; Gavilanes, José G.; Martínez del Pozo, Álvaro; Slotte, J. PeterSticholysin II (StnII) is a pore-forming toxin that uses sphingomyelin (SM) as the recognition molecule in targeting membranes.After StnII monomers bind to SM, several toxin monomers act in concert to oligomerize into a functional pore. The regulation of StnII binding to SM, and the subsequent pore-formation process, is not fully understood. In this study, we examined how the biophysical properties of bilayers, originating from variations in the SM structure, from the presence of sterol species, or from the presence of increasingly polyunsaturated glycerophospholipids,affected StnII-induced pore formation. StnII-induced pore formation, as determined from calcein permeabilization, was fastest in the pure unsaturated SM bilayers. In 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/saturated SM bilayers (4:1 molar ratio), pore formation became slower as the chain length of the saturated SMs increased from 14 up to 24 carbons. In the POPC/palmitoyl-SM (16:0-SM) 4:1 bilayers, SM could not support pore formation by StnII if dimyristoyl-PC was included at 1:1 stoichiometry with 16:0-SM, suggesting that free clusters of SM were required for toxin binding and/or pore formation. Cholesterol and other sterols facilitated StnII-induced pore formation markedly, but the efficiency did not appear to correlate with the sterol structure. Benzyl alcohol was more efficient than sterols in enhancing the pore-formation process, suggesting that the effect on pore formation originated from alcohol-induced alteration of the hydrogen-bonding network in the SM-containing bilayers. Finally, we observed that pore formation by StnII was enhanced in the PC/16:0-SM 4:1 bilayers, in which the PC was increasingly unsaturated. We conclude that the physical state of bilayer lipids greatly affected pore formation by StnII. Phase boundaries were not required for pore formation, although SM in a gel state attenuated pore formation.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 The behaviour of sea anemone actinoporins at the water-membrane interface(Biochimica et Biophysica Acta - Biomembranes, 2011) García Ortega, Lucía; Alegre Cebollada, Jorge; García Linares, Sara; Bruix, Marta; Martínez del Pozo, Álvaro; Gavilanes, José G.Actinoporins constitute a group of small and basic α-pore forming toxins produced by sea anemones. They display high sequence identity and appear as multigene families. They show a singular behaviour at the water-membrane interface: In aqueous solution, actinoporins remain stably folded but, upon interaction with lipid bilayers, become integral membrane structures. These membranes contain sphingomyelin, display phase coexistence, or both. The water soluble structures of the actinoporins equinatoxin II (EqtII) and sticholysin II (StnII) are known in detail. The crystalline structure of a fragaceatoxin C (FraC) nonamer has been also determined. The three proteins fold as a β-sandwich motif flanked by two α-helices, one of them at the N-terminal end. Four regions seem to be especially important: A cluster of aromatic residues, a phosphocholine binding site, an array of basic amino acids, and the N-terminal α-helix. Initial binding of the soluble monomers to the membrane is accomplished by the cluster of aromatic amino acids, the array of basic residues, and the phosphocholine binding site. Then, the N-terminal α-helix detaches from the β-sandwich, extends, and lies parallel to the membrane. Simultaneously, oligomerization occurs. Finally, the extended N-terminal α-helix penetrates the membrane to build a toroidal pore. This model has been however recently challenged by the cryo-EM reconstruction of FraC bound to phospholipid vesicles. Actinoporins structural fold appears across all eukaryotic kingdoms in other functionally unrelated proteins. Many of these proteins neither bind to lipid membranes nor induce cell lysis. Finally, studies focusing on the therapeutic potential of actinoporins also abound.Item One single salt bridge explains the different cytolytic activities shown by actinoporins sticholysin I and II from the venom of Stichodactyla helianthus(Archives of Biochemistry and Biophysics, 2017) Rivera de Torre, Esperanza; Palacios Ortega, Juan; García Linares, Sara; Gavilanes, José G.; Martínez del Pozo, ÁlvaroSticholysins I and II (StnI and StnII), α-pore forming toxins from the sea anemone Stichodactyla helianthus, are water-soluble toxic proteins which upon interaction with lipid membranes of specific composition bind to the bilayer, extend and insert their N-terminal α-helix, and become oligomeric integral membrane structures. The result is a pore that leads to cell death by osmotic shock. StnI and StnII show 93% of sequence identity, but also different membrane pore-forming activities. The hydrophobicity profile along the first 18 residues revealed differences which were canceled by substituting StnI amino acids 2 and 9. Accordingly, the StnID9A mutant, and the corresponding StnIE2AD9A variant, showed enhanced hemolytic activity. They also revealed a key role for an exposed salt bridge between Asp9 and Lys68. This interaction is not possible in StnII but appears conserved in the other two well-characterized actinoporins, equinatoxin II and fragaceatoxin C. The StnII mutant A8D showed that this single replacement was enough to transform StnII into a version with impaired pore-forming activity. Overall, the results show the key importance of this salt bridge linking the N-terminal stretch to the β-sandwich core. A conclusion of general application for the understanding of salt bridges role in protein design, folding and stability.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 Cholesterol stimulates and ceramide inhibits Sticholysin II-induced pore formation in complex bilayer membranes(BBA - Biochimica et Biophysica Acta, 2015) Alm, Ida; García Linares, Sara; Gavilanes, José G.; Martínez del Pozo, Álvaro; Slotte, J. PeterThe pore forming capacity of Sticholysin II (StnII; isolated from Stichodactyla helianthus) in bilayer membranes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), palmitoylsphingomyelin (PSM) and either cholesterol or palmitoyl ceramide (PCer) has been examined. The aim of the study was to elucidate how the presence of differently ordered PSM domains affected StnII oligomerization and pore formation. Cholesterol is known to enhance pore formation by StnII, and our results confirmed this and provide kinetic information for the process. The effect of cholesterol on bilayer permeabilization kinetics was concentration-dependent. In the concentration regime used (2.5–10 nmol cholesterol in POPC:PSM 80:20 by nmol), cholesterol also increased the acyl chain order in the fluid PSM domain and thus decreased bilayer fluidity, suggesting that fluidity per se was not responsible for cholesterol's effect. Addition of PCer (2.5–10 nmol) to the POPC:PSM (80:20 by nmol) bilayers attenuated StnII-induced pore formation, again in a concentration-dependent fashion. This addition also led to the formation of a PCer-rich gel phase. Addition of cholesterol to PCer-containing membranes could partially reduce the inhibitory effect of PCer on StnII pore formation. We conclude that the physical state of PSM (as influenced by either cholesterol or PCer) affected StnII binding and pore formation under the conditions examined.Item Toxin-induced pore formation is hindered by intermolecular hydrogen bonding in sphingomyelin bilayers(Biochimica et Biophysica Acta - Biomembranes, 2016) García Linares, Sara; Palacios Ortega, Juan; Yasuda, Tomokazu; Åstrand, Mia; Gavilanes, José G.; Martínez del Pozo, Álvaro; Slotte, J. PeterSticholysin 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.Item Actinoporinas:un veneno letal(2016) García Linares, Sara; Rivera de Torre, EsperanzaEn 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.