Person:
García Linares, Sara

First Name

Sara

Last Name

García Linares

URI

https://hdl.handle.net/20.500.14352/85452

Affiliation

Universidad Complutense de Madrid

Faculty / Institute

Ciencias Biológicas

Department

Bioquímica y Biología Molecular

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Now showing 1 - 10 of 15

Structural foundations of sticholysin functionality
(BBA - Proteins and Proteomics, 2021) Palacios Ortega, Juan; García Linares, Sara; Rivera de Torre, Esperanza; Heras Márquez, Diego; Gavilanes, José G.; Peter, Slotte; Martínez del Pozo, Álvaro
Actinoporins constitute a family of α pore-forming toxins produced by sea anemones. The soluble fold of these proteins consists of a β-sandwich flanked by two α-helices. Actinoporins exert their activity by specifically recognizing sphingomyelin at their target membranes. Once there, they penetrate the membrane with their N-terminal α-helices, a process that leads to the formation of cation-selective pores. These pores kill the target cells by provoking an osmotic shock on them. In this review, we examine the role and relevance of the structural features of actinoporins, down to the residue level. We look at the specific amino acids that play significant roles in the function of actinoporins and their fold. Particular emphasis is given to those residues that display a high degree of conservation across the actinoporin sequences known to date. In light of the latest findings in the field, the membrane requirements for pore formation, the effect of lipid composition, and the process of pore formation are also discussed.
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. Peter
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.
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. Peter
Sticholysin 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.
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, Álvaro
Sea 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.
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.
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, Álvaro
Sticholysins 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.
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, Sara
Venoms 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.
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, Alvaro
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.
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, Álvaro
Actinoporins 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.
Sticholysin I–II oligomerization in the absence of membranes
(FEBS Letters, 2022) García Linares, Sara; Amigot Sánchez, Rafael; García Montoya, Carmen; Alfonso, Carlos; Luque Ortega, Juan Román; Gavilanes, José G.; Martínez del Pozo, Álvaro; Palacios Ortega, Juan
Sticholysins are pore-forming toxins produced by the sea anemone Stichodactyla helianthus. When they encounter a sphingomyelin-containing membrane, these proteins bind to it and oligomerize, a process that ends in pore formation. Mounting evidence indicates that StnII can favour the activity of StnI. Previous results have shown that these two isotoxins can oligomerize together. Furthermore, StnII appeared to potentiate the activity of StnI through the membrane-binding step of the process. Hence, isotoxin interaction should occur prior to membrane encounter. Here, we have used analytical ultracentrifugation to investigate the oligomerization of Stns in solution, both separately and together. Our results indicate that while StnI seems to be more prone to oligomerize in water solution than StnII, a small percentage of StnII in StnI–StnII mixtures promotes oligomerization.