RESUMEN
Spanish or Spanish-speaking scientists represent a remarkably populated group within the scientific community studying pore-forming proteins. Some of these scientists, ourselves included, focus on the study of actinoporins, a fascinating group of metamorphic pore-forming proteins produced within the venom of several sea anemones. These toxic proteins can spontaneously transit from a water-soluble fold to an integral membrane ensemble because they specifically recognize sphingomyelin in the membrane. Once they bind to the bilayer, they subsequently oligomerize into a pore that triggers cell-death by osmotic shock. In addition to sphingomyelin, some actinoporins are especially sensible to some other membrane components such as cholesterol. Our group from Universidad Complutense of Madrid has focused greatly on the role played by sterols in this water-membrane transition, a question which still remains only partially solved and constitutes the main core of the article below.
Asunto(s)
Venenos de Cnidarios , Anémonas de Mar , Animales , Colesterol/metabolismo , Porinas/metabolismo , Esfingomielinas/metabolismo , Agua/metabolismoRESUMEN
Induction of CD8+ T cell responses against tumor cells and intracellular pathogens is an important goal of modern vaccinology. One approach of translational interest is the use of liposomes encapsulating pore-forming proteins (PFPs), such as Listeriolysin O (LLO), which has shown efficacy at priming strong and sustained CD8+ T cell responses. Recently, we have demonstrated that Sticholysin II (StII), a PFP from the sea anemone Stichodactyla helianthus, co-encapsulated into liposomes with ovalbumin (OVA) was able to stimulate, antigen presenting cells, antigen-specific CD8+ T cells and anti-tumor activity in mice. In the present study, we aimed to compare StII and LLO in terms of their abilities to stimulate dendritic cells and to induce major histocompatibility complex (MHC) class I restricted T cell responses against OVA. Interestingly, StII exhibited similar abilities to LLO in vitro of inducing dendritic cells maturation, as measured by increased expression of CD40, CD80, CD86 and MHC-class II molecules, and of stimulating OVA cross-presentation to a CD8+ T cell line. Remarkably, using an ex vivo Enzyme-Linked ImmunoSpot Assay (ELISPOT) to monitor gamma interferon (INF-γ) producing effector memory CD8+ T cells, liposomal formulations containing either StII or LLO induced comparable frequencies of OVA-specific INF-γ producing CD8+ T cells in mice that were sustained in time. However, StII-containing liposomes stimulated antigen-specific memory CD8+ T cells with a higher potential to secrete IFN-γ than liposomes encapsulating LLO. This StII immunostimulatory property further supports its use for the rational design of T cell vaccines against cancers and intracellular pathogens. In summary, this study indicates that StII has immunostimulatory properties similar to LLO, despite being evolutionarily distant PFPs.
Asunto(s)
Linfocitos T CD8-positivos , Linfocitos T Citotóxicos , Animales , Toxinas Bacterianas , Venenos de Cnidarios , Células Dendríticas , Proteínas de Choque Térmico , Proteínas Hemolisinas , Ratones , Ratones Endogámicos C57BL , OvalbúminaRESUMEN
Sticholysin I (StI) is a toxin produced by the sea anemone Stichodactyla helianthus and belonging to the actinoporins family. Upon binding to sphingomyelin-containing membranes StI forms oligomeric pores, thereby leading to cell death. According to recent controversial experimental evidences, the pore architecture of actinoporins is a debated topic. Here, we investigated the StI topology in membranes by site-directed spin labeling and electron paramagnetic resonance spectroscopy. The results reveal that StI in membrane exhibits an oligomeric architecture with heterogeneous stoichiometry of predominantly eight or nine protomers, according to the available structural models. The StI topology resembles the conic pore structure reported for the actinoporin fragaceatoxin C. Our data show that StI coexists in two membrane-associated conformations, with the N-terminal segment either attached to the protein core or inserted in the membrane forming the pore. This finding suggests a 'pre-pore' to 'pore' transition determined by a conformational change that detaches the N-terminal segment.
Asunto(s)
Venenos de Cnidarios/metabolismo , Animales , Venenos de Cnidarios/química , Compuestos Orgánicos/química , Compuestos Orgánicos/metabolismo , Estructura Secundaria de Proteína , Anémonas de MarRESUMEN
Sticholysin I and II (Sts: St I and St II) are proteins of biomedical interest that form pores upon the insertion of their N-terminus in the plasma membrane. Peptides spanning the N-terminal residues of StI (StI1-31) or StII (StII1-30) can mimic the permeabilizing ability of these toxins, emerging as candidates to rationalize their potential biomedical applications. These peptides have different activities that correlate with their hydrophobicity. However, it is not clear how this property contributes to peptide folding in solution or upon binding to membranes. Here we compared the conformational properties of these peptides and shorter versions lacking the hydrophobic segment 1-11 of StI (StI12-31) or 1-10 of StII (StII11-30). Folding of peptides was assessed in solution and in membrane mimetic systems and related with their ability to bind to membranes and to permeabilize lipid vesicles. Our results suggest that the differences in activity among peptides could be ascribed to their different folding propensity and different membrane binding properties. In solution, StII1-30 tends to acquire α-helical conformation coexisting with self-associated structures, while StI1-31 remains structureless. Both peptides fold as α-helix in membrane; but StII1-30 also self-associates in the lipid environment, a process that is favored by its higher affinity for membrane. We stress the contribution of the non-polar/polar balance of the 1-10 amino acid sequence of the peptides as a determining factor for different self-association capabilities. Such difference in hydrophobicity seems to determine the molecular path of peptides folding upon binding to membranes, with an impact in their permeabilizing activity. This study contributes to a better understanding of the molecular mechanisms underlying the permeabilizing activity of Sts N-terminal derived peptides, with connotation for the exploitation of these small molecules as alternative of the full-length toxins in clinical settings.
Asunto(s)
Venenos de Cnidarios/química , Membranas Artificiales , Pliegue de Proteína , Compuestos Orgánicos/química , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
Cross-presentation is an important mechanism for the differentiation of effector cytotoxic T lymphocytes (CTL) from naïve CD8+ T-cells, a key response for the clearance of intracellular pathogens and tumors. The liposomal co-encapsulation of the pore-forming protein sticholysin II (StII) with ovalbumin (OVA) (Lp/OVA/StII) induces a powerful OVA-specific CTL activation and an anti-tumor response in vivo. However, the pathway through which the StII contained in this preparation is able to induce antigen cross-presentation and the type of professional antigen presenting cells (APCs) involved have not been elucidated. Here, the ability of mouse bone marrow-derived dendritic cells (BM-DCs) and macrophages (BM-MΦs) stimulated with Lp/OVA/StII to activate SIINFEKL-specific B3Z CD8+ T cells was evaluated in the presence of selected inhibitors. BM-MΦs, but not BM-DCs were able to induce SIINFEKL-specific B3Z CD8+ T cell activation upon stimulation with Lp/OVA/StII. The cross-presentation of OVA was markedly decreased by the lysosome protease inhibitors, leupeptin and cathepsin general inhibitor, while it was unaffected by the proteasome inhibitor epoxomicin. This process was also significantly reduced by phagocytosis and Golgi apparatus function inhibitors, cytochalasin D and brefeldin A, respectively. These results are consistent with the concept that BM-MΦs internalize these liposomes through a phagocytic mechanism resulting in the cross-presentation of the encapsulated OVA by the vacuolar pathway. The contribution of macrophages to the CTL response induced by Lp/OVA/StII in vivo was determined by depleting macrophages with clodronate-containing liposomes. CTL induction was almost completely abrogated in mice depleted of macrophages, demonstrating the relevance of these APCs in the antigen cross-presentation induced by this formulation.
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Venenos de Cnidarios/metabolismo , Células Dendríticas/fisiología , Macrófagos/fisiología , Linfocitos T Citotóxicos/inmunología , Vacuolas/metabolismo , Animales , Antígenos/inmunología , Antígenos CD8/metabolismo , Células Cultivadas , Venenos de Cnidarios/química , Reactividad Cruzada , Femenino , Leupeptinas/farmacología , Liposomas/química , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunologíaRESUMEN
Sticholysin II (StII) is a pore-forming toxin of biomedical interest that belongs to the actinoporin protein family. Sticholysins are currently under examination as an active immunomodulating component of a vaccinal platform against tumoral cells and as a key element of a nucleic acids delivery system to cell cytosol. These proteins form pores in the plasma membrane leading to ion imbalance and cell lysis. However, the intracellular mechanisms triggered by actinoporins upon binding to membranes and its consequences for cell death are barely understood. Here, we have examined the cytotoxicity and intracellular responses induced by StII upon binding to human B-cell lymphoma Raji in vitro. StII cytotoxicity involves a functional actin cytoskeleton, induces cellular swelling, lysis and the concomitant release of cytosol content. In addition, StII induces calcium release mainly from the Endoplasmic Reticulum, activates Mitogen-Activated Protein Kinase ERK and impairs mitochondrial membrane potential. Furthermore, StII stimulates the expression of receptor interacting protein kinase 1 (RIP1), normally related to different forms of regulated cell death such as apoptosis and necroptosis. In correspondence, necrostatin-1, an inhibitor of this kinase, reduces StII cytotoxicity. However, the mechanism of cell death activated by StII does not involve caspases activation, typical molecular features of apoptosis and pyroptosis. Our results suggest that, beyond pore-formation and cell lysis, StII-induced cytotoxicity could involve other regulated intracellular mechanisms connected to RIP1-MEK1/2 -ERK1/2- pathways. This opens new perspectives and challenges the general point of view that these toxins induce a completely unregulated mechanism of necrotic cell death. This study contributes to a better understanding of the molecular mechanisms involved in toxin-cell interaction and the implications for cell functioning, with connotation for the exploitations of these toxins in clinical settings.
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Muerte Celular/efectos de los fármacos , Venenos de Cnidarios/toxicidad , Citotoxinas/toxicidad , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Línea Celular , Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Sticholysin II is a pore-forming toxin produced by the sea anemone Stichodactyla helianthus that belongs to the actinoporin protein family. The high affinity of actinoporins for sphingomyelin (SM)-containing membranes has been well documented. However, the molecular determinants that define this affinity have not been fully clarified. Here, we have examined the binding and permeabilizing activity of StII to different single and mixed lipidic systems by combining lipid monolayers, liposomes, and permeabilizing assays. This study characterizes the contribution of ceramide-derived compounds for StII-membrane interaction. Molecular dynamics simulations revealed a differential binding mode of StII with the polar head group of SM and PC. The electrostatic interaction energies were the major energetic contributors to the better affinity of StII for SM compared to PC, while the van der Waals interaction energies were the major driving forces of the better affinity of StII for SM respect to Cer. Furthermore, the presence of sugar residues in glycosphingolipids modulated binding and pore-formation by actinoporins probably by hindering StII to reach relevant structural motifs in membrane for binding or inducing a non-competent adsorption to membrane. Our results demonstrate that StII-membrane interaction, leading to pore formation, may critically respond to changes in lipid head group properties, and the access to SM interfacial structural motif.
Asunto(s)
Venenos de Cnidarios/metabolismo , Simulación de Dinámica Molecular , Anémonas de Mar/química , Esfingomielinas/metabolismo , Termodinámica , Animales , Venenos de Cnidarios/química , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/química , Liposomas/química , Esfingomielinas/químicaRESUMEN
Pore-forming toxins (PFTs) form holes in membranes causing one of the most catastrophic damages to a target cell. Target organisms have evolved a regulated response against PFTs damage including cell membrane repair. This ability of cells strongly depends on the toxin concentration and the properties of the pores. It has been hypothesized that there is an inverse correlation between the size of the pores and the time required to repair the membrane, which has been for long a non-intuitive concept and far to be completely understood. Moreover, there is a lack of information about how cells react to the injury triggered by eukaryotic PFTs. Here, we investigated some molecular events related with eukaryotic cells response against the membrane damage caused by sticholysin II (StII), a eukaryotic PFT produced by a sea anemone. We evaluated the change in the cytoplasmic potassium, identified the main MAPK pathways activated after pore-formation by StII, and compared its effect with those from two well-studied bacterial PFTs: aerolysin and listeriolysin O (LLO). Strikingly, we found that membrane recovery upon StII damage takes place in a time scale similar to LLO in spite of the fact that they form pores by far different in size. Furthermore, our data support a common role of the potassium ion, as well as MAPKs in the mechanism that cells use to cope with these toxins injury.
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Venenos de Cnidarios/toxicidad , Células Eucariotas/efectos de los fármacos , Proteínas Citotóxicas Formadoras de Poros/toxicidad , Potasio/metabolismo , Anémonas de Mar/patogenicidad , Animales , Células Cultivadas , Cricetinae , Células Eucariotas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
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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Porinas/metabolismo , Isoformas de Proteínas/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida , Hemólisis , Lípidos de la Membrana/metabolismo , Fosfolípidos/metabolismo , Porinas/química , Isoformas de Proteínas/química , Anémonas de MarRESUMEN
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 Δ(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 pH7.0) 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.
Asunto(s)
Membrana Dobles de Lípidos , Porinas/farmacología , Esfingomielinas/metabolismo , Enlace de Hidrógeno , Esfingomielinas/química , Resonancia por Plasmón de SuperficieRESUMEN
Actinoporins (APs) from sea anemones are ~20 kDa pore forming toxins with a ß-sandwich structure flanked by two α-helices. The molecular mechanism of APs pore formation is composed of several well-defined steps. APs bind to membrane by interfacial binding site composed of several aromatic amino acid residues that allow binding to phosphatidylcholine and specific recognition of sphingomyelin. Subsequently, the N-terminal α-helix from the ß-sandwich has to be inserted into the lipid/water interphase in order to form a functional pore. Functional studies and single molecule imaging revealed that only several monomers, 3-4, oligomerise to form a functional pore. In this model the α-helices and surrounding lipid molecules build toroidal pore. In agreement, AP pores are transient and electrically heterogeneous. On the contrary, crystallized oligomers of actinoporin fragaceatoxin C were found to be composed of eight monomers with no lipids present between the adjacent α-helices. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Maur Dalla Serra and Franco Gambale.
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Venenos de Cnidarios/química , Perforina/química , Porinas/química , Anémonas de Mar/química , Animales , Venenos de Cnidarios/metabolismo , Perforina/metabolismo , Porinas/metabolismo , Estructura Secundaria de Proteína , Anémonas de Mar/metabolismoRESUMEN
Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT exclusively found in sea anemones. As for actinoporins, it has been proposed that the presence of sphingomyelin (SM) and the coexistence of lipid phases increase binding to the target membrane. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, presence of lipid domains) on actinoporins' activity or which regions of the membrane are the most favorable platforms for protein insertion. To gain insight into the role of SM on the interaction of St I to lipid membranes we studied their binding to monolayers of phosphatidylcholine (PC) and SM in different proportions. Additionally, the effect of acyl chain length and unsaturation, two features related to membrane fluidity, was evaluated on St I binding to monolayers. This study revealed that St I binds and penetrates preferentially and with a faster kinetic to liquid-expanded films with high lateral mobility and moderately enriched in SM. A high content of SM induces a lower lateral diffusion and/or liquid-condensed phases, which hinder St I binding and penetration to the lipid monolayer. Furthermore, the presence of lipid domain borders does not appear as an important factor for St I binding to the lipid monolayer.
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Fluidez de la Membrana/fisiología , Lípidos de la Membrana/metabolismo , Esfingomielinas/metabolismo , Animales , Venenos de Cnidarios/metabolismo , Cinética , Membranas/metabolismo , Compuestos Orgánicos/metabolismo , Fosfatidilcolinas/metabolismo , Anémonas de MarRESUMEN
Sea anemone actinoporins constitute an optimum model to investigate mechanisms of membrane pore formation. All actinoporins of known structure show a general fold of a ß-sandwich motif flanked by two α-helices. The crucial structure for pore formation seems to be the helix located at the N-terminal end. The role of several other protein regions in membrane attachment is also well established. However, not much is known about the protein residues involved in the oligomerization required for pore formation. Previous detailed analysis of the soluble three-dimensional structures of different wild-type and mutant actinoporins from Stychodactyla helianthus suggested residues which could be involved in this oligomerization. One of these stretches contains a conserved sequence compatible with an integrin-binding RGD motif. The results presented now deal with mutants affecting this motif in the well-characterized actinoporin sticholysin II. Small modifications along this three-residue sequence had profound effects on its solubility. Just a single methyl group yielded an RAD mutant version with a highly diminished haemolytic activity and altered oligomerization behaviour. The results obtained are discussed in terms of a key role for the RGD motif in maintaining the actinoporins' pore-competent state of protein oligomerization.
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Venenos de Cnidarios/química , Venenos de Cnidarios/genética , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Venenos de Cnidarios/toxicidad , Secuencia Conservada , Hemólisis/efectos de los fármacos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Proteínas Citotóxicas Formadoras de Poros/toxicidad , Conformación Proteica , Estructura Cuaternaria de Proteína , Anémonas de Mar/química , Anémonas de Mar/genéticaRESUMEN
Sticholysin I and Sticholysin II (StI and StII) are two potent hemolysins which form pores in natural and model membranes at nanomolar concentrations. These proteins were purified from the aqueous extract of the sea anemone Stichodactyla helianthus, Ellis 1768, by gel filtration and ionic exchange chromatography. This procedure rendered StI and StII with high purity (purification factors: 36 and 50, respectively) but a low yield of hemolytic activity, HA (<3%). Additionally, these toxins exhibited very low phospholipase activity (10(-3)U/mg of protein). In this work, a mixture StI-StII was obtained (yield >95%, with an increase in specific activity: 14 times) from the animal extract using an oxidized phospholipid-based affinity chromatographic matrix binding phospholipases. Cytolysin identification in the mixture was performed by immunoblotting and N-terminal sequence analyses. Phospholipase A2 (PLA2) activity of StI-StII was relatively high (1.85U/mg) and dependent of Ca(2+). The activity resulted optimum when was measured with the mostly unsaturated soybean phosphatidylcholine (PC), when compared to the less unsaturated egg PC or completely saturated dipalmitoyl PC, in the presence of 40mM Ca(2+) at pH 8.0. This Ca(2+) concentration did not exert any effect on binding of StI-StII with soybean PC monolayers. Then, PLA2 activity seems not be required to binding to membranes.
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Venenos de Cnidarios/metabolismo , Proteínas Hemolisinas/metabolismo , Fosfolipasas A2/metabolismo , Anémonas de Mar/química , Anémonas de Mar/enzimología , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Cromatografía de Afinidad , Venenos de Cnidarios/química , Venenos de Cnidarios/aislamiento & purificación , Proteínas Hemolisinas/química , Proteínas Hemolisinas/aislamiento & purificación , Datos de Secuencia Molecular , Compuestos Orgánicos/química , Compuestos Orgánicos/aislamiento & purificación , Compuestos Orgánicos/metabolismo , Fosfolipasas A2/química , Fosfolipasas A2/aislamiento & purificación , Alineación de SecuenciaRESUMEN
Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.
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Venenos de Cnidarios/farmacología , Lípidos/química , Microdominios de Membrana/metabolismo , Anémonas de Mar/metabolismo , Animales , Rastreo Diferencial de Calorimetría , Microscopía de Fuerza Atómica , Compuestos Orgánicos/farmacología , Anémonas de Mar/químicaRESUMEN
Sticholysin II (St II) is the most potent cytolysin produced by the sea anemone Stichodactyla helianthus, exerting hemolytic activity via pore formation in membranes. The toxin's N-terminus contains an amphipathic α-helix that is very likely involved in pore formation. We have previously demonstrated that the synthetic peptide StII(1-30) encompassing the 1-30 segment of St II forms pores of similar radius to that of the protein (around 1 nm), being a good model of toxin functionality. Here we have studied the functional and conformational properties of fluorescent analogs of StII(1-30) in lipid membranes. The analogs were obtained by replacing Leu residues at positions 2, 12, 17, and 24 with the intrinsically fluorescent amino acid Trp (StII(1-30L2W), StII(1-30L12W), StII(1-30L17W), or StII(1-30L24W), respectively). The exchange by Trp did not significantly modify the activity and conformation of the parent peptide. The blue-shift and intensity enhancement of fluorescence in the presence of membrane indicated that Trp at position 2 is more deeply buried in the hydrophobic region of the bilayer. These experiments, as well as assays with water-soluble or spin-labeled lipid-soluble fluorescence quenchers suggest an orientation of StII(1-30) with its N-terminus oriented towards the hydrophobic core of the bilayer while the rest of the peptide is more exposed to the aqueous environment, as hypothesized for sticholysins.