Cytotoxicity of equinatoxin II from the sea anemoneActinia equina involves ion channel formation and an increase in intracellular calcium activity

Zorec, Robert; Tester, Mark; Maček, Peter; Mason, William T.

doi:10.1007/bf01868608

Cited by 79 publications

(39 citation statements)

References 37 publications

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“…Indeed, rapid elevations of cytosolic Ca 2ϩ levels following membrane injury alter the cytoskeleton and its contacts with the membrane (27,45). To detect possible increases in cytosolic Ca 2ϩ associated with EqtII-induced membrane permeabilization, we treated the cells with the nonfluorescent acetoxymethyl ester of Fluo4, which is cleaved inside cells to yield the free, fluorescent form of the calcium indicator (46). As shown in Fig.…”

Section: Equinatoxin II Membrane Permeabilization Is Accompanied By Cmentioning

confidence: 99%

Oligomerization and Pore Formation by Equinatoxin II Inhibit Endocytosis and Lead to Plasma Membrane Reorganization

García‐Sáez

Buschhorn

Keller

et al. 2011

Journal of Biological Chemistry

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Pore-forming toxins have evolved to induce membrane injury by formation of pores in the target cell that alter ion homeostasis and lead to cell death. Many pore-forming toxins use cholesterol, sphingolipids, or other raft components as receptors. However, the role of plasma membrane organization for toxin action is not well understood. In this study, we have investigated cellular dynamics during the attack of equinatoxin II, a poreforming toxin from the sea anemone Actinia equina, by combining time lapse three-dimensional live cell imaging, fluorescence recovery after photobleaching, FRET, and fluorescence crosscorrelation spectroscopy. Our results show that membrane binding by equinatoxin II is accompanied by extensive plasma membrane reorganization into microscopic domains that resemble coalesced lipid rafts. Pore formation by the toxin induces Ca 2؉ entry into the cytosol, which is accompanied by hydrolysis of phosphatidylinositol 4,5-bisphosphate, plasma membrane blebbing, actin cytoskeleton reorganization, and inhibition of endocytosis. We propose that plasma membrane reorganization into stabilized raft domains is part of the killing strategy of equinatoxin II.Pore-forming proteins (PFPs) 4 have been developed by many organisms ranging from bacteria and parasites to animals to disrupt membrane integrity of the target cell. The purpose of membrane permeabilization in many cases is to compromise cell survival, as it happens with toxins like ␣-toxin, actinoporins, or the proteins of the Bcl-2 family and perforin in human cells. However, it can also be related with the escape of certain bacteria from the phagosome, like listeriolysin, or with protein delivery into the cytosol, as in the case of colicins (1-3).PFPs are secreted by the producer cell in soluble form and bind to target cells via specific receptors. Then they undergo a conformational change that exposes hydrophobic residues and allows their insertion into the plasma membrane and oligomerization into a pore. The nature, stoichiometry, and size of that pore depend on the PFP (1). In vitro studies suggest that the toxins of the actinoporin family form a tetramer (4 -6), although it could also have a structure similar to those recently reported for cytolysin A or fragaceatoxin C (7, 8), with a higher oligomeric state. The nature of the pore is believed to be toroidal, with lipids exposed to the pore lumen (9). In red blood cells and on artificial lipid vesicles, actinoporins formed pores ϳ2 nm in diameter (10, 11).Many PFPs use molecules associated with lipid rafts as receptors (12). Rafts are lipid/protein domains enriched in cholesterol and sphingolipids that act as signaling platforms in the plasma membrane of the cell. During the last years, there has been extensive debate over what rafts are and their functional role. Different domains of raft nature have been observed in several temporal and spatial scales. As a consequence, there are many different kinds of domains that have been associated with rafts (13).Some examples of raft-associated toxin...

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Section: Equinatoxin II Membrane Permeabilization Is Accompanied By Cmentioning

confidence: 99%

Oligomerization and Pore Formation by Equinatoxin II Inhibit Endocytosis and Lead to Plasma Membrane Reorganization

García‐Sáez

Buschhorn

Keller

et al. 2011

Journal of Biological Chemistry

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“…Equinatoxin II increased membrane electrical conductance, indicating that the cytotoxic action of equinatoxin II involves an increase in the permeability of membranes to Ca 2? (Zorec et al 1990). …”

Section: Introductionmentioning

confidence: 99%

The effect of sea anemone (H. magnifica) venom on two human breast cancer lines: death by apoptosis

2013

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Venom from the sea anemone, Heteractis magnifica, has multiple biological effects including, cytotoxic, cytolytic and hemolytic activities. In this study, cytotoxicity induced by H. magnifica venom was investigated using the crystal violet assay on human breast cancer T47D and MCF7 cell lines and normal human breast 184B5 cell line. Apoptosis was also assayed via Annexin V-flourescein isothiocyanate and propidium iodide (PI) staining followed by flow cytometric analysis. Cell cycle progression and mitochondria membrane potential were studied via flow cytometry following PI and JC-1 staining respectively. H. magnifica venom induced significant reductions in viable cell numbers and increases in apoptosis in T47D and MCF7 in dose-dependent manners. A significant apoptosisrelated increase in the sub G1 peak of the cell cycle in both breast cancer cell lines was also observed. Moreover, treatment by venom cleaved caspase-8, caspase-9, and activated caspase-3. Overall, H. magnifica venom was highly cytotoxic to T47D and MCF7 human breast cancer cells, and the phenomenon could be the killing phenomenon via the death receptor-mediated and the mitochondria-mediated apoptotic pathways. Consequently, H. magnifica venom has potential for the development of a breast cancer therapeutic.

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“…Actinoporins act on cellular and model lipid membranes by forming cation-selective pores with a hydrodynamic diameter of ϳ2 nm. The mechanism of pore formation involves at least two steps: binding of the water soluble monomer to the membrane; and subsequent oligomerization of three to four monomers on the surface of the membrane, leading to the formation of a functional pore (10,(12)(13)(14)(15)(16). The number of monomers in the final pore was deduced from cross-linking (12) and kinetic experiments (16).…”

mentioning

confidence: 99%

Two-step Membrane Binding by Equinatoxin II, a Pore-forming Toxin from the Sea Anemone, Involves an Exposed Aromatic Cluster and a Flexible Helix

Hong¹,

Gutiérrez-Aguirre²,

Barlič³

et al. 2002

Journal of Biological Chemistry

197

235

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Equinatoxin II (EqtII) belongs to a unique family of 20-kDa pore-forming toxins from sea anemones. These toxins preferentially bind to membranes containing sphingomyelin and create cation-selective pores by oligomerization of 3-4 monomers. In this work we have studied the binding of EqtII to lipid membranes by the use of lipid monolayers and surface plasmon resonance (SPR). The binding is a two-step process, separately mediated by two regions of the molecule. An exposed aromatic cluster involving tryptophans 112 and 116 mediates the initial attachment that is prerequisite for the next step. Steric shielding of the aromatic cluster or mutation of Trp-112 and -116 to phenylalanine significantly reduces the toxin-lipid interaction. The second step is promoted by the N-terminal amphiphilic helix, which translocates into the lipid phase. The two steps were distinguished by the use of a double cysteine mutant having the N-terminal helix fixed to the protein core by a disulfide bond. The kinetics of membrane binding derived from the SPR experiments could be fitted to a two-stage binding model. Finally, by using membraneembedded quenchers, we showed that EqtII does not insert deeply in the membrane. The first step of the EqtII binding is reminiscent of the binding of the evolutionarily distant cholesterol-dependant cytolysins, which share a similar structural motif in the membrane attachment domain.Targeting and attachment of proteins to membranes is one of the key steps in many cellular processes (1-3). Protein-membrane interactions have been studied intensively in recent years with many different examples of proteins and membranes. These interactions can be promoted at the lipid-water interface by lipid anchors, electrostatic forces or surface-exposed aromatic and aliphatic residues (1, 2, 4). Compared with protein-protein interactions, details of protein-membrane interactions are poorly defined. Some of the best characterized examples are a phospholipase C pleckstrin homology domain specific for phosphatidylinositol trisphosphate (5) and small protein kinase-C-conserved (C2) domains specific for zwitterionic, particularly phosphatidylcholine membranes (6).Another group of proteins interacting with lipid membranes are pore-forming toxins (PFT) 1 (7-10), which bind to membranes before eliciting their toxic effects via the formation of transmembrane pores. The most studied PFT are bacterial since this group includes important virulence factors. Few examples of eukaryotic PFT have been well characterized, exceptions being the actinoporins, cytolysins found exclusively in sea anemones (10, 11). Members of this family have properties distinct from other PFT: they are composed of 175-179 amino acids, contain no cysteine residues, have pIϾ9.5, and show a preference for sphingomyelin (SM)-containing membranes. Actinoporins act on cellular and model lipid membranes by forming cation-selective pores with a hydrodynamic diameter of ϳ2 nm. The mechanism of pore formation involves at least two steps: binding of the water soluble m...

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Cytotoxicity of equinatoxin II from the sea anemoneActinia equina involves ion channel formation and an increase in intracellular calcium activity

Cited by 79 publications

References 37 publications

Oligomerization and Pore Formation by Equinatoxin II Inhibit Endocytosis and Lead to Plasma Membrane Reorganization

Oligomerization and Pore Formation by Equinatoxin II Inhibit Endocytosis and Lead to Plasma Membrane Reorganization

The effect of sea anemone (H. magnifica) venom on two human breast cancer lines: death by apoptosis

Two-step Membrane Binding by Equinatoxin II, a Pore-forming Toxin from the Sea Anemone, Involves an Exposed Aromatic Cluster and a Flexible Helix

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