Molecular Mechanism of Sphingomyelin-Specific Membrane Binding and Pore Formation by Actinoporins

Bakrač, Biserka; Anderluh, Gregor

doi:10.1007/978-1-4419-6327-7_9

Cited by 50 publications

(47 citation statements)

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“…It has been shown that EqtII binds specifically to sphingomyelin, a raft lipid, and that it oligomerizes to form pores (4,16). Our FRET experiments on living cells also show EqtII/EqtII interactions within the toxin-induced domains.…”

Section: Discussionsupporting

confidence: 63%

“…EqtII binds specifically to SM, a lipid that has been involved in the formation of rafts (16,40). Moreover, other toxins of the same family have been found associated with the fraction of detergent-resistant membranes in isolation experiments (17).…”

Section: Equinatoxin II Clustering Reorganizes the Plasma Membrane Anmentioning

confidence: 99%

“…EqtII belongs to the family of actinoporins and, like the other members of the family, forms pores in sphingomyelin-containing membranes by oligomerization and insertion of an N-terminal ␣-helix that contributes to the structure of the pore (1,16). Using model membranes in in vitro experiments, we and others have shown that the presence of liquid ordered, raft-like lipid domains promotes EqtII binding and pore activity, with an enrichment of the toxin at the domain interface (19,20).…”

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confidence: 99%

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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: Discussionsupporting

confidence: 63%

Section: Equinatoxin II Clustering Reorganizes the Plasma Membrane Anmentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…Thus, high affinity recognition of sphingomyelin (SM) 3 is crucial for specific attachment to a membrane, but the subsequent effects observed also depend on the physical properties derived from its particular composition and not only from its SM content (23, 32). In fact, although still controversial, the presence of cholesterol and the coexistence of different phases in the membrane seem to be important factors, if not for binding then at least for the final formation of the pore (23,28,29,(32)(33)(34)(35)(36).…”

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confidence: 99%

Synergistic Action of Actinoporin Isoforms from the Same Sea Anemone Species Assembled into Functionally Active Heteropores

Rivera-de-Torre

García‐Linares

Alegre-Cebollada

et al. 2016

Journal of Biological Chemistry

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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.Actinoporins, single polypeptide chains of around 175 amino acids, constitute a family of toxic proteins produced by different sea anemone species. They show basic isoelectric point values and are usually cysteineless (1-4). Actinoporins belong to a much larger group of widely distributed proteins, known as pore-forming toxins, whose toxic activity relies on the formation of pores within biological membranes (5-9). All poreforming toxins show a very similar dual behavior by which they remain mostly monomeric and stably folded in aqueous solution but become oligomeric integral proteins when encountering membranes (2-4, 10 -23).The incorporation of actinoporins into the membrane largely depends on lipid bilayer composition and membrane physicochemical state (18, 24 -29). Both factors influence the conformational changes occurring during the transition from the water media to the inserted states of the protein (30, 31). Thus, high affinity recognition of sphingomyelin (SM) 3 is crucial for specific attachment to a membrane, but the subsequent effects observed also depend on the physical properties derived from its particular composition and not only from its SM content (23, 32). In fact, although still controversial, the presence of cholesterol and the coexistence of different phases in the membrane seem to be important factors, if not for binding then at least for the final formation of the pore (23,28,29,(32)(33)(34)(35)(36).Actinoporins have been isolated from more than 20 different sea anemone species (1, 3, 37-41) in agreement with their rather ubiquitous distribution within the Actinaria order (1). They display high sequence identities (between 60 and 80%) and appear as multigene families, ...

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“…Although numerous experiments support a critical role for sphingomyelin as the membrane attachment site for sea anemone antinoporins (Bakrač and Anderluh 2010), some reports suggested otherwise. For example, De los Ríos et al (1998) and Caaveiro et al (2001) tested the lytic action of StnII and EqtII with model membranes having different lipid compositions.…”

Section: Introductionmentioning

confidence: 99%

Identification of a target protein of Hydra actinoporin-like toxin-1 (HALT-1) using GST affinity purification and SILAC-based quantitative proteomics

Ameirika

Hong

Hwang

2017

Toxicon

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Hydra actinoporin-like toxin-1 (HALT-1) is a 20.8 kDa pore-forming toxin isolated from Hydra magnipapillata. HALT-1 shares structural similarity with actinoporins, a family that is well known for its haemolytic and cytolytic activity. However, the precise pore-forming mechanism of HALT-1 remains an open question since little is known about the specific target binding for HALT-1. For this reason, a comprehensive proteomic analysis was performed using affinity purification and SILAC-based mass spectrometry to identify potential protein-protein interactions between mammalian HeLa cell surface proteins and HALT-1. A total of 4 mammalian proteins was identified, of which only folate receptor alpha was further verified by ELISA. Our preliminary results highlight an alternative-binding mode of HALT-1 to the human plasma membrane. This is the first evidence showing that HALT-1, an actinoporin-like protein, binds to a membrane protein, the folate receptor alpha. This study would advance our understanding of the molecular basis of toxicity of pore-forming toxins and provide new insights in the production of more potent inhibitors for the toxin-membrane receptor interactions.3

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Molecular Mechanism of Sphingomyelin-Specific Membrane Binding and Pore Formation by Actinoporins

Cited by 50 publications

References 62 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

Synergistic Action of Actinoporin Isoforms from the Same Sea Anemone Species Assembled into Functionally Active Heteropores

Identification of a target protein of Hydra actinoporin-like toxin-1 (HALT-1) using GST affinity purification and SILAC-based quantitative proteomics

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