Biophysical and biochemical strategies to understand membrane binding and pore formation by sticholysins, pore-forming proteins from a sea anemone

Álvarez, Carlos; Ros, Uris; Valle, Aisel; Pedrera, Lohans; Soto, C.; Hervis, Yadira P.; Cabezas, Sheila; Valiente, Pedro A.; Pazos, Fabiola; Lanio, María E.

doi:10.1007/s12551-017-0316-0

Cited by 20 publications

(8 citation statements)

References 103 publications

(227 reference statements)

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“…The increase in surface pressure (Δπ) by the association of peptides with previously formed lipid monolayers at the air-water interface can be employed to characterize their ability to interact with organized lipids (17,48,61). This method has the advantage that it does not require fluorescent amino acids or probes to detect peptide-lipid interactions, thus complementing fluorescence based lipid binding assays (62).…”

Section: Iii4 Stii1-30 Activity In Membrane Systems Is Enhanced By Pementioning

confidence: 99%

Membrane remodeling by the lytic fragment of sticholysin II: implications for the toroidal pore model

Mesa-Galloso

Valiente

Epand

et al. 2019

Preprint

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Sticholysins are pore-forming toxins of biomedical interest and represent a prototype of proteins acting through the formation of protein-lipid or toroidal pores. Peptides spanning the N-terminus of sticholysins can mimic their permeabilizing activity and together with the full-length toxins have been used as a tool to understand the mechanism of pore formation in membranes. However, the lytic mechanism of these peptides and the lipid shape modulating their activity are not completely clear. In this paper, we combine molecular dynamics (MD) simulations and experimental biophysical tools to dissect different aspects of the pore-forming mechanism of StII1-30, a peptide derived from the N-terminus of sticholysin II. With this combined approach, membrane curvature induction and flip-flop movement of the lipids were identified as two important membrane remodeling steps mediated by StII1-30-pore forming activity.Pore-formation by this peptide was enhanced by the presence of the negatively-curved lipid phosphatidylethanolamine (PE) in membranes. This lipid emerged not only as a facilitator of membrane interactions but also as a structural element of the StII1-30-pore that is recruited to the pore ring upon its assembly. Collectively, these new findings support a toroidal model for the architecture of the pore formed by this peptide and provide new molecular insight into the role of PE as a membrane component that easily accommodates into the ring of toroidal pores aiding in its stabilization. This study contributes to a better understanding of the molecular mechanism underlying the permeabilizing activity of StII1-30 and peptides or proteins acting via a toroidal pore mechanism and offers an informative framework for the optimization of the biomedical application of this and similar molecules.

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Section: Iii4 Stii1-30 Activity In Membrane Systems Is Enhanced By Pementioning

confidence: 99%

Membrane remodeling by the lytic fragment of sticholysin II: implications for the toroidal pore model

Mesa-Galloso

Valiente

Epand

et al. 2019

Preprint

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“…The present study showed the identification of a pore-forming protein in the A. dowii venom. Among proteins previously described in sea anemones to have pore-forming capacity, actinoporins stand out owing to their proven ability to form pores in the cell membrane, which are directly responsible for osmotic imbalance and cell death [15,58]. The membrane-binding activity of actinoporins depends on the lipid composition, while a high concentration of sphingomyelin has been proven to facilitate pore formation [24,59,60].…”

Section: Discussionmentioning

confidence: 99%

Identification of a pore-forming protein from sea anemone Anthopleura dowii Verrill (1869) venom by mass spectrometry

Ramírez-Carreto

Pérez-García

Salazar-García

et al. 2019

J. Venom. Anim. Toxins incl. Trop. Dis

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Background: Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17. Methods: Cytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18. Results: The amount of protein at which the venom produced 50% hemolysis (HU 50 ) was determined in hemolysis assays using erythrocytes from sheep (HU 50 = 10.7 ± 0.2 μg), goat (HU 50 = 13.2 ± 0.3 μg), rabbit (HU 50 = 34.7 ± 0.5 μg), and human (HU 50 = 25.6 ± 0.6 μg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC 50 ) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 μg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: A. dowii Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.

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“…Among PFTs, APs have aroused the interest of the scientific community due to their biomedical or biotechnological potential to build immunotoxins [ 12 , 13 , 14 ], vaccine platforms [ 7 , 8 ], and nanopore-based biosensors [ 15 , 16 , 17 ]. Fragaceatoxin C (FraC) produced by Actinia fragacea [ 18 , 19 ], equinatoxin II (EqTII) from Actinia equina [ 20 , 21 ] and sticholysins (Sts), I (StI), and II (StII) purified from Stichodactyla helianthus [ 22 , 23 , 24 , 25 ] are the most extensively studied toxins in this family. APs exist as isoforms in most sea anemones which exhibit diverse pI, molecular weight, and piercing activity.…”

Section: Actinoporins Are Potent Toxins Produced By Sea Anemonesmentioning

confidence: 99%

Panorama of the Intracellular Molecular Concert Orchestrated by Actinoporins, Pore-Forming Toxins from Sea Anemones

Álvarez

Soto

Cabezas

et al. 2021

Toxins

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Actinoporins (APs) are soluble pore-forming proteins secreted by sea anemones that experience conformational changes originating in pores in the membranes that can lead to cell death. The processes involved in the binding and pore-formation of members of this protein family have been deeply examined in recent years; however, the intracellular responses to APs are only beginning to be understood. Unlike pore formers of bacterial origin, whose intracellular impact has been studied in more detail, currently, we only have knowledge of a few poorly integrated elements of the APs’ intracellular action. In this review, we present and discuss an updated landscape of the studies aimed at understanding the intracellular pathways triggered in response to APs attack with particular reference to sticholysin II, the most active isoform produced by the Caribbean Sea anemone Stichodactyla helianthus. To achieve this, we first describe the major alterations these cytolysins elicit on simpler cells, such as non-nucleated mammalian erythrocytes, and then onto more complex eukaryotic cells, including tumor cells. This understanding has provided the basis for the development of novel applications of sticholysins such as the construction of immunotoxins directed against undesirable cells, such as tumor cells, and the design of a cancer vaccine platform. These are among the most interesting potential uses for the members of this toxin family that have been carried out in our laboratory.

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Biophysical and biochemical strategies to understand membrane binding and pore formation by sticholysins, pore-forming proteins from a sea anemone

Cited by 20 publications

References 103 publications

Membrane remodeling by the lytic fragment of sticholysin II: implications for the toroidal pore model

Membrane remodeling by the lytic fragment of sticholysin II: implications for the toroidal pore model

Identification of a pore-forming protein from sea anemone Anthopleura dowii Verrill (1869) venom by mass spectrometry

Panorama of the Intracellular Molecular Concert Orchestrated by Actinoporins, Pore-Forming Toxins from Sea Anemones

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