Binding of sea anemone pore-forming toxins sticholysins I and II to interfaces—Modulation of conformation and activity, and lipid–protein interaction

Álvarez, Carlos; Casallanovo, Fábio; Shida, Cláudio S.; Nogueira, L.; Martínez, Diana; Tejuca, Mayra; Pazos, Fabiola; Lanio, María E.; Menestrina, Gianfranco; Lissi, E. A.; Schreier, Shirley

doi:10.1016/s0009-3084(02)00181-0

Cited by 40 publications

(42 citation statements)

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“…Selective Trp fluorescence quenching by different agents, such hydrophilic neutral quencher (acrylamide) or lipid-confined phosphatidyltype quenchers [bis(9,10-dibromostearoyl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-(1-pyrenedecanoyl)-snglycero-3-phosphocholine] have been used to assess the exposure of the emitting centers to the solvent and/or to the lipid environment. Quenching studies together with binding isotherms offer complementary information on toxin binding to membranes (Macek et al 1995;Martinez et al 2001;Alvarez et al 2003).…”

Section: Spectroscopic Methods To Study the Binding Of Sts To Model Mmentioning

confidence: 99%

“…Usually, the protein or peptide CD spectra are acquired in solution and titrated with increasing amounts of vesicles. As in fluorescence studies, CD spectroscopy in the presence of vesicles should be carried out in translucent solutions to minimize light scattering; therefore, it is advisable to use homogeneous small vesicles (SUV; Alvarez et al 2003). Another approach to assess the conformational changes resulting from toxin binding to membranes is to use other less legitimized systems than liposomal vesicles, such as (1) the solvent trifluoroethanol (Casallanovo et al 2006), which is a medium that induces peptide conformational changes similar to those occurring in membranes, and (2) detergent micelles (Lanio et al 2002(Lanio et al , 2003(Lanio et al , 2007.…”

Section: Spectroscopic Methods To Study the Binding Of Sts To Model Mmentioning

confidence: 99%

“…The first step in this sequence of events is binding of the soluble monomers to the membrane. As this step does not involve significant protein conformational changes, the structure of the membrane-bound monomer is similar to that of the solution (Menestrina et al 1999;Alvarez et al 2003;Alegre-Cebollada et al 2007). Once the toxin binds to the membrane, the association of several monomers and the transfer of the amino-terminal region from the body of the protein to the hydrophobic core of the bilayer must occur.…”

Section: The Mechanism Of Pore Formation By Actinoporins In Membranesmentioning

confidence: 99%

“…This activity is strongly dependent on the lipid composition of the membrane favored by the presence of SM, cholesterol and non-bilayer-forming lipids (Alvarez-Valcarcel et al 2001;Alvarez et al 2003;Martinez et al 2007;Pedrera et al 2015). In turn, this activity modifies the membrane properties by promoting the mixing of the lipid phases and an increase in the order of the system; therefore, the interaction of Sts with lipids entails a remodeling of membrane domains that probably facilitates the action of the toxin (Ros et al 2013).…”

Section: The Pore Architecture Of Actinoporinsmentioning

confidence: 99%

“…Understanding the assembly mechanism of actinoporins has dramatically increased after determination of the crystal structure of FraC, which complemented a body of biochemical and biophysical data previously obtained by different groups (Tejuca et al 1996;Alvarez-Valcarcel et al 2001;Hong et al 2002;Alvarez et al 2003;Anderluh et al 2003;Rojko et al 2013;Antonini et al 2014;Subburaj et al 2015). However, the exact sequence of events that takes place during the assembly of actinoporins and the identification of the functionally relevant intermediates in the membrane remain under debate.…”

Section: Computational Insights Into the Mechanism Of Actinoporin Pormentioning

confidence: 99%

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

et al. 2017

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Actinoporins constitute a unique class of poreforming toxins found in sea anemones that are able to bind and oligomerize in membranes, leading to cell swelling, impairment of ionic gradients and, eventually, to cell death. In this review we summarize the knowledge generated from the combination of biochemical and biophysical approaches to the study of sticholysins I and II (Sts, StI/II), two actinoporins largely characterized by the Center of Protein Studies at the University of Havana during the last 20 years. These approaches include strategies for understanding the toxin structure-function relationship, the protein-membrane association process leading to pore formation and the interaction of toxin with cells. The rational combination of experimental and theoretical tools have allowed unraveling, at least partially, of the complex mechanisms involved in toxin-membrane interaction and of the molecular pathways triggered upon this interaction. The study of actinoporins is important not only to gain an understanding of their biological roles in anemone venom but also to investigate basic molecular mechanisms of protein insertion into membranes, protein-lipid interactions and the modulation of protein conformation by lipid binding. A deeper knowledge of the basic molecular mechanisms involved in Sts-cell interaction, as described in this review, will support the current investigations conducted by our group which focus on the design of immunotoxins against tumor cells and antigen-releasing systems to cell cytosol as Stsbased vaccine platforms.

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Section: Spectroscopic Methods To Study the Binding Of Sts To Model Mmentioning

confidence: 99%

Section: Spectroscopic Methods To Study the Binding Of Sts To Model Mmentioning

confidence: 99%

Section: The Mechanism Of Pore Formation By Actinoporins In Membranesmentioning

confidence: 99%

Section: The Pore Architecture Of Actinoporinsmentioning

confidence: 99%

Section: Computational Insights Into the Mechanism Of Actinoporin Pormentioning

confidence: 99%

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

et al. 2017

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Model peptides mimic the structure and function of the N‐terminus of the pore‐forming toxin sticholysin II

Casallanovo¹,

Oliveira²,

Souza³

et al. 2005

Biopolymers

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To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane environment, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behavior. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P1-30 was estimated by measuring the permeability to PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St II conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation.

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Functional and topological studies with Trp‐containing analogs of the peptide StII_1–30 derived from the N‐terminus of the pore forming toxin sticholysin II: contribution to understand its orientation in membrane

et al. 2013

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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.

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Binding of sea anemone pore-forming toxins sticholysins I and II to interfaces—Modulation of conformation and activity, and lipid–protein interaction

Cited by 40 publications

References 19 publications

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

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

Model peptides mimic the structure and function of the N‐terminus of the pore‐forming toxin sticholysin II

Functional and topological studies with Trp‐containing analogs of the peptide StII_1–30 derived from the N‐terminus of the pore forming toxin sticholysin II: contribution to understand its orientation in membrane

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Binding of sea anemone pore-forming toxins sticholysins I and II to interfaces—Modulation of conformation and activity, and lipid–protein interaction

Cited by 40 publications

References 19 publications

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

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

Model peptides mimic the structure and function of the N‐terminus of the pore‐forming toxin sticholysin II

Functional and topological studies with Trp‐containing analogs of the peptide StII1–30 derived from the N‐terminus of the pore forming toxin sticholysin II: contribution to understand its orientation in membrane

Contact Info

Product

Resources

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Functional and topological studies with Trp‐containing analogs of the peptide StII_1–30 derived from the N‐terminus of the pore forming toxin sticholysin II: contribution to understand its orientation in membrane