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Balázs Lukács,
Mónika Sztretye,
János Almássy,
Sándor Sárközi,
Beatrix Dienes, Kamel Mabrouk,
Cecilia Simut,
László Szabó,
Péter Szentesi,
Michel De Waard,
Michel Ronjat,
István Jóna,
László Csernoch
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ABSTRACT: The 33 amino acid scorpion toxin maurocalcine (MCa) has been shown to modify the gating of the skeletal-type ryanodine receptor (RyR1). Here we explored the effects of MCa and its mutants ([Ala(8)]MCa, [Ala(19)]MCa, [Ala(20)]MCa, [Ala(22)]MCa, [Ala(23)]MCa, and [Ala(24)]MCa) on RyR1 incorporated into artificial lipid bilayers and on elementary calcium release events (ECRE) in rat and frog skeletal muscle fibers. The peptides induced long-lasting subconductance states (LLSS) on RyR1 that lasted for several seconds. However, their average length and frequency were decreased if the mutation was placed farther away in the 3D structure from the critical (24)Arg residue. The effect was strongly dependent on the direction of the current through the channel. If the direction was similar to that followed by calcium during release, the peptides were 8- to 10-fold less effective. In fibers long-lasting calcium release events were observed after the addition of the peptides. The average length of these events correlated well with the duration of LLSS. These data suggest that the effect of the peptide is governed by the large charged surface formed by residues Lys(20), Lys(22), Arg(23), Arg(24), and Lys(8). Our observations also indicate that the results from bilayer experiments mimic the in situ effects of MCa on RyR1.
Biophysical Journal 08/2008; 95(7):3497-509. · 3.65 Impact Factor
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ABSTRACT: Maurocalcine (MCa), initially identified from a Tunisian scorpion venom, defines a new member of the family of cell penetrating peptides by its ability to efficiently cross the plasma membrane. The initiating mechanistic step required for the cell translocation of a cell penetrating peptide implicates its binding onto cell surface components such as membrane lipids and/or heparan sulfate proteoglycans. Here we characterized the interaction of wild-type MCa and MCa K20A, a mutant analogue with reduced cell-penetration efficiency, with heparin (HP) and heparan sulfates (HS) through surface plasma resonance. HP and HS bind both to MCa, indicating that heparan sulfate proteoglycans may represent an important entry route of the peptide. This is confirmed by the fact that (i) both compounds bind with reduced affinity to MCa K20A and (ii) the cell penetration of wild-type or mutant MCa coupled to fluorescent streptavidin is reduced by about 50% in mutant Chinese hamster ovary cell lines lacking either all glycosaminoglycans (GAGs) or just HS. Incubating MCa with soluble HS, HP, or chondroitin sulfates also inhibits the cell penetration of MCa-streptavidin complexes. Analyses of the cell distributions of MCa/streptavidin in several Chinese hamster ovary cell lines show that the distribution of the complex coincides with the endosomal marker Lyso-Tracker red and is not affected by the absence of GAGs. The distribution of MCa/streptavidin is not coincident with that of transferrin receptors nor affected by a dominant-negative dynamin 2 K44A mutant, an inhibitor of clathrin-mediated endocytosis. However, entry of the complex is greatly diminished by amiloride, indicating the importance of macropinocytosis in MCa/streptavidin entry. It is concluded that (i) interaction of MCa with GAGs quantitatively improves the cell penetration of MCa, and (ii) GAG-dependent and -independent MCa penetration rely similarly on the macropinocytosis pathway.
Journal of Biological Chemistry 08/2008; 283(35):24274-84. · 4.77 Impact Factor
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ABSTRACT: Maurocalcine (MCa) is a 33-amino acid residue peptide that was initially identified in the Tunisian scorpion Scorpio maurus palmatus. This peptide triggers interest for three main reasons. First, it helps unravelling the mechanistic basis of Ca(2+) mobilization from the sarcoplasmic reticulum because of its sequence homology with a calcium channel domain involved in excitation-contraction coupling. Second, it shows potent pharmacological properties because of its ability to activate the ryanodine receptor. Finally, it is of technological value because of its ability to carry cell-impermeable compounds across the plasma membrane. Herein, we characterized the molecular determinants that underlie the pharmacological and cell-penetrating properties of maurocalcine. We identify several key amino acid residues of the peptide that will help the design of cell-penetrating analogues devoid of pharmacological activity and cell toxicity. Close examination of the determinants underlying cell penetration of maurocalcine reveals that basic amino acid residues are required for an interaction with negatively charged lipids of the plasma membrane. Maurocalcine analogues that penetrate better have also stronger interaction with negatively charged lipids. Conversely, less effective analogues present a diminished ability to interact with these lipids. These findings will also help the design of still more potent cell penetrating analogues of maurocalcine.
Biochimica et Biophysica Acta 11/2007; 1768(10):2528-40. · 4.66 Impact Factor
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ABSTRACT: We have previously shown that MCa (maurocalcine), a toxin from the venom of the scorpion Maurus palmatus, binds to RyR1 (type 1 ryanodine receptor) and induces strong modifications of its gating behaviour. In the present study, we investigated the ability of MCa to bind to and modify the gating process of cardiac RyR2. By performing pull-down experiments we show that MCa interacts directly with RyR2 with an apparent affinity of 150 nM. By expressing different domains of RyR2 in vitro, we show that MCa binds to two domains of RyR2, which are homologous with those previously identified on RyR1. The effect of MCa binding to RyR2 was then evaluated by three different approaches: (i) [(3)H]ryanodine binding experiments, showing a very weak effect of MCa (up to 1 muM), (ii) Ca(2+) release measurements from cardiac sarcoplasmic reticulum vesicles, showing that MCa up to 1 muM is unable to induce Ca(2+) release, and (iii) single-channel recordings, showing that MCa has no effect on the open probability or on the RyR2 channel conductance level. Long-lasting opening events of RyR2 were observed in the presence of MCa only when the ionic current direction was opposite to the physiological direction, i.e. from the cytoplasmic face of RyR2 to its luminal face. Therefore, despite the conserved MCa binding ability of RyR1 and RyR2, functional studies show that, in contrast with what is observed with RyR1, MCa does not affect the gating properties of RyR2. These results highlight a different role of the MCa-binding domains in the gating process of RyR1 and RyR2.
Biochemical Journal 10/2007; 406(2):309-15. · 4.90 Impact Factor
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Sylvie Boisseau, Kamel Mabrouk,
Narendra Ram,
Nicolas Garmy,
Véronique Collin,
Abir Tadmouri,
Mohamad Mikati,
Jean-Marc Sabatier,
Michel Ronjat,
Jacques Fantini,
Michel De Waard
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ABSTRACT: Maurocalcine (MCa) is a 33-amino acid residue peptide toxin initially isolated from the scorpion Scorpio maurus maurus. Its structural and functional features make it resembling many Cell Penetrating Peptides. In particular, MCa exhibits a characteristic positively charged face that may interact with membrane lipids. External application of MCa is known to produce Ca2+-release from intracellular stores within seconds. MCa binds directly to the skeletal muscle isoform of the ryanodine receptor, an intracellular channel target of the endoplasmic reticulum, and induces long-lasting channel openings in a mode of smaller conductance. The binding sites for MCa have been mapped within the cytoplasmic domain of the ryanodine receptor. In this manuscript, we further investigated how MCa proceeds to cross biological membranes in order to reach its target. A biotinylated derivative of MCa (MCab) was chemically synthesized, coupled to a fluorescent streptavidin indicator (Cy3 or Cy5) and the cell penetration of the entire complex followed by confocal microscopy and FACS analysis. The data provide evidence that MCa allows the penetration of the macro proteic complex and therefore may be used as a vector for the delivery of proteins in the cytoplasm as well as in the nucleus. Using both FACS and confocal analysis, we show that the cell penetration of the fluorescent complex is observed at concentrations as low as 10 nM, is sensitive to membrane potential and is partly inhibited by heparin. We also show that MCa interacts with the disialoganglioside GD3, the most abundant charged lipid in natural membranes. Despite its action on ryanodine receptor, MCa showed no sign of cell toxicity on HEK293 cells suggesting that it may have a wider application range. These data indicate that MCa may cross the plasma membrane directly by cell translocation and has a promising future as a carrier of various drugs and agents of therapeutic, diagnostic and technological value.
Biochimica et Biophysica Acta 04/2006; 1758(3):308-19. · 4.66 Impact Factor
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Eric Estève, Kamel Mabrouk,
Alain Dupuis,
Sophia Smida-Rezgui,
Xavier Altafaj,
Didier Grunwald,
Jean-Claude Platel,
Nicolas Andreotti,
Isabelle Marty,
Jean-Marc Sabatier,
Michel Ronjat,
Michel De Waard
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ABSTRACT: Maurocalcine (MCa) is a 33-amino-acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. External application of MCa to cultured myotubes is known to produce Ca2+ release from intracellular stores. MCa binds directly to the skeletal muscle isoform of the ryanodine receptor, an intracellular channel target of the endoplasmic reticulum, and induces long lasting channel openings in a mode of smaller conductance. Here we investigated the way MCa proceeds to cross biological membranes to reach its target. A biotinylated derivative of MCa was produced (MCa(b)) and complexed with a fluorescent indicator (streptavidine-cyanine 3) to follow the cell penetration of the toxin. The toxin complex efficiently penetrated into various cell types without requiring metabolic energy (low temperature) or implicating an endocytosis mechanism. MCa appeared to share the same features as the so-called cell-penetrating peptides. Our results provide evidence that MCa has the ability to act as a molecular carrier and to cross cell membranes in a rapid manner (1-2 min), making this toxin the first demonstrated example of a scorpion toxin that translocates into cells.
Journal of Biological Chemistry 05/2005; 280(13):12833-9. · 4.77 Impact Factor
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Eric Estève, Kamel Mabrouk,
Alain Dupuis,
Sophia Smida-Rezgui,
Xavier Altafaj,
Didier Grunwald,
Jean-Claude Platel,
Nicolas Andreotti,
Isabelle Marty,
Jean-Marc Sabatier,
Michel Ronjat,
Michel De Waard
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ABSTRACT: Maurocalcine (MCa) is a 33-amino-acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. External application of MCa to cultured myotubes is known to produce Ca2+ release from intracellular stores. MCa binds directly to the skeletal muscle isoform of the ryanodine receptor, an intracellular
channel target of the endoplasmic reticulum, and induces long lasting channel openings in a mode of smaller conductance. Here
we investigated the way MCa proceeds to cross biological membranes to reach its target. A biotinylated derivative of MCa was
produced (MCab) and complexed with a fluorescent indicator (streptavidine-cyanine 3) to follow the cell penetration of the toxin. The toxin
complex efficiently penetrated into various cell types without requiring metabolic energy (low temperature) or implicating
an endocytosis mechanism. MCa appeared to share the same features as the so-called cell-penetrating peptides. Our results
provide evidence that MCa has the ability to act as a molecular carrier and to cross cell membranes in a rapid manner (1–2
min), making this toxin the first demonstrated example of a scorpion toxin that translocates into cells.
Journal of Biological Chemistry 03/2005; 280(13):12833-12839. · 4.77 Impact Factor
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Besma Jouirou,
Amor Mosbah,
Violeta Visan,
Stephan Grissmer,
Sarrah M'Barek,
Ziad Fajloun,
Jurphaas Van Rietschoten,
Christiane Devaux,
Hervé Rochat,
Guy Lippens,
Mohamed El Ayeb,
Michel De Waard, Kamel Mabrouk,
Jean-Marc Sabatier
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ABSTRACT: CoTX1 (cobatoxin 1) is a 32-residue toxin with three disulphide bridges that has been isolated from the venom of the Mexican scorpion Centruroides noxius Hoffmann. Here we report the chemical synthesis, disulphide bridge organization, 3-D (three-dimensional) solution structure determination, pharmacology on K+ channel subtypes (voltage-gated and Ca2+-activated) and docking-simulation experiments. An enzyme-based cleavage of the synthetic folded/oxidized CoTX1 indicated half-cystine pairs between Cys3-Cys22, Cys8-Cys27 and Cys12-Cys29. The 3-D structure of CoTX1 (solved by 1H-NMR) showed that it folds according to the common alpha/beta scaffold of scorpion toxins. In vivo, CoTX1 was lethal after intracerebroventricular injection to mice (LD50 value of 0.5 microg/mouse). In vitro, CoTX1 tested on cells expressing various voltage-gated or Ca2+-activated (IKCa1) K+ channels showed potent inhibition of currents from rat K(v)1.2 ( K(d) value of 27 nM). CoTX1 also weakly competed with 125I-labelled apamin for binding to SKCa channels (small-conductance Ca2+-activated K+ channels) on rat brain synaptosomes (IC50 value of 7.2 microM). The 3-D structure of CoTX1 was used in docking experiments which suggests a key role of Arg6 or Lys10, Arg14, Arg18, Lys21 (dyad), Ile23, Asn24, Lys28 and Tyr30 (dyad) residues of CoTX1 in its interaction with the rat K(v)1.2 channel. In addition, a [Pro7,Gln9]-CoTX1 analogue (ACoTX1) was synthesized. The two residue replacements were selected aiming to restore the RPCQ motif in order to increase peptide affinity towards SKCa channels, and to alter the CoTX1 dipole moment such that it is expected to decrease peptide activity on K(v) channels. Unexpectedly, ACoTX1 exhibited an activity similar to that of CoTX1 towards SKCa channels, while it was markedly more potent on IKCa1 and several voltage-gated K+ channels.
Biochemical Journal 02/2004; 377(Pt 1):37-49. · 4.90 Impact Factor
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ABSTRACT: We have modelled the conformation of the III-IV loop of the Ca(v)2.1 subunit of P/Q calcium channels, a loop that is implicated in fast voltage-dependent inactivation. Change in channel inactivation requires its direct interaction with the I-II loop. This interaction occurs with an affinity in the order of 70 nm. Intracellular injection of a 40-mer III-IV loop-derived peptide produces an increase in the rate of fast inactivation. This alteration in channel kinetic is also accompanied by a hyperpolarizing shift in the steady-state voltage-dependence of inactivation. None of these effects are observed in the presence of a beta subunit, suggesting the existence of a competitive mechanism of action between the beta subunit and the III-IV loop. Amino acid sequence comparison using BLAST reveals that the III-IV loop shares 53% identity with the gamma subunit of G proteins. Because of the pivotal contribution of the III-IV loop to inactivation, an atomic model of the III-IV loop was generated by both homology modelling and molecular mechanics calculations. Using the X-ray structures of the betagamma dimer of the heterotrimeric G-proteins as templates, the III-IV loop is predicted to contain a well-structured alpha-helix at the amino-terminus with both the N- and C-termini having the same orientation in the plane of the inner lipid bilayer. We provide a hypothetical working model in which we propose that the III-IV loop interacts with the I-II loop via its Gbetagamma binding domain.
European Journal of Neuroscience 08/2002; 16(2):219-28. · 3.63 Impact Factor
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ABSTRACT: Because encapsulation of antiviral drugs in liposomes resulted generally in improved activity against retroviral replication in vivo, the antiviral effects of free-SPC3 and liposome-associated SPC3 were compared in cultured human lymphocytes infected with HIV-1. SPC3 was entrapped in various liposomal formulations, either different in size (mean diameter of 100 and 250 nm), SPC3 concentration or cholesterol content. Liposome-associated SPC3 were tested for both inhibition of cell-cell fusion and infection with HIV-1 clones. SPC3 inhibited HIV-1-induced fusion at a micromolar concentration range. When associated with liposomes, SPC3 was found to be about 10-fold more potent than free SPC3 in inhibiting syncytium formation. Continuous treatment with free SPC3 also inhibited virus production in a dose-dependent manner, with inhibition of HIV infection of C8166 T-cells or human peripheral blood lymphocytes (PBLs) at micromolar concentrations. Liposomal entrapment was found to increase the antiviral efficacy of SPC3 by more than 10- and 5-fold in C8166 and PBLs, respectively. These data suggest that the liposome approach may be used to improve SPC3 antiviral efficacy.
Antiviral Research 07/2002; 54(3):175-88. · 4.30 Impact Factor
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Sandrine Geib,
Guillaume Sandoz, Kamel Mabrouk,
Alessandra Matavel,
Pascale Marchot,
Toshinori Hoshi,
Michel Villaz,
Michel Ronjat,
Raymond Miquelis,
Christian Lévêque,
Michel de Waard
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ABSTRACT: Native high-voltage-gated calcium channels are multi-subunit complexes comprising a pore-forming subunit Ca(v) and at least two auxiliary subunits alpha(2)delta and beta. The beta subunit facilitates cell-surface expression of the channel and contributes significantly to its biophysical properties. In spite of its importance, detailed structural and functional studies are hampered by the limited availability of native beta subunit. Here, we report the purification of a recombinant calcium-channel beta(4) subunit from bacterial extracts by using a polyhistidine tag. The purified protein is fully functional since it binds on the alpha1 interaction domain, its main Ca(v)-binding site, and regulates the activity of P/Q calcium channel expressed in Xenopus oocytes in a similar way to the beta(4) subunit produced by cRNA injection. We took advantage of the functionality of the purified material to (i) develop an efficient surface-plasmon resonance assay of the interaction between two calcium channel subunits and (ii) measure, for the first time, the affinity of the recombinant His-beta(4) subunit for the full-length Ca(v)2.1 channel. The availability of this purified material and the development of a surface-plasmon resonance assay opens two immediate research perspectives: (i) drug screening programmes applied to the Ca(v)/beta interaction and (ii) crystallographic studies of the calcium-channel beta(4) subunit.
Biochemical Journal 06/2002; 364(Pt 1):285-92. · 4.90 Impact Factor
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ABSTRACT: We have investigated the molecular mechanisms whereby the I-II loop controls voltage-dependent inactivation in P/Q calcium channels. We demonstrate that the I-II loop is localized in a central position to control calcium channel activity through the interaction with several cytoplasmic sequences; including the III-IV loop. Several experiments reveal the crucial role of the interaction between the I-II loop and the III-IV loop in channel inactivation. First, point mutations of two amino acid residues of the I-II loop of Ca(v)2.1 (Arg-387 or Glu-388) facilitate voltage-dependent inactivation. Second, overexpression of the III-IV loop, or injection of a peptide derived from this loop, produces a similar inactivation behavior than the mutated channels. Third, the III-IV peptide has no effect on channels mutated in the I-II loop. Thus, both point mutations and overexpression of the III-IV loop appear to act similarly on inactivation, by competing off the native interaction between the I-II and the III-IV loops of Ca(v)2.1. As they are known to affect inactivation, we also analyzed the effects of beta subunits on these interactions. In experiments in which the beta(4) subunit is co-expressed, the III-IV peptide is no longer able to regulate channel inactivation. We conclude that (i) the contribution of the I-II loop to inactivation is partly mediated by an interaction with the III-IV loop and (ii) the beta subunits partially control inactivation by modifying this interaction. These data provide novel insights into the mechanisms whereby the beta subunit, the I-II loop, and the III-IV loop altogether can contribute to regulate inactivation in high voltage-activated calcium channels.
Journal of Biological Chemistry 04/2002; 277(12):10003-13. · 4.77 Impact Factor
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ABSTRACT: We have investigated the molecular mechanisms whereby the I-II loop controls voltage-dependent inactivation in P/Q calcium
channels. We demonstrate that the I-II loop is localized in a central position to control calcium channel activity through
the interaction with several cytoplasmic sequences; including the III-IV loop. Several experiments reveal the crucial role
of the interaction between the I-II loop and the III-IV loop in channel inactivation. First, point mutations of two amino
acid residues of the I-II loop of Cav2.1 (Arg-387 or Glu-388) facilitate voltage-dependent inactivation. Second, overexpression of the III-IV loop, or injection
of a peptide derived from this loop, produces a similar inactivation behavior than the mutated channels. Third, the III-IV
peptide has no effect on channels mutated in the I-II loop. Thus, both point mutations and overexpression of the III-IV loop
appear to act similarly on inactivation, by competing off the native interaction between the I-II and the III-IV loops of
Cav2.1. As they are known to affect inactivation, we also analyzed the effects of β subunits on these interactions. In experiments
in which the β4 subunit is co-expressed, the III-IV peptide is no longer able to regulate channel inactivation. We conclude that (i) the
contribution of the I-II loop to inactivation is partly mediated by an interaction with the III-IV loop and (ii) the β subunits
partially control inactivation by modifying this interaction. These data provide novel insights into the mechanisms whereby
the β subunit, the I-II loop, and the III-IV loop altogether can contribute to regulate inactivation in high voltage-activated
calcium channels.
Journal of Biological Chemistry 03/2002; 277(12):10003-10013. · 4.77 Impact Factor
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ABSTRACT: Pi1 is a 35-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the chactidae scorpion Pandinus imperator. Due to its very low abundance in the venom, we have chemically synthesized this toxin in order to study its biological activity. Enzyme-based proteolytic cleavage of the synthetic Pi1 (sPi1) demonstrates half-cystine pairings between Cys4–Cys25, Cys10–Cys30, Cys14–Cys32 and Cys20–Cys35, which is in agreement with the disulfide bridge organization initially reported on the natural toxin. In vivo, intracerebroventricular injection of sPi1 in mice produces lethal effects with an LD50 of 0.2 µg per mouse. In vitro, the application of sPi1 induces drastic inhibition of Shaker B (IC50 of 23 nm) and rat Kv1.2 channels (IC50 of 0.44 nm) heterologously expressed in Xenopus laevis oocytes. No effect was observed on rat Kv1.1 and Kv1.3 currents upon synthetic peptide application. Also, sPi1 is able to compete with 125I-labeled apamin for binding onto rat brain synaptosomes with an IC50 of 55 pm. Overall, these results demonstrate that sPi1 displays a large spectrum of activities by blocking both SK- and Kv1-types of K+ channels; a selectivity reminiscent of that of maurotoxin, another structurally related four disulfide-bridged scorpion toxin that exhibits a different half-cystine pairing pattern.
European Journal of Biochemistry. 12/2001; 267(16):5149 - 5155.
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Ziad Fajloun,
Gilles Ferrat,
Edmond Carlier,
Mohamed Fathallah,
Catherine Lecomte,
Guillaume Sandoz,
Eric di Luccio, Kamel Mabrouk,
Christian Legros,
Herve Darbon,
Herve Rochat,
Jean-Marc Sabatier,
Michel De Waard
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ABSTRACT: Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpionScorpio maurus palmatus. The toxin displays an exceptionally wide range of pharmacological activity since it binds onto small conductance Ca2+-activated K+channels and also blocks Kv channels (Shaker, Kv1.2 and Kv1.3). MTX possesses 53–68% sequence identity with HsTx1 and Pi1, two other K+ channel short chain scorpion toxins cross-linked by four disulfide bridges. These three toxins differ from other K+/Cl−/Na+ channel scorpion toxins cross-linked by either three or four disulfide bridges by the presence of an extra half-cystine residue
in the middle of a consensus sequence generally associated with the formation of an α/β scaffold (an α-helix connected to
an antiparallel β-sheet by two disulfide bridges). Because MTX exhibits an uncommon disulfide bridge organization among known
scorpion toxins (C1-C5, C2-C6, C3-C4, and C7-C8 instead of C1-C4, C2-C5, and C3-C6 for three-disulfide-bridged toxins or C1-C5,
C2-C6, C3-C7, and C4-C8 for four-disulfide-bridged toxins), we designed and chemically synthesized an MTX analog with three
instead of four disulfide bridges ([Abu19,Abu34]MTX) and in which the entire consensus motif of scorpion toxins was restored by the substitution of the two half-cystines
in positions 19 and 34 (corresponding to C4 and C8) by two isosteric α-aminobutyrate (Abu) derivatives. The three-dimensional
structure of [Abu19,Abu34]MTX in solution was solved by1H NMR. This analog adopts the α/β scaffold with now conventional half-cystine pairings connecting C1-C5, C2-C6, and C3-C7
(with C4 and C8 replaced by Abu derivatives). This novel arrangement in half-cystine pairings that concerns the last disulfide
bridge results mainly in a reorientation of the α-helix regarding the β-sheet structure. In vivo, [Abu19,Abu34]MTX remains lethal in mice as assessed by intracerebroventricular injection of the peptide (LD50 value of 0.25 μg/mouse). The structural variations are also accompanied by changes in the pharmacological selectivity of
the peptide, suggesting that the organization pattern of disulfide bridges should affect the three-dimensional presentation
of certain key residues critical to the blockage of K+ channel subtypes.
Journal of Biological Chemistry 05/2000; 275(18):13605-13612. · 4.77 Impact Factor
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ABSTRACT: The V3 loop is one of the variable domains of the HIV-1 surface envelope glycoprotein gp120 that mainly generates isolate-specific neutralizing antibodies. These anti-V3 antibodies presumably interfere with the function of the V3 loop that is thought to be involved in virus-cell fusion. In an approach to neutralize HIV infection, monomeric linear or cyclic V3 loop-related peptides have experimentally been used to impair the fusion process. The reported results are contradictory, including peptide-induced enhancement of viral infectivity. In order to develop a new strategy, synthetic polymeric constructions (SPCs) including the V3 loop consensus sequence of HIV-1 North American/European isolates (i.e. GPGRAF) have been synthesized and tested for antiviral activity. The rationale for using SPCs instead of monomeric peptides was a presumed enhancement of ligand avidity due to multivalence. Among the SPCs tested, SPC3 (eight GPGRAF motifs radially branched on an uncharged polylysine core matrix) was found to inhibit the infection of human CD4+ lymphocytes and macrophages, as well as CD4–/galactosylceramide (GalCer)-expressing epithelial cells by distantly related laboratory strains and clinical isolates of HIV-1 and HIV-2. SPC3 affected HIV-1 infection by two distinct mechanisms, depending on the cell-type: (i) postbinding inhibition of HIV-1 entry into CD4+ lymphocytes; and (ii) prevention of GalCer-mediated HIV-1 attachment to the surface of CD4–/GalCer+ cells. SPC3 may therefore represent the first of a novel class of anti-HIV therapeutic agents able to neutralize a wide range of HIV isolates in both CD4+ and CD4– susceptible cells. The antiviral properties of this peptide are currently being evaluated in HIV-1-infected patients (phase II clinical trials).
Perspectives in Drug Discovery and Design 11/1996; 5(1):243-250.
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ABSTRACT: We have recently reported a basic domain-mediated neurotoxic activity of HIV-1 Tat [1991, J. Virol. 65, 961–965]. Here we have tested the neurotoxicity in vivo of several Rev-related synthetic peptides and found that only those mimicking the basic regions of Rev from HIV-1, HIV-2 and SIV were lethal to mice. In contrast, the homologous domain of HTLV-1 Rex was found to be inactive for lethal activity. Analysis of the tropism of these peptides for phospholipids has demonstrated a direct interaction of the basic domain-containing peptides, except Rex, with acidic — but not neutral — phospholipids. As determined by circular dichroism, a possible correlation between the conformation of the basic regions and the toxicity is discussed.
FEBS Letters.
