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1.
Eur J Pharmacol ; 794: 162-172, 2017 Jan 05.
Article in English | MEDLINE | ID: mdl-27743884

ABSTRACT

The NADPH oxidase proteins catalyse the formation of superoxide anion which act as signalling molecules in physiological and pathological processes. Nox1-dependent NADPH oxidase is expressed in heart, lung, colon, blood vessels and brain. Different strategies involving Nox1 inhibition based on diphenylene iodonium derivatives are currently tested for colorectal cancer therapy. Here, after peptides screening on Nox1-dependent NADPH oxidase assay in HT-29 cells, we identify a peptide (referred to as NF02), cell-active, that potently block Nox1-dependent reactive oxygen species generation. Study of DEPMPO adduct formation by electron paramagnetic resonance showed that NF02 has no superoxide scavenging activity and no impact on cellular reactive oxygen species-producing enzymes such xanthine oxidase. NF02 was not cytotoxic, inhibited reactive oxygen species production of reconstituted Nox1/Noxo1/Noxa1 complex in HEK293 and did not decrease Nox2 dependent cellular NADPH oxidase reactive oxygen species production. Finally, NF02 inhibited cell migration and invasion of colorectal cancer cells which is consistent with the described impact of Nox1 inhibitors on cell migration. NF02 peptide is a new NADPH oxidase inhibitor specific for Nox1 over Nox2 and xanthine oxidase which might represent a useful Nox1 tool with potential therapeutic insights.


Subject(s)
Cell Movement/drug effects , Enzyme Inhibitors/pharmacology , NADPH Oxidases/antagonists & inhibitors , Oligopeptides/pharmacology , Amino Acid Sequence , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Humans , NADPH Oxidase 1 , NADPH Oxidases/metabolism , Neoplasm Invasiveness , Oligopeptides/chemistry
2.
PLoS One ; 10(5): e0127264, 2015.
Article in English | MEDLINE | ID: mdl-25938421

ABSTRACT

Small-conductance, Ca2+ activated K+ channels (SK channels) are expressed at high levels in brain regions responsible for learning and memory. In the current study we characterized the contribution of SK2 channels to synaptic plasticity and to different phases of hippocampal memory formation. Selective SK2 antisense-treatment facilitated basal synaptic transmission and theta-burst induced LTP in hippocampal brain slices. Using the selective SK2 antagonist Lei-Dab7 or SK2 antisense probes, we found that hippocampal SK2 channels are critical during two different time windows: 1) blockade of SK2 channels before the training impaired fear memory, whereas, 2) blockade of SK2 channels immediately after the training enhanced contextual fear memory. We provided the evidence that the post-training cleavage of the SK2 channels was responsible for the observed bidirectional effect of SK2 channel blockade on memory consolidation. Thus, Lei-Dab7-injection before training impaired the C-terminal cleavage of SK2 channels, while Lei-Dab7 given immediately after training facilitated the C-terminal cleavage. Application of the synthetic peptide comprising a leucine-zipper domain of the C-terminal fragment to Jurkat cells impaired SK2 channel-mediated currents, indicating that the endogenously cleaved fragment might exert its effects on memory formation by blocking SK2 channel-mediated currents. Our present findings suggest that SK2 channel proteins contribute to synaptic plasticity and memory not only as ion channels but also by additionally generating a SK2 C-terminal fragment, involved in both processes. The modulation of fear memory by down-regulating SK2 C-terminal cleavage might have applicability in the treatment of anxiety disorders in which fear conditioning is enhanced.


Subject(s)
Fear/physiology , Hippocampus/metabolism , Memory/physiology , Small-Conductance Calcium-Activated Potassium Channels/metabolism , Animals , Long-Term Potentiation/physiology , Male , Mice , Mice, Inbred C57BL
4.
Infect Disord Drug Targets ; 12(4): 316-25, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22697129

ABSTRACT

A new bacteriocin, lacticin LC14, produced by Lactococcus lactis BMG6.14, was isolated and characterized. It was purified to homogeneity from overnight broth culture by ammonium sulfate precipitation, Sep-Pak chromatography, and two steps of reversed-phase HPLC. Lacticin LC14 showed bactericidal-type antimicrobial activity against several lactic acid bacteria and pathogenic strains including Listeria monocytogenes. It was inactivated by proteinase K and pronase E, but was resistant to papain, lysozyme, lipase and catalase. Lacticin LC14 was heat resistant, stable over a wide range of pH (2-10) and after treatment by solvents and detergents. Its N-terminal end was found unreactive towards Edman sequencing. Based on MALDI-TOF mass spectrometry, its molecular mass was 3333.7 Da. LC14 amino acid composition revealed a high proportion of hydrophobic residues, but no modified ones. LC14 may be able to challenge other well known other bacteriocins in probiotic and therapeutic applications.


Subject(s)
Bacteriocins/isolation & purification , Lactococcus lactis/metabolism , Amino Acid Sequence , Amino Acids/analysis , Bacteriocins/analysis , Bacteriocins/chemistry , Bacteriocins/pharmacology , Lactococcus lactis/isolation & purification
5.
J Biol Chem ; 287(21): 17331-17342, 2012 May 18.
Article in English | MEDLINE | ID: mdl-22433862

ABSTRACT

Maurocalcine is the first demonstrated example of an animal toxin peptide with efficient cell penetration properties. Although it is a highly competitive cell-penetrating peptide (CPP), its relatively large size of 33 amino acids and the presence of three internal disulfide bridges may hamper its development for in vitro and in vivo applications. Here, we demonstrate that several efficient CPPs can be derived from maurocalcine by replacing Cys residues by isosteric 2-aminobutyric acid residues and sequence truncation down to peptides of up to 9 residues in length. A surprising finding is that all of the truncated maurocalcine analogues possessed cell penetration properties, indicating that the maurocalcine is a highly specialized CPP. Careful examination of the cell penetration properties of the truncated analogues indicates that several maurocalcine-derived peptides should be of great interest for cell delivery applications where peptide size matters.


Subject(s)
Cell-Penetrating Peptides/pharmacology , Scorpion Venoms/pharmacology , Animals , CHO Cells , Cell-Penetrating Peptides/chemical synthesis , Cell-Penetrating Peptides/chemistry , Cricetinae , Cricetulus , Scorpion Venoms/chemical synthesis , Scorpion Venoms/chemistry
6.
PLoS One ; 6(8): e22386, 2011.
Article in English | MEDLINE | ID: mdl-21857926

ABSTRACT

Respiratory distress syndrome is responsible for 40 to 60 percent mortality. An over mortality of about 10 percent could result from additional lung injury and inflammation due to the life-support mechanical ventilation, which stretches the lung. It has been recently demonstrated, in vitro, that pharmacological activation of the alpha 7 nicotinic receptors (α7-nAChR) could down regulate intracellular mediators involved in lung cell inflammatory response to stretch. Our aim was to test in vivo the protective effect of the pharmacological activation of the α7-nAChR against ventilator-induced lung injury (VILI). Anesthetized rats were ventilated for two hours with a high stretch ventilation mode delivering a stroke volume large enough to generate 25-cmH(2)O airway pressure, and randomly assigned to four groups: pretreated with parenteral injection of saline or specific agonist of the α7-nAChR (PNU-282987), or submitted to bilateral vagus nerve electrostimulation while pre-treated or not with the α7-nAChR antagonist methyllycaconitine (MLA). Controls ventilated with a conventional stroke volume of 10 mL/kg gave reference data. Physiological indices (compliance of the respiratory system, lung weight, blood oxygenation, arterial blood pressure) and lung contents of inflammatory mediators (IL-6 measured by ELISA, substance P assessed using HPLC) were severely impaired after two hours of high stretch ventilation (sham group). Vagal stimulation was able to maintain the respiratory parameters close to those obtained in Controls and reduced lung inflammation except when associated to nicotinic receptor blockade (MLA), suggesting the involvement of α7-nAChR in vagally-mediated protection against VILI. Pharmacological pre-treatment with PNU-282987 strongly decreased lung injury and lung IL-6 and substance P contents, and nearly abolished the increase in plasmatic IL-6 levels. Pathological examination of the lungs confirmed the physiological differences observed between the groups. In conclusion, these data suggest that the stimulation of α7-nAChR is able to attenuate VILI in rats.


Subject(s)
Benzamides/pharmacology , Bridged Bicyclo Compounds/pharmacology , Receptors, Nicotinic/metabolism , Ventilator-Induced Lung Injury/prevention & control , Aconitine/analogs & derivatives , Aconitine/pharmacology , Anesthesia , Animals , Enzyme-Linked Immunosorbent Assay , Interleukin-6/blood , Interleukin-6/metabolism , Lung/drug effects , Lung/metabolism , Lung/physiopathology , Male , Nicotinic Agonists/pharmacology , Nicotinic Antagonists/pharmacology , Random Allocation , Rats , Rats, Sprague-Dawley , Substance P/metabolism , Ventilator-Induced Lung Injury/metabolism , Ventilator-Induced Lung Injury/physiopathology , alpha7 Nicotinic Acetylcholine Receptor
7.
J Pept Sci ; 17(3): 200-10, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21308876

ABSTRACT

Maurotoxin (MTX) is a 34-residue toxin that was isolated initially from the venom of the scorpion Scorpio maurus palmatus. Unlike the other toxins of the α-KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C(1) C(5) , C(2) C(6) , C(3) C(4) , and C(7) C(8) (instead of the conventional C(1) C(5) , C(2) C(6) , C(3) C(7) , and C(4) C(8) , herein referred to as Pi1-like) that does not prevent its folding along the classic α/ß scaffold of scorpion toxins. MTX(Pi1) is an MTX variant with a conventional pattern of disulfide bridging without any primary structure alteration of the toxin. Here, using MTX and/or MTX(Pi1) as models, we investigated how the type of folding influences toxin recognition of the Shaker B potassium channel. Amino acid residues of MTX that were studied for Shaker B recognition were selected on the basis of their homologous position in charybdotoxin, a three disulfide-bridged scorpion toxin also active on this channel type. These residues favored either an MTX- or MTX(Pi1) -like folding. Our data indicate clearly that Lys(23) and Tyr(32) (two out of ten amino acid residues studied) are the most important residues for Shaker B channel blockage by MTX. For activity on SKCa channels, the same amino acid residues also affect, directly or indirectly, the recognition of SK channels. The molecular modeling technique and computed docking indicate the existence of a correlation between the half cystine pairings of the mutated analogs and their activity on the Shaker B K(+) channel. Overall, mutations in MTX could, or could not, change the reorganization of disulfide bridges of this molecule without affecting its α/ß scaffold. However, changing of the peptide backbone (cross linking disulfide bridges from MTX-like type vs MTX(Pi1) -like type) appears to have less impact on the molecule activity than mutation of certain key amino acids such as Lys(23) and Tyr(32) in this toxin.


Subject(s)
Potassium Channels, Voltage-Gated/chemistry , Potassium Channels, Voltage-Gated/metabolism , Scorpion Venoms/chemistry , Scorpion Venoms/metabolism , Animals , Electrophysiology , In Vitro Techniques , Point Mutation , Potassium Channels, Voltage-Gated/genetics , Protein Binding , Protein Conformation , Rats , Scorpion Venoms/genetics , Synaptosomes/metabolism
8.
J Physiol ; 588(Pt 8): 1309-19, 2010 Apr 15.
Article in English | MEDLINE | ID: mdl-20176632

ABSTRACT

Overdistension of lung tissue during mechanical ventilation causes cytokine release, which may be facilitated by the autonomic nervous system. We used mechanical ventilation to cause lung injury in rats, and studied how cervical section of the vagus nerve, or substance P (SP) antagonism, affected the injury. The effects of 40 or 25 cmH(2)O high airway pressure injurious ventilation (HV(40) and HV(25)) were studied and compared with low airway pressure ventilation (LV) and spontaneous breathing (controls). Lung mechanics, lung weight, gas exchange, lung myeloperoxidase activity, lung concentrations of interleukin (IL)-1 beta and IL-6, and amounts of lung SP were measured. Control rats were intact, others were bivagotomized, and in some animals we administered the neurokinin-1 (NK-1) receptor blocking agent SR140333. We first determined the durations of HV(40) and HV(25) that induced the same levels of lung injury and increased lung contents of IL-1 beta and IL-6. They were 90 min and 120 min, respectively. Both HV(40) and HV(25) increased lung SP, IL-1 beta and IL-6 levels, these effects being markedly reduced by NK-1 receptor blockade. Bivagotomy reduced to a lesser extent the HV(40)- and HV(25)-induced increases in SP but significantly reduced cytokine production. Neither vagotomy nor NK-1 receptor blockade prevented HV(40)-induced lung injury but, in the HV(25) group, they made it possible to maintain lung injury indices close to those measured in the LV group. This study suggests that both neuronal and extra-neuronal SP might be involved in ventilator-induced lung inflammation and injury. NK-1 receptor blockade could be a pharmacological tool to minimize some adverse effects of mechanical ventilation.


Subject(s)
Cytokines/metabolism , Lung Injury/prevention & control , Lung/metabolism , Neurokinin-1 Receptor Antagonists , Animals , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Lung Injury/metabolism , Models, Animal , Piperidines/pharmacology , Quinuclidines/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Neurokinin-1/drug effects , Receptors, Neurokinin-1/metabolism , Respiration, Artificial/adverse effects , Vagotomy , Vagus Nerve/physiology
9.
Curr Pharm Des ; 14(24): 2503-18, 2008.
Article in English | MEDLINE | ID: mdl-18781998

ABSTRACT

Animal venoms are rich natural sources of bioactive compounds, including peptide toxins acting on the various types of ion channels, i.e. K(+), Na(+), Cl(-) and Ca(2+). Among K+ channel-acting toxins, those selective for voltage-gated K(+) (Kv) channels are widely represented and have been isolated from the venoms of numerous animal species, such as scorpions, sea anemones, snakes, marine cone snails and spiders. The toxins characterized hitherto contain between 22 and 60 amino acid residues, and are cross-linked by two to four disulfide bridges. Depending on their types of fold, toxins can be classified in eight structural categories, which showed a combination of beta-strands, helices, or a mixture of both. The main architectural motifs thereof are referred to as alpha/beta scaffold and inhibitor cystine knot (ICK). A detailed analysis of toxin structures and pharmacological selectivities indicates that toxins exhibiting a similar type of fold can exert their action on several subtypes of Kv channels, whereas a particular Kv channel can be targeted by toxins that possess unrelated folds. Therefore, it appears that the ability of structurally divergent toxins to interact with a particular Kv channel relies onto a similar spatial distribution of amino acid residues that are key to the toxin-channel interaction (rather than the type of toxin fold). The diversity of Kv channel blockers and their therapeutic value in the potential treatment of a number of specific human diseases, especially autoimmune disorders, inflammatory neuropathies and cancer, are reviewed.


Subject(s)
Drug Design , Peptides/pharmacology , Potassium Channel Blockers/pharmacology , Potassium Channels, Voltage-Gated/metabolism , Venoms/analysis , Amino Acid Sequence , Animals , Humans , Models, Molecular , Molecular Sequence Data , Peptides/isolation & purification , Peptides/therapeutic use , Potassium Channel Blockers/isolation & purification , Potassium Channel Blockers/therapeutic use , Potassium Channels, Voltage-Gated/genetics , Potassium Channels, Voltage-Gated/physiology , Protein Conformation , Sequence Alignment
10.
J Biol Chem ; 283(40): 27048-56, 2008 Oct 03.
Article in English | MEDLINE | ID: mdl-18621738

ABSTRACT

Maurocalcine is a 33-mer peptide initially isolated from the venom of a Tunisian scorpion. It has proved itself valuable as a pharmacological activator of the ryanodine receptor and has helped the understanding of the molecular basis underlying excitation-contraction coupling in skeletal muscles. Because of its positively charged nature, it is also an innovative vector for the cell penetration of various compounds. We report a novel maurocalcine analog with improved properties: (i) the complete loss of pharmacological activity, (ii) preservation of the potent ability to carry cargo molecules into cells, and (iii) coupling chemistries not affected by the presence of internal cysteine residues of maurocalcine. We did this by replacing the six internal cysteine residues of maurocalcine by isosteric 2-aminobutyric acid residues and by adding an additional N-terminal biotinylated lysine (for a proof of concept analog) or an N-terminal cysteine residue (for a chemically competent coupling analogue). Additional replacement of a glutamate residue by alanyl at position 12 further improves the potency of these analogues. Coupling to several cargo molecules or nanoparticles are presented to illustrate the cell penetration potency and usefulness of these pharmacologically inactive analogs.


Subject(s)
Drug Carriers/pharmacology , Scorpion Venoms/pharmacology , Animals , CHO Cells , Cricetinae , Cricetulus , Disulfides/chemistry , Drug Carriers/chemical synthesis , Drug Carriers/chemistry , Humans , Mice , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Scorpion Venoms/chemical synthesis , Scorpion Venoms/genetics , Scorpions/genetics , Scorpions/metabolism
11.
J Antibiot (Tokyo) ; 61(2): 89-93, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18408329

ABSTRACT

Bacteriocin J46 is a 27-residue polypeptide produced by Lactococcus lactis subsp. cremoris J46 in fermented milk. The natural form of J46 (nJ46) exhibits a broad antimicrobial spectrum. Herein, we produced the synthetic form of J46 (sJ46) by solid-phase chemical synthesis. The biochemical and physico-chemical properties of sJ46, as well as its antimicrobial activity, were found to be identical to those of its natural counterpart nJ46. It showed a potent antimicrobial activity against both lactic acid bacteria and other Gram-positive microorganisms. (1)H-NMR conformational analysis of sJ46 indicates that it adopts a flexible random coil structure.


Subject(s)
Anti-Bacterial Agents/chemical synthesis , Bacteriocins/chemical synthesis , Lactococcus lactis/chemistry , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacteriocins/chemistry , Bacteriocins/pharmacology , Microbial Sensitivity Tests , Milk/microbiology , Molecular Conformation
12.
Proteins ; 61(4): 1010-23, 2005 Dec 01.
Article in English | MEDLINE | ID: mdl-16247791

ABSTRACT

Animal toxins are highly reticulated and structured polypeptides that adopt a limited number of folds. In scorpion species, the most represented fold is the alpha/beta scaffold in which an helical structure is connected to an antiparallel beta-sheet by two disulfide bridges. The intimate relationship existing between peptide reticulation and folding remains poorly understood. Here, we investigated the role of disulfide bridging on the 3D structure of HsTx1, a scorpion toxin potently active on Kv1.1 and Kv1.3 channels. This toxin folds along the classical alpha/beta scaffold but belongs to a unique family of short-chain, four disulfide-bridged toxins. Removal of the fourth disulfide bridge of HsTx1 does not affect its helical structure, whereas its two-stranded beta-sheet is altered from a twisted to a nontwisted configuration. This structural change in HsTx1 is accompanied by a marked decrease in Kv1.1 and Kv1.3 current blockage, and by alterations in the toxin to channel molecular contacts. In contrast, a similar removal of the fourth disulfide bridge of Pi1, another scorpion toxin from the same structural family, has no impact on its 3D structure, pharmacology, or channel interaction. These data highlight the importance of disulfide bridging in reaching the correct bioactive conformation of some toxins.


Subject(s)
Disulfides/analysis , Scorpion Venoms/chemistry , Amino Acid Sequence , Animals , Magnetic Resonance Spectroscopy , Mice , Models, Molecular , Peptides/chemistry , Protein Conformation , Protein Structure, Secondary , Scorpion Venoms/toxicity , Scorpions
13.
Peptides ; 26(7): 1095-108, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15949626

ABSTRACT

The small-conductance Ca2+-activated K+ (SKCa) channels modulate cytosolic Ca2+ concentration in excitable and non-excitable tissues by regulating the membrane potential and are responsible of slow action potential after hyperpolarization that inhibits cell firing. Among these, human SKCa2 and SKCa3 channels differ in the pore region by only two residues: Ala331 and Asn367 (human small-conductance calcium-activated potassium channel, hSKCa2) instead of Val485 and His521 (hSKCa3). To design highly selective blockers of hSKCa channels, a number of known hSKCa2 and/or hSKCa3-active peptides (i.e. scorpion toxins and analogs thereof) were analyzed for their interactions and selectivities toward these channels. Molecular models of hSKCa2 and hSKCa3 channels (S5-H5-S6 portion) were generated, and scorpion toxins/peptides of unsolved three-dimensional (3D) structures were modeled. Models of toxin-channel complexes were generated by the bimolecular complex generation with global evaluation, and ranking (BiGGER) docking software and selected by using a screening method of the docking solutions. A high degree of correlation was found to exist between docking energies and experimental Kd values of peptides that blocked hSKCa2 and/or hSKCa3 channels, suggesting it could be appropriate to predict Kd values of other bioactive peptides. The best scoring complexes were also used to identify key residues of both interacting partners, indicating that such an approach should help the design of more active and/or selective peptide blockers of targeted ion channels.


Subject(s)
Models, Molecular , Neuropeptides/chemistry , Potassium Channels, Calcium-Activated/chemistry , Potassium Channels/chemistry , Scorpion Venoms/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Computer Simulation , Humans , Molecular Sequence Data , Neuropeptides/antagonists & inhibitors , Peptides/chemistry , Potassium Channels, Calcium-Activated/antagonists & inhibitors , Protein Conformation , Small-Conductance Calcium-Activated Potassium Channels
14.
Proteins ; 60(3): 401-11, 2005 Aug 15.
Article in English | MEDLINE | ID: mdl-15971207

ABSTRACT

Scorpion toxins interact with their target ion channels through multiple molecular contacts. Because a "gain of function" approach has never been described to evaluate the importance of the molecular contacts in defining toxin affinity, we experimentally examined whether increasing the molecular contacts between a toxin and an ion channel directly impacts toxin affinity. For this purpose, we focused on two scorpion peptides, the well-characterized maurotoxin with its variant Pi1-like disulfide bridging (MTX(Pi1)), used as a molecular template, and butantoxin (BuTX), used as an N-terminal domain provider. BuTX is found to be 60-fold less potent than MTX(Pi1) in blocking Kv1.2 (IC(50) values of 165 nM for BuTX versus 2.8 nM for MTX(Pi1)). Removal of its N-terminal domain (nine residues) further decreases BuTX affinity for Kv1.2 by 5.6-fold, which is in agreement with docking simulation data showing the importance of this domain in BuTX-Kv1.2 interaction. Transfer of the BuTX N-terminal domain to MTX(Pi1) results in a chimera with five disulfide bridges (BuTX-MTX(Pi1)) that exhibits 22-fold greater affinity for Kv1.2 than MTX(Pi1) itself, in spite of the lower affinity of BuTX as compared to MTX(Pi1). Docking experiments performed with the 3-D structure of BuTX-MTX(Pi1) in solution, as solved by (1)H-NMR, reveal that the N-terminal domain of BuTX participates in the increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicate that acting on molecular contacts between a toxin and a channel is an efficient strategy to modulate toxin affinity.


Subject(s)
Computational Biology/methods , Kv1.2 Potassium Channel/chemistry , Potassium Channels, Voltage-Gated/chemistry , Proteomics/methods , Scorpion Venoms/chemistry , Amino Acid Sequence , Animals , Circular Dichroism , Cysteine/chemistry , Disulfides/chemistry , Electrophysiology , Inhibitory Concentration 50 , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Peptides/chemistry , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Scorpions , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trifluoroacetic Acid/chemistry
15.
J Biol Chem ; 280(13): 12833-9, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15653689

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.


Subject(s)
Cell Membrane/drug effects , Cell Membrane/metabolism , Scorpion Venoms/metabolism , Amino Acid Sequence , Biological Transport , Biotinylation , Calcium/chemistry , Carrier Proteins/chemistry , Cell Differentiation , Cell Line , Cell-Penetrating Peptides , Endocytosis , Endoplasmic Reticulum/metabolism , Gene Products, tat/metabolism , Humans , Kinetics , Microscopy, Confocal , Models, Molecular , Molecular Sequence Data , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Peptides/chemistry , Protein Conformation , Protein Isoforms , Protein Transport , Ryanodine/metabolism , Ryanodine Receptor Calcium Release Channel/chemistry , Sarcoplasmic Reticulum/metabolism , Scorpion Venoms/pharmacokinetics , Signal Transduction , Temperature , Time Factors
16.
Biochem J ; 385(Pt 1): 95-104, 2005 Jan 01.
Article in English | MEDLINE | ID: mdl-15588251

ABSTRACT

OSK1 (alpha-KTx3.7) is a 38-residue toxin cross-linked by three disulphide bridges that was initially isolated from the venom of the Asian scorpion Orthochirus scrobiculosus. OSK1 and several structural analogues were produced by solid-phase chemical synthesis, and were tested for lethality in mice and for their efficacy in blocking a series of 14 voltage-gated and Ca2+-activated K+ channels in vitro. In the present paper, we report that OSK1 is lethal in mice by intracerebroventricular injection, with a LD50 (50% lethal dose) value of 2 microg/kg. OSK1 blocks K(v)1.1, K(v)1.2, K(v)1.3 channels potently and K(Ca)3.1 channel moderately, with IC50 values of 0.6, 5.4, 0.014 and 225 nM respectively. Structural analogues of OSK1, in which we mutated positions 16 (Glu16-->Lys) and/or 20 (Lys20-->Asp) to amino acid residues that are conserved in all other members of the alpha-KTx3 toxin family except OSK1, were also produced and tested. Among the OSK1 analogues, [K16,D20]-OSK1 (OSK1 with Glu16-->Lys and Lys20-->Asp mutations) shows an increased potency on K(v)1.3 channel, with an IC50 value of 0.003 nM, without loss of activity on K(Ca)3.1 channel. These data suggest that OSK1 or [K16,D20]-OSK1 could serve as leads for the design and production of new immunosuppressive drugs.


Subject(s)
Potassium Channel Blockers/pharmacology , Potassium Channels/metabolism , Scorpion Venoms/chemical synthesis , Scorpion Venoms/pharmacology , Scorpions/chemistry , Toxins, Biological/pharmacology , Amino Acid Sequence , Animals , Cell Line, Tumor , Humans , Inhibitory Concentration 50 , Injections, Intraventricular , Lethal Dose 50 , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Potassium Channel Blockers/chemical synthesis , Potassium Channel Blockers/chemistry , Potassium Channel Blockers/toxicity , Scorpion Venoms/chemistry , Scorpion Venoms/toxicity , Toxins, Biological/chemical synthesis , Toxins, Biological/chemistry , Toxins, Biological/toxicity
17.
J Biol Chem ; 279(53): 55690-6, 2004 Dec 31.
Article in English | MEDLINE | ID: mdl-15498765

ABSTRACT

Maurotoxin (MTX) and HsTx1 are two scorpion toxins belonging to the alpha-KTx6 structural family. These 34-residue toxins, cross-linked by four disulfide bridges, share 59% sequence identity and fold along the classical alpha/beta scaffold. Despite these structural similarities, they fully differ in their pharmacological profiles. MTX is highly active on small (SK) and intermediate (IK) conductance Ca(2+)-activated (K(+)) channels and on voltage-gated Kv1.2 channel, whereas HsTx1 potently blocks voltage-gated Kv1.1 and Kv1.3 channels only. Here, we designed and chemically produced MTX-HsTx1, a chimera of both toxins that contains the N-terminal helical region of MTX (sequence 1-16) and the C-terminal beta-sheet region of HsTx1 (sequence 17-34). The three-dimensional structure of the peptide in solution was solved by (1)H NMR. MTX-HsTx1 displays the activity of MTX on SK channel, whereas it exhibits the pharmacological profile of HsTx1 on Kv1.1, Kv1.2, Kv1.3, and IK channels. These data demonstrate that the helical region of MTX exerts a key role in SK channel recognition, whereas the beta-sheet region of HsTx1 is crucial for activity on all other channel types tested.


Subject(s)
Potassium Channels, Calcium-Activated/chemistry , Potassium Channels, Voltage-Gated/chemistry , Scorpion Venoms/pharmacology , Amino Acid Sequence , Animals , Binding, Competitive , Brain/metabolism , Cell Line , Disulfides/chemistry , Dose-Response Relationship, Drug , Electrophysiology , Kv1.1 Potassium Channel , Kv1.2 Potassium Channel , Kv1.3 Potassium Channel , Magnetic Resonance Spectroscopy , Mice , Molecular Sequence Data , Peptides/chemistry , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Rats , Scorpion Venoms/chemistry , Sequence Analysis, Protein , Small-Conductance Calcium-Activated Potassium Channels , Static Electricity , Synaptosomes/metabolism , Time Factors
18.
Toxicon ; 43(8): 909-14, 2004 Jun 15.
Article in English | MEDLINE | ID: mdl-15208024

ABSTRACT

Ion channel-acting toxins are mainly short peptides generally present in minute amounts in the venoms of diverse animal species such as scorpions, snakes, spiders, marine cone snails and sea anemones. Interestingly, these peptides have evolved over time on the basis of clearly distinct architectural motifs present throughout the animal kingdom, but display convergent molecular determinants and functional homologies. As a consequence of this conservation of some key determinants, it has also been evidenced that toxin targets display some common evolutionary origins. Indeed, these peptides often target ion channels and ligand-gated receptors, though other interacting molecules such as enzymes have been further evidenced. In this review, we provide an overview of some selected peptides from various animal species that act on specific K+ conducting voltage-gated ion channels. In particular, we emphasize our global analysis on the structural determinants of these molecules that are required for the recognition of a particular ion channel pore structure, a property that should be correlated to the blocking efficacy of the K+ efflux out of the cell during channel opening. A better understanding of these molecular determinants is valuable to better specify and derive useful peptide pharmacological properties.


Subject(s)
Peptides/chemistry , Potassium Channels/metabolism , Toxins, Biological/chemistry , Toxins, Biological/metabolism , Models, Chemical , Protein Conformation , Structure-Activity Relationship
19.
J Biol Chem ; 278(33): 31095-104, 2003 Aug 15.
Article in English | MEDLINE | ID: mdl-12783861

ABSTRACT

Maurotoxin (MTX) is a 34-residue toxin that has been isolated initially from the venom of the scorpion Scorpio maurus palmatus. It presents a large number of pharmacological targets, including small conductance Ca2+-activated and voltage-gated K+ channels. Contrary to other toxins of the alpha-KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C1-C5, C2-C6, C3-C4, and C7-C8 (instead of the conventional C1-C5, C2-C6, C3-C7, and C4-C8, herein referred to as Pi1-like) that does not prevent its folding along the classic alpha/beta scaffold of scorpion toxins. Here, we developed an innovative strategy of chemical peptide synthesis to produce an MTX variant (MTXPi1) with a conventional pattern of disulfide bridging without any alteration of the toxin chemical structure. This strategy was used solely to address the impact of half-cystine pairings on MTX structural properties and pharmacology. The data indicate that MTXPi1 displays some marked changes in affinities toward the target K+ channels. Computed docking analyses using molecular models of both MTXPi1 and the various voltage-gated K+ channel subtypes (Shaker B, Kv1.2, and Kv1.3) were found to correlate with MTXPi1 pharmacology. A functional map detailing the interaction between MTXPi1 and Shaker B channel was generated in line with docking experiments.


Subject(s)
Disulfides/chemistry , Scorpion Venoms/chemistry , Scorpion Venoms/toxicity , Scorpions/chemistry , Amino Acid Sequence , Animals , Apamin/metabolism , Apamin/pharmacology , Binding Sites , Binding, Competitive , Iodine Radioisotopes , Membrane Potentials/drug effects , Molecular Sequence Data , Oocytes/physiology , Potassium Channels/chemistry , Potassium Channels/metabolism , Protein Structure, Tertiary , Rats , Scorpion Venoms/metabolism , Sequence Analysis, Protein , Shaker Superfamily of Potassium Channels , Synaptosomes/drug effects , Xenopus
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