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Ziad Abdallah Fajloun

Full professor
Life & Earth Sciences department - Section III - Tripoli
Speciality: Neurobiology, Pharmaceutical Science, Biochemistry
Specific Speciality: Neurosciences Biology Biotherapy

Teaching 6 Taught Courses
(2014-2015) BIOA 410 - Virology

M1 Cellular and Molecular Biology - Section 3

(2014-2015) BIOA 411 - Virology Lab

M1 Cellular and Molecular Biology - Section 3

(2014-2015) BioA 428 - Neurophysiology

M1 Cellular and Molecular Biology - Section 2

(2014-2015) BioA 429 - Neurophysiology Lab

M1 Cellular and Molecular Biology - Section 2

(2014-2015) Biol 331 - Physiology of Higher Integrative Functions

BS Earth and life sciences

(2014-2015) Biol 351 - Physiology of Higher Integrative Functions Lab

BS Earth and life sciences

Education
2001 - 2005: Habilitation a Diriger des Recherches

Universite Aix-Marseille
Neurobiologie/Biochimie

Professeur

1997 - 2001: PhD - These de doctorat

Universite Aix-Marseille
Neurosciences

PhD

1993 - 1997: Maitrise

Universite Libanaise
Biologie Animale

Master

Conferences 24 participations
4èmes Journées Scientifiques du Médicament

Colloquium
2014-06-19 to 2014-06-20

Journées Scientifiques de l’Ecole Doctorale Biologie et Santé

Colloquium
2013-12-09 to 2013-12-10

6èmes Journées Scientifiques de la STM

Congress
2013-10-22 to 2013-10-24

Printemps de la Cardiologie

Congress
2013-04-18 to 2013-04-19

The World Congress of Herpetology 7 in Vancouver

Congress
2012-08-08 to 2012-08-14

ATSB Congress

Congress
2012-03-21 to 2012-03-24

CMH2 de Marakeche

Congress
2011-05-23 to 2011-05-27

Journée Internationale de Biotechnologies 2010

Symposium
2010-12-19 to 2010-12-22

16ème journée de l’Association Tunisienne des sciences biologiques,

Symposium
2005-03-20 to 2005-05-23

EMBO course : « Multimensionnel NMR In Structural Biology »

Congress
2004-08-10 to 2004-10-20

11ème Rencontres en Toxinologie : « Toxinogénèse naturelle, toxinogénèse anthropique

Conference
2003-12-11 to 2003-12-12

5ème Colloque de l'Institut Fédératif Jean Roche de Biologie des Interactions Cellulaires

Colloquium
2003-05-03 to 2003-05-03

27ème Congrés Mondial Vétérinaire WORLD TUNISIA

Congress
2002-12-25 to 2002-12-29

12ème Réunion du Groupe Français des Peptides et Protéines

Symposium
2002-03-03 to 2002-03-08

Journée du Réseau d’Etude du Médicament

Symposium
2001-12-14 to 2001-12-14

4ème Colloque de l'Institut Fédératif Jean Roche de Biologie des Interactions Cellulaires

Colloquium
2001-10-15 to 2001-10-16

XXVIIIth Forum des jeunes chercheurs

Congress
2001-09-03 to 2001-09-07

Biophysical Society, 45th Annual Meeting, Boston

Congress
2001-02-17 to 2001-02-21

XIIIth World Congress on Animal, Plant and Microbial Toxins

Congress
2000-09-18 to 2000-09-20

Colloque "Canaux Ioniques", 11ème Edition

Colloquium
2000-03-13 to 2000-03-14

3ème Colloque de l'Institut Fédératif Jean Roche de Biologie des Interactions Cellulaires

Colloquium
1999-05-11 to 1999-05-12

Colloque "Canaux Ioniques", 10ème Edition, La Londe les Maures

Colloquium
1999-04-13 to 1999-04-14

11ème Réunion Peptides et Protéines

Colloquium
1999-03-09 to 1999-03-13

7ème Rencontres en Toxinologie, "Avancées Biotechnologiques Associées aux Toxines - 1999

Conference

Publications 36 publications
Claudine Accary, Aziza Mantash, Yassine Mallem, Ziad Fajloun, Assem Elkak Separation and biological activities of phospholipase A2 (Mb-PLA2) from the venom of Montivipera bornmuelleri, a Lebanese viper Journal of Liquid Chromatography & Related Technologies 2014

Phospholipase A2 (PLA2), a common toxic component of snake venom, which is responsible of a wide spectrum of biological and toxicological effects including bactericidal, anticoagulant, hemolytic as well as neurotoxic and cardiotoxicity activities. In the present work, phospholipase A2 named (Mb-PLA2) was isolated from the venom of Montivipera bornmuelleri a lebanese snake, by two-step procedure consisting of gel filtration chromatography on Biogel P60 and reverse phase high pressure liquid chromatography (RP-HPLC) on Restek Ultra II C18 column. The experimentally determined molecular weight of the Mb-PLA2 was 13650 Da, which was determined by electrospray ionization mass spectrometry and close to 14400 Da as judged by SDS-PAGE electrophoresis. The isolated Mb-PLA2 was evaluated for bactericidal activity against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Mb-PLA2 showed strong antibacterial activity against the Gram positive bacteria S. aureus, followed by the Gram negative bacteria, E. coli then P. aeruginosa. Moreover, the Mb-PLA2 exhibited hemolytic activity toward human erythrocyte as well as an anticoagulant activity when evaluated on human plasma.

Claudine Accary, Souad Hraoui-Bloquet, Monzer Hamze, Yassine Mallem, Fawaz El Omar, Jean-Marc Sabatier, Jean-Claude Desfontis and Ziad Fajloun Protein content analysis and antimicrobial activity of the crude venom of Montivipera bornmuelleri; a viper from Lebanon. Infectious Disorders - Drug Targets 2014

Viperidae snakes venoms represent a source of efficient bioactive components that have already led to the development of several new drugs. In this work, we analyzed the protein content of the Montivipera bornmuelleri crude venom using LC-ESI-MS, sephadex G-75 gel filtration and SDS-PAGE and demonstrated the presence of proteins with molecular masses corresponding to metalloprotease III, serine-protease and PLA2 in three fractions collected after gel filtration. Equally, we examined the antimicrobial effect of the venom that showed an important potency, as bactericidal agent, based on MBC and MIC values obtained, against Staphylococcus aureus and Morganella morganii bacteria. However, no activity was registered against Enterococcus faecalis, being the most resistant bacteria, neither against Aspergillus flavus and Penicillium digitatum fungal. Furthermore, on eleven other bacterial strains and the Candida albicans fungus, the venom has shown an intermediate efficacy by slightly reducing the growth. Our data concerning the Montivipera bornmuelleri venom give evidence of a rich and complex content aiding the exploration of new bioactive molecules for biopharmaceuticals purposes.

C. Accary, M. Rima, A. Kouzahya, W. Hleihel, Jean Claude Desfontis, Z. Fajloun, S. Hraoui-Bloquet Effect of the Montivipera bornmuelleri snake venom on human blood: coagulation disorders and hemolytic activities Open Journal of Hematology. 2014

Viper’s venom is a source of biopharmaceutical compounds, hence the need to assess the effect of this animal extract on human blood. Here, we studied the blood coagulation disorders and hemolytic activities of the venom of M. bornmulleri viper. The pro-coagulant and anticoagulant effects are analyzed with venom concentrations ranging from 0.4 to 0.0031 mg/mL. Thus, the PT is way above the normal value indicating an anticoagulant activity whereas for the aPTT, the high concentration of the venom showed an anticoagulant activity, but a pro-coagulant effect was occurred when the venom concentration decreases to 0.05 and/or 0.025 mg/mL. Hemolytic tests, performed in suspension (30% RBCs) and on blood agar plate (5% RBCs), show that an increased concentration of the venom going until 1.6 mg cannot produce a hemolytic effect, even in the presence of Ca2+ (hemolysis < 0.5%). Also, on the blood agar plate no hemolytic area appeared even with 0.04 mg of the lyophilized venom. Otherwise, the venom was able to induce a low hemolytic activity (hemolysis = 1.3 %) by acting on L-α-PC used as substrate. In this case, the destruction of erythrocytes increased proportionally to the added amount of phospholipids which are hydrolyzed to fatty acids and lysophospholipids (two toxic substances for RBCs), probably due to the presence of PLA2 in the venom and which are known by their ability to hydrolyze lecithin

Accary C., Hraoui-Bloquet S., Hamze M., Sadek R., Hleihel W., Desfontis J.-C. and Fajloun Z Preliminary proteomic analysis and biological characterization of the crude venom of Montivipera bornmuelleri; a viper from Lebanon. Recent Advances in Biomedical & Chemical Engineering and Materials Science 2014

Viperidae snakes venoms represent a source of efficient bioactive components that have already led to the development of several new drugs. Here, we analyze the proteome of the Montivipera bornmuelleri venom using liquid chromatography coupled to electrospray ionisation mass spectrometry, sephadex G-75 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have demonstrated the presence of proteins with molecular masses corresponding to metalloprotease III, serine-protease and phospholipase A2 in three fractions collected after gel filtration. In parallel, the crude venom was explored for its biological properties; it presents an antibiotic activity on different strains of bacteria revealing a potential effect against Staphylococcus aureus and Morganella morganii strains. In human plasma, it showed pro-coagulant and anti-coagulant activities at different concentrations. Indeed, for venom concentrations ranging from 0.1 to 3.125μg/mL, the prothrombine time is way above the normal value indicating an anti-coagulant activity whereas for the activated partial thromboplastine time, the high concentration of the venom showed an anti-coagulant activity, but a pro-coagulant effect was occurred when the venom concentration decreases to 50 and 25μg/mL. Also, the venom reveals an anti-platelet aggregation effect when the test is not interrupted by the formation of blood clots associated with the pro-coagulant propriety and finally, it shows an ability to induce an inflammatory response by acting on L-α-phosphatidylcholine used as substrate. Our data concerning the Montivipera bornmuelleri venom give evidence of a rich and complex content aiding the exploration of new bioactive molecules for biopharmaceuticals purposes.

Claudine Accary, Souad Hraoui-Bloquet, Monzer Hamze, Riyad Sadek, Walid Hleihel, Jean Claude Desfontis and Ziad Fajloun Exploration des propriétés biologiques du venin du serpent Montivipera Bornmuelleri. The Lebanese Association For The Advancement Of Science (LAAS) 2014

Autre thème de la conférence, l’exploration des propriétés biologiques du venin du serpent Montivipera Bornmuelleri. Les venins de serpents contiennent une variété de toxines hautement sélectives qui se sont révélées être une excellente source de molécules pour le développement d’agents thérapeutiques. Le travail des chercheurs consiste à la caractérisation biochimique et à l’évaluation des propriétés biologiques du venin de Montivipera Bornmuelleri, une vipère du Liban. La teneur en protéines du venin a été étudiée. Le résultat est que le venin brut de cette vipère est doté d’u ne activité antibactérienne, il affecte la cascade de coagulation sanguine par une double action contradictoire, il ne détruit pas les globules rouges et possède un effet relaxant sur l’aorte isolée. Ce venin est une source potentielle de molécules bioacti ves qui méritent d’être mieux exploitées.

Mohamad Rima, Claudine Accary, Katia Haddad, Riyad Sadek, Souad Hraoui-Bloquet, Jean Claude Desfontis and Ziad Fajloun Identification of L-Amino Acid Oxidase (Mb-LAAO) with antibacterial activity in the Venom of Montivipera bornmuelleri, a Viper from Lebanon Infectious Disorders - Drug Targets 2013

The L-amino acid oxidase (LAAO) is a multifunctional enzyme, able to partake in different activities including antibacterial activity. In this study, a novel LAAO (Mb-LAAO) was isolated from the venom of M. bornmuelleri snake using size exclusion chromatography followed by RP-HPLC and partially characterized. However, the molecular weight of the Mb-LAAO determined by ESI-MS and SDS-PAGE was 59 960.4 Da. Once the enzymatic activity test confirming the enzyme's identity (transformation of L-leucine) was done, the Mb-LAAO was evaluated for its antibacterial activity against Gram-negative bacteria. It showed a remarkable effect against M. morganii and K. pneumoniae. Moreover, no cytotoxic activity was observed for Mb-LAAO against human erythrocytes arguing for an exploration of its pharmaceutical interest.

Souad Hraoui-Bloquet, Riyad Sadek 1 , Claudine Accary 3 , Walid Hleihel 2 and Ziad Fajloun An ecological study of the montane Montivipera bornmuelleri (Werner, 1898) viper including preliminary biochemical characterization of venom Lebanese Science Journal 2012

This study concentrates on the Lebanon mountain viper Montiv ipera bornmuelleriendemic to the Lebanese mountains, in terms of ecology, geographic distribution and preliminary biochemical characte rization of its venom. Here, one shows that M. bornmuelleri lives in habitats dominated by a variety of mountain plant species at altitudes ranging from 1900 m to 2200 m. This viper exists in association with endemic lacertid lizards in the Oyoun Al Simane area of Kfardebian in the Sannine mountain and with other snakes (such as Platyceps najadum (Müller, 1878) and Hemorrhois ravergieri (Ménétries, 1832) in the Makmel (Bcharre) mountain. Preliminary analysis of this viper’s venom was conducted using liquid chromatography coupled to electrospr ay ionisation mass spectrometry (LC-ESI-MS). The analysis revealed the presence of some bioactive molecules in the crude product. This work constitutes a preliminary study of a research project involving the extraction and characterization of bioactive molecules and potential therapeutic uses of the venom.

Lasta S, Ouzari H, Andreotti N, Fajloun Z, Mansuelle P, Boudabous A, Sampieri F, Sabatier JM Lacticin LC14, a New Bacteriocin Produced by Lactococcus lactis BMG6.14: Isolation, Purification and Partial Characterization Infect Disord Drug Targets 2012

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.

Fajloun Z1, Andreotti N, Fathallah M, Sabatier JM, De Waard M. Analysis of the interacting surface of maurotoxin with the voltage-gated Shaker B K+ channel. J. Pept. Sci 2011

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/β 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/β 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.

Olfa Kallech-Ziri, Jose Luis, Ziad Faljoun, Jean-Marc Sabatier, Maxime Lehmann, Mohamed El Ayeb, Naziha Marrakchi and Erwann Loret - See more at: http://www.eurekaselect.com/85279/article/structure-function-relationships-kts-disintegrins-and-design-antiangiogenic-drugs#sthash.4tkbCDGn.dpuf Structure Function Relationships of KTS Disintegrins and Design of Antiangiogenic Drugs. Letters in Drug Design & Discovery 2010

Disintegrins are natural toxins found in snake venom having anti angiogenic activity. The KTS disintegrins group appears to be the most interesting as specific blocking agent of tumor growth. We report the chemical synthesis of three KTS disintegrins (Lebestatin, Obtustatin and Viperistatin) carried out with different substitution. The activity of these synthetic peptides (41 aa) and native Lebestatin purified from venom was compared with cell adhesion and migration assays. Synthetic and native Lebestatin inhibit cell adhesion of PC12 cells at 0.2 nM and integrin-dependent migration of CHO cells at 1 nM. This study shows that Lebestatin has the highest activity followed by Obtustatin and then Viperistatin in the two assays. Circular dichroism spectra of these KTS disintegrins show that their folding is similar. Molecular modeling shows that two arginines (8 and 24) and two lysines (21 and 32) have probably their chemical function interacting with integrins in protruding from the surface of Lebestatin. This study should help to design a lead compound to discover a new family of anti-angiogenic drugs. - See more at: http://www.eurekaselect.com/85279/article/structure-function-relationships-kts-disintegrins-and-design-antiangiogenic-drugs.

Lasta S, Fajloun Z, Mansuelle P, Sabatier JM, Boudabous A, Sampieri F. Chemical synthesis of lactococcin B and functional evaluation of the N-terminal domain using a truncted synthetic analogue Arch Inst Pasteur Tunis 2008

The lactococcin B (LnB) is a hydrophobic, positively charged bacteriocin, produced by Lactococcus lactis ssp. cremoris 9B4. It consists of a peptidic chain made up of 47 amino acid residues, and inhibits Lactococcus exclusively. In order to study its biological activity a synthetic lactococcin B (LnBs) was obtained by solid-phase chemical synthesis using a Fmoc strategy. LnBs was shown to be indistinguishable from the natural peptide. In addition, a synthetic (7-47) LnBst analogue was obtained by withdrawal of peptidyl-resin after the 41 cycle of LnBs peptide chain assembly. The synthetic N-terminal truncated (7-47) LnBst analogue was found to be inactive on indicator strains. Our results strongly suggest that the first six N-terminal amino acid residues are involved in the bactericidal activity of LnB.

Lasta S, Fajloun Z, Darbon H, Mansuelle P, Andreotti N, Sabatier JM, Boudabous A, Sampieri F. Chemical synthesis and characterization of J46 peptide, an atypical class IIa bacteriocin from Lactococcus lactis subsp. cremoris J46 Strain J Antibiotics 2008

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.

Pimentel C1, M'Barek S, Visan V, Grissmer S, Sampieri F, Sabatier JM, Darbon H, Fajloun Z. Chemical synthesis and 1H-NMR 3D structure determination of AgTx2-MTX chimera, a new potential blocker for Kv1.2 channel, derived from MTX and AgTx2 scorpion toxins. Protein Science 2008

Agitoxin 2 (AgTx2) is a 38-residue scorpion toxin, cross-linked by three disulfide bridges, which acts on voltage-gated K(+) (Kv) channels. Maurotoxin (MTX) is a 34-residue scorpion toxin with an uncommon four-disulfide bridge reticulation, acting on both Ca(2+)-activated and Kv channels. A 39-mer chimeric peptide, named AgTx2-MTX, was designed from the sequence of the two toxins and chemically synthesized. It encompasses residues 1-5 of AgTx2, followed by the complete sequence of MTX. As established by enzyme cleavage, the new AgTx2-MTX molecule displays half-cystine pairings of the type C1-C5, C2-C6, C3-C7, and C4-C8, which is different from that of MTX. The 3D structure of AgTx2-MTX solved by (1)H-NMR, revealed both alpha-helical and beta-sheet structures, consistent with a common alpha/beta scaffold of scorpion toxins. Pharmacological assays of AgTx2-MTX revealed that this new molecule is more potent than both original toxins in blocking rat Kv1.2 channel. Docking simulations, performed with the 3D structure of AgTx2-MTX, confirmed this result and demonstrated the participation of the N-terminal domain of AgTx2 in its increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicated that replacement of the N-terminal domain of MTX by the one of AgTx2 in the AgTx2-MTX chimera results in a reorganization of the disulfide bridge arrangement and an increase of affinity to the Kv1.2 channel.

Guieu, R., Fenouillet, E., Devaux, C., Fajloun, Z., Carrega, L., Pham, T., Sauze, N., Marguet, D CD26 modulates nociception via dipeptidyl-peptidase IV activity. Life Sci Journal 2006

BACKGROUND: CD26 is a multifunctional cell surface glycoprotein expressed by T and B cells. It exhibits a dipeptidyl-peptidase activity (DPP-IV) that cleaves the penultimate proline from the N-terminus of polypeptides, thereby regulating their activity and concentration. METHODS: Using CD26-/- mice resulting from targeted inactivation of the gene, we examined the consequences of a DPP-IV defect on behavioural response to nociceptive stimuli and concentration of the pain modulator peptides substance P (SP) and endomorphin 2, two DPP-IV substrates. RESULTS: CD26 inactivation induced a three-fold decrease in circulating endopeptidase activity while that found in brain extracts was normal, albeit very weak. CD26-/- mice had high SP concentrations in plasma (3.4+/-1 pg/ml versus 1.5+/-0.3 pg/ml, P<10(-3)) but not in brain extracts (35+/-12 pg/ml versus 32+/-9 pg/ml, P>0.05). Endomorphin-2 levels in the two groups were in the same range for plasma and brain extracts. CD26-/- mice displayed short latencies to nociceptive stimuli (hot plate test: 6.6+/-1.2 s versus 8.6+/-1.5 s, P<10(-4); tail pinch test: 3.1+/-0.6 s versus 4.2+/-0.8 s, P<10(-3)). Administration of an SP (NK1) receptor antagonist or DPP-IV to CD26-/- mice normalised latencies. DPP-IV inhibitors decreased latencies only in CD26+/+ mice. CONCLUSIONS: Our observations represent the first fundamental evidence showing that DPP-IV influences pain perception via modulation of the peripheral SP concentration. Our work also highlights the role of peripheral NK1 receptors in nociception.

Jean-Marc Sabatier, Ziad Fajloun Oxydation de peptides Patent Number EP1628997 A2 2006

Le pliage/oxydation d'un peptide réduit ou partiellement réduit pour former un peptide à pont disulfure est réalisé(e): par dissolution du peptide dans un solvant organique de réduction par l'oxyde utilisé seul ou dans un mélange avec de l'eau; adjonction d'un tampon alcalin aqueux à la solution; et récupération du peptide à pont disulfure résultant. Le solvant organique de réduction par l'oxyde préféré est le diméthylsulfoxyde, qui est utilisé autant que possible sous forme de solution aqueuse à 10 à 50 %. L'adjonction du tampon alcalin aqueux, qui est de préférence un tampon de tris-HCI 0,2 M, s'effectue de préférence au cours d'une période de 5 à 90 minutes suivant la dissolution du peptide réduit dans le solvant organique de réduction par l'oxyde. La méthode permet, d'une part d'oxyder des peptides réduits, qui sont insolubles dans des conditions alcalines, d'autre part d'oxyder complètement des peptides réduits pouvant former des espèces oxydées stables mais inactives s'ils sont traités uniquement avec le diméthylsulfoxyde.

M'Barek S1, Chagot B, Andreotti N, Visan V, Mansuelle P, Grissmer S, Marrakchi M, El Ayeb M, Sampieri F, Darbon H, Fajloun Z, De Waard M, Sabatier JM. Acting on the number of molecular contacts between Maurotoxin and Kv1.2 channel impacts ligand affinity Proteins Journal 2005

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.

Jouirou B, Mosbah A, Visan V, Grissmer S, M'Barek S, Fajloun Z, Van Rietschoten J, Devaux C, Rochat H, Lippens G, El Ayeb M, De Waard M, Mabrouk K, Sabatier JM. Cobatoxin 1 from Centruroides noxius scorpion venom : Chemical synthesis, 3-D structure in solution, pharmacology and docking on K+ channels. Biochem. J 2004

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.

M'Barek S, Fajloun Z, Cestèle S, Devaux C, Mansuelle P, Mosbah A, Jouirou B, Mantegazza M, Van Rietschoten J, El Ayeb M, Rochat H, Sabatier JM, Sampieri First chemical synthesis of a scorpion -toxin affecting sodium channels : the AaH I toxin of Androctonus australis hector. J. Peptide Sci 2004

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.

Visan V, Fajloun Z, Sabatier JM, Grissmer S. Mapping of maurotoxin (MTX) binding sites on hKv1.2, hKv1.3 and hIKCa1 channels. Mol. Pharmacology 2004

Maurotoxin (MTX) is a potent blocker of human voltage-activated Kv1.2 and intermediate-conductance calcium-activated potassium channels, hIKCa1. Because its blocking affinity on both channels is similar, although the pore region of these channels show only few conserved amino acids, we aimed to characterize the binding sites of MTX in these channels. Investigating the pH(o) dependence of MTX block on current through hKv1.2 channels, we concluded that the block is less pH(o) - sensitive than for hIKCa1 channels. Using mutant cycle analysis and computer docking, we tried to identify the amino acids through which MTX binds to hKv1.2 and hIKCa1 channels. We report that MTX interacts with hKv1.2 mainly through six strong interactions. Lys(23) from MTX protrudes into the channel pore interacting with the GYGD motif, whereas Tyr(32) and Lys(7) interact with Val(381), Asp(363), and Glu(355), stabilizing the toxin onto the channel pore. Because only Val(381), Asp(363), and the GYGD motif are conserved in hIKCa1 channels, and the replacement of His(399) from hKv1.3 channels with a threonine makes this channel MTX-sensitive, we concluded that MTX binds to all three channels through the same amino acids. Glu(355), although important, is not essential in MTX recognition. A negatively charged amino acid in this position could better stabilize the toxin-channel interaction and could explain the pH(o) sensitivity of MTX block on current through hIKCa1 versus hKv1.2 channels.

Castle NA, London DO, Creech C, Fajloun Z, Stocker JW, Sabatier JM. Maurotoxin, a potent inhibitor of intermediate conductance Ca2+-activated potassium channels. Mol. Pharmacology 2003

Maurotoxin, a 34-amino acid toxin from Scorpio maurus scorpion venom, was examined for its ability to inhibit cloned human SK (SK1, SK2, and SK3), IK1, and Slo1 calcium-activated potassium (K(Ca)) channels. Maurotoxin was found to produce a potent inhibition of Ca(2+)-activated (86)Rb efflux (IC(50), 1.4 nM) and inwardly rectifying potassium currents (IC(50), 1 nM) in CHO cells stably expressing IK1. In contrast, maurotoxin produced no inhibition of SK1, SK2, and SK3 small-conductance or Slo1 large-conductance K(Ca) channels at up to 1 microM in physiologically relevant ionic strength buffers. Maurotoxin did inhibit (86)Rb efflux (IC(50), 45 nM) through, and (125)I-apamin binding (K(i), 10 nM) to SK channels in low ionic strength buffers (i.e., 18 mM sodium, 250 mM sucrose), which is consistent with previous reports of inhibition of apamin binding to brain synaptosomes. Under similar low ionic strength conditions, the potency for maurotoxin inhibition of IK1 increased by approximately 100-fold (IC(50), 14 pM). In agreement with its ability to inhibit recombinant IK1 potassium channels, maurotoxin was found to potently inhibit the Gardos channel in human red blood cells and to inhibit the K(Ca) in activated human T lymphocytes without affecting the voltage-gated potassium current encoded by Kv1.3. Maurotoxin also did not inhibit Kv1.1 potassium channels but potently blocked Kv1.2 (IC(50), 0.1 nM). Mutation analysis indicates that similar amino acid residues contribute to the blocking activity of both IK1 and Kv1.2. The results from this study show that maurotoxin is a potent inhibitor of the IK1 subclass of K(Ca) potassium channels and may serve as a useful tool for further defining the physiological role of this channel subtype.

M'Barek S, Lopez-Gonzalez , Andreotti N, di Luccio E, Visan V, Grissmer S, Judge S, El Ayeb M, Darbon H, Rochat H, Sampieri F, Béraud E, Fajloun Z, De Waard M, Sabatier JM. A maurotoxin with constrained standard disulfide bridging – Innovative strategy of chemical synthesis, pharmacology and docking on K+ channels. J. Biol. Chem 2003

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.

M'Barek S, Mosbah A, Sandoz G, Fajloun Z, Olamendi-Portugal T, Rochat H, Sampieri F, Guijarro JI, Mansuelle P, Delepierre M, De Waard M, Sabatier JM. Synthesis and characterization of Pi4, a scorpion toxin from Pandinus imperator that acts on K+ channels. Eur. J. Biochem 2003

Pi4 is a 38-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the Chactidae scorpion Pandinus imperator. Together with maurotoxin, Pi1, Pi7 and HsTx1, Pi4 belongs to the alpha KTX6 subfamily of short four-disulfide-bridged scorpion toxins acting on K+ channels. Due to its very low abundance in venom, Pi4 was chemically synthesized in order to better characterize its pharmacology and structural properties. An enzyme-based cleavage of synthetic Pi4 (sPi4) indicated half-cystine pairings between Cys6-Cys27, Cys12-32, Cys16-34 and Cys22-37, which denotes a conventional pattern of scorpion toxin reticulation (Pi1/HsTx1 type). In vivo, sPi4 was lethal after intracerebroventricular injection to mice (LD50 of 0.2 microg per mouse). In vitro, addition of sPi4 onto Xenopus laevis oocytes heterologously expressing various voltage-gated K+ channel subtypes showed potent inhibition of currents from rat Kv1.2 (IC50 of 8 pm) and Shaker B (IC50 of 3 nm) channels, whereas no effect was observed on rat Kv1.1 and Kv1.3 channels. The sPi4 was also found to compete with 125I-labeled apamin for binding to small-conductance Ca(2+)-activated K+ (SK) channels from rat brain synaptosomes (IC50 value of 0.5 microm). sPi4 is a high affinity blocker of the Kv1.2 channel. The toxin was docked (BIGGER program) on the Kv channel using the solution structure of sPi4 and a molecular model of the Kv1.2 channel pore region. The model suggests a key role for residues Arg10, Arg19, Lys26 (dyad), Ile28, Lys30, Lys33 and Tyr35 (dyad) in the interaction and the associated blockage of the Kv1.2 channel.

di Luccio E, Matavel A, Opi S, Regaya I, Sandoz G, M'barek S, Carlier E, Estève E, Carrega L, Fajloun Z, Rochat H, Loret E, de Waard M, Sabatier JM. Evolution of Maurotoxin conformation and blocking efficacy towards Shaker B channels during the course of folding and oxidation in vitro. Biochem. J. 2002

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K(+) channels, including the voltage-gated Shaker B subtype. In the present study, we have investigated over 80 h: (1) the time-course of folding of synthetic MTX (sMTX) by CD analysis; (2) the kinetics of disulphide bridge formation by MS; and (3) the potency of MTX in blocking Shaker B currents during the combined process of its in vitro folding and oxidation. From the CD data, we show that stable secondary structures of sMTX evolve sequentially over time, with the appearance of the alpha-helix within 5 h, followed by the formation of the beta-sheet within 22 h. Using MS analysis, the sMTX intermediates were also found to appear sequentially from the least (one-disulphide-bridged sMTX) to the most oxidized species (native-like, four-disulphide-bridged sMTX). The time course of formation of secondary structures coincides mainly with the occurrence of one-disulphide-bridged sMTX for the alpha-helix and two- or three-disulphide-bridged sMTX for the beta-sheet. On-line electrophysiological recordings, which measure sMTX blocking efficacy on K(+) currents during its folding and oxidation, were performed on Shaker B channels expressed in Xenopus oocytes. Unexpectedly, the results demonstrate that sMTX is highly potent at the initial stage of oxidation, whereas its blocking activity can be transiently and dramatically reduced at later stages during the course of folding/oxidation before it reaches full bioactivity. These data suggest that formation of disulphide bridges can both physically stabilize and alter the bioactive three-dimensional structure of sMTX.

Fajloun Z, Ferrat G, Carlier E, M'Barek S, Regaya I, Fathallah M, Rochat H, Darbon H, de Waard M, Sabatier JM. Synthesis, 3-D structure and pharmacology of a reticulated chimera derived from maurotoxin and Tsk scorpion toxins. Biochem. Biophys. Res. Comm. 2002

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulfide bridges that acts on both Ca(2+)-activated (SK) and voltage-gated (Kv) K(+) channels. A 38-mer chimera of MTX, Tsk-MTX, has been synthesized by the solid-phase method. It encompasses residues from 1 to 6 of Tsk at N-terminal, and residues from 3 to 34 of MTX at C-terminal. As established by enzyme cleavage, Tsk-MTX displays half-cystine pairings of the type C1-C5, C2-C6, C3-C7 and C4-C8 which, contrary to MTX, correspond to a disulfide bridge pattern common to known scorpion toxins. The 3-D structure of Tsk-MTX, solved by (1)H NMR, demonstrates that it adopts the alpha/beta scaffold of scorpion toxins. In vivo, Tsk-MTX is lethal by intracerebroventricular injection in mice (LD(50) value of 0.2 microg/mouse). In vitro, Tsk-MTX is as potent as MTX, or Tsk, to interact with apamin-sensitive SK channels of rat brain synaptosomes (IC(50) value of 2.5 nM). It also blocks voltage-gated K(+) channels expressed in Xenopus oocytes, but is inactive on rat Kv1.3 contrary to MTX.

Di Luccio, E., Fajloun, Z., Mouhat, S., Rochat, H., De Waard, M., Sabatier, J-M. Etude des relations structure-fonction de la maurotoxine. ELSEVIER / MASSON 2002

Carlier E, Fajloun Z, Mansuelle P, Fathallah M, Mosbah A, Oughideni R, Sandoz G, Di Luccio E, Geib S, Regaya I, Brocard J, Rochat H, Darbon H, Devaux C, Sabatier JM, de Waard M. Disulfide bridge reorganization induced by proline mutations in Maurotoxin. FEBS Lett. 2001

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus, and characterized. Together with Pi1 and HsTx1, MTX belongs to a family of short-chain four-disulfide-bridged scorpion toxins acting on potassium channels. However, contrary to other members of this family, MTX exhibits an uncommon disulfide bridge organization of the type C1-C5, C2-C6, C3-C4 and C7-C8, versus C1-C5, C2-C6, C3-C7 and C4-C8 for both Pi1 and HsTx1. Here, we report that the substitution of MTX proline residues located at positions 12 and/or 20, adjacent to C3 (Cys(13)) and C4 (Cys(19)), results in conventional Pi1- and HsTx1-like arrangement of the half-cystine pairings. In this case, this novel disulfide bridge arrangement is without obvious incidence on the overall three-dimensional structure of the toxin. Pharmacological assays of this structural analog, [A(12),A(20)]MTX, reveal that the blocking activities on Shaker B and rat Kv1.2 channels remain potent whereas the peptide becomes inactive on rat Kv1.3. These data indicate, for the first time, that discrete point mutations in MTX can result in a marked reorganization of the half-cystine pairings, accompanied with a novel pharmacological profile for the analog.

di Luccio E, Azulay DO, Regaya I, Fajloun Z, Sandoz G, Mansuelle P, Kharrat R, Fathallah M, Carrega L, Estève E, Rochat H, De Waard M, Sabatier JM. Parameters affecting oxidation/folding of Maurotoxin, a four disulfide-bridged scorpion toxin. Biochem. J. 2001

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K(+) channel subtypes. MTX adopts a disulphide bridge organization of the type C1-C5, C2-C6, C3-C4 and C7-C8, and folds according to the common alpha/beta scaffold reported for other known scorpion toxins. Here we have investigated the process and kinetics of the in vitro oxidation/folding of reduced synthetic L-MTX (L-sMTX, where L-MTX contains only L-amino acid residues). During the oxidation/folding of reduced L-sMTX, the oxidation intermediates were blocked by iodoacetamide alkylation of free cysteine residues, and analysed by MS. The L-sMTX intermediates appeared sequentially over time from the least (intermediates with one disulphide bridge) to the most oxidized species (native-like, four-disulphide-bridged L-sMTX). The mathematical formulation of the diffusion-collision model being inadequate to accurately describe the kinetics of oxidation/folding of L-sMTX, we have formulated a derived mathematical description that better fits the experimental data. Using this mathematical description, we have compared for the first time the oxidation/folding of L-sMTX with that of D-sMTX, its stereoisomer that contains only D-amino acid residues. Several experimental parameters, likely to affect the oxidation/folding process, were studied further; these included temperature, pH, ionic strength, redox potential and concentration of reduced toxin. We also assessed the effects of some cellular enzymes, peptidylprolyl cis-trans isomerase (PPIase) and protein disulphide isomerase (PDI), on the folding pathways of reduced L-sMTX and D-sMTX. All the parameters tested affect the oxidative folding of sMTX, and the kinetics of this process were indistinguishable for L-sMTX and D-sMTX, except when stereospecific enzymes were used. The most efficient conditions were found to be: 50 mM Tris/HCl/1.4 mM EDTA, pH 7.5, supplemented by 0.5 mM PPIase and 50 units/ml PDI for 0.1 mM reduced compound. These data represent the first report of potent stereoselective effects of cellular enzymes on the oxidation/folding of a scorpion toxin.

Shakkottai VG, Regaya I, Wulff H, Fajloun Z, Tomita H, Fathallah M, Cahalan MD, Gargus JJ, Sabatier JM, Chandy KG. Design and characterization of a highly selective peptide inhibitor of the small conductance calcium-activated K+ channel, SKCa2 J. Biol. Chem. 2001

Apamin-sensitive small conductance calcium-activated potassium channels (SKCa1-3) mediate the slow afterhyperpolarization in neurons, but the molecular identity of the channel has not been defined because of the lack of specific inhibitors. Here we describe the structure-based design of a selective inhibitor of SKCa2. Leiurotoxin I (Lei) and PO5, peptide toxins that share the RXCQ motif, potently blocked human SKCa2 and SKCa3 but not SKCa1, whereas maurotoxin, Pi1, Tskappa, and PO1 were ineffective. Lei blocked these channels more potently than PO5 because of the presence of Ala(1), Phe(2), and Met(7). By replacing Met(7) in the RXCQ motif of Lei with the shorter, unnatural, positively charged diaminobutanoic acid (Dab), we generated Lei-Dab(7), a selective SKCa2 inhibitor (K(d) = 3.8 nm) that interacts with residues in the external vestibule of the channel. SKCa3 was rendered sensitive to Lei-Dab(7) by replacing His(521) with the corresponding SKCa2 residue (Asn(367)). Intracerebroventricular injection of Lei-Dab(7) into mice resulted in no gross central nervous system toxicity at concentrations that specifically blocked SKCa2 homotetramers. Lei-Dab(7) will be a useful tool to investigate the functional role of SKCa2 in mammalian tissues.

Fajloun Z, Kharrat R, Chen L, Lecomte C, Di Luccio E, Bichet D, El Ayeb M, Rochat H, Allen PD, Pessah IN, De Waard M, Sabatier JM. Chemical synthesis and characterization of Maurocalcine, a scorpion toxin that activates Ca2+ release channel/ryanodine receptors FEBS Lett. 2000

Maurocalcine is a novel toxin isolated from the venom of the chactid scorpion Scorpio maurus palmatus. It is a 33-mer basic peptide cross-linked by three disulfide bridges, which shares 82% sequence identity with imperatoxin A, a scorpion toxin from the venom of Pandinus imperator. Maurocalcine is peculiar in terms of structural properties since it does not possess any consensus motif reported so far in other scorpion toxins. Due to its low concentration in venom (0.5% of the proteins), maurocalcine was chemically synthesized by means of an optimized solid-phase method, and purified after folding/oxidation by using both C18 reversed-phase and ion exchange high-pressure liquid chromatographies. The synthetic product (sMCa) was characterized. The half-cystine pairing pattern of sMCa was identified by enzyme-based cleavage and Edman sequencing. The pairings were Cys3-Cys17, Cys10-Cys21, and Cys16-Cys32. In vivo, the sMCa was lethal to mice following intracerebroventricular inoculation (LD(50), 20 microg/mouse). In vitro, electrophysiological experiments based on recordings of single channels incorporated into planar lipid bilayers showed that sMCa potently and reversibly modifies channel gating behavior of the type 1 ryanodine receptor by inducing prominent subconductance behavior.

Mosbah A, Kharrat R, Fajloun Z, Renisio JG, Blanc E, Sabatier JM, El Ayeb M, Darbon H. A new fold in the scorpion toxin family, associated with an activity on a ryanodine-sensitive calcium channel. Proteins Journal 2000

We determined the structure in solution by (1)H two-dimensional NMR of Maurocalcine from the venom of Scorpio maurus. This toxin has been demonstrated to be a potent effector of ryanodyne-sensitive calcium channel from skeletal muscles. This is the first description of a scorpion toxin which folds following the Inhibitor Cystine Knot fold (ICK) already described for numerous toxic and inhibitory peptides, as well as for various protease inhibitors. Its three dimensional structure consists of a compact disulfide-bonded core from which emerge loops and the N-terminus. A double-stranded antiparallel beta-sheet comprises residues 20-23 and 30-33. A third extended strand (residues 9-11) is perpendicular to the beta-sheet. Maurocalcine structure mimics the activating segment of the dihydropyridine receptor II-III loop and is therefore potentially useful for dihydropyridine receptor/ryanodine receptor interaction studies. Proteins 2000;40:436-442.

Fajloun Z1, Ferrat G, Carlier E, Fathallah M, Lecomte C, Sandoz G, di Luccio E, Mabrouk K, Legros C, Darbon H, Rochat H, Sabatier JM, De Waard M. Synthesis, 1H-NMR structure and activity of a three disulfide-bridged Maurotoxin analog designed to restore the consensus motif of scorpion toxins. J. Biol. Chem. 2000

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus. The toxin displays an exceptionally wide range of pharmacological activity since it binds onto small conductance Ca(2+)-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 alpha/beta scaffold (an alpha-helix connected to an antiparallel beta-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 ([Abu(19),Abu(34)]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 alpha-aminobutyrate (Abu) derivatives. The three-dimensional structure of [Abu(19), Abu(34)]MTX in solution was solved by (1)H NMR. This analog adopts the alpha/beta 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 alpha-helix regarding the beta-sheet structure. In vivo, [Abu(19),Abu(34)]MTX remains lethal in mice as assessed by intracerebroventricular injection of the peptide (LD(50) value of 0. 25 microg/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.

Fajloun Z1, Carlier E, Lecomte C, Geib S, Di Luccio E, Bichet D, Mabrouk K, Rochat H, De Waard M, Sabatier JM. Chemical synthesis and characterization of Pi1, a scorpion toxin from Pandinus imperator active on K+ channels. Eur. J. Biochem. 2000

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 microgram 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.

Fajloun Z1, Mosbah A, Carlier E, Mansuelle P, Sandoz G, Fathallah M, di Luccio E, Devaux C, Rochat H, Darbon H, De Waard M, Sabatier JM. Maurotoxin vs Pi1/HsTx1 : Towards new insights in the understanding of their distinct disulfide bridge patterns. J. Biol. Chem. 2000

Maurotoxin (MTX) is a scorpion toxin acting on several K(+) channel subtypes. It is a 34-residue peptide cross-linked by four disulfide bridges that are in an "uncommon" arrangement of the type C1-C5, C2-C6, C3-C4, and C7-C8 (versus C1-C5, C2-C6, C3-C7, and C4-C8 for Pi1 or HsTx1, two MTX-related scorpion toxins). We report here that a single mutation in MTX, in either position 15 or 33, resulted in a shift from the MTX toward the Pi1/HsTx1 disulfide bridge pattern. This shift is accompanied by structural and pharmacological changes of the peptide without altering the general alpha/beta scaffold of scorpion toxins.

Carlier E, Avdonin V, Geib S, Fajloun Z, Kharrat R, Rochat H, Sabatier JM, Hoshi T, De Waard M. Effect of maurotoxin, a four disulfide-bridged toxin from the chactoid scorpion Scorpio maurus, on Shaker K+ channels. J Pept Res. 2000

Maurotoxin is a 34-residue toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus palmatus and contains four disulfide bridges that are normally found in long-chain toxins of 60-70 amino acid residues, which affect voltage-gated sodium channels. However, despite the unconventional disulfide-bridge pattern of maurotoxin, the conformation of this toxin remains similar to that of other toxins acting on potassium channels. Here, we analyzed the effects of synthetic maurotoxin on voltage-gated Shaker potassium channels (ShB) expressed in Xenopus oocytes. Maurotoxin produces a strong, but reversible, inhibition of the ShB K+ current with an IC50 of 2 nM. Increasing concentrations of the toxin induce a progressively higher block at saturating concentrations. At nonsaturating concentrations of the toxin (5-20 nM), the channel block appears slightly more pronounced at threshold potentials suggesting that the toxin may have a higher affinity for the closed state of the channel. At the single channel level, the toxin does not modify the unitary current amplitude, but decreases ensemble currents by increasing the number of depolarizing epochs that failed to elicit any opening. A point mutation of Lys23 to alanine in maurotoxin produces a 1000-fold reduction in the IC50 of block by the toxin suggesting the importance of this charged residue for the interaction with the channel. Maurotoxin does not affect K+ currents carried by Kir2.3 channels in oocytes or Na+ currents carried by the alphaIIa channel expressed in CHO cells.

Carlier E, Mabrouk K, Moulard M, Fajloun Z, Rochat H, De Waard M, Sabatier JM. Ion channel activation by SPC3, a peptide derived from the HIV-1 gp 120 V3 loop. J. Peptide Res. 2000

SPC3 is a multibranched peptide containing eight identical GPGRAF motifs which are derived from the human immunodeficiency virus (HIV)-1 gp120 V3 loop consensus sequence. This molecule was reported to prevent the infection of CD4+ cells by various HIV-1 and HIV-2 strains. However, the molecular mode of action of SPC3 remains unclear. Here, we investigated the possibility that SPC3 could interact with alpha/beta-chemokine receptors following observations that, first, the V3 loop is likely to be involved in alpha/beta-chemokine receptor-dependent HIV entry and, second, natural ligands of these receptors are potent inhibitors of cell infection. To address this point, we examined the effects of SPC3 on Xenopus oocytes either uninjected or expressing exogenous human CXCR4 alpha-chemokine receptors. Extracellular applications of micromolar concentrations of SPC3 onto Xenopus oocytes trigger potent inward chloride currents which can be inhibited by increasing extracellular Ca2+ concentration. This effect can be blocked by chloride channel antagonists and is highly specific to SPC3 as it is not triggered by structural analogs of SPC3. The SPC3-induced chloride conductance in oocytes is alpha/beta-chemokine receptor dependent because: (i) SPC3 alters the sensitivity of this channel to external applications of human recombinant MIP-1alpha, a natural ligand of human CCR5 receptor, and (ii) the amplitude of the inward current could be increased by the expression of exogenous human CXCR4 chemokine receptor. The effect of SPC3 appears to rely on the activation of a phospholipase A2 signaling pathway, but is not affected by changes in cytosolic Ca2+ concentration, or by alterations in Gi/Go protein, adenylate cyclase, phospholipase C or protein kinase C activity. Altogether, the data indicate that SPC3 is capable of activating a surface alpha/beta-chemokine-like receptor-mediated signaling pathway in competent cells, thereby triggering, either directly or indirectly, a Ca2+-inactivated chloride conductance.

Lecomte C, Ferrat G, Fajloun Z, Van Rietschoten J, Rochat H, Martin-Eauclaire MF, Darbon H, Sabatier JM. Chemical synthesis and structure-activity of TsK, a novel scorpion toxin acting on apamin-sensitive SK channel. J. Peptide Res. 1999

Tityus kappa (Ts kappa), a novel toxin from the venom of the scorpion Tityus serrulatus, is a 35-residue polypeptide cross-linked by three disulphide bridges and acts on small-conductance calcium-activated potassium channels (SK channels). Ts K was chemically synthesized using the solid-phase method and characterized. The synthetic product, sTs kappa, was indistinguishable from the natural toxin when tested in vitro in competition assay with radiolabelled apamin for binding to rat brain synaptosomes (IC50 = 3 nM). The sTs kappa was further tested in vivo for lethal activity to mice following intracerebroventricular inoculation (LD50 = 70 ng per mouse). The half-cystine pairings were formerly established by enzyme-based cleavage of sTs kappa; they were between Cys7-Cys28, Cys13-CyS33 and Cys17-Cys35, which is a disulphide bridge pattern similar to that of other short scorpion toxins. According to previous studies on SK channel-acting toxins, the putative influence of certain basic residues of Ts kappa (i.e. Arg6, Arg9, Lys18, Lys19) in its pharmacological activity was investigated using synthetic point-mutated analogues of the toxin with an Ala substitution at these positions. Data from binding assay, together with conformational analysis of the synthetic analogues by 1H-NMR, suggest that Arg6, and to a lesser extent Arg9, are important residues for an high-affinity interaction of this toxin with SK channels; interestingly these residues are located outside the alpha-helical structure, whereas the pharmacologically important basic residues from other SK channel-specific toxins had been located inside the alpha-helix.

Supervision 9 Supervised Students
Chimie Analytique

Josephine AL-ALAM
Title of the PhD thesis topic: Search of pesticides and other organic contaminants (PCBs, phthalates ...) in biological samples; application to biomonitoring of air quality and the quality of food.

Biotherapie

Diana CHAKER
Title of the PhD thesis topic: Evaluation of the impact of progenitor endothelial cells and monocytes on the differentiation of mesenchymal stem cell into mature hepatocytes.

Neurosciences

Mohamad RIMA
Title of the PhD thesis topic: studies of the interaction between the proteins of the postsynaptic density and the beta subunit of voltage-dependent calcium channel.

Physiopathology

Abdallah DIB
Title of the PhD thesis topic: INFLUENCE DES FACTEURS DE RISQUES ENVIRONNEMENTAUX DURANT LA GROSSESSE: Programmation fœtale de la structure et de la fonction vasculaire

Neurosciences

Jeanne d'Arc AL-BACHA
Title of the PhD thesis topic: studies of pro(renin) receptors or PRR involved in the function of renin-angiotensin system.

Physiopathology

Asma ALAMEDDINE
Title of the PhD thesis topic: The cardiovascular effects of salidroside in the Goto-Kakizaki diabetic rat model

Neurosciences

Joseph KHOURY
Title of the PhD thesis topic: Implication du VEGF-C dans les propriétés métastatiques des tumeurs en réponse aux traitements antiangiogéniques

Immunothrapy

Walid WARDA
Title of the PhD thesis topic: Développement d’une biothérapie utilisant un récepteur chimérique (CAR) exprimé par des lymphocytes T et ciblant le CD123 dans la leucémie dérivée des cellules dendritiques plasmocytoïdes

Neurosciences

Samy RIMA
Title of the PhD thesis topic: Apprentissages associatifs et signaux de récompense dans le cortex visuel du primate

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