Fontecilla-Camps JC


Full name : Fontecilla-Camps Juan C

First name : Juan C

Mail : Metalloproteins Unit, Institut de Biologie Structurale J.-P. Ebel,CEA-CNRS-Universite Joseph Fourier, 38027 Grenoble

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Country : France

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References (14)

Title : Structure-activity analysis of aging and reactivation of human butyrylcholinesterase inhibited by analogues of tabun. - Carletti_2009_Biochem.J_421_97
Author(s) : Carletti E , Aurbek N , Gillon E , Loiodice M , Nicolet Y , Fontecilla-Camps JC , Masson P , Thiermann H , Nachon F , Worek F
Ref : Biochemical Journal , 421 :97 , 2009
Abstract : hBChE [human BChE (butyrylcholinesterase)] naturally scavenges OPs (organophosphates). This bioscavenger is currently in Clinical Phase I for pretreatment of OP intoxication. Phosphylated ChEs (cholinesterases) can undergo a spontaneous time-dependent process called 'aging' during which the conjugate is dealkylated, leading to creation of an enzyme that cannot be reactivated. hBChE inhibited by phosphoramidates such as tabun displays a peculiar resistance to oxime-mediated reactivation. We investigated the basis of oxime resistance of phosphoramidyl-BChE conjugates by determining the kinetics of inhibition, reactivation (obidoxime {1,1'-(oxybis-methylene) bis[4-(hydroxyimino) methyl] pyridinium dichloride}, TMB-4(Trimedoxime) [1,3-trimethylene-bis(4-hydroxyiminomethylpyridinium) dibromide], HL 7 {1-[[[4-(aminocarbonyl) pyridinio]methoxy]methyl]-2,4-bis-[(hydroxyimino)methyl] pyridinium dimethanesulfonate)}, HI-6 {1-[[[4-(aminocarbonyl) pyridinio] methoxy] methyl]-2-[(hydroxyimino)methyl]pyridinium dichloride monohydrate} and aging, and the crystal structures of hBChE inhibited by different N-monoalkyl and N,N-dialkyl tabun analogues. The refined structures of aged hBChE conjugates show that aging proceeds through O-dealkylation of the P(R) enantiomer of N,N-diethyl and N-propyl analogues, with subsequent formation of a salt bridge preventing reactivation, similarly to a previous observation made on tabun-ChE conjugates. Interestingly, the N-methyl analogue projects its amino group towards the choline-binding pocket, so that aging proceeds through deamination. This orientation results from a preference of hBChE's acyl-binding pocket for larger than 2-atoms linear substituents. The correlation between the inhibitory potency and the N-monoalkyl chain length is related to increasingly optimized interactions with the acyl-binding pocket as shown by the X-ray structures. These kinetics and X-ray data lead to a structure-activity relationship that highlights steric and electronic effects of the amino substituent of phosphoramidate. This study provides the structural basis to design new oximes capable of reactivating phosphoramidyl-hBChE conjugates after intoxication, notably when hBChE is used as pretreatment, or to design BChE-based catalytic bioscavengers.
ESTHER : Carletti_2009_Biochem.J_421_97
PubMedSearch : Carletti_2009_Biochem.J_421_97
PubMedID: 19368529
Gene_locus related to this paper: human-BCHE

Title : X-Ray structures of native and soman-aged human butyrylcholinesterase. -
Author(s) : Nachon F , Nicolet Y , Masson P , Lockridge O , Fontecilla-Camps JC
Ref : Cholinergic Mechanisms, CRC Press :165 , 2004

Title : Poster (93) How do the crystal structures of human butyrylcholinesterase compare to torpedo californica acetylcholinesterases structures? -
Author(s) : Nachon F , Nicolet Y , Masson P , Lockridge O , Fontecilla-Camps JC
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :369 , 2004

Title : Poster (49) Crystallographic basis for substrate\/product exchange in cholinesterases. -
Author(s) : Nicolet Y , Lockridge O , Masson P , Fontecilla-Camps JC , Nachon F
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :347 , 2004

Title : Photoreversible inhibition of cholinesterases: catalytic serine-labeled caged butyrylcholinesterase - Loudwig_2003_Chembiochem_4_762
Author(s) : Loudwig S , Nicolet Y , Masson P , Fontecilla-Camps JC , Bon S , Nachon F , Goeldner M
Ref : Chembiochem , 4 :762 , 2003
Abstract : The photoregulation of the catalytic activity of butyrylcholinesterase (BChE) was investigated by treating the enzyme with a newly developed carbamylating reagent, N-methyl-N-(2-nitrophenyl)carbamoyl chloride, which has proved to be an efficient photoremovable alcohol-protecting group. BChE was efficiently inhibited in a time- and concentration-dependent manner, and the enzyme could be protected against inhibition by active-site-specific ligands (that is, tacrine). The inactivation kinetics showed a biphasic character, which can be analyzed as the result of the existence of two conformational states in solution. Pseudo-irreversible inactivation of BChE, which results from catalytic serine carbamylation, was suggested by recovery of the enzyme activity after dilution and was demonstrated by X-ray crystallography. Remarkably, the 3D structure of the carbamylated BChE conjugate showed a nonambiguous carbamylation of the catalytic serine residue as the only chemical modification on the protein. The photoreversibility of the enzyme inactivation was analyzed by irradiating the inactivated enzyme at 365 nm and was shown to reach completion in some conditions. The efficient and specific "caging" of BChE, together with the availability of carbamylated BChE crystals, will offer a unique possibility to study the catalytic properties of this enzyme by kinetic crystallography after cryophotolytic uncaging of the enzyme conjugate crystals.
ESTHER : Loudwig_2003_Chembiochem_4_762
PubMedSearch : Loudwig_2003_Chembiochem_4_762
PubMedID: 12898628

Title : Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products - Nicolet_2003_J.Biol.Chem_278_41141
Author(s) : Nicolet Y , Lockridge O , Masson P , Fontecilla-Camps JC , Nachon F
Ref : Journal of Biological Chemistry , 278 :41141 , 2003
Abstract : Cholinesterases are among the most efficient enzymes known. They are divided into two groups: acetylcholinesterase, involved in the hydrolysis of the neurotransmitter acetylcholine, and butyrylcholinesterase of unknown function. Several crystal structures of the former have shown that the active site is located at the bottom of a deep and narrow gorge, raising the question of how substrate and products enter and leave. Human butyrylcholinesterase (BChE) has attracted attention because it can hydrolyze toxic esters such as cocaine or scavenge organophosphorus pesticides and nerve agents. Here we report the crystal structures of several recombinant truncated human BChE complexes and conjugates and provide a description for mechanistically relevant non-productive substrate and product binding. As expected, the structure of BChE is similar to a previously published theoretical model of this enzyme and to the structure of Torpedo acetylcholinesterase. The main difference between the experimentally determined BChE structure and its model is found at the acyl binding pocket that is significantly bigger than expected. An electron density peak close to the catalytic Ser(198) has been modeled as bound butyrate.
ESTHER : Nicolet_2003_J.Biol.Chem_278_41141
PubMedSearch : Nicolet_2003_J.Biol.Chem_278_41141
PubMedID: 12869558
Gene_locus related to this paper: human-BCHE

Title : Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase: expression, purification, characterization and crystallization - Nachon_2002_Eur.J.Biochem_269_630
Author(s) : Nachon F , Nicolet Y , Viguie N , Masson P , Fontecilla-Camps JC , Lockridge O
Ref : European Journal of Biochemistry , 269 :630 , 2002
Abstract : Human butyrylcholinesterase (BChE; EC is of particular interest because it hydrolyzes or scavenges a wide range of toxic compounds including cocaine, organophosphorus pesticides and nerve agents. The relative contribution of each N-linked glycan for the solubility, the stability and the secretion of the enzyme was investigated. A recombinant monomeric BChE lacking four out of nine N-glycosylation sites and the C-terminal oligomerization domain was stably expressed as a monomer in CHO cells. The purified recombinant BChE showed catalytic properties similar to those of the native enzyme. Tetragonal crystals suitable for X-ray crystallography studies were obtained; they were improved by recrystallization and found to diffract to 2.0 A resolution using synchrotron radiation. The crystals belong to the tetragonal space group I422 with unit cell dimensions a = b = 154.7 A, c = 124.9 A, giving a Vm of 2.73 A3 per Da (estimated 60% solvent) for a single molecule of recombinant BChE in the asymmetric unit. The crystal structure of butyrylcholinesterase will help elucidate unsolved issues concerning cholinesterase mechanisms in general.
ESTHER : Nachon_2002_Eur.J.Biochem_269_630
PubMedSearch : Nachon_2002_Eur.J.Biochem_269_630
PubMedID: 11856322

Title : Critical role of micelles in pancreatic lipase activation revealed by small angle neutron scattering - Pignol_2000_J.Biol.Chem_275_4220
Author(s) : Pignol D , Ayvazian L , Kerfelec B , Timmins P , Crenon I , Hermoso J , Fontecilla-Camps JC , Chapus C
Ref : Journal of Biological Chemistry , 275 :4220 , 2000
Abstract : In the duodenum, pancreatic lipase (PL) develops its activity on triglycerides by binding to the bile-emulsified oil droplets in the presence of its protein cofactor pancreatic colipase (PC). The neutron crystal structure of a PC-PL-micelle complex (Hermoso, J., Pignol, D., Penel, S., Roth, M., Chapus, C., and Fontecilla-Camps, J. C. (1997) EMBO J. 16, 5531-5536) has suggested that the stabilization of the enzyme in its active conformation and its adsorption to the emulsified oil droplets are mediated by a preformed lipase-colipase-micelle complex. Here, we correlate the ability of different amphypathic compounds to activate PL, with their association with PC-PL in solution. The method of small angle neutron scattering with D(2)O/H(2)O contrast variation was used to characterize a solution containing PC-PL complex and taurodeoxycholate micelles. The resulting radius of gyration (56 A) and the match point of the solution indicate the formation of a ternary complex that is similar to the one observed in the neutron crystal structure. In addition, we show that either bile salts, lysophospholipids, or nonionic detergents that form micelles with radii of gyration ranging from 13 to 26 A are able to bind to the PC-PL complex, whereas smaller micelles or nonmicellar compounds are not. This further supports the notion of a micelle size-dependent affinity process for lipase activation in vivo.
ESTHER : Pignol_2000_J.Biol.Chem_275_4220
PubMedSearch : Pignol_2000_J.Biol.Chem_275_4220
PubMedID: 10660587

Title : Structure of fasciculin 2 from green mamba snake venom: evidence for unusual loop flexibility - le_1996_Acta.Crystallogr.D.Biol.Crystallogr_52_87
Author(s) : le Du MH , Housset D , Marchot P , Bougis PE , Navaza J , Fontecilla-Camps JC
Ref : Acta Crystallographica D Biol Crystallogr , 52 :87 , 1996
Abstract : The crystal structure of the snake toxin fasciculin 2, a potent acetylcholinesterase inhibitor from the venom of the green mamba (Dendroaspis angusticeps), has been determined by the molecular-replacement method, using the fasciculin 1 model and refined to 2.0 A resolution. The introduction of an overall anisotropic temperature factor improved significantly the quality of the electron-density map. It suggests, as it was also indicated by the packing, that the thermal motion along the unique axis direction is less pronounced than on the (ab) plane. The final crystallographic R factor is 0.188 for a model having r.m.s. deviations from ideality of 0.016 A for bond lengths and 2.01 degrees for bond angles. As fasciculin 1, fasciculin 2 belongs to the three-finger class of Elapidae toxins, a structural group that also contains the alpha-neurotoxins and the cardiotoxins. Although the two fasciculins have, overall, closely related structures, the conformation of loop I differs appreciably in the two molecules. The presence of detergent in crystallization medium in the case of fasciculin 2 appears to be responsible for the displacement of the loop containing Thr9. This conformational change also results in the formation of a crystallographic dimer that displays extensive intermolecular interactions.
ESTHER : le_1996_Acta.Crystallogr.D.Biol.Crystallogr_52_87
PubMedSearch : le_1996_Acta.Crystallogr.D.Biol.Crystallogr_52_87
PubMedID: 15299729

Title : Lipase activation by nonionic detergents. The crystal structure of the porcine lipase-colipase-tetraethylene glycol monooctyl ether complex - Hermoso_1996_J.Biol.Chem_271_18007
Author(s) : Hermoso J , Pignol D , Kerfelec B , Crenon I , Chapus C , Fontecilla-Camps JC
Ref : Journal of Biological Chemistry , 271 :18007 , 1996
Abstract : The crystal structure of the ternary porcine lipase-colipase-tetra ethylene glycol monooctyl ether (TGME) complex has been determined at 2.8 A resolution. The crystals belong to the cubic space group F23 with a = 289.1 A and display a strong pseudo-symmetry corresponding to a P23 lattice. Unexpectedly, the crystalline two-domain lipase is found in its open configuration. This indicates that in the presence of colipase, pure micelles of the nonionic detergent TGME are able to activate the enzyme; a process that includes the movement of an N-terminal domain loop (the flap). The effects of TGME and colipase have been confirmed by chemical modification of the active site serine residue using diisopropyl p-nitrophenylphosphate (E600). In addition, the presence of a TGME molecule tightly bound to the active site pocket shows that TGME acts as a substrate analog, thus possibly explaining the inhibitory effect of this nonionic detergent on emulsified substrate hydrolysis at submicellar concentrations. A comparison of the lipase-colipase interactions between our porcine complex and the human-porcine complex (van Tilbeurgh, H., Egloff, M.-P., Martinez, C., Rugani, N., Verger, R., and Cambillau, C.(1993) Nature 362, 814-820) indicates that except for one salt bridge interaction, they are conserved. Analysis of the superimposed complexes shows a 5.4 degrees rotation on the relative position of the N-terminal domains excepting the flap that moves in a concerted fashion with the C-terminal domain. This flexibility may be important for the binding of the complex to the water-lipid interface.
ESTHER : Hermoso_1996_J.Biol.Chem_271_18007
PubMedSearch : Hermoso_1996_J.Biol.Chem_271_18007
PubMedID: 8663362
Gene_locus related to this paper: pig-1plip

Title : Isoform purification of gastric lipases. Towards crystallization - Moreau_1992_J.Mol.Biol_225_147
Author(s) : Moreau H , Abergel C , Carriere F , Ferrato F , Fontecilla-Camps JC , Cambillau C , Verger R
Ref : Journal of Molecular Biology , 225 :147 , 1992
Abstract : Several isoforms of rabbit and human gastric lipases have been purified. These isoforms have the same apparent molecular weight (Mr approximately 50,000), but very different isoelectric points. Some of these isoforms were purified: pI 7.2 and 6.5 in the case of rabbit gastric lipase; and pI 7.4 and 7.2 in that of human gastric lipase. All the purified isoforms were found to have the same specific lipase activity (around 1200 units per mg of protein, measured on tributyrin as substrate). The isoforms of dog gastric lipase are more closely related, and could not be separated. Partial enzymatic deglycosylation of human gastric lipase reduced the apparent molecular weight from Mr approximately 50,000 to Mr approximately 43,000 and induced a change in the isoelectrofocusing pattern and the emergence of a new isoform (pI 7.3). It is concluded that the charge heterogeneity of gastric lipases is at least partly due to the glycan moiety of the molecule, which amounts to approximately 14% of the total molecular weight. Several crystallization trials on purified native preparations of rabbit and human gastric lipases were unsuccessful, whereas crystals were obtained from native dog gastric lipase and all the purified isoforms of rabbit and human gastric lipases, some of which were crystallographically characterized.
ESTHER : Moreau_1992_J.Mol.Biol_225_147
PubMedSearch : Moreau_1992_J.Mol.Biol_225_147
PubMedID: 1583687

Title : 1.9-A resolution structure of fasciculin 1, an anti- acetylcholinesterase toxin from green mamba snake venom - le Du_1992_J.Biol.Chem_267_22122
Author(s) : le Du MH , Marchot P , Bougis PE , Fontecilla-Camps JC
Ref : Journal of Biological Chemistry , 267 :22122 , 1992
Abstract : The crystal structure of fasciculin 1, a potent acetylcholinesterase inhibitor from green mamba snake venom, has been solved by the multiple isomorphous replacement method complemented with anomalous scattering and subsequently refined at 1.9-A resolution. The overall structure of fasciculin is similar to those of the short alpha-neurotoxins and cardiotoxins, with a dense core rich in disulfide bridges and three long loops disposed as the central fingers of a hand. A comparison of these three prototypic toxin types shows that fasciculin 1 has structural features that are intermediate between those of the other two molecules. Its core region, which can be defined as a continuous stretch of conserved residues, is very similar to that of erabutoxin b, whereas the orientation of its long loops resembles that of cardiotoxin VII4. This result introduces a new element in the study of phylogenetic relationships of snake toxins and suggests that, after divergency from an ancestral gene, convergent evolution may have played an important factor in the evolution of these proteins. In fasciculin 1, several arginine and lysine residues are well ordered and relatively exposed to the solvent medium and may play a role in the binding to the peripheral site of acetylcholinesterases.
ESTHER : le Du_1992_J.Biol.Chem_267_22122
PubMedSearch : le Du_1992_J.Biol.Chem_267_22122
PubMedID: 1429564

Title : Crystallization and preliminary X-ray study of a recombinant cutinase from Fusarium solani pisi - Abergel_1990_J.Mol.Biol_215_215
Author(s) : Abergel C , Martinez C , Fontecilla-Camps JC , Cambillau C , de Geus P , Lauwereys M
Ref : Journal of Molecular Biology , 215 :215 , 1990
Abstract : Recombinant cutinase from Fusarium solani pisi is expressed and excreted with very high yields in Escherichia coli cultures. Cutinase was crystallized at 20 degrees C using the vapour diffusion technique, with polyethylene glycol 6000 as precipitant. Best crystals were obtained at pH 7.0 with polyethylene glycol 6000 as precipitant. Best crystals were obtained at pH 7.0 with polyethylene glycol at 15 to 20%. They are monoclinic, with space group P2(1) and cell dimensions a = 35.1 A, b = 67.4 A, c = 37.05 A and beta = 94.0 degrees; they diffract beyond 1.5 A resolution. The asymmetric unit contains one molecule of 22,000 Da (Vm = 1.98 A3/Da; 38% water).
ESTHER : Abergel_1990_J.Mol.Biol_215_215
PubMedSearch : Abergel_1990_J.Mol.Biol_215_215
PubMedID: 2213880

Title : Crystals of fasciculin 2 from green mamba snake venom. Preparation and preliminary x-ray analysis - le Du_1989_J.Biol.Chem_264_21401
Author(s) : le Du MH , Marchot P , Bougis PE , Fontecilla-Camps JC
Ref : Journal of Biological Chemistry , 264 :21401 , 1989
Abstract : Fasciculin 2 from the venom of the green mamba, Dendroaspis angusticeps, has been crystallized. The crystals are tetragonal, with unit cell dimensions a = 48.9 A and c = 82.0 A, space group P 41 21 2 or P 43212. Density measurements and pseudocentering of the hko zone indicate that there are 16 molecules in the unit cell.
ESTHER : le Du_1989_J.Biol.Chem_264_21401
PubMedSearch : le Du_1989_J.Biol.Chem_264_21401
PubMedID: 2592383