Thierry Foulon

Publications (19)43.67 Total impact

• Source

  Available from: Sereno Denis

  Article: Antibacterial and leishmanicidal activities of temporin-SHd, a 17-residue long membrane-damaging peptide.

  Feten Abbassi, Zahid Raja, Bruno Oury, Elodie Gazanion, Christophe Piesse, Denis Sereno, Pierre Nicolas, Thierry Foulon, Ali Ladram
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  ABSTRACT: Temporins are a family of short antimicrobial peptides (8-17 residues) that mostly show potent activity against Gram-positive bacteria. Herein, we demonstrate that temporin-SHd, a 17-residue peptide with a net charge of +2 (FLPAALAGIGGILGKLF(amide)), expressed a broad spectrum of antimicrobial activity. This peptide displayed potent antibacterial activities against Gram-negative and Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus strains, as well as antiparasitic activity against promastigote and the intracellular stage (amastigote) of Leishmania infantum, at concentration not toxic for the macrophages. Temporin-SHd that is structured in a non-amphipathic α-helix in anionic membrane-mimetic environments, strongly and selectively perturbs anionic bilayer membranes by interacting with the polar head groups and acyl region of the phospholipids, with formation of regions of two coexisting phases: one phase rich in peptide and the other lipid-rich. The disruption of lipid packing within the bilayer may lead to the formation of transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. To our knowledge, Temporin-SHd represents the first known 17-residue long temporin expressing such broad spectrum of antimicrobial activity including members of the trypanosomatidae family. Additionally, since only a few shorter members (13 residues) of the temporin family are known to display antileishmanial activity (temporins-TA, -TB and -SHa), SHd is an interesting tool to analyze the antiparasitic mechanism of action of temporins.
  Biochimie 10/2012; · 3.02 Impact Factor
• Chapter: Chapter 6 Arginyl Aminopeptidase

  Sandrine Cadel, Christophe Piesse, Cécile Gouzy-Darmon, Paul Cohen, Thierry Foulon
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  ABSTRACT: Aminopeptidase B (Ap-B; EC.3.4.11.6) was originally defined as an exopeptidase able to trim out basic amino acid residues from the Nt2-terminus of peptides. Purification of Ap-B from rat testes showed that this enzyme is a monomeric 72 kDa Zn2+-dependent exopeptidase, which selectively removes Arg and/or Lys residues from the NH2-terminus of various peptides. In vitro, Ap-B exhibits a weak ability to hydrolyze leukotriene A4 into leukotriene B4, a lipid mediator of inflammation. The in vivo bi-functionality of Ap-B remains to be demonstrated. Elucidation of the rat, human and mouse Ap-B primary structures allows its classification in the Ml family of Zn2+-aminopeptidases and reveals a structural relationship with leukotriene A4 hydrolase, an important enzyme of the arachidonic pathway. The human Ap-B gene (rnpep) is localized on chromosome 1 band q32 in a high transcript density chromosomal region. The gene is bracketed by tim17a and elf3, which encode a pre-protein translocase of the inner mitochondrial membrane and an ETS family transcription factor, respectively. The recent description of the mouse genome allows to localize the mouse Ap-B encoding gene on chromosome 1, in a putative inversed synthenic region. Ap-B is widely distributed in a number of rat and human tissues. Ap-B expression level varies depending on the cells or tissues considered and likely in a species-dependent manner. Both the constitutive and the regulated pathways secrete the enzyme. Moreover, in PC12 cells, the protein is associated, as an active form, to the external face of the plasma membrane. Although the physiological function of Ap-B remains an open question, several data strongly support the hypothesis that Ap-B could participate in the final stages of precursor processing mechanisms and thereby in some inflammatory processes and tumour developments. Key wordsZn2+-metallopeptidase–aminopeptidase B–leukotriene A4 hydrolase–prohormone processing
  07/2011: pages 113-126;
• Article: Mutation in the substrate-binding site of aminopeptidase B confers new enzymatic properties.

  Viet-Laï Pham, Cécile Gouzy-Darmon, Julien Pernier, Chantal Hanquez, Vivian Hook, Margery C Beinfeld, Pierre Nicolas, Catherine Etchebest, Thierry Foulon, Sandrine Cadel
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  ABSTRACT: Aminopeptidase B (Ap-B) catalyzes the cleavage of arginine and lysine residues at the N-terminus of various peptide substrates. In vivo, it participates notably in the miniglucagon and cholecystokinin 8 processing, but the complete range of physiological functions of Ap-B remains to be discovered. Ap-B is a member of the M1 family of Zn(2+)-metallopeptidases that are characterized by two highly conserved motives, GXMEN (potential substrate binding site) and HEXXHX(18)E (Zn(2+)-binding site). In this study, mutagenesis and molecular modelling were used to investigate the enzymatic mechanism of Ap-B. Nineteen rat Ap-B mutants of the G(298)XM(300)E(301)N(302) motif and one mutant of the HEIS(328)HX(18)E motif were expressed in Escherichia coli. All mutations except G(298)P, G(298)S, and S(328)A abolished the aminopeptidase activity. The S(328)A mutant mimics the sequence of bovine Ap-B Zn(2+)-binding site, which differs from those of other mammalian Ap-B. This mutant conserved a canonical Ap-B activity. G(298)S and G(298)P mutants exhibit new enzymatic properties such as changes in their profile of inhibition and their sensitivity to Cl(-) anions. Moreover, the G(298)P mutant exhibits new substrate specificity. A structural analysis using circular dichroism, fluorescence spectroscopy, molecular modelling and dynamics was performed to investigate the role that residue G(298) plays in the catalytic mechanism of Ap-B. Our results show that G(298) is essential to Ap-B activity and participates to the substrate specificity of the enzyme.
  Biochimie 01/2011; 93(4):730-41. · 3.02 Impact Factor
• Article: Temporin-SHf, a New Type of Phe-rich and Hydrophobic Ultrashort Antimicrobial Peptide

  Feten Abbassi, Olivier Lequin, Christophe Piesse, Nicole Goasdoué, Thierry Foulon, Pierre Nicolas, Ali Ladram
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  ABSTRACT: Because issues of cost and bioavailability have hampered the development of gene-encoded antimicrobial peptides to combat infectious diseases, short linear peptides with high microbial cell selectivity have been recently considered as antibiotic substitutes. A new type of short antimicrobial peptide, designated temporin-SHf, was isolated and cloned from the skin of the frog Pelophylax saharica. Temporin-SHf has a highly hydrophobic sequence (FFFLSRIFa) and possesses the highest percentage of Phe residues of any known peptide or protein. Moreover, it is the smallest natural linear antimicrobial peptide found to date, with only eight residues. Despite its small size and hydrophobicity, temporin-SHf has broad-spectrum microbicidal activity against Gram-positive and Gram-negative bacteria and yeasts, with no hemolytic activity. CD and NMR spectroscopy combined with restrained molecular dynamics calculations showed that the peptide adopts a well defined non-amphipathic α-helical structure from residue 3 to 8, when bound to zwitterionic dodecyl phosphocholine or anionic SDS micelles. Relaxation enhancement caused by paramagnetic probes showed that the peptide adopts nearly parallel orientations to the micelle surface and that the helical structure is stabilized by a compact hydrophobic core on one face that penetrates into the micelle interior. Differential scanning calorimetry on multilamellar vesicles combined with membrane permeabilization assays on bacterial cells indicated that temporin-SHf disrupts the acyl chain packing of anionic lipid bilayers, thereby triggering local cracks and microbial membrane disintegration through a detergent-like effect probably via the carpet mechanism. The short length, compositional simplicity, and broad-spectrum activity of temporin-SHf make it an attractive candidate to develop new antibiotic agents.
  Journal of Biological Chemistry 05/2010; 285(22):16880-16892. · 4.77 Impact Factor
• Article: Temporin-SHf, a new type of phe-rich and hydrophobic ultrashort antimicrobial peptide.

  Feten Abbassi, Olivier Lequin, Christophe Piesse, Nicole Goasdoué, Thierry Foulon, Pierre Nicolas, Ali Ladram
  [show abstract] [hide abstract]
  ABSTRACT: Because issues of cost and bioavailability have hampered the development of gene-encoded antimicrobial peptides to combat infectious diseases, short linear peptides with high microbial cell selectivity have been recently considered as antibiotic substitutes. A new type of short antimicrobial peptide, designated temporin-SHf, was isolated and cloned from the skin of the frog Pelophylax saharica. Temporin-SHf has a highly hydrophobic sequence (FFFLSRIFa) and possesses the highest percentage of Phe residues of any known peptide or protein. Moreover, it is the smallest natural linear antimicrobial peptide found to date, with only eight residues. Despite its small size and hydrophobicity, temporin-SHf has broad-spectrum microbicidal activity against Gram-positive and Gram-negative bacteria and yeasts, with no hemolytic activity. CD and NMR spectroscopy combined with restrained molecular dynamics calculations showed that the peptide adopts a well defined non-amphipathic alpha-helical structure from residue 3 to 8, when bound to zwitterionic dodecyl phosphocholine or anionic SDS micelles. Relaxation enhancement caused by paramagnetic probes showed that the peptide adopts nearly parallel orientations to the micelle surface and that the helical structure is stabilized by a compact hydrophobic core on one face that penetrates into the micelle interior. Differential scanning calorimetry on multilamellar vesicles combined with membrane permeabilization assays on bacterial cells indicated that temporin-SHf disrupts the acyl chain packing of anionic lipid bilayers, thereby triggering local cracks and microbial membrane disintegration through a detergent-like effect probably via the carpet mechanism. The short length, compositional simplicity, and broad-spectrum activity of temporin-SHf make it an attractive candidate to develop new antibiotic agents.
  Journal of Biological Chemistry 03/2010; 285(22):16880-92. · 4.77 Impact Factor
• Article: Cathepsin L plays a major role in cholecystokinin production in mouse brain cortex and in pituitary AtT-20 cells: protease gene knockout and inhibitor studies.

  Margery C Beinfeld, Lydiane Funkelstein, Thierry Foulon, Sandrine Cadel, Kouki Kitagawa, Thomas Toneff, Thomas Reinheckel, Christoph Peters, Vivian Hook
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  ABSTRACT: Cholecystokinin (CCK) is a peptide neurotransmitter whose production requires proteolytic processing of the proCCK precursor to generate active CCK8 neuropeptide in brain. This study demonstrates the significant role of the cysteine protease cathepsin L for CCK8 production. In cathepsin L knockout (KO) mice, CCK8 levels were substantially reduced in brain cortex by an average of 75%. To evaluate the role of cathepsin L in producing CCK in the regulated secretory pathway of neuroendocrine cells, pituitary AtT-20 cells that stably produce CCK were treated with the specific cathepsin L inhibitor, CLIK-148. CLIK-148 inhibitor treatment resulted in decreased amounts of CCK secreted from the regulated secretory pathway of AtT-20 cells. CLIK-148 also reduced cellular levels of CCK9 (Arg-CCK8), consistent with CCK9 as an intermediate product of cathepsin L, shown by the decreased ratio of CCK9/CCK8. The decreased CCK9/CCK8 ratio also suggests a shift in the production to CCK8 over CCK9 during inhibition of cathepsin L. During reduction of the PC1/3 processing enzyme by siRNA, the ratio of CCK9/CCK8 was increased, suggesting a shift to the cathepsin L pathway for the production of CCK9. The changes in ratios of CCK9 compared to CCK8 are consistent with dual roles of the cathepsin L protease pathway that includes aminopeptidase B to remove NH2-terminal Arg or Lys, and the PC1/3 protease pathway. These results suggest that cathepsin L functions as a major protease responsible for CCK8 production in mouse brain cortex, and participates with PC1/3 for CCK8 production in pituitary cells.
  Peptides 08/2009; 30(10):1882-91. · 2.43 Impact Factor
• Article: Secretory vesicle aminopeptidase B related to neuropeptide processing: molecular identification and subcellular localization to enkephalin- and NPY-containing chromaffin granules.

  Shin-Rong Hwang, Audrey O'Neill, Steven Bark, Thierry Foulon, Vivian Hook
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  ABSTRACT: Biosynthesis of peptide hormones and neurotransmittters involves proteolysis of proprotein precursors by secretory vesicle cathepsin L. Cathepsin L generates peptide intermediates with basic residues at their NH(2)-termini, indicating that Arg/Lys aminopeptidase is needed to generate the smaller biologically active peptide. Therefore, this study identified the Arg/Lys aminopeptidase that is present in secretory vesicles of adrenal medulla and neuroendocrine tissues, achieved by molecular cloning and localization in 'model' neuropeptide-containing secretory vesicles (bovine). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA defined its primary sequence that allowed selection of antisera for immunolocalization studies. AP-B was present in secretory vesicles that contain cathepsin L with the neuropeptides enkephalin and neuropeptide Y. The AP-B in several neuroendocrine tissues was detected by western blots. Recombinant bovine AP-B showed preference for Arg-methylcoumarinamide substrate. AP-B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP-B showed similar activities for Arg-(Met)enkephalin (ME) and Lys-ME neuropeptide substrates to generate ME, while rat AP-B preferred Arg-ME. Furthermore, AP-B possesses an acidic pH optimum of 5.5-6.5 that is similar to the internal pH of secretory vesicles. The significant finding of the secretory vesicle localization of AP-B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides.
  Journal of Neurochemistry 04/2007; 100(5):1340-50. · 4.06 Impact Factor
• Source

  Available from: Margery C Beinfeld

  Article: Aminopeptidase B, a glucagon-processing enzyme: site directed mutagenesis of the Zn2+-binding motif and molecular modelling.

  Viet-Laï Pham, Marie-Sandrine Cadel, Cécile Gouzy-Darmon, Chantal Hanquez, Margery C Beinfeld, Pierre Nicolas, Catherine Etchebest, Thierry Foulon
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  ABSTRACT: Aminopeptidase B (Ap-B; EC 3.4.11.6) catalyzes the cleavage of basic residues at the N-terminus of peptides and processes glucagon into miniglucagon. The enzyme exhibits, in vitro, a residual ability to hydrolyze leukotriene A4 into the pro-inflammatory lipid mediator leukotriene B4. The potential bi-functional nature of Ap-B is supported by close structural relationships with LTA4 hydrolase (LTA4H ; EC 3.3.2.6). A structure-function analysis is necessary for the detailed understanding of the enzymatic mechanisms of Ap-B and to design inhibitors, which could be used to determine the complete in vivo functions of the enzyme. The rat Ap-B cDNA was expressed in E. coli and the purified recombinant enzyme was characterized. 18 mutants of the H325EXXHX18E348 Zn2+-binding motif were constructed and expressed. All mutations were found to abolish the aminopeptidase activity. A multiple alignment of 500 sequences of the M1 family of aminopeptidases was performed to identify 3 sub-families of exopeptidases and to build a structural model of Ap-B using the x-ray structure of LTA4H as a template. Although the 3D structures of the two enzymes resemble each other, they differ in certain details. The role that a loop, delimiting the active center of Ap-B, plays in discriminating basic substrates, as well as the function of consensus motifs, such as RNP1 and Armadillo domain are discussed. Examination of electrostatic potentials and hydrophobic patches revealed important differences between Ap-B and LTA4H and suggests that Ap-B is involved in protein-protein interactions. Alignment of the primary structures of the M1 family members clearly demonstrates the existence of different sub-families and highlights crucial residues in the enzymatic activity of the whole family. E. coli recombinant enzyme and Ap-B structural model constitute powerful tools for investigating the importance and possible roles of these conserved residues in Ap-B, LTA4H and M1 aminopeptidase catalytic sites and to gain new insight into their physiological functions. Analysis of Ap-B structural model indicates that several interactions between Ap-B and proteins can occur and suggests that endopeptidases might form a complex with Ap-B during hormone processing.
  BMC Biochemistry 02/2007; 8:21. · 1.99 Impact Factor
• Article: Aminopeptidase B, a glucagon-processing enzyme: site directed mutagenesis of the Zn²⁺-binding motif and molecular modelling

  Viet-Laï Pham, Marie-Sandrine Cadel, Cécile Gouzy-Darmon, Chantal Hanquez, Margery Beinfeld, Pierre Nicolas, Catherine Etchebest, Thierry Foulon
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  ABSTRACT: Abstract Background Aminopeptidase B (Ap-B; EC 3.4.11.6) catalyzes the cleavage of basic residues at the N-terminus of peptides and processes glucagon into miniglucagon. The enzyme exhibits, in vitro , a residual ability to hydrolyze leukotriene A₄into the pro-inflammatory lipid mediator leukotriene B₄. The potential bi-functional nature of Ap-B is supported by close structural relationships with LTA₄hydrolase (LTA₄H ; EC 3.3.2.6). A structure-function analysis is necessary for the detailed understanding of the enzymatic mechanisms of Ap-B and to design inhibitors, which could be used to determine the complete in vivo functions of the enzyme. Results The rat Ap-B cDNA was expressed in E. coli and the purified recombinant enzyme was characterized. 18 mutants of the H³²⁵EXXHX₁₈E³⁴⁸Zn²⁺-binding motif were constructed and expressed. All mutations were found to abolish the aminopeptidase activity. A multiple alignment of 500 sequences of the M1 family of aminopeptidases was performed to identify 3 sub-families of exopeptidases and to build a structural model of Ap-B using the x-ray structure of LTA₄H as a template. Although the 3D structures of the two enzymes resemble each other, they differ in certain details. The role that a loop, delimiting the active center of Ap-B, plays in discriminating basic substrates, as well as the function of consensus motifs, such as RNP1 and Armadillo domain are discussed. Examination of electrostatic potentials and hydrophobic patches revealed important differences between Ap-B and LTA₄H and suggests that Ap-B is involved in protein-protein interactions. Conclusion Alignment of the primary structures of the M1 family members clearly demonstrates the existence of different sub-families and highlights crucial residues in the enzymatic activity of the whole family. E. coli recombinant enzyme and Ap-B structural model constitute powerful tools for investigating the importance and possible roles of these conserved residues in Ap-B, LTA₄H and M1 aminopeptidase catalytic sites and to gain new insight into their physiological functions. Analysis of Ap-B structural model indicates that several interactions between Ap-B and proteins can occur and suggests that endopeptidases might form a complex with Ap-B during hormone processing.
  BMC Biochemistry. 01/2007;
• Article: Two Novel Metallopeptidases with a Specificity for Basic Residues Functional Properties, Structure, and Cellular Distributiona

  THIERRY FOULON, SANDRINE CADEL, VALÉRIE CHESNEAU, MURIEL DRAOUI, ANNIK PRAT, PAUL COHEN
  Annals of the New York Academy of Sciences 12/2006; 780(1):106 - 120. · 3.15 Impact Factor
• Article: Miniglucagon (MG)-generating endopeptidase, which processes glucagon into MG, is composed of N-arginine dibasic convertase and aminopeptidase B.

  Ghislaine Fontés, Anne-Dominique Lajoix, François Bergeron, Sandrine Cadel, Annik Prat, Thierry Foulon, René Gross, Stéphane Dalle, Dung Le-Nguyen, Florence Tribillac, Dominique Bataille
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  ABSTRACT: Miniglucagon (MG), the C-terminal glucagon fragment, processed from glucagon by the MG-generating endopeptidase (MGE) at the Arg17-Arg18 dibasic site, displays biological effects opposite to that of the mother-hormone. This secondary processing occurs in the glucagon- and MG-producing alpha-cells of the islets of Langerhans and from circulating glucagon. We first characterized the enzymatic activities of MGE in culture media from glucagon and MG-secreting alphaTC1.6 cells as made of a metalloendoprotease and an aminopeptidase. We observed that glucagon is a substrate for N-arginine dibasic convertase (NRDc), a metalloendoprotease, and that aminopeptidase B cleaves in vitro the intermediate cleavage products sequentially, releasing mature MG. Furthermore, immunodepletion of either enzyme resulted in the disappearance of the majority of MGE activity from the culture medium. We found RNAs and proteins corresponding to both enzymes in different cell lines containing a MGE activity (mouse alphaTC1.6 cells, rat hepatic FaO, and rat pituitary GH4C1). Using confocal microscopy, we observed a granular immunostaining of both enzymes in the alphaTC1.6 and native rat alpha-cells from islets of Langerhans. By immunogold electron microscopy, both enzymes were found in the mature secretory granules of alpha-cells, close to their substrate (glucagon) and their product (MG). Finally, we found NRDc only in the fractions from perfused pancreas that contain glucagon and MG after stimulation by hypoglycemia. We conclude that MGE is composed of NRDc and aminopeptidase B acting sequentially, providing a molecular basis for this uncommon regulatory process, which should be now addressed in both physiological and pathophysiological situations.
  Endocrinology 02/2005; 146(2):702-12. · 4.46 Impact Factor
• Article: Expression of aminopeptidase B in the developing and adult rat retina.

  Christophe Piesse, Sandrine Cadel, Cécile Gouzy-Darmon, Jean-Claude Jeanny, Véronique Carrière, Didier Goidin, Laurent Jonet, Danielle Gourdji, Paul Cohen, Thierry Foulon
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  ABSTRACT: Aminopeptidase B (Ap-B), a ubiquitous enzyme, catalyses the amino-terminal cleavage of basic residues of peptide or protein substrates, indicating a role in precursor processing. The physiological function of Ap-B still remains an open question, even though its activity suggests that it could be involved in inflammatory processes and proliferation of tumor cells. This study was conducted to determine the expression of Ap-B in the developing and adult retina as a path to envisage physiological roles of Ap-B. RT-PCR and in situ hybridization were used to detect expression of Ap-B mRNA and activity tests, Western blotting and immunofluorescence microscopy were performed to identify and localize the enzyme in the rat retina. These biochemical and morphological methods show that Ap-B is expressed in the retina from embryo to adult. Expression level is restricted to specific layers (pigmented epithelium, outer and inner plexiform layers and ganglion cell layer) and is developmentally regulated. Moreover, a preliminary analysis indicates that Ap-B, the glucose transporter GLUT3 and choline acetyltransferase (ChAT) share a similar expression pattern in retina. Altogether, Ap-B appears predominantly expressed in neuronal cells lying in retinal layers containing neuritic extensions and synaptic junctions. Such expression is up-regulated during ontogenesis allowing to hypothesized that Ap-B participates in processes accompanying retinal neuronal cell differentiation.
  Experimental Eye Research 12/2004; 79(5):639-48. · 3.26 Impact Factor
• Article: Expression and purification of rat recombinant aminopeptidase B secreted from baculovirus-infected insect cells.

  Sandrine Cadel, Cécile Gouzy-Darmon, Stéphane Petres, Christophe Piesse, Viet-Laï Pham, Margery C Beinfeld, Paul Cohen, Thierry Foulon
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  ABSTRACT: Aminopeptidase B (Ap-B) is a ubiquitous enzyme and its physiological function still remains an open question. This Zn2+ -exopeptidase catalyzes the amino-terminal cleavage of basic residues of peptide or protein substrates, indicating a role in precursor processing. In addition, the enzyme exhibits a residual capacity to hydrolyze leukotriene A4 (LTA4) into the pro-inflammatory lipid mediator leukotriene B4 (LTB4) in vitro. This potential bi-functional nature of Ap-B is supported by a close structural relationship with LTA4 hydrolase, which hydrolyzes LTA4 into LTB4, in vivo, and exhibits an aminopeptidase activity, in vitro. Structural studies are necessary for the detailed understanding of the bi-functional enzymatic mechanism of Ap-B. In this study, we report cDNA cloning, baculovirus expression, and purification of the rat Ap-B (rAp-B). The Ap-B cDNA was constructed from extracted rat testes total RNA and introduced into the pBAC1 baculovirus transfer vector to generate recombinant baculoviruses. rAp-B expression, with or without COOH-hexahistidine tag, was tested in two different insect cell hosts (Sf9 and H5). The enzyme is secreted into the insect cell culture medium, which allowed a rapid purification of the protein. The His-tagged rAp-B was purified using metal affinity resin while the native recombinant rAp-B was partially purified using a single step DEAE Trisacryl ion exchange column. Although the recombinant rAp-B exhibits biochemical properties equivalent to those of the rat testes purified protein, the presence of the histidine-tag seems to partially inhibit the exopeptidase activity. However, this report shows that baculovirus-infected cells are a useful system to produce rat Ap-B for use in studying enzymatic mechanisms in vitro and 3D structure.
  Protein Expression and Purification 08/2004; 36(1):19-30. · 1.59 Impact Factor
• Article: Human aminopeptidase B (rnpep) on chromosome 1q32.2: complementary DNA, genomic structure and expression.

  Christophe Piesse, Martin Tymms, Emirena Garrafa, Cécile Gouzy, Michel Lacasa, Sandrine Cadel, Paul Cohen, Thierry Foulon
  [show abstract] [hide abstract]
  ABSTRACT: Aminopeptidase B (APB) is a Zn(2+)-metalloexopeptidase, which selectively removes Arg and/or Lys residues from the N-terminus of several peptide substrates. Several data strongly support the hypothesis that this enzyme could participate in the final stages of precursor processing mechanisms and/or in particular inflammatory processes and tumor developments. Therefore, we have cloned the complementary DNA encoding the human APB, a 658-residues protein, containing the canonical "HEXXH(X(18))E", a signature allowing its classification in the M1 family of metallopeptidases. The genomic structure of the human APB gene (rnpep; 1q32.1-q32.2) was also determined. rnpep is bracketed by pre-protein translocase of the inner mitochondrial membrane gene and ETS family transcription factor ELF3 gene. It spans more than 24 kbp and contains 11 exons ranging from 109 to 574 bp. Finally, expression of the human APB messenger RNA (mRNA) was investigated using a pre-made dot-blot. This mRNA seems to be ubiquitous although its expression level varies depending of the cells or tissues considered.
  Gene 07/2002; 292(1-2):129-40. · 2.34 Impact Factor
• Article: Production, purification, and characterization of recombinant prohormone convertase 5 from baculovirus-infected insect cells.

  Brian M Cain, Daesety Vishnuvardhan, Wenge Wang, Thierry Foulon, Sandrine Cadel, Paul Cohen, Margery C Beinfeld
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  ABSTRACT: The discovery of the prohormone convertase (PC) family of enzymes has provided several good candidates (PC1, PC2, and PC5) for the enzymes responsible for the endoproteolytic cleavage of procholecystokinin (pro-CCK). Determination of the role of individual pro-hormone convertases in the processing of pro-CCK is complicated because several of these enzymes are found in endocrine tumor cells expressing CCK mRNA and in identified neurons in the brain. Production of active recombinant PC5 permits the determination of its ability to cleave substrates related to pro-CCK. Active PC5, secreted from baculovirus-infected Sf9 cells, was partially purified by ion-exchange chromatography. Western blot analysis confirmed the presence of the active form of the enzyme in infected cell media and its absence from uninfected cell media. The enzyme is most active at acidic pH 6.5 and is maximally activated by 5 mM calcium. PC5 was able to cleave both monobasic and dibasic substrates without a requirement for a basic residue at P-4 and it displayed a K(m) in the micromolar range. The enzyme was inhibited by EDTA, 1,10-phenanthroline, and p-CMS, as well as by two specific PC inhibitors. This is the first reported preparation of active recombinant PC5. Like the other members of its family, it has the correct catalytic characteristics in vitro to play a role in the processing of neuropeptide precursor proteins into their final bioactive forms.
  Protein Expression and Purification 04/2002; 24(2):227-33. · 1.59 Impact Factor
• Article: Organization of a molecular biology workshop in Tunisia

  Ali Ladram, Jean-Jacques Montagne, Mohamed Amiche, Thierry Foulon, Sandrine Cadel, Marie-Christine Chauviere, Cécile Gouzy, Paul Cohen, Hamadi Boussetta
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  ABSTRACT: A five day laboratory course in Molecular Biology for PhD students is proposed. The aim of this program, which can be taught by a relatively small staff of experienced scientists, was to introduce basic experimental techniques which are of general use in various fields of biology and biomedicine. These include restriction enzyme purification, restriction mapping, polymerase chain reaction, cloning and DNA sequencing.
  Biochemical Education. 12/1998; 27(1):2 - 6.
• Article: Expression and retinoid modulation of N-arginine dibasic convertase and an aminopeptidase-B in human neuroblastoma cell lines

  Muriel Draoui, Lorenza Bellincampi, Véronique Hospital, Sandrine Cadel, Thierry Foulon, Annik Prat, Nicole Barré, Uwe Reichert, Gerry Melino, Paul Cohen
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  ABSTRACT: Under retinoic acid exposure, the three SK-N-BE(2)-derived human neuroblastoma cell lines, BE(2)-NA, BE(2)-SA and BE(2)-M17 undergo mainly differentiation, apoptosis or continue to proliferate, respectively. We have used this model system to study the modulation of the transcriptional expression of putative processing enzymes, two novel metallopeptidases; i.e. N-arginine dibasic convertase (NRD convertase; EC 3.4.24.61) and an aminopeptidase-B after exposure of the cells either to retinoic acid or to synthetic retinoid analogs. The data indicate that the two respective enzymes are differently modulated in the various cell lines. Whereas aminopeptidase-B expression is enhanced in most cases, NRD convertase appears to undergo opposite regulation in proliferating versus differentiating neuroblastoma cells. It is concluded that both genes might contain retinoic acid regulatory elements (RARE) in their promoters.
  Journal of Neuro-Oncology 12/1996; 31(1):99-106. · 3.21 Impact Factor
• Article: Aminopeptidase-B in the rat testes: isolation, functional properties and cellular localization in the seminiferous tubules

  Sandrine Cadel, Adrian R. Pierotti, Thierry Foulon, Christophe Cre´minon, Nicole Barre´, Dominique Segre´tain, Paul Cohen
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  ABSTRACT: An aminopeptidase of the B-type, with an apparent Mr 72 000 and pI = 4.9, was isolated from rat testes and characterized. The enzyme was able to remove only Arg and/or Lys residues from l-amino acid β-naphthylamide derivatives and from the N-terminus of several peptides. No cleavage occurred in the case of Arg-Pro bonds as found in bradykinin and substance P. The enzyme was sensitive to cysteinyl reagents and to aminopeptidase inhibitors, such as bestatin, amastatin and arphamenines A and B. The aminopeptidase activity, tested with l-Arg β-naphthylamide and with Arg0-Met-enkephalin as substrates, was inhibited by o-phenanthroline, and restored by Zn2+ suggesting its metallopeptidase character. The partial characterization of an aminopeptidase-B activity in rat brain cortex identified a protein which is biochemically and immunologically related to the testis enzyme. By immunohistochemistry, the aminopeptidase-B was found to be particularly abundant in the seminiferous tubules at late stages of spermatogenesis and was clearly detected in a restricted area of elongated spermatids. Remarkably, the enzyme was observed to concentrate massively in the residual bodies. Since this aminopeptidase-B was able in vitro to trim out N-terminal Arg and/or Lys residues from peptides mimicking processing intermediates, it is proposed that this enzyme may be involved in propeptide and proprotein processing mechanisms in the course of spermatid differentiation.
  Molecular and Cellular Endocrinology.
• Source

  Available from: endojournals.org

  Article: Miniglucagon-generating endopeptidase, which processes glucagon into miniglucagon, is composed of NRD convertase and aminopeptidase B

  Ghislaine Fontés, Anne-Dominique Lajoix, François Bergeron, Sandrine Cadel, Annik Prat, Thierry Foulon, René Gross, Stéphane Dalle, Dung Le-Nguyen, Florence Tribillac, Dominique Bataille