F Schuber

University of Strasbourg, Strasburg, Alsace, France

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Publications (126)397.23 Total impact

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    ABSTRACT: Bovine CD38/NAD(+) glycohydrolase catalyzes the hydrolysis of NAD(+) to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism. Our recent crystallographic study of its Michaelis complex and covalently-trapped intermediates provided insights into the modalities of substrate binding and the molecular mechanism of bCD38. The aim of the present work was to determine the precise role of key conserved active site residues (Trp118, Glu138, Asp147, Trp181 and Glu218) by focusing mainly on the cleavage of the nicotinamide-ribosyl bond. We analyzed the kinetic parameters of mutants of these residues which reside within the bCD38 subdomain in the vicinity of the scissile bond of bound NAD(+). To address the reaction mechanism we also performed chemical rescue experiments with neutral (methanol) and ionic (azide, formate) nucleophiles. The crucial role of Glu218, which orients the substrate for cleavage by interacting with the N-ribosyl 2'-OH group of NAD(+), was highlighted. This contribution to catalysis accounts for almost half of the reaction energy barrier. Other contributions can be ascribed notably to Glu138 and Asp147 via ground-state destabilization and desolvation in the vicinity of the scissile bond. Key interactions with Trp118 and Trp181 were also proven to stabilize the ribooxocarbenium ion-like transition state. Altogether we propose that, as an alternative to a covalent acylal reaction intermediate with Glu218, catalysis by bCD38 proceeds through the formation of a discrete and transient ribooxocarbenium intermediate which is stabilized within the active site mostly by electrostatic interactions.
    Biochimica et Biophysica Acta 04/2014; · 4.66 Impact Factor
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    ABSTRACT: Bovine CD38/NAD+ glycohydrolase catalyzes the hydrolysis of NAD+ to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism. Our recent crystallographic study of its Michaelis complex and covalently-trapped intermediates provided insights into the modalities of substrate binding and the molecular mechanism of bCD38. The aim of the present work was to determine the precise role of key conserved active site residues (Trp118, Glu138, Asp147, Trp181 and Glu218) by focusing mainly on the cleavage of the nicotinamide-ribosyl bond. We analyzed the kinetic parameters of mutants of these residues which reside within the bCD38 subdomain in the vicinity of the scissile bond of bound NAD+. To address the reaction mechanism we also performed chemical rescue experiments with neutral (methanol) and ionic (azide, formate) nucleophiles. The crucial role of Glu218, which orients the substrate for cleavage by interacting with the N-ribosyl 2’-OH group of NAD+, was highlighted. This contribution to catalysis accounts for almost half of the reaction energy barrier. Other contributions can be ascribed notably to Glu138 and Asp147 via ground-state destabilization and desolvation in the vicinity of the scissile bond. Key interactions with Trp118 and Trp181 were also proven to stabilize the ribooxocarbenium ion-like transition state. Altogether we propose that, as an alternative to a covalent acylal reaction intermediate with Glu218, catalysis by bCD38 proceeds through the formation of a discrete and transient ribooxocarbenium intermediate which is stabilized within the active site mostly by electrostatic interactions.
    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 01/2014; · 3.73 Impact Factor
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    ABSTRACT: Schistosoma mansoni NAD(+) catabolizing enzyme (SmNACE), a distant homolog of mammalian CD38, shows significant structural and functional analogy to the members of the CD38/ADP-ribosyl cyclase family. The hallmark of SmNACE is the lack of ADP-ribosyl cyclase activity that might be ascribed to subtle changes in its active site. To better characterize the residues of the active site we determined the kinetic parameters of nine mutants encompassing three acidic residues: (i) the putative catalytic residue Glu202 and (ii) two acidic residues within the 'signature' region (the conserved Glu124 and the downstream Asp133), (iii) Ser169, a strictly conserved polar residue and (iv) two aromatic residues (His103 and Trp165). We established the very important role of Glu202 and of the hydrophobic domains overwhelmingly in the efficiency of the nicotinamide-ribosyl bond cleavage step. We also demonstrated that in sharp contrast with mammalian CD38, the 'signature' Glu124 is as critical as Glu202 for catalysis by the parasite enzyme. The different environments of the two Glu residues in the crystal structure of CD38 and in the homology model of SmNACE could explain such functional discrepancies. Mutagenesis data and 3D structures also indicated the importance of aromatic residues, especially His103, in the stabilization of the reaction intermediate as well as in the selection of its conformation suitable for cylization to cyclic ADP-ribose. Finally, we showed that inhibition of SmNACE by the natural product cyanidin requires the integrity of Glu202 and Glu124, but not of His103 and Trp165, hence suggesting different recognition modes for substrate and inhibitor.
    Biochimica et Biophysica Acta 09/2013; · 4.66 Impact Factor
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    ABSTRACT: A more complete understanding of the mechanism of action of TLR agonists has fueled the investigation of new synthetic immunoadjuvants. In this context, we designed and synthesized glycolipids of the type Pam(2)Cys-α-Galactose as novel immunoadjuvants. Their synthesis required modifying a hydrophobic tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety, i.e. the minimal structure required for TLR2 agonist activity, by addition of a hydrophilic head, either an α-Galactosylpyranose or an α-Galactosylfuranose to gain respectively Pam(2)CGalp and Pam(2)CGalf. While preparing a carbohydrate building block, an unexpected stereoselectivity was observed during a halide ion-catalytic process on a protected galactofuranose: the alpha anomer was obtained with surprisingly high selectivity (α/β ratio>9) and with good isolated yield (51%). The TLR2 binding properties of Pam(2)CGalp and Pam(2)CGalf were then fully evaluated. Their efficiency in triggering the proliferation of BALB/c mouse splenocytes was also compared to that of Pam(2)CAG and Pam(3)CAG, two well-established ligands of TLRs. Moreover, the maturation state of murine dendritic cells previously incubated with either Pam(2)CGalp or Pam(2)CGalf was monitored by flow cytometry and compared to that induced by lipopolysaccharide. Pam(2)CGalp and Pam(2)CGalf were found to be equivalent TLR2 agonists, and induced splenocyte proliferation and DC maturation. With very similar activity, Pam(2)CGalp and Pam(2)CGalf were also 10-fold to 100-fold better than Pam(2)CAG and Pam(3)CAG at inducing B cell proliferation. This represents the first time a glucidic head has been added to the tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety whilst maintaining the immunomodulating activity. This should greatly enrich the data available on Pam(2)C structure/activity relationships.
    European journal of medicinal chemistry 03/2012; 51:174-83. · 3.27 Impact Factor
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    ABSTRACT: Bovine CD38/NAD(+)glycohydrolase (bCD38) catalyses the hydrolysis of NAD(+) into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR). We solved the crystal structures of the mono N-glycosylated forms of the ecto-domain of bCD38 or the catalytic residue mutant Glu218Gln in their apo state or bound to aFNAD or rFNAD, two 2'-fluorinated analogs of NAD(+). Both compounds behave as mechanism-based inhibitors, allowing the trapping of a reaction intermediate covalently linked to Glu218. Compared to the non-covalent (Michaelis) complex, the ligands adopt a more folded conformation in the covalent complexes. Altogether these crystallographic snapshots along the reaction pathway reveal the drastic conformational rearrangements undergone by the ligand during catalysis with the repositioning of its adenine ring from a solvent-exposed position stacked against Trp168 to a more buried position stacked against Trp181. This adenine flipping between conserved tryptophans is a prerequisite for the proper positioning of the N1 of the adenine ring to perform the nucleophilic attack on the C1' of the ribofuranoside ring ultimately yielding cADPR. In all structures, however, the adenine ring adopts the most thermodynamically favorable anti conformation, explaining why cyclization, which requires a syn conformation, remains a rare alternate event in the reactions catalyzed by bCD38 (cADPR represents only 1% of the reaction products). In the Michaelis complex, the substrate is bound in a constrained conformation; the enzyme uses this ground-state destabilization, in addition to a hydrophobic environment and desolvation of the nicotinamide-ribosyl bond, to destabilize the scissile bond leading to the formation of a ribooxocarbenium ion intermediate. The Glu218 side chain stabilizes this reaction intermediate and plays another important role during catalysis by polarizing the 2'-OH of the substrate NAD(+). Based on our structural analysis and data on active site mutants, we propose a detailed analysis of the catalytic mechanism.
    PLoS ONE 01/2012; 7(4):e34918. · 3.73 Impact Factor
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    ABSTRACT: Synthetic and molecularly defined constructs containing the minimal components to mimic and amplify the physiological immune response are able to induce an efficient cytotoxic response. In the current study this approach was applied to the development of highly versatile liposomal constructs to co-deliver peptide epitopes in combination with TLR agonists in order to induce a specific anti-tumor cellular immune response against ErbB2 protein-expressing tumor cells. Liposomes containing ErbB2 p63-71 cytotoxic T lymphocyte (CTL) and HA307-319 T- helper (Th) peptide epitopes associated to innovative synthetic TLR2/1 (Pam(3)CAG) or TLR2/6 agonists (Pam(2)CAG and Pam(2)CGD), were injected in mice bearing ErbB2 protein-expressing tumor cells. Mannosylated ligands were also incorporated into the constructs to target antigen-presenting cells. We showed that the TLR2/6 agonists were more efficient than the TLR2/1 agonists for the eradication of tumors expressing ErbB2 protein. Furthermore, mannose-targeted liposomes displayed higher therapeutic efficiency against tumor allowing treatment with decreased quantities of both TLR ligands and peptide epitopes. Our results validated that antigen-associated mannosylated liposomes combined with efficient TLR ligands are effective vectors for vaccination against tumor. In this study we developed useful tools to evaluate the vaccination efficiency of various adjuvants and/or targeting molecules and their potential synergy.
    Biomaterials 07/2011; 32(20):4574-83. · 8.31 Impact Factor
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    ABSTRACT: CD38 is a multifunctional enzyme which is ubiquitously distributed in mammalian tissues. It is involved in the conversion of NAD(P)(+) into cyclic ADP-ribose, NAADP(+) and ADP-ribose and the role of these metabolites in multiple Ca(2+) signaling pathways makes CD38 a novel potential pharmacological target. The dire paucity of CD38 inhibitors, however, renders the search for new molecular tools highly desirable. We report that human CD38 is inhibited at low micromolar concentrations by flavonoids such as luteolinidin, kuromanin and luteolin (IC(50) <10 μM). Docking studies provide some clues on the mode of interaction of these molecules with the active site of CD38.
    Bioorganic & medicinal chemistry letters 07/2011; 21(13):3939-42. · 2.65 Impact Factor
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    ABSTRACT: Schistosomiasis is a major tropical parasitic disease. For its treatment, praziquantel remains the only effective drug available and the dependence on this sole chemotherapy emphasizes the urgent need for new drugs to control this neglected disease. In this context, the newly characterized Schistosoma mansoni NAD(+) catabolizing enzyme (SmNACE) represents a potentially attractive drug target. This potent NAD(+)glycohydrolase, which is localized to the outer surface (tegument) of the adult parasite, is presumably involved in the parasite survival by manipulating the host's immune regulatory pathways. In an effort to identify SmNACE inhibitors, we have developed a sensitive and robust fluorometric high-throughput screening assay. The implementation of this assay to the screening of a highly diverse academic chemical library of 14,300 molecules yielded, after secondary assays and generation of dose-response curves, the identification of two natural product inhibitors, cyanidin and delphinidin. These confirmed hits inhibit SmNACE with IC(50) values in the low micromolar range. To rationalize the structure-activity relationship, several related flavonoids were tested, thereby leading to the identification of 15 additional natural product inhibitors. A selection of representative flavonoid inhibitors indicated that although they also inhibit the homologous human CD38, a selectivity in favor of SmNACE could be reached. Docking studies indicated that these inhibitors mimic the binding mode of the enzyme substrate NAD(+) and suggested the pharmacophoric features required for SmNACE active site recognition.
    Bioorganic & medicinal chemistry 09/2010; 18(22):7900-10. · 2.82 Impact Factor
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    ABSTRACT: Cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate are ubiquitous calcium-mobilizing messengers produced by the same family of multifunctional enzymes, the ADP-ribosyl cyclases. Not all ADP-ribosyl cyclases have been identified, and how production of different messengers is achieved is incompletely understood. Here, we report the cloning and characterization of a novel ADP-ribosyl cyclase (SpARC4) from the sea urchin, a key model organism for the study of calcium-signaling pathways. Like several other members of the ADP-ribosyl cyclase superfamily, SpARC4 is a glycoprotein targeted to the plasma membrane via a glycosylphosphatidylinositol anchor. However, unlike most other members, SpARC4 shows a remarkable preference for producing cyclic ADP-ribose over nicotinic acid adenine dinucleotide phosphate. Mutation of a single residue (tyrosine 142) within a noncanonical active site reversed this striking preference. Our data highlight further diversification of this unusual enzyme family, provide mechanistic insight into multifunctionality, and suggest that different ADP-ribosyl cyclases are fine-tuned to produce specific calcium-mobilizing messengers.
    Journal of Biological Chemistry 04/2010; 285(26):19900-9. · 4.65 Impact Factor
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    ABSTRACT: Click chemistry represents a new bioconjugation strategy that can be used to conveniently attach various ligands to the surface of preformed liposomes. This efficient and chemoselective reaction involves a Cu(I)-catalyzed azide-alkyne cycloaddition, which can be performed under mild experimental conditions in aqueous media. Here, we describe the application of a model click reaction to the conjugation, in a single step of unprotected alpha-1-thiomannosyl ligands, functionalized with an azide group to liposomes containing a terminal alkyne-functionalized lipid anchor. Excellent coupling yields were obtained in the presence of bathophenanthrolinedisulphonate, a water soluble copper-ion chelator, acting as a catalyst. No vesicle leakage was triggered by this conjugation reaction and the coupled mannose ligands were exposed at the surface of the liposomes. The major limitation of Cu(I)-catalyzed click reactions is that this conjugation is restricted to liposomes made of saturated (phospho)lipids. Efficient copper-free azide-alkyne click reactions are, however, being developed, which should alleviate this constraint in the future.
    Methods in molecular biology (Clifton, N.J.) 01/2010; 605:267-77. · 1.29 Impact Factor
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    ABSTRACT: Bovine CD38, a type II glycoprotein, contains two potential N-glycosylation sites (Asn-201 and Asn-268) in its extracellular domain. This contrasts with the other mammalian members of the ADP-ribosyl cyclase family, such as human CD38 and BST-1/CD157, in which four such sites are present. Our study was designed to determine the occupancy of these sites in a recombinant form of this ecto-enzyme and to evaluate its impact on the protein stability and catalytic functions. To that end we have successfully expressed the hydrosoluble ecto-domain of bovine CD38 (bCD38; residues 32-278), and corresponding glycosylation mutants, in the methylotrophic yeast Pichia pastoris. The secreted proteins were purified to homogeneity by affinity chromatography on immobilized Cibacron blue. We found by site-directed mutagenesis and mass spectrometry that bCD38 was a monoglycosylated protein at Asn-201. The expression yield of the deglycosylated mutants was not significantly affected, indicating that glycosylation at Asn-201 was not required for a proper processing and secretion of this protein by P. pastoris. Significant alterations in the kinetic parameters of NAD(+) were observed for the deglycosylated mutants. The thermostability of the recombinant enzyme was also influenced by mutation at position 201. Interestingly both parameters were dependent on the nature of the mutant and a stable deglycosylated mutant N201D of bCD38 could be produced that can be further used for structural studies.
    Protein Expression and Purification 10/2009; 70(2):151-7. · 1.43 Impact Factor
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    ABSTRACT: We have designed chemically defined diepitope constructs consisting of liposomes displaying at their surface synthetic oligosaccharides mimicking the O-antigen of the Shigella flexneri 2a lipopolysaccharide (B-cell epitope) and influenza hemagglutinin peptide HA 307-319 (Th epitope). Using well controlled and high-yielding covalent bioconjugation reactions, the two structurally independent epitopes were coupled to the lipopeptide Pam(3)CAG, i.e. a TLR2 ligand known for its adjuvant properties, anchored in preformed vesicles. The synthetic construct containing a pentadecasaccharide corresponding to three O-antigen repeating units triggered T-dependent anti-oligosaccharide and anti-S. flexneri 2a LPS antibody responses when administered i.m. to BALB/c mice. Moreover, the long-lasting anti-LPS antibody response afforded protection against a S. flexneri 2a challenge. These results show that liposome diepitope constructs could be attractive alternatives in the development of synthetic carbohydrate-based vaccines.
    Vaccine 07/2009; 27(39):5419-26. · 3.77 Impact Factor
  • B. Heurtault, F. Schuber, B. Frisch
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    ABSTRACT: Once it has been administered, an active principle still has to face many physiological barriers on the way to its target, and this may significantly affect its efficiency. These different barriers depend to a great extent on the active ingredient itself and on the way it is administered. They may be constituted by enzymes, an acidic or basic pH, or cell membranes that must be crossed. As a consequence, the active principle may be degraded or distributed to organs other than the therapeutic target. This can reduce the efficiency of the administered dose, or even lead to toxicity with regard to organs other than the target. For example, this situation is observed in trials for the oral administration of insulin (for treating type I diabetes). One point is that this molecule is weakly absorbed by the digestive epithelium (first barrier). Secondly, it undergoes enzymatic degradation by gastric proteases (second barrier). As a consequence, the free form of the molecule cannot be administered orally. This is why insulin is mainly administered subcutaneously, so that it attains the blood circulation directly. However, such a means of administration requires specific training of the patient. This example shows that lack of efficiency and/or difficulties in using certain molecules are not necessarily due to their pharmacology, but rather in some cases to their physicochemical properties.
    Nanoscience: Nanobiotechnology and Nanobiology. 01/2009;
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    ABSTRACT: An important challenge for the development of new generations of vaccines is the efficient delivery of antigens to antigen presenting cells such as dendritic cells. In the present study we compare the interaction of plain and targeted liposomes, containing mono-, di-, and tetraantennary mannosyl lipid derivatives, with human monocyte-derived immature dendritic cells (iDCs). Whereas efficient mannose receptor-mediated endocytosis by iDCs was observed for the mannosylated liposomes, in contrast, only nonspecific interaction with little uptake was observed with plain liposomes. In accordance with the clustering effect, liposomes prepared with multibranched mannosylated lipids displayed higher binding affinity for the mannose receptor than vesicles containing the monomannosylated analogs. Importantly, we have found that dimannosylated ligands present at the surface of the liposomes were as efficient as tetramannosylated ones to engage in multidentate interactions with the mannose receptor of iDCs, resulting in both cases in an effective uptake/endocytosis. This result will greatly facilitate, from a practical standpoint, the design of mannose-targeted vaccination constructs. Moreover, we showed that mannose-mediated uptake of liposomes did not result in an activation of iDCs. Altogether, our results suggest that antigen-associated targeted liposomes containing diantennary mannosylated lipids could be effective vectors for vaccines when combined with additional DC activation signals.
    Bioconjugate Chemistry 12/2008; 19(12):2385-93. · 4.58 Impact Factor
  • Francis SCHUBER, Pierre TRAVO, Marc PASCAL
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    ABSTRACT: Steady-state analysis of product inhibition indicates that the most probable minimum kinetic mechanism for soluble calf spleen NAD glycohydrolase is ping-pong bi-bi, reducing to an ordered uni-bi for hydrolysis alone. In the hydrolysis of NAD nicotinamide is released first. Isotope exchange, transglycosidation reaction and nucleophilic competition with methanol show that the formation of the intermediary enzyme · adenosine-diphosphoribosyl complex (E-ADP-Rib) is rate limiting.The NAD hydrolysis is subject to inhibition by excess of substrate, which is markedly accentuated by increasing concentrations of nicotinamide. Kinetic analysis of this behaviour is in favour of the formation of an apparent dead-end complex of NAD with the Michaelis complex.The reactivies of methanol and nicotinamide compared to water with the intermediate E-ADP-Rib suggest the occurrence of an intermediary oxocarbonium ion in the reaction catalyzed by NAD glycohydrolase.
    European Journal of Biochemistry. 06/2008; 69(2):593 - 602.
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    ABSTRACT: ADP-ribosyl cyclases catalyze the transformation of nicotinamide adenine dinucleotide (NAD+) into the calcium-mobilizing nucleotide second messenger cyclic adenosine diphosphoribose (cADP-ribose) by adenine N1-cyclization onto the C-1' ' position of NAD+. The invertebrate Aplysia californica ADP-ribosyl cyclase is unusual among this family of enzymes by acting exclusively as a cyclase, whereas the other members, such as CD38 and CD157, also act as NAD+ glycohydrolases, following a partitioning kinetic mechanism. To explore the intramolecular cyclization reaction, the novel nicotinamide 2-fluoroadenine dinucleotide (2-fluoro-NAD+) was designed as a sterically very close analogue to the natural substrate NAD+, with only an electronic perturbation at the critical N1 position of the adenine base designed to impede the cyclization reaction. 2-Fluoro-NAD+ was synthesized in high yield via Lewis acid catalyzed activation of the phosphoromorpholidate derivative of 2-fluoroadenosine 5'-monophosphate and coupling with nicotinamide 5'-monophosphate. With 2-fluoro-NAD+ as substrate, A. californica ADP-ribosyl cyclase exhibited exclusively a NAD+ glycohydrolase activity, catalyzing its hydrolytic transformation into 2-fluoro-ADP-ribose, albeit at a rate ca. 100-fold slower than for the cyclization of NAD+ and also, in the presence of methanol, into its methanolysis product beta-1' '-O-methyl 2-fluoro-ADP-ribose with a preference for methanolysis over hydrolysis of ca. 100:1. CD38 likely converted 2-fluoro-NAD+ exclusively into the same product. We conclude that A. californica ADP-ribosyl cyclase can indeed be classified as a multifunctional enzyme that also exhibits a classical NAD+ glycohydrolase function. This alternative pathway that remains, however, kinetically cryptic when using NAD+ as substrate can be unmasked with a dinucleotide analogue whose conversion into the cyclic derivative is blocked. 2-Fluoro-NAD+ is therefore a useful molecular tool allowing dissection of the kinetic scheme for this enzyme.
    Biochemistry 05/2007; 46(13):4100-9. · 3.38 Impact Factor
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    ABSTRACT: Schistosoma mansoni NAD(P)+ catabolizing enzyme (SmNACE) is a new member of the ADP-ribosyl cyclase family. In contrast to all the other enzymes that are involved in the production of metabolites that elicit Ca2+ mobilization, SmNACE is virtually unable to transform NAD+ into the second messenger cyclic ADP-ribose (cADPR). Sequence alignments revealed that one of four conserved residues within the active site of these enzymes was replaced in SmNACE by a histidine (His103) instead of the highly conserved tryptophan. To find out whether the inability of SmNACE to catalyze the canonical ADP-ribosyl cyclase reaction is linked to this change, we have replaced His103 with a tryptophan. The H103W mutation in SmNACE was indeed found to restore ADP-ribosyl cyclase activity as cADPR amounts for 7% of the reaction products (i.e., a value larger than observed for other members of this family such as CD38). Introduction of a Trp103 residue provides some of the binding characteristics of mammalian ADP-ribosyl cyclases such as increased affinity for Cibacron blue and slow-binding inhibition by araF-NAD+. Homology modeling of wild-type and H103W mutant three-dimensional structures, and docking of substrates within the active sites, provides new insight into the catalytic mechanism of SmNACE. Both residue side chains share similar roles in the nicotinamide-ribose bond cleavage step leading to an E.ADP-ribosyl reaction intermediate. They diverge, however, in the evolution of this intermediate; His103 provides a more polar environment favoring the accessibility to water and hydrolysis leading to ADP-ribose at the expense of the intramolecular cyclization pathway resulting in cADPR.
    Biochemistry 11/2006; 45(39):11867-78. · 3.38 Impact Factor
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    ABSTRACT: Cross-linking of CD38 on hematopoietic cells induces activation, proliferation and differentiation of mature T and B cells and mediates apoptosis of myeloid and lymphoid progenitor cells. In addition to acting as a signaling receptor, CD38 is also an enzyme capable of producing several calcium-mobilizing metabolites, including cyclic adenosine diphosphate ribose (cADPR). It has been previously postulated that the calcium-mobilizing metabolites produced by CD38 may regulate its receptor-based activities. To test this hypothesis, we examined whether the enzyme activity of CD38 controls the apoptosis of an anti-CD38-stimulated leukemic B cell. We show that anti-CD38-induced apoptosis of Ba/F3 cells, a murine pro-B cell line, is not affected by blocking the calcium-mobilizing activity of cADPR or by inhibiting intracellular or extracellular calcium mobilization. In addition, we demonstrate that blocking CD38 enzyme activity with 2'-deoxy-2'-fluoro-nicotinamide arabinoside adenine dinucleotide has no effect on apoptosis and that Ba/F3 cells expressing catalytically inactive mutant forms of CD38 still undergo apoptosis upon CD38 cross-linking. Instead, we find that anti-CD38-induced apoptosis is dependent on tyrosine kinase and caspase activation, and that this process appears to be potentiated by the presence of membrane microdomains. Thus, the receptor-mediated functions of CD38 can be separated from its enzyme activity in a murine leukemic cell line, suggesting that CD38 plays multiple, but independent, biologic roles.
    International Immunology 08/2006; 18(7):1029-42. · 3.14 Impact Factor
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    ABSTRACT: cADPR (cADP-ribose), a metabolite of NAD+, is known to modulate intracellular calcium levels and to be involved in calcium-dependent processes, including synaptic transmission, plasticity and neuronal excitability. However, the enzyme that is responsible for producing cADPR in the cytoplasm of neural cells, and particularly at the synaptic terminals of neurons, remains unknown. In the present study, we show that endogenous concentrations of cADPR are much higher in embryonic and neonate mouse brain compared with the adult tissue. We also demonstrate, by comparing wild-type and Cd38-/- tissues, that brain cADPR content is independent of the presence of CD38 (the best characterized mammalian ADP-ribosyl cyclase) not only in adult but also in developing tissues. We show that Cd38-/- synaptosome preparations contain high ADP-ribosyl cyclase activities, which are more important in neonates than in adults, in line with the levels of endogenous cyclic nucleotide. By using an HPLC method and adapting the cycling assay developed initially to study endogenous cADPR, we accurately examined the properties of the synaptosomal ADP-ribosyl cyclase. This intracellular enzyme has an estimated K(m) for NAD+ of 21 microM, a broad optimal pH at 6.0-7.0, and the concentration of free calcium has no major effect on its cADPR production. It binds NGD+ (nicotinamide-guanine dinucleotide), which inhibits its NAD+-metabolizing activities (K(i)=24 microM), despite its incapacity to cyclize this analogue. Interestingly, it is fully inhibited by low (micromolar) concentrations of zinc. We propose that this novel mammalian ADP-ribosyl cyclase regulates the production of cADPR and therefore calcium levels within brain synaptic terminals. In addition, this enzyme might be a potential target of neurotoxic Zn2+.
    Biochemical Journal 05/2006; 395(2):417-26. · 4.65 Impact Factor
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    ABSTRACT: We have developed liposome-based synthetic constructs incorporating peptide epitope(s) (ErbB2 p63-67 CTL which is overexpressed in many tumors and/or HA 307-319 T-helper) and lipopeptide adjuvants (Pam3CysSerSer, Pam3CysAlaGly) in order to elicit an anti-tumor immune response. The epitopes, derivatized with a linker containing a cysteine residue, were conjugated on preformed vesicles (dia. approximately 100 nm) containing lipopeptides functionalized with thiol reactive groups (maleimide or bromoacetyl). The therapeutic efficacy of these constructs was evaluated on a Balb/c mice tumor model inoculated with syngenic murine renal carcinoma (Renca) cells expressing human ErbB2 (Her2/neu) receptor. A successful therapeutic vaccination was obtained which was antigen specific. Furthermore, it appeared that the nature of the polar head group of the lipopeptide adjuvant and also its type of functionalization influence the efficacy of the construct. In our study, the best results were obtained with formulations containing a Pam3CSS anchor in association with the CTL and Th epitopes. Considering these promising results studies are in progress with a new generation of liposomes that incorporate a neutral lipid--lacking adjuvant properties--that serves as anchor of the peptide epitopes and new adjuvants synthesized in our laboratory, which are screened for their antitumour activity in a therapeutic setting.
    Journal of Liposome Research 02/2006; 16(3):205-13. · 1.91 Impact Factor

Publication Stats

2k Citations
397.23 Total Impact Points

Institutions

  • 1976–2014
    • University of Strasbourg
      • • Laboratoire de Conception et Application de Molécules Bioactives
      • • Faculté de pharmacie
      Strasburg, Alsace, France
  • 1998–2013
    • French National Centre for Scientific Research
      • Institute for Molecular and Cellular Biology (IBMC)
      Lutetia Parisorum, Île-de-France, France
    • Roche
      Bâle, Basel-City, Switzerland
  • 2012
    • University of California, Los Angeles
      • Department of Biological Chemistry
      Los Angeles, CA, United States
  • 1998–2006
    • Trudeau Institute
      Saranac Lake, New York, United States
  • 1999
    • Palo Alto Institute for Research and Education
      Palo Alto, California, United States
  • 1997
    • Laboratoire de Chimie de Coordination.
      Tolosa de Llenguadoc, Midi-Pyrénées, France
  • 1995
    • Università degli Studi di Torino
      • Dipartimento di Scienza e Tecnologia del Farmaco
      Torino, Piedmont, Italy