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1.
PLoS Pathog ; 16(5): e1008539, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32459815

RESUMO

NAD, a key co-enzyme required for cell metabolism, is synthesized via two pathways in most organisms. Since schistosomes apparently lack enzymes required for de novo NAD biosynthesis, we evaluated whether these parasites, which infect >200 million people worldwide, maintain NAD homeostasis via the NAD salvage biosynthetic pathway. We found that intracellular NAD levels decline in schistosomes treated with drugs that block production of nicotinamide or nicotinamide mononucleotide-known NAD precursors in the non-deamidating salvage pathway. Moreover, in vitro inhibition of the NAD salvage pathway in schistosomes impaired egg production, disrupted the outer membranes of both immature and mature parasites and caused loss of mobility and death. Inhibiting the NAD salvage pathway in schistosome-infected mice significantly decreased NAD levels in adult parasites, which correlated with reduced egg production, fewer liver granulomas and parasite death. Thus, schistosomes, unlike their mammalian hosts, appear limited to one metabolic pathway to maintain NAD-dependent metabolic processes.


Assuntos
Interações Hospedeiro-Parasita/fisiologia , NAD/metabolismo , Schistosoma mansoni/fisiologia , Esquistossomose mansoni/metabolismo , Animais , Feminino , Camundongos , Reprodução/fisiologia , Esquistossomose mansoni/patologia
2.
ACS Chem Biol ; 12(7): 1787-1795, 2017 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-28481502

RESUMO

SmNACE is a NAD catabolizing enzyme expressed on the outer tegument of S. mansoni, a human parasite that is one of the major agents of the neglected tropical disease schistosomiasis. Recently, we identified aroylhydrazone derivatives capable of inhibiting the recombinant form of the enzyme with variable potency (IC50 ranging from 88 µM to 33 nM). In the present study, we investigated the mechanism of action of the least potent micromolar inhibitor (compound 1) and the most potent nanomolar inhibitor (compound 2) in the series on both the recombinant and native SmNACE enzymes. Using mass spectroscopy, spectrophotometry, and activity assays under different experimental conditions, we demonstrated that the >3 log gain in potency against recombinant SmNACE by this class of compounds is dependent on the formation of a coordination complex with metal cations, such as Ni(II), Zn(II), and Fe(II), that are loaded on the protein surface. Testing the compounds on live parasites, we observed that only the weak micromolar compound 1 was active on the native enzyme. We showed that S. mansoni effectively sequesters the metal from the coordination complex, resulting in the loss of inhibitory activity of the potent nanomolar compound 2. Importantly, the modeling of the transition complex between Zn(II) and compound 2 enabled the discovery of a new metal-independent aroylhydrazone analogue, which is now the most potent and selective inhibitor of native SmNACE known.


Assuntos
Complexos de Coordenação/farmacologia , Metais/metabolismo , Schistosoma mansoni/enzimologia , Animais , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Schistosoma mansoni/metabolismo , Zinco/química
3.
J Med Chem ; 58(8): 3582-92, 2015 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-25803425

RESUMO

The blood fluke Schistosoma mansoni is the causative agent of the intestinal form of schistosomiasis (or bilharzia). Emergence of Schistosoma mansoni with reduced sensitivity to praziquantel, the drug currently used to treat this neglected disease, has underlined the need for development of new strategies to control schistosomiasis. Our ability to screen drug libraries for antischistosomal compounds has been hampered by the lack of validated S. mansoni targets. In the present work, we describe a virtual screening approach to identify inhibitors of S. mansoni NAD(+) catabolizing enzyme (SmNACE), a receptor enzyme suspected to be involved in immune evasion by the parasite at the adult stage. Docking of commercial libraries into a homology model of the enzyme has led to the discovery of two in vitro micromolar inhibitors. Further structure-activity relationship studies have allowed a 3-log gain in potency, accompanied by a largely enhanced selectivity for the parasitic enzyme over the human homologue CD38.


Assuntos
Antiparasitários/química , Inibidores Enzimáticos/química , Proteínas de Helminto/antagonistas & inibidores , NAD/metabolismo , Schistosoma mansoni/enzimologia , Esquistossomose mansoni/tratamento farmacológico , Esquistossomose mansoni/parasitologia , ADP-Ribosil Ciclase 1/metabolismo , Animais , Antiparasitários/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Helminto/metabolismo , Humanos , Simulação de Acoplamento Molecular , Schistosoma mansoni/efeitos dos fármacos , Esquistossomose mansoni/enzimologia , Relação Estrutura-Atividade
4.
Biochim Biophys Acta ; 1844(7): 1317-31, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24721563

RESUMO

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.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Aminoácidos/genética , Mutação/genética , NAD/metabolismo , ADP-Ribosil Ciclase 1/química , ADP-Ribosil Ciclase 1/genética , Animais , Catálise , Domínio Catalítico , Bovinos , Glicosilação , Hidrólise , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Especificidade por Substrato
5.
Biochim Biophys Acta ; 1834(12): 2520-7, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24035885

RESUMO

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 cyclization 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.


Assuntos
Proteínas de Helminto/química , Hidrolases/química , Schistosoma mansoni/enzimologia , ADP-Ribosil Ciclase 1/química , ADP-Ribosil Ciclase 1/genética , Aminoácidos , Animais , Antocianinas/química , Domínio Catalítico , Proteínas de Helminto/genética , Humanos , Hidrolases/antagonistas & inibidores , Hidrolases/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , NAD/antagonistas & inibidores , NAD/química , NAD/genética , Estrutura Terciária de Proteína , Schistosoma mansoni/genética , Homologia Estrutural de Proteína
6.
PLoS One ; 7(4): e34918, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22529956

RESUMO

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.


Assuntos
ADP-Ribosil Ciclase 1/química , ADP-Ribosil Ciclase/química , ADP-Ribosil Ciclase/metabolismo , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/metabolismo , Monofosfato de Adenosina/química , Substituição de Aminoácidos , Animais , Catálise , Domínio Catalítico , Bovinos , Cristalografia por Raios X , Glicosilação , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , NAD/análogos & derivados , NAD/química , NAD/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato
7.
Bioorg Med Chem Lett ; 21(13): 3939-42, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21641214

RESUMO

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.


Assuntos
ADP-Ribosil Ciclase 1/antagonistas & inibidores , Inibidores Enzimáticos/química , Flavonoides/farmacologia , Antocianinas/química , Antocianinas/farmacologia , Domínio Catalítico , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Flavonoides/química , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular
8.
Bioorg Med Chem ; 18(22): 7900-10, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20951593

RESUMO

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.


Assuntos
Inibidores Enzimáticos/química , Flavonoides/química , NAD+ Nucleosidase/química , Schistosoma mansoni/enzimologia , Esquistossomicidas/química , ADP-Ribosil Ciclase 1/antagonistas & inibidores , ADP-Ribosil Ciclase 1/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Simulação por Computador , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Flavonoides/síntese química , Flavonoides/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , NAD+ Nucleosidase/metabolismo , Esquistossomicidas/síntese química , Esquistossomicidas/farmacologia , Relação Estrutura-Atividade
9.
J Biol Chem ; 285(26): 19900-9, 2010 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-20385553

RESUMO

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.


Assuntos
ADP-Ribosil Ciclase/metabolismo , ADP-Ribose Cíclica/metabolismo , NADP/análogos & derivados , ADP-Ribosil Ciclase/genética , Sequência de Aminoácidos , Animais , Blastômeros/citologia , Blastômeros/metabolismo , Western Blotting , Linhagem Celular , Clonagem Molecular , Humanos , Cinética , Microinjeções , Microscopia Confocal , Dados de Sequência Molecular , Mutação , NADP/metabolismo , Homologia de Sequência de Aminoácidos , Strongylocentrotus purpuratus/enzimologia , Strongylocentrotus purpuratus/genética , Strongylocentrotus purpuratus/metabolismo , Transfecção , Tirosina/genética , Tirosina/metabolismo , Xenopus laevis/embriologia
10.
Protein Expr Purif ; 70(2): 151-7, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19818407

RESUMO

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.


Assuntos
ADP-Ribosil Ciclase 1/química , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/biossíntese , Sequência de Aminoácidos , Animais , Catálise , Bovinos , Estabilidade Enzimática , Glicosilação , Cinética , Mutagênese Sítio-Dirigida , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
11.
Biochemistry ; 46(13): 4100-9, 2007 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-17341094

RESUMO

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.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , NAD+ Nucleosidase/metabolismo , NAD/análogos & derivados , Animais , Aplysia/enzimologia , Simulação por Computador , Cinética , Modelos Químicos , Modelos Moleculares , NAD/síntese química , NAD/química , NAD/metabolismo
12.
Biochemistry ; 45(39): 11867-78, 2006 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-17002287

RESUMO

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.


Assuntos
ADP-Ribosil Ciclase/genética , Proteínas de Helminto/genética , Mutação de Sentido Incorreto , NAD/metabolismo , Schistosoma mansoni/genética , ADP-Ribosil Ciclase/metabolismo , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Cálcio/metabolismo , Catálise , ADP-Ribose Cíclica/genética , ADP-Ribose Cíclica/metabolismo , Proteínas de Helminto/metabolismo , Humanos , Schistosoma mansoni/enzimologia
13.
Int Immunol ; 18(7): 1029-42, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16720618

RESUMO

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.


Assuntos
ADP-Ribosil Ciclase 1/imunologia , ADP-Ribosil Ciclase/imunologia , Apoptose/imunologia , Linfócitos B/imunologia , Sinalização do Cálcio/imunologia , ADP-Ribosil Ciclase/antagonistas & inibidores , ADP-Ribosil Ciclase/metabolismo , ADP-Ribosil Ciclase 1/antagonistas & inibidores , ADP-Ribosil Ciclase 1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linfócitos B/enzimologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , ADP-Ribose Cíclica/imunologia , ADP-Ribose Cíclica/metabolismo , Inibidores Enzimáticos/farmacologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Capeamento Imunológico/efeitos dos fármacos , Capeamento Imunológico/imunologia , Leucemia de Células B/imunologia , Leucemia de Células B/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Microdomínios da Membrana/enzimologia , Microdomínios da Membrana/imunologia , Camundongos , NAD/análogos & derivados , NAD/farmacologia
14.
Biochem J ; 395(2): 417-26, 2006 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-16411897

RESUMO

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+.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , ADP-Ribosil Ciclase/metabolismo , Envelhecimento/fisiologia , Encéfalo/enzimologia , Sinaptossomos/enzimologia , ADP-Ribosil Ciclase/antagonistas & inibidores , ADP-Ribosil Ciclase 1/deficiência , Animais , Animais Recém-Nascidos , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , ADP-Ribose Cíclica/metabolismo , Nucleotídeos de Guanina/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Camundongos , Camundongos Knockout , NAD/análogos & derivados , NAD/farmacologia , Sinaptossomos/efeitos dos fármacos , Zinco/farmacologia
16.
Biochemistry ; 44(33): 11082-97, 2005 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-16101292

RESUMO

ADP-ribosyl cyclases are structurally conserved enzymes that are best known for catalyzing the production of the calcium-mobilizing metabolite, cyclic adenosine diphosphate ribose (cADPR), from nicotinamide adenine dinucleotide (NAD(+)). However, these enzymes also produce adenosine diphosphate ribose (ADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP(+)), both of which have been shown to modulate calcium mobilization in vitro. We have now characterized a new member of the cyclase family from Schistosoma mansoni, a member of the Platyhelminthes phylum. We show that the novel NAD(P)(+) catabolizing enzyme (NACE) expressed by schistosomes is structurally most closely related to the cyclases cloned from Aplysia but also shows significant homology with the mammalian cyclases, CD38 and CD157. NACE expression is developmentally regulated in schistosomes, and the GPI-anchored protein is localized to the outer tegument of the adult schistosome. Importantly, NACE, like all members of the cyclase family, is a multifunctional enzyme and catalyzes NAD(+) glycohydrolase and base-exchange reactions to produce ADPR and NAADP(+). However, despite being competent to generate a cyclic product from NGD(+), a nonphysiologic surrogate substrate, NACE is so far the only enzyme in the cyclase family that is unable to produce significant amounts of cADPR (<0.02% of reaction products) using NAD(+) as the substrate. This suggests that the other calcium-mobilizing metabolites produced by NACE may be more important for calcium signaling in schistosomes. Alternatively, the function of NACE may be to catabolize extracellular NAD(+) to prevent its use by host enzymes that utilize this source of NAD(+) to facilitate immune responses.


Assuntos
ADP-Ribosil Ciclase/metabolismo , Aplysia/genética , Cálcio/metabolismo , ADP-Ribose Cíclica/biossíntese , Expressão Gênica/genética , Schistosoma mansoni , ADP-Ribosil Ciclase/genética , ADP-Ribosil Ciclase/imunologia , Animais , Aplysia/enzimologia , Sequência de Bases , Sinalização do Cálcio/genética , Clonagem Molecular , Dados de Sequência Molecular , NAD/metabolismo , NAD+ Nucleosidase/metabolismo , NADP/análogos & derivados , NADP/metabolismo , Filogenia , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Schistosoma mansoni/imunologia
17.
Eur J Biochem ; 271(5): 1025-34, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15009214

RESUMO

CD38 is a transmembrane glycoprotein that functions as an ectoenzyme and as a receptor. Based on the structural similarity between CD38 and ADP-ribosyl cyclase from Aplysia californica, it was hypothesized that CD38 is expressed as a homodimer on the surface of cells. Indeed, CD38 dimers have been reported, however, the structural requirements for their stabilization on the plasma membrane are unknown. We demonstrate that the majority of CD38 is assembled as noncovalently associated homodimers on the surface of B cells. Analysis of CD38 mutants, expressed in Ba/F3 cells, revealed that truncation of the cytoplasmic region or mutation of a single amino acid within the alpha1-helix of CD38 decreased the stability of the CD38 homodimers when solubilized in detergent. Cells expressing the unstable CD38 homodimers had diminished expression of CD38 on the plasma membrane and the half-lives of these CD38 mutant proteins on the plasma membrane were significantly reduced. Together, these results show that CD38 is expressed as noncovalently associated homodimers on the surface of murine B cells and suggest that appropriate assembly of CD38 homodimers may play an important role in stabilizing CD38 on the plasma membrane of B cells.


Assuntos
ADP-Ribosil Ciclase/química , ADP-Ribosil Ciclase/metabolismo , Antígenos CD/química , Antígenos CD/metabolismo , Linfócitos B/metabolismo , Estrutura Quaternária de Proteína , ADP-Ribosil Ciclase/genética , ADP-Ribosil Ciclase 1 , Animais , Antígenos CD/genética , Linfócitos B/citologia , Linhagem Celular , Membrana Celular/metabolismo , Detergentes , Dimerização , Glicosídeo Hidrolases/metabolismo , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Estrutura Terciária de Proteína , Baço/citologia
18.
J Biol Chem ; 278(42): 40670-8, 2003 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-12909645

RESUMO

Cyclic ADP-ribose, a metabolite of NAD+, is known to modulate intracellular calcium levels and signaling in various cell types, including neural cells. The enzymes responsible for producing cyclic ADP-ribose in the cytoplasm of mammalian cells remain unknown; however, two mammalian enzymes that are capable of producing cyclic ADP-ribose extracellularly have been identified, CD38 and CD157. The present study investigated whether an ADP-ribosyl cyclase/NAD+-glycohydrolase independent of CD38 is present in brain tissue. To address this question, NAD+ metabolizing activities were accurately examined in developing and adult Cd38-/- mouse brain protein extracts and cells. Low ADP-ribosyl cyclase and NAD+-glycohydrolase activities (in the range of pmol of product formed/mg of protein/min) were detected in Cd38-/- brain at all developmental stages studied. Both activities were found to be associated with cell membranes. The activities were significantly higher in Triton X-100-treated neural cells compared with intact cells, suggesting an intracellular location of the novel cyclase. The cyclase and glycohydrolase activities were optimal at pH 6.0 and were inhibited by zinc, properties which are distinct from those of CD157. Both activities were enhanced by guanosine 5'-O-(3-thiotriphosphate), a result suggesting that the novel enzyme may be regulated by a G protein-dependent mechanism. Altogether our results indicate the presence of an intracellular membrane-bound ADP-ribosyl cyclase/NAD+-glycohydrolase distinct from CD38 and from CD157 in mouse brain. This novel enzyme, which is more active in the developing brain than in the adult tissue, may play an important role in cyclic ADP-ribose-mediated calcium signaling during brain development as well as in adult tissue.


Assuntos
ADP-Ribosil Ciclase/genética , ADP-Ribosil Ciclase/metabolismo , Antígenos CD/genética , Encéfalo/metabolismo , NAD+ Nucleosidase/química , ADP-Ribosil Ciclase 1 , Animais , Cálcio/metabolismo , Membrana Celular/metabolismo , Cloretos/farmacologia , Cromatografia Líquida de Alta Pressão , Detergentes/farmacologia , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana , Camundongos , Neurônios/metabolismo , Octoxinol/farmacologia , Transdução de Sinais , Fatores de Tempo , Zinco/metabolismo , Compostos de Zinco/farmacologia
19.
Biochem J ; 370(Pt 1): 175-83, 2003 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-12403647

RESUMO

CD38 is a transmembrane glycoprotein that is expressed in many tissues throughout the body. In addition to its major NAD+-glycohydrolase activity, CD38 is also able to synthesize cyclic ADP-ribose, an endogenous calcium-regulating molecule, from NAD+. In the present study, we have compared ADP-ribosyl cyclase and NAD+-glycohydrolase activities in protein extracts of brains from developing and adult wild-type and Cd38 -/- mice. In extracts from wild-type brain, cyclase activity was detected spectrofluorimetrically, using nicotinamide-guanine dinucleotide as a substrate (GDP-ribosyl cyclase activity), as early as embryonic day 15. The level of cyclase activity was similar in the neonate brain (postnatal day 1) and then increased greatly in the adult brain. Using [14C]NAD+ as a substrate and HPLC analysis, we found that ADP-ribose is the major product formed in the brain at all developmental stages. Under the same experimental conditions, neither NAD+-glycohydrolase nor GDP-ribosyl cyclase activity could be detected in extracts of brains from developing or adult Cd38 -/- mice, demonstrating that CD38 is the predominant constitutive enzyme endowed with these activities in brain at all developmental stages. The activity measurements correlated with the level of CD38 transcripts present in the brains of developing and adult wild-type mice. Using confocal microscopy we showed, in primary cultures of hippocampal cells, that CD38 is expressed by both neurons and glial cells, and is enriched in neuronal perikarya. Intracellular NAD+-glycohydrolase activity was measured in hippocampal cell cultures, and CD38-dependent cyclase activity was higher in brain fractions enriched in intracellular membranes. Taken together, these results lead us to speculate that CD38 might have an intracellular location in neural cells in addition to its plasma membrane location, and may play an important role in intracellular cyclic ADP-ribose-mediated calcium signalling in brain tissue.


Assuntos
ADP-Ribosil Ciclase/metabolismo , Antígenos CD/metabolismo , Hipocampo/enzimologia , ADP-Ribosil Ciclase/genética , ADP-Ribosil Ciclase 1 , Animais , Antígenos CD/genética , Sequência de Bases , Western Blotting , Sinalização do Cálcio , Células Cultivadas , Centrifugação , Primers do DNA , Glicoproteínas de Membrana , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , NAD/metabolismo , RNA Mensageiro/genética
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