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M Véron

Publications and source records attributed to M Véron.

At least 55 records · Page 3Linked to original sources
PubMed1994

Adenosine 5'-diphosphate binding and the active site of nucleoside diphosphate kinase.

The X-ray structure of nucleoside diphosphate kinase (NDP kinase) from the slime mold Dictyostelium discoideum has been determined to 2.2-A resolution and refined to an R-factor of 0.19 with and without bound ADP-Mg2+. The nucleotide binds near His 122, a residue which becomes phosphorylated during the catalytic cycle. The mode of binding is different from that observed in other phosphokinases, and it involves no glycine-rich sequence. The adenine base makes only nonpolar contacts with the protein. It points outside, explaining the lack of specificity of NDP kinase toward the base. The ribose 2'- and 3'-hydroxyls and the pyrophosphate moiety are H-bonded to polar side chains. A Mg2+ ion bridges the alpha- to the beta-phosphate which approaches the imidazole group of His 122 from the N delta side. The geometry at the active site in the ADP-Mg2+ complex suggests a mechanism for catalysis whereby the gamma-phosphate of a nucleoside triphosphate can be transferred onto His 122 with a minimum of atomic motion.

Adenosine Diphosphate↗
PubMed1994

Protein kinases share a common structural motif outside the conserved catalytic domain.

A comparison of the sequences of the mammalian and Dictyostelium catalytic subunits of cAMP-dependent protein kinase revealed extensive sequence similarities through the catalytic core and the carboxy terminal tail. The amino terminal sequences however differ dramatically. The large difference in size, 73 kDa for the Dictyostelium enzyme versus 40 kDa is due to an extension in the N-terminus. The mouse enzyme has at its amino-terminus a long amphipatic helix, the A-helix, that precedes the catalytic core, covering the surface of both lobes of the enzyme. Dictyostelium does in fact, have a similar motif but it is remote from the catalytic core, in the N-terminal extension. On the basis of molecular modeling, it is proposed that residues 77-98 correspond to a structural motif similar to the A-helix in mouse catalytic subunit. Sequences encoding similar putative motifs contiguous to the catalytic core can be recognized in many other protein kinases and is particularly prominent in all of the non-receptor tyrosine kinases. In the case of Src, this A-helix motif appears to serve as the linker between the conserved catalytic core and the SH2 domain. The interaction between the A-helix motif and the core is described, and the general occurrence of this structure within the protein kinase family is discussed.

Amino Acid Sequence↗
PubMed1993

Crystal structure of the Awd nucleotide diphosphate kinase from Drosophila.

BACKGROUND: Nucleotide diphosphate kinase (NDP kinase) is a phosphate transfer enzyme involved in cell regulation and in animal development. Drosophila NDP kinase is the product of the abnormal wing disc (awd) developmental gene, a point mutation in which can produce the killer of prune (K-pn) conditional lethal phenotype. The highly homologous mammalian genes control metastasis and a human NDP kinase acts as a transcription factor. RESULTS: The X-ray structure of the Awd protein prepared from Drosophila was solved at 2.4 A resolution by molecular replacement from the homologous Dictyostelium protein. Both are hexamers, and both have the same fold and the same active site. Subunit contacts differ as a result of sequence changes in the carboxy-terminal segment and in the loop that is the site of the K-pn mutation. CONCLUSIONS: Regulatory properties of animal NDP kinases depend on interactions with other macromolecules, such as DNA and the product of the Drosophila prune gene. The Awd structure suggests an allosteric mechanism of action of NDP kinase where DNA is the effector and the protein undergoes a major conformational change, possibly dissociating to dimers.

Amino Acid Sequence↗
PubMed1993

Separate nuclear genes encode cytosolic and mitochondrial nucleoside diphosphate kinase in Dictyostelium discoideum.

We have previously isolated cDNA clones for the gip17 gene encoding the cytosolic nucleoside diphosphate (NDP) kinase from Dictyostelium discoideum, and partial cDNAs for guk, a second member of the NDP kinase gene family (Wallet, V., Mutzel, R., Troll, H., Barzu, O., Wurster, B., Véron, M., and Lacombe, M. L. (1990) J. Natl. Cancer Inst. 80, 1199-1202). We now characterize genomic DNA clones for both NDP kinase genes, and we show that guk defines a nuclear-encoded mitochondrial NDP kinase. Isolated D. discoideum mitochondria contain 3% of the total cellular NDP kinase activity. Antibodies which specifically recognize and inhibit the activity of either cytosolic or mitochondrial NDP kinase unambiguously distinguish between these activities. The nascent mitochondrial NDP kinase contains a presequence of 57 amino acids that is removed during import into the organelle as shown by determination of the NH2 terminus of the mature protein from mitochondria. The genes for mitochondrial and cytosolic NDP kinases contain four and two introns, respectively. The positions of the of the introns in the gene for the cytosolic enzyme match exactly the positions of the second and fourth introns in the coding region of its mitochondrial homologue. From these results we conclude that the isozymes diverged from a common ancestor, and we discuss possible phylogenetic pathways for the evolution of cytosolic and organelle NDP kinases.

Amino Acid Sequence↗
PubMed1993

Equilibrium dissociation and unfolding of nucleoside diphosphate kinase from Dictyostelium discoideum. Role of proline 100 in the stability of the hexameric enzyme.

The Killer-of-prune (K-pn) mutation in Drosophila corresponds to a Pro-Ser substitution in nucleoside diphosphate kinase (Lascu, I. Chaffotte, A., Limbourg-Bouchon, B., and Véron, M. (1992) J. Biol. Chem. 267, 12775-12781). We investigated the role of the equivalent proline (Pro100) in the formation and stability of the Dictyostelium nucleoside diphosphate kinase hexamers. Mutations to serine or glycine had only little effect on the properties of the native enzyme. However, the mutant drastically affected the subunit interaction in the hexamer and the ability of the isolated subunits to associate in vitro. While the wild-type hexamer inactivated and unfolded concomitantly at 5-6 M urea, the mutant proteins dissociated to monomers at 0.5-2 M urea and unfolded at 2.5-4 M urea. At intermediate urea concentrations, the unique species present in solution was a folded, partially active monomer as shown by size-exclusion chromatography, UV, fluorescence, and CD spectroscopy. Proline 100 is located in a loop involved in subunits contact. Altered conformation of the loop in P100S and P100S mutants demonstrates its crucial role in subunit assembly. We propose to explain the conditional dominance of the K-pn mutation by the presence of a monomeric form of the enzyme that would have deleterious effects in vivo.

Amino Acid Sequence↗
PubMed1993

An unusual catalytic subunit for the cAMP-dependent protein kinase of Dictyostelium discoideum.

The cAMP-dependent protein kinase (cAPK) plays an essential role during differentiation and fruit morphogenesis in Dictyostelium discoideum. The presence of an open reading frame on the gene, pkaC (previously named either Dd PK2 or Dd PK3 by different groups), predicts a 73-kDa polypeptide with 54% similarity to the catalytic subunits of cAPKs from other organisms. Using anti-peptide antibodies, we show that the pkaC gene product, PkaC, is a 73-kDa polypeptide. Despite the fact that PkaC is about twice the size of its mammalian counterparts, it possesses all of the properties required of a catalytic subunit. It is physically associated with the regulatory subunit, and this association results in an inhibition of the catalytic activity which is reverted by cAMP. PkaC copurifies with cAPK activity, and an increased cAPK activity is observed in cells overexpressing PkaC. We conclude that PkaC is a catalytic subunit of the Dictyostelium discoideum cAPK and discuss the unusual features of this protein with the highest molecular weight of known cAPKs.

Amino Acid Sequence↗
PubMed1993

Assay of nucleoside diphosphate kinase in microtiter plates using a peroxidase-coupled method.

A sensitive assay for nucleoside diphosphate kinase which utilizes microtiter plates is described. ATP, formed in the reaction between dGTP and ADP, is trapped by the glycerol kinase reaction. A colored compound is generated by glycerol-3-phosphate oxidase and peroxidase. This assay is useful for testing a large number of samples generated by chromatographic techniques or for screening purposes.

Adenosine Diphosphate↗
PubMed1993

Activation of the prespore and spore cell pathway of Dictyostelium differentiation by cAMP-dependent protein kinase and evidence for its upstream regulation by ammonia.

Expression of a dominant inhibitor of the Dictyostelium cAMP-dependent protein kinase in prespore cells blocks their differentiation into spore cells. The resultant structures comprise a normal stalk supporting a bolus of cells that fail to express a sporulation-specific gene and that show greatly reduced levels of expression of several prespore-specific genes. The latter result suggests that in addition to activating spore formation, the cAMP-dependent protein kinase may play a role in initial prespore cell differentiation. Development of the strain expressing the dominant inhibitor is hypersensitive to the inhibitory effects of ammonia, the molecule that is believed to repress entry into culmination during normal development. This result supports a model whereby a decrease in ambient ammonia concentration at culmination acts to elevate intracellular cAMP and hence induce terminal differentiation.

Ammonia↗
PubMed1993

Relatedness of three species of "false neisseriae," Neisseria caviae, Neisseria cuniculi, and Neisseria ovis, by DNA-DNA hybridizations and fatty acid analysis.

DNA-DNA hybridization was used to determine the levels of genomic relatedness of the three species of "false neisseriae," Neisseria caviae, Neisseria cuniculi, and Neisseria ovis. The reference strains of these species exhibited high levels of intraspecies relatedness (93 to 100% for N. caviae, 79 to 100% for N. cuniculi, and 68 to 100% for N. ovis) but low levels of interspecific relatedness (less than 34%) to each other and to various species belonging to the beta subclass of the Proteobacteria (Kingella kingae, Neisseria gonorrhoeae, Neisseria meningitidis, and Oligella urethralis) or to the gamma subclass (Branhamella catarrhalis, Kingella indologenes, Moraxella atlantae, Moraxella bovis, Moraxella lacunata subsp. lacunata, Moraxella lacunata subsp. liquefaciens, Moraxella nonliquefaciens, Moraxella osloensis, and Moraxella phenylpyruvica). However, the levels of DNA-DNA hybridization for the three species of "false neisseriae" were significantly higher with the species belonging to the gamma subclass (average, 13.7%) than with the species belonging to the beta subclass (average, 4.5%). These data suggest that N. caviae, N. cuniculi, and N. ovis are three separate genomic species in the gamma subclass. An ascendant hierarchical classification based only on fatty acid profiles distinguished four main classes containing (i) most of the "classical moraxellae," the "false neisseriae," and B. catarrhalis, (ii) only Acinetobacter spp., (iii) M. nonliquefaciens and "misnamed moraxellae" (M. atlantae, M. osloensis, and M. phenylpyruvica), and (iv) the "true neisseriae," the three Kingella species, and O. urethralis. Fatty acids that distinguish these four classes were identified. The fatty acid profiles of the two strains of Psychrobacter immobilis which we studied are not very similar to the profiles of the other taxa. Our results support the hypothesis that the three species of "false neisseriae," B. catarrhalis, the "classical moraxellae," and Acinetobacter spp. should be included in the same family.

Bacterial Typing Techniques↗
PubMed1993

Molecular epidemiology of chronic pulmonary colonisation by Pseudomonas aeruginosa in cystic fibrosis.

The epidemiology of pulmonary colonisation by Pseudomonas aeruginosa was studied in 21 patients with cystic fibrosis (CF) by field inversion gel electrophoresis. DraI-DNA restriction patterns were analysed for 187 P. aeruginosa isolates from these patients. The results revealed that the strains present in individual patients varied during the course of chronic colonisation; the emergence of new strains often was associated with periods of antibiotic therapy. Patients often were colonised by more than one strain (two or three strains were present in 54% of the patients) and the strains obtained from unrelated patients were highly heterogeneous, in contrast to those isolated from a pair of twins. These results demonstrate the heterogeneity and variability of P. aeruginosa isolates in the pulmonary flora of chronically infected CF patients.

Adolescent↗
PubMed1993

Developmental regulation of hexosamine biosynthesis by protein phosphatases 2A and 2C in Blastocladiella emersonii.

Extracts of the aquatic fungus Blastocladiella emersonii were found to contain protein phosphatases type 1, type 2A, and type 2C with properties analogous to those found in mammalian tissues. The activities of all three protein phosphatases are developmentally regulated, increasing during sporulation, with maximum level in zoospores. Protein phosphatases 2A and 2C, present in zoospore extracts, catalyze the dephosphorylation of L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), a key regulatory enzyme in hexosamine biosynthesis. The protein phosphatase inhibitor okadaic acid induces encystment and inhibits germ tube formation but does not affect the synthesis of the chitinous cell wall. These results strongly suggest that phosphatase 2C is responsible for the dephosphorylation of amidotransferase in vivo. This dephosphorylation is inhibited by uridine-5'-diphospho-N-acetylglucosamine, the end product of hexosamine synthesis and the substrate for chitin synthesis. This result demonstrates a dual role of uridine-5'-diphospho-N-acetylglucosamine by inhibiting the activity of the phosphorylated form of amidotransferase and by preventing its dephosphorylation by protein phosphatases.

Blastocladiella↗
PubMed1993

Interacting signalling pathways regulating prestalk cell differentiation and movement during the morphogenesis of Dictyostelium.

Analysis of the expression patterns of two genes encoding extracellular matrix proteins shows there to be an unexpectedly complex pattern of prestalk cell differentiation and movement during the morphogenesis of Dictyostelium. The organism employs both cell sorting and positional differentiation to generate a patterned structure but these two mechanisms are used at different times during development. During slug formation prestalk cells arise at scattered positions within the aggregate and then move to its apex to form the tip. In contrast, during culmination, stalk cell differentiation occurs in a positionally localized manner at the entrance to the stalk tube. Two interacting signalling pathways regulate the differentiation of prestalk and stalk cells. Prestalk cell differentiation is induced by DIF, a chlorinated hexaphenone, and a repression mechanism prevents DIF acting to induce premature stalk cell differentiation during slug migration. At culmination intracellular cAMP levels rise, the cAMP dependent protein kinase (PKA) is activated and the block to stalk cell differentiation is lifted. Activation of PKA is also necessary in order that prestalk cells move to the entrance of the stalk tube at culmination. Thus, in Dictyostelium, PKA plays a role both in the regulation of cellular differentiation and in morphogenetic cell movement.

Animals↗
PubMed1993

Serum bactericidal test as a prognostic indicator in acute pulmonary exacerbations of cystic fibrosis.

The serum bactericidal test has been used for many years for optimal assessment of the efficacy of antibiotic therapy in patients with infective endocarditis and other bacterial infections. Its capacity to predict the bacteriological outcome of acute pulmonary exacerbations in patients with cystic fibrosis was evaluated. A total of 54 courses of intravenous antibiotic therapy were analyzed in 22 patients, whose ages ranged from 4 months to 24 years (mean age: 10 years). The serum bactericidal activity of blood samples, taken at expected peak and trough antibiotic levels on day 4 of therapy, were determined against the potentially pathogenic strains isolated in sputum at the time of admission. For 104 isolates (64 Pseudomonas aeruginosa, 28 Staphylococcus aureus, and 12 Haemophilus influenzae strains), the peak and trough bactericidal titers were compared to bacteriological outcome. Bacteriological success was defined as a decrease of 2 log10 units or more in the bacterial density in sputum between days 0 and 7 of therapy. At peak antibiotic levels, serum bactericidal titers of 1:128 or more were 96% (all isolates) and 89% (P aeruginosa isolates), predictive of cure, whereas serum bactericidal titers of less than 1:16 were 100% predictive of failure for all infecting bacteria. In patients aged less than 18 years, the best peak titer for predicting success was 1:64, with a predictive value of 96% for titers of 1:64 or greater. The peak titer that best predicted success in patients aged 18 years or more was 1:128, with a predictive value of only 83% for titers of 1:128 or greater.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1992

Biochemical characterization of thioredoxin 1 from Dictyostelium discoideum.

Multiple genes for thioredoxins (TRX) have been demonstrated in Dictyostelium discoideum. We expressed the cDNA for one of these genes (DdTrx1) in E. coli and purified the protein to homogeneity. The interaction with classic substrates as well as TRX reductases was analysed. It reacted with every tested substrate: insulin, NADP-dependent malate dehydrogenase and fructose-1,6-bisphosphatase. With a S0.5 of 20 microM, the reactivity with the fructose-1,6-bisphosphatase is the highest ever found for a heterologous TRX. DdTRX1 itself is accepted as a substrate by the chloroplast ferredoxin-dependent TRX reductase, as well as by the E. coli NADPH-dependent TRX reductase. Thus, the Dictyostelium TRX is functionally promiscuous. Its reactivity with insulin, chloroplast NADP-dependent malate dehydrogenase and ferredoxin-dependent TRX reductase resemble those of other TRX. It is, however, clearly different in its good interaction with chloroplast fructose-1,6-bisphosphatase and in its poor interaction with E. coli NADP-dependent TRX reductase.

Animals↗