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  Director : Michel VERON (mveron@pasteur.fr)



We study the role of NDP kinase in the phosphorylation of nucleoside analogs used in anti-HIV therapies. We have characterized the interaction beween human type B-NDP kinase and single stranded DNA and studied its role in the regulation of the transcription of the c-myc oncogene. We have determined the oligiomeric structure of NEMO, an essential element of the NF-kB signal transduction pathway. We have continued our previous studies on cAMP dependent protein kinase from Dictyostelium discoideum and started a characterization of the PKA from Plasmodium falciparum. We study the mecanism of recombination in retrovirus in vitro.



Catalytic mechanism of NDP kinase Benoit schneider, Sarah gallois-montbrun et Dominique deville-bonne

Nucleoside Diphosphate Kinase (NDP Kinase) catalyzes the transfer of a phosphate from a nucleoside triphosphate to a nucleoside diphosphate by a ping-pong mechanism involving the transient phosphorylation of a his residue. The 3'-OH group of the ribose of the nucleotide substrate plays an important role in the"sustrate assisted" reaction mechanism.
We study the reaction mechanism by using point mutants of active site residues. Mutants H122G and H122A of Dictyostelium NDP kinase are inactive but they still catalyze the synthesis of imidazole-phosphate from ATP and a exogenous nucleophile as imidazole (collaboration D. Herschlag, Standford, USA). We have studied the ionisation strate of Lys 16 and Tyr 56 participating in the phosphotransfer. While both residues interact in the active form of the wild type enzyme, Tyr 56 has an unusual pK in the K16A mutant. However, the substitution of these "catalytic" residues has less effect on the activity than the absence of the 3'-OH group of the substrate. We have synthesized a stable analog of the phosphorylated intermediate by chemically modifying the cysteine in the active site of the H122C mutant. This phosphorylated enzyme has an increased affinity for NDPs as compared to NTPs.

In vitro study of the phoshorylation of nucleotide analogs used against AIDS by NDP kinaseBenoit schneider, Sarah gallois-montbrun, Annett Kreimeyer et Dominique deville-bonne

Akthough new inhibitors are now used in clinics, nucleotide analogs substituted in the 3'OH position of ribose like AZT are still used in all current therapies against AIDS. They are delivered to the paptients as uncharged nucleoside so that they enter the cell, and they need to be phosphorylated into triphospho-derivatives by several cellular kinases before they can act as chain terminator blocking the viral reverse transcriptase. The last step in this activation pathway is catalyzed by NDP kinase.
We study the reactivity of recombinant human NDP kinase with several analogs currently used in clinics (ddI, ddC, AZT, d4T), both at the biochemical and at the structural level (collaboration with L. Mulard, Unité de Chimie Organique, Institut Pasteur for the synthesis of phosphorylated nucleotiude analogs and with J. Janin, LEBS, Gif-sur-Yvette, France, for the X ray structural studies). We showed that phosphorylation of the analogs is much slower than for their natural counterpart, indicating that NDP kinase may be a limiting step in the activation process. Both kinectic and binding constants were determined. Combining biochemichal and structural data, we have proposed a precise mechanistic model for the phosphorylation of anti AIDS nucleotide analogs by NDP kinase.
3TC is a L-derivative of thiacytidine recently introduced in therapeutic protocols for AIDS and hepatitis B and b-L-desoxynucleosides (b-L-thymidine and b-L-2'-desoxycytidine) have recently been recognizded as specific anti-hepatitis B agents. We have shown that both nucleotides and desoxynucleotides in -L configuration and in particular 3TC are very poor substrates for NDP kinase, (collaboration with A. Faraj and J.P. Sommadossi, NOVORIO-CNRS and Université Montpellier II). This suggests that new yet unidentified kinases are responsible for the synthesis of the L-XTP and L-dXTP in the cell.
Ribavirine, a guanosine analog which is the only nucleoside analog used in clinics against RNA virus, is a good drug candidate against dengue, a viral desease responsible for hemorragic fever. Ribavirine is active in the cell as a triphospho-derivative. We want to increase the drug efficiency of ribavirine by screening new derivatives that would be active at lower doses with less toxicity. We are currently analyzing several ribavirine analogs for their phosphorylation by NDP kinase and for their affinity with their putative cellular target, the viral capping enzyme (collaboration with L.Mulard, Unité de Chimie Organique, Institut Pasteur and B. Canard, ESIL,CRNS, Marseille).

New analogs and mutant NDP kinase with increased phosphorylation capacity. Benoit schneider, Véronique Giaccomoni-fernandez, Céline boulard, Dominique deville-bonne

We have designed new reverse transcriptase inhibitoirs by modification of currently used nucleoside inhibitors with a borano (BH3) group of the a-phosphate (collaboration with S. Sarfati and L. Mulard, Unité de Chimie Organique, Institut Pasteur). The biochemical properties of the borano-derivatives of AZT and d4T were characterized in vitro on NDP kinase and reverste transcriptase. The X-ray structure of the analog/NDP kinase complexes allows to explain the increased reactivity of the borano-derivatives. The efficiency of these new compounds at the cellular level is being investigated (collaboration with P. Clayette, CEA).
Using protein ingeniering, we want to modify NDP kinase active site to make it a better enzyme to phosphorylate nucleoside analogs. The phosphorylation of AZT-DP and d4T-DP in vitro by a mutant human NDP kinase is increased by a factor > 10. Current studies are aimed at determining if cells transformed with this mutant have increased levels of triphospho-derivatives along with increased sensitivity to the drug.

In vitro recombination in retrovirusesAbdeladim Moumen, Lucette POLOMACK, Matteo Negroni, Henri BUC (NB: H. Buc is Professor emeritous attached to CNRS - URA 1960)

Homologous recombination is a major source of genetic variability in retroviruses. In these viruses, most recombination is generated by template switching during reverse transcription between the two copies of genomic RNA present in the viral particle, a process known as "copy choice" or strand transfer. The impact of recombination on the dynamics of retroviral infections is dramatically illustrated with the spreading of the AIDS pandemic where at least 10% of the infectious strains of HIV originate through recombination among different viral subtypes.
We study the mechanism of retroviral recombination in a reconstituted system, using purified nucleic acids and proteins. We focus particularly on the role of RNA secondary structures which we have recently suggested to play an important role in the recombination process. The role of the viral nucleocapsid protein, a major cofactor of the reverse transcription complex, has been extensively investigated. This protein, known to enhance the frequency of recombination in vitro, carries an RNA chaperone activity. Our results strongly suggest a crucial implication of the structures of the genomic RNAs in copy choice recombination. We are currently trying to define the structural determinants for efficient copy choice, both from the standpoint of the RNA templates and of the reverse transcriptase.

Interaction of human NDP kinase type B with single strand DNA Sharona Raveh, Fei WE et Fabrice Agou

NDPK-B is an isoform of human NDP kinase which is involved in the regulation of the expression of the c-myc oncogene. We have used recombinant NDPK-B to show that this protein binds to single strand DNA. The binding occurs has no sequence specificity but occurs only with this isozyme and not with 88% identical NDPK-A. We have crosslinked synthetic oligonucleotides with NDPK-B by laser UV irradiation of the complex. We have then identified the DNA/protein contacts by MALDI-TOF spectrometry after proteolysis of the complex and separation of the peptides on reverse phase-HPLC (collaboration with J. Rossier, Ecole de Physique et Chimie de Paris). We next probed whether the oligomeric form of NDPK-B present in the nucleus of cultured cells was a dimer or an hexamer by using a bifunctional crosslinker specific of cysteine residues. We propose that NDPK-B is an "architectural transcription factor" interacting with single stranded DNA stretches arising from sequences forming triplex or tetraplex structures.

Biochimical properties of NEMO, an essential element of the NF-kB signal transduction pathwayFabrice Agou et Stephane Goffinont

NEMO (NF-kB Essential MOdulator) has been recently characterized as a crucial element in the NF-kB signal transduction pathway. NEMO plays crucial a role upstream of IKK kinases in the activation pathway.
We have purified recombinant NEMO in the pesence of non-ionic detergents and studied its quaternary structure. The purification of a C-terminal fragment showed that a coiled-coil domain is responsible for the formation of dimers and trimers. Recombinant NEMO is associated with the E. coli DnaK (Hsp 70) chaperone, suggesting a role for this type of protein in the oligomerisation of NEMO in vivo. The presence of imers and trilmers of NEMO in vivo has also been demonstrated in several cultured cell lines. These results are of particular interest considering the recent discovery that mutations in the gene encoding NEMO are the cause of at least two human pathologies (collaboration with A. Israel, Unité de Biologie Moléculaire de l'Expression Génique, Institut Pasteur).

cAMP dependant proteine kinase from Dictyostelium and plasmodium falciparumFrançois Traincart

Dictyostelium discoideum amoebae enter a development cycle in response to food starvation. After a few hours, the cells form streams and migrate in response to cAMP signals to form a multicellular aggregate . In a second phase, the latter develops into a fruting body formed of a stalk supporting a mass of spores.
Collaborating with Carmen Buchrieser (Laboratoire de Génomique des microorganismes pathogènes, Institut Pasteur),we have contributed to the Dictyostelium genome project by sequencing 4 YAC clones covering a fraction of chromosome 6. This project is a European Union network coordinated by the Sanger Center (Hinxton, UK).
We have shown that cAMP dependent protein kinase (PKA) plays a central role in Dictyostelium development. The C-terminal amino-acids of the catalytic subunit (C) are necessary for enzymatic activity. Contrary to most of the catalytic core, this C-terminal region is not conserved between mammals and eukaryotic pathogens for which PKA could be a potential therapeutic target. This is the case of Plasmodium falciparum where have demonstrated a PKA activity and shown that it is necessary for parasite maturation (collaboration G. Langsley, Unité de Signalisation Immunoparasitaire, Institut Pasteur). We have raised antibodies against a C-terminal peptide of Dictyostelium PKA which are strong specific inhibitors of the enzymatic activity, suggesting that this sequence is a good candidate for raising species specific PKA inhibitors.
Premiminary results of immunofluorescence indicate that Dictyostelium NDP kinase is enriched in submembranous cell structures. We are currently studying its role in cytosqueletton reorganisation using the many experimental possibilities of the Dictyostelium model.
In a different domain, we have started to collaborate with highschool teachers in order to use Dictyostelium as a model system to be used during practicals in senior highschool classes. The first results are vey encouraging and we now plan to extend this approach to other classes including in the primary school.

A new cAMP sensor using the R subunit of Dictyostelium PKABenoit schneider

A fusion protein was constructed by insertion at random of GFP in the coding sequence of the regulatory subunit (R) of Dictyostelium PKA. The fusion protein was selected for its cAMP binding properties as well as its inhibitory effect on the C subunit. A fluorescence energy transfer (FRET) occurs between GFP and a fluorescent derivative of cGMP bound to the cAMP binding site of the R subunit. This could constitute a "sensor" that could be used to measure the cAMP concentration in cell free extracts. (collaboration with C. Reymond, Lausanne University, Switerland).

Phosphorylation of the E. coli CheY protein Sandra Da-RE, Jeff Stock, Dominique deville-Bonne

Bacterial chemotaxis is controlled by two-component systems involving cascades of phosphorylation by histidine-kinases. In E. coli. CheY, " the response regulator", is phosphorylated on a Asp by the histidine-kinase CheA. Chey can be phosphorylated in vitro by small molecules acting as phosphate donnor, like acetylphosphate or phosphoramidate. Using a fluorescent stopped flow, we study the phosphorylation of CheY or of its mutants by these small molecules as a model for phospho-transfer reactions (collaboration with J. Stock, Princeton, USA).


Figure 1 : The active site of NDP kinase
The Figure shows a dTDP molecule bound in the active site of Dictyostelium NDP kinase.The catalytic histidine (H122) is shown, as well as several residues important for binding of the substrate and catalysis. The crucial hydrogen bond between the ribose 3'OH and the O7 of the _-phosphate's is also shown.

Figure 2 : Dictyostelium discoideum development cycle
The different phases of morphogenesis from the multicellular aggregate to the fruiting body

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  Office staff Researchers Scientific trainees Other personnel

AYTAC Marie-Dominique (Institut Pasteur) – mdaytac@pasteur.fr

AGOU Fabrice (Institut Pasteur) – fagou@pasteur.fr

DEVILLE-BONNE Dominique (Université Pierre et Marie Curie) – ddeville@pasteur.fr

NEGRONI Matteo (Institut Pasteur) - matteo@pasteur.fr

GALLOIS-MONTBRUN Sarah (DEA) (Université Paris VI)

MOUMEN Abdeladim (Thèse) (Université Paris XI)

SCHNEIDER Benoit (Thèse) (Université Paris VII/ENS Cachan)

BOULARD Céline (Technicienne supérieure, IP)

CORTES Marie-Thérèse (Institut Pasteur – agent de laboratoire, IP)

GIACOMONI-FERNANDES Véronique (Technicienne supérieure, IP) – verofern@pasteur.fr

GOFFINONT Stéphane (Assistant Ingénieur CNRS) – sgoffi@pasteur.fr

TRAINCARD François (Ingénieur, IP) – traincar@pasteur.fr

POLOMACK Lucette (Technicienne supérieure, IP)


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