Unit: Enzymatic Regulation of Cell Activities
Director: Michel VERON
The main projects of the laboratory are : (A) Study of the phosphorylation of nucleoside analogs used in anti-viral therapies. (B) Biochemical characterization of NEMO, an essential regulatory protein of the NF-kB signal transduction pathway. (C) Mechanistic studies on recombination in retrovirus.
In vitro study of the phoshorylation of antiviral nucleotide analogs. Sarah gallois-montbrun and Dominique deville-bonne
Nucleoside analogs like AZT and d4T used in association with other drugs in multi-therapy protocols against AIDS are delivered to the patients as nucleosides in order to allow cell penetration. They need to be phosphorylated into triphospho-derivatives by cellular kinases to act as chain terminators blocking the viral reverse transcriptase and the last step in this activation pathway is catalyzed by NDP kinase. We have studied 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 J. Janin, LEBS, Gif-sur-Yvette, France). Both kinetic and binding constants were determined showing that the analog phosphorylation is much slower than for their natural counterpart. We have proposed a mechanistic model for the phosphorylation of anti-AIDS nucleotide analogs by NDP kinase.
Ribavirin, a guanosine analog which is used in clinics against RNA virus, is active in the cell as a triphospho-derivative. We have screened new ribavirin derivatives modified on the sugar for increased drug efficiency. The 2'-deoxy derivative of ribavirin showing a phosphorylation rate by NDP kinase and an inhibition of hepatitis C polymerase similar to ribavirin could constitute an interesting alternative to ribavirin (coll. L. Mulard, Unité de Chimie Organique, IP and B. Canard, ESIL, CNRS, Marseille).
A human NDP kinase was ingineered with increased activity for nucleoside analogs. The L55H-N115S mutant shows a change in "specificity factor" of more than 300 for 3'OH substituted analogs. Current studies are aimed at determining whether the expression of this mutant protein in cultured cells could change their sensitivity to nucleoside analogs (coll.J. Balzarini, Rega Institut, Leuwen).
We are also studying the role of PhosphoGlycerate Kinase in the phosphorylation of nucleoside analogues as well as human UMP-CMP Kinase which was recently cristallized (coll. P. Alzari, IP).
Biochemical studies of the protein NEMO, an essential component of the NF-kB signalling pathway. François TRAINCARD, Emilie Vinolo, Elisabeth FONTAN, Jeanne CHIARAVALLI et Fabrice Agou
In response to a variety of stimuli including the pro-inflammatory cytokines (TNFα, IL-1) or endotoxines (LPS), cells activate genes involved in the inflammatory and immune responses as well as in oncogenesis and apoptosis which are under the control of the NF-kB transcription factor. The activation of this pathway is modulated by a high molecular weight protein complex IKK, containing two protein kinases, and a non-catalytic scaffolding protein called NEMO. We investigate the molecular mechanism by which NEMO promotes the activation of the IKK kinases. (coll. A. Israël, Unité de Biologie Moléculaire de l'Expression Génique, Institut Pasteur).
We studied a series of mutant proteins deleted in one or several of the subdomains composing NEMO, and we showed that the oligomerization of the protein is necessary for its biological function. A "minimum oligomerization domain" composed of two coiled coils (CC2 and LZ) was defined and a molecular model was proposed in which the helices corresponding to a trimeric NEMO associate into a six-helix bundle. Based on this model, we designed synthetic peptides with the aim of disrupting the oligomerization of NEMO thus preventing the activation of the NF-kB pathway. One of these peptides corresponding to the LZ coiled-coil, specifically inhibit NF-kB pathway with an IC50 of 3 μM and also stimulated apoptosis in cultured cells. We are currently trying to isolate peptides with higher affinity that could lead to the development of anti-cancer drugs.
NEMO is also involved in several human genetical diseases, including EDA-ID and IP (http://www.nfed.org/; http://imgen.bcm.tmc.edu/IPIF/) a disease of the immune system resulting from point mutations in the coding sequence of NEMO. We have started to study the biochemical properties of the mutant protein A281G, localized in the minimal oligomerization domain of NEMO, which has been identified in an EDA-ID patient. A better knowledge of the structure-function relationships of pathogenic NEMO would allow the developement of new therapeutic approaches of theses diseases.
Mechanisms of genetic recombination in retroviruses. Roman GALETTO, Véronique GIACOMONI and Matteo NEGRONI
Homologous recombination is a major source of genetic variability in retroviruses. During their extracellular life, retroviruses store genetic information as a RNA molecule, which is present in two copies within each viral particle. Genetic recombination occurs in retroviruses mostly through template switching during reverse transcription between these two copies of genomic RNA, a process known as "copy choice" or "strand transfer". The impact of recombination on the dynamics of retroviral infections has been dramatically illustrated for the spreading of the AIDS pandemic. Indeed, at least 10% of the infectious strains of HIV originate through recombination among different viral subtypes (intersubtype recombinants). Some intersubtype recombinant strains comprise forms efficiently spreading throughout the world.
We study the mechanism of retroviral recombination using, in parallel, purified nucleic acids and proteins (in vitro system), and an original system where infection of cells in culture is limited to one cycle (ex vivo system). We focus on the role of the structures of the genomic RNA, since previous results from our laboratory indicated that they are a major determinant in the recombination process. Template switching preferentially occurs within hairpin regions of the genome both in vitro and ex vivo. The folding of the acceptor RNA in vitro, (the one onto which DNA synthesis is continued after the transfer) constitutes the crucial parameter for the efficiency of template switching. These observations have led us to propose a mechanism accounting for strand transfer within hairpin regions of genomic RNAs where the transfer would proceed through a mechanism reminiscent of "branch migration" taking place during DNA-DNA recombination. Our current efforts are now to validate this mechanism under our ex vivo conditions. We will also focus on the role of the sequence divergence on copy choice by studying recombination between isolates from different HIV-1 subtypes, and of the viral RNA chaperone nucleocapside in the process. In collaboration with E. Art's laboratory (Cleveland) we will study the relationship between the composition of the viral population generated after a single infectious cycle and the one observed after multiple infection cycles of cells in culture.
Keywords: NDP kinase, Analogues de nucléotides, Thérapies anti-virales, HIV, Recombinaison, Rétrovirus, Signal transduction, NEMO, NF-kB