Unit: Enzymatic Regulation of Cell Activities
Director: Michel VERON
The main projects of the laboratory in 2004 have been : (A) In vitro study of the phoshorylation of antiviral nucleotide analogs; (B) Biochemical and structural studies of the NF-κ B Essential MOdulator (NEMO) protein, an essential component of the NF-κ B signaling pathway and his alteration in patients having a mutated NEMO gene ; (C) Study of genetic recombination in HIV.
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 (coll. 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 (1).
Ribavirin, a guanosine analog which is used in clinics against RNA virus, is also active in the cell as a triphospho-derivative. We have screened new ribavirin derivatives modified on the sugar for more increased drug efficiency. A 2'-deoxy derivative of ribavirin was efficiently phosphorylated by NDP kinase and had an inhibitor effect towards hepatitis C polymerase (coll. L. Mulard, Unité de Chimie Organique, IP and B. Canard, ESIL, CNRS, Marseille) but we could not show antiviral effect in cell culture infected by several RNA virus (Coll. J. Balzarini, Rega Institut, Leuwen)
A human NDP kinase was engineered with increased activity for nucleoside analogs. In vitro, the L55H-N115S mutant shows a change in "specificity factor" of more than 300 for 3'OH substituted analogs. Current studies, aimed at determining whether the expression of this mutant protein in cultured cells could change their sensitivity to nucleoside analogs, were not conclusive (coll. J. Balzarini, Rega Institut, Leuwen).
We have also studied the role of PhosphoGlycerate Kinase in the phosphorylation of Lnucleoside analogs (2) as well as human UMP-CMP Kinase (3) which was recently cristallized (coll. P. Alzari, IP).
Biochemical studies of the NF-κ B Essential MOdulator (NEMO) protein, an essential component of the NF-κ B signaling pathway. (Elisabeth FONTAN, François TRAINCARD, Jeanne CHIARAVALLI, Emilie vinolo et Fabrice Agou)
In response to a wide variety of stimuli including the pro-inflammatory cytokines (TNF-alpha, IL-1) or endotoxines (LPS), most of cells activate a series of genes involved in the inflammatory and immune responses as well as in tumorogenesis and apoptosis. These genes are under the control of the NF-κ B transcription factor. The activation of the NF-κ B pathway is modulated by a high molecular weight protein complex, containing two IKK protein kinases, and a regulatory/non catalytic protein called NEMO. We investigate biochemical and structural properties of NEMO and the molecular mechanism by which it promotes the activation of the IKK kinases (coll. A. Israël, Unité de Biologie Moléculaire de l'Expression Génique, IP).
We have demonstrated that NEMO trimerizes through its C-terminal domain that includes several subdomains (4). These subdomains were purified and their association properties were characterized by gel filtration, fluorescence polarization, circular dichroïsm, analytical centrifugation and limited proteolysis. We showed that the minimal trimerization domain comprises the CC2-LZ coiled-coil subdomain and that the isolated CC2 and LZ domains bind to each other to form a stable hetero-trimer. On the basis of a structural model for the oligomeric fold of NEMO (4), we rationally designed cell-permeable peptides corresponding to optimal portions of CC2 or LZ subdomains that mimic the contact area between NEMO subunits. These peptides were delivered to cells to block the NF-κ B pathway by disrupting NEMO oligomerization. Peptide transduction was monitored by FACS and their effect on LPS-induced NF-κ B activation was quantified using a NF-κ B dependent β galactosidase assay in stably transfected pre-B 70Z/3 lymphocytes. Our results show that the LZ peptide and, to a lesser extent, the CC2 peptide inhibit NF-κ B activation following a variety of pro-inflammatory stimulation (LPS, IL-1, TNF-alpha) with IC50 values in the µM range (5). Control peptides including mutated peptides as well as heterologous coiled-coil peptides had no inhibitory effect. We also showed that the peptides inhibit NEMO oligomerization in vitro and that they interact with the protein in vivo. In addition, they show specificity for the NF-κ B pathway since they have no effect on the MAP Kinase/ERK and P38 pathways. Finally, we showed that both CC2 and LZ peptides significantly induce apoptosis in cancer cell. Altogether these results provide "proof of concept" for a new and promising strategy to inhibit the NF-κ B pathway in cancer therapy and in inflammation by targeting NEMO's alteration of its oligomerization.
NEMO is also associated with two human genetical diseases, linked to the X chromosome, including ectodermal dysplasia anhidrotic with immunodeficiency (EDA-ID) and Incontinentia pigmenti (IP). Some of the mutations found in patients are single replacements in the coding sequence of NEMO. We have started to study the A288G mutation identified in a EDA-ID patient, and located within the CC2 motif of NEMO in order to search for differences in the biochemical properties of the mutated protein (Coll. G. Courtois, Hopital St. Louis and JL. Cazanova, Hopital Necker).
Study of genetic recombination in HIV. (Roman Galetto, Véronique Giacomoni, Etienne Simon-Lorière and Matteo Negroni)
Homologous recombination is a major source of genetic variability in retroviruses. In HIV, three to thirty recombination events have been observed during a single infectious cycle, depending on the cell type involved. 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 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 by 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 have also studied the mechanism of retroviral recombination using purified nucleic acids and proteins (in vitro system) and, in parallel, 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 (6). Recently we have shown that, in infected cells, besides the structure of the RNA, also its sequence plays a major role in the generation of the major hot spot we have been studying in the env gene. Currently, using the ex vivo system, we study recombination involving isolates from different subtypes of the M group of HIV-1, in the absence of selective pressure. A main goal is the definition of a map of the most frequently generated recombinant forms. In the longer term this study is aimed at characterising the early steps of generation of the recombinant forms found in the epidemics, a study developed in collaboration with the laboratories of Eric Arts (Cleveland) and of Jeff DeStefano (College Park).
Keywords: Anti-viral therapy, Nucleotides analogs, NDP kinase, Signal transducttion, NF-kB, NEMO, Retrovirus, HIV, Recombination