|Enzymatic Regulation of Cellular Activities - CNRS URA 2185|
|HEAD||VERON Michel / email@example.com|
|MEMBERS||Secrétariat TRAN Catherine / firstname.lastname@example.org
Chercheurs Dr AGOU Fabrice / Chef de labo IP (email@example.com), Dr FONTAN Elisabeth / CR IP (firstname.lastname@example.org), Dr NEGRONI Matteo / CR IP (email@example.com), Dr RAMIREZ Berta Cecilia / CR1 CNRS (firstname.lastname@example.org), Prof. VERON Michel / DR1 CNRS (email@example.com)
Stagiaires Dr GALETTO Roman / Post-doctorant (firstname.lastname@example.org), Dr GRUBISHA Olivera / Post-doctorant (email@example.com), Dr KAMINSKA Monika / Post-doctorant (firstname.lastname@example.org), SIMON-LORIERE Etienne / Etudiant en thèse (email@example.com)
Ingénieurs, Techniciens, Administratifs AYCIN Pinar / Aide de labo IP (firstname.lastname@example.org), CHIARAVALLI Jeanne / Ing. Rech IP CDD (email@example.com), GIACOMONI Véronique / Techn. Sup. IP (firstname.lastname@example.org), TRAINCARD François / Ing. Rech. IP (email@example.com)
NEMO, an essential regulator of the NF-kB signal transduction pathway (PI, Fabrice Agou).
The NF-B signal tranduction pathway is of crucial importance for the cellular response to a great variety of extracellular stimuli and its role in apoptosis and cancer is now universally recognized. Thus, inhibitors of the pathway are of great interest as potential pro-apoptotic anti-cancerous agents. A central regulatory role of the pathway is played by the IKK complex which comprises two protein kinases and a non catalytic scaffolding protein named NEMO (for NF-kB essential regulator). We study NEMO in vitro, by structural and biochemical approaches and, in cellulo, using cell biology techniques.
NEMO oligomerization is necessary for its activity and we identified the « minimum oligomerization domain » formed of the CC2 and LZ coiled-coil (Fig. 1). Using glycerol gradient centrifugation and FRET, we showed that the IKK complex corresponds to a dynamic assembly of its elements, and that stimulation of the NF-kB pathway in cultured cells results in oligomerization and recruitment of a NEMO in a high molecular weight complex comprising kinase dimers.
Several small protein from the ankyrin family were selected using the “ribosome display” method, for their high affinity for the CC2-LZ domain. One of these ankyrins facilitated the crystallisation of the CC2-LZ and the resolution of the 3D structure of this domain is under way (Col. S. Duquerroy, Unité de Virologie structurale). The solution structure of the Zinc finger domain at the C-terminus of NEMO was also determined (Col. F. Cordier, Unité de RMN des Biomolécules).
Several synthetic peptides mimicking the LZ coiled-coil have strong and specific inhibitory effects on both the oligomerization of NEMO in vitro and the activation of the pathway in cultured cells, with IC50 in the M range (Col. F. Baleux, Unité de Chimie Organique). One of these peptides also induces apoptosis of malignant cells isolated from patients with acute myeloid leukemia (AML) and myelodysplasic syndromes (MDS). These results provide a proof of concept for the search of inhibitors of NEMO oligomerization as potential reagents to specifically stimulate apoptosis of cancer cells.
NEMO is ubiquitylated and also binds to K63 polyubiquitin chains. We have identified the site for K63 polyubiquitin binding within the oligomerization domain of NEMO, suggesting a possible functional link between oligomerization and ubiquitin binding in the process of kinase activation of the IKK kinase complex.
NEMO is involved in several rare human genetical diseases including EDA-ID, a dermatological pathology linked to immuno-deficiency. Several NEMO mutants found in patients were produced as recombinant proteins. Their biochemical properties show an altered stability of the oligomer, again pointing to a strong link between oligomerization and NEMO function.
Mechanisms of generation of recombinant forms in HIV (PI, Matteo Negroni)
A peculiar feature of the HIV virus is the impressive genetic diversity of the infectious forms spread worldwide. A major source of this variability is due to recombination that, in retroviruses, arises during reverse transcription as a consequence of template switching, by the reverse transcriptase, between the two copies of genomic RNA present in the viral particle ("copy choice"). By reshuffling large regions of the viral genome, copy choice can bring on the same molecule multiple mutations, increasing considerably genetic complexity of the viral population. Apart from speeding up the generation of resistant strains in response to antiviral treatment, genetic variability is thought to lead the virus to escape from immune control. We study the mechanism of copy choice and its consequences on viral evolution.
Using a reconstituted in vitro system, we first showed that strand exchange was not necessarily due to pauses of reverse transcription but that it was frequently related to the presence of a secondary structure in the acceptor RNA. We proposed that copy choice in structured regions of the genomic RNA could resemble branch migration occurring during DNA-DNA recombination. We developed an original system where recombination was studied after a single infection cycle of cells in culture allowing to study for the first time recombination within the infected cell. Indeed, by allowing the characterisation of recombination products in the absence of any selection, this system provides a snapshot of the recombination products. The mechanism proposed from in vitro studies can also explain the observations made in cellulo.
More recently, the recombination products generated in the env gene using this system between primary isolates from subtypes of group M from HIV 1 have been analyzed. We demonstrated that the recombination yield is very variable along the sequence and we defined the local constraints of sequence that allow two divergent strains to recombine in a given region of the genome.
Fontan, E., Traincard, F., Lévy, S. G., Yamaoka, S., Véron, M. and Agou, F. (2007) NEMO oligomerization in the dynamic assembly of the IkappaB kinase complex. FEBS J., 274, 2540-2551.
Vinolo, E., Sebban, H., Chaffotte, A., Israël, A., Courtois, G., Véron M. and Agou, F. (2006) A point mutation in NEMO associated with anhidrotic ectodermal dysplasia with immunodeficiency pathology results in destabilization of the oligomer and reduces lipopolysaccharide- and tumor necrosis factor-mediated NF-kappa B activation. J. Biol. Chem., 281, 6334-6348.
Galetto, R., Giacomoni, V., Véron, M. and Negroni, M. (2006) Dissection of a circumscribed recombination hot spot in HIV-1 after a single infectious cycle. J. Biol. Chem., 281, 2711-2720.
Baird, H. A., Galetto, R., Gao, Y., Simon-Lorière, E., Abreha, M., Archer, J., Fan, J. Robertson, D. L., Arts, E. J., and Negroni, M. (2006) Sequence determinants of breakpoint location during HIV-1 intersubtype recombination. Nucleic Acids Res., 34, 5203-5216.
Agou, F., Courtois, G., Chiaravalli, J., Baleux, F., Coïc, Y.-M., Traincard, F., Israël, A. and Véron, M. (2004), Inhibition of NF-κB activation by peptides targeting NEMO oligomerization. J. Biol. Chem. , 279, 54248-54257.
Activity Reports 2007 - Institut Pasteur
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