Unit: Enzymatic Regulation of Cell Activities

Director: Michel VERON

The laboratory is organized around two main projects: (i) The study of the biochemical and structural properties of the NF-κ B Essential MOdulator (NEMO) protein, an essential component of the NF-κB signaling pathway and his alteration in patients having a mutated NEMO gene (PI, Fabrice Agou); Study of the mechanism of generation of HIV recombinant forms, a main source of variability in this virus, and the impact of recombination on viral replication ability (PI, Matteo Negroni).

A. Biochemical studies of the NF-κB Essential MOdulator (NEMO) protein, an essential component of the NF-κB signaling pathway (Elisabeth Fontan, François Traincard, Jeanne Chiaravalli, Monika kaminska, Emilie Vinolo, Emmanuel Wyler et Fabrice Agou)

In response to a wide variety of stimuli including the pro-inflammatory cytokines (TNF-α , 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-κB transcription factor. The activation of the NF-κB 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.

We have demonstrated that NEMO trimerizes through its C-terminal domain that includes several subdomains. 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, 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-κB pathway by targeting NEMO oligomerization. Peptide transduction was monitored by FACS and their effect on LPS-induced NF-κB activation was quantified using a NF-κB dependent α-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-κB activation following a variety of pro-inflammatory stimulation (LPS, IL-1, TNF-α, PMA) with IC50 values in the µM range. 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 as well as in cellulo. In addition, they show specificity for the NF-κB 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. Recently, we isolated using the Ribosome Display a new family of ankyrins that bind to the oligomerization domain. Ankyrins constitute an attractive class of stable and small repeat proteins that provide variable and modular binding surfaces to a target protein. After several rounds of selection, we isolated high affinity ankyrins in the low nM range. When expressed in cells, ankyrins inhibit specifically the IKK activation through a specific interaction with the endogenous NEMO. Altogether these results provide the "proof of concept" for a new and promising strategy to inhibit the NF-κB 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 studied the EDA-ID-related A288G mutation which is located within the CC2 motif of NEMO. The mutation does not dramatically change the native-like state of the trimer or NEMO ubiquitination, but temperature-induced unfolding studied by circular dichroism showed that it leads to an important loss in oligomer stability. Fluorescence studies showed that the tyrosine located in the adjacent zinc finger (ZF) domain exhibits an alteration in its spectral properties. This is probably due to a conformational change of this domain, providing evidence for a close interaction between the oligomerization domain and the ZF. Relatedly, functional complementation assays using NEMO-deficient pre-B and T lymphocytes showed that the pathogenic mutation reduced TNF-α and LPS-induced NF-κB activation, providing understanding as to how a single point mutation in NEMO leads to the observed EDA-ID phenotype.

B. Generation and selection oh HIV-1 recombinant forms: structural and genetic aspects (Román Galetto, Etienne Simon-Lorière, Véronique Giacomoni, Cecilia Ramírez & 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". 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, known as CRF, for circulating recombinant forms.

We study the mechanisms fostering the generation of recombinant forms using mostly an original system of infection of cells in culture that limits infection to a single cycle. This approach allows us to characterize the recombinant population generated in the absence of selection, leading to conclusive results on the mechanisms of recombination. Using this system we have dissected a recombination hot spot found in the env gene of the LAI isolate which correlates with the presence of a hairpin structure in the genomic RNA. We have determined that several parameters contribute to the existence of this hot spot, and we have proposed a mechanism accounting for strand transfer within hairpin regions of genomic RNAs. According to this mechanism, the transfer would proceed through a mechanism reminiscent of "branch migration" taking place during DNA-DNA recombination. Recently we have shown that, in infected cells, besides the structure of the RNA also the presence of a perfect sequence identity between donor and acceptor RNAs is required, a single discordant residue triggering a dramatic drop in recombination in the hot spot.

Currently we study recombination between isolates from different subtypes of the M group of HIV-1, in the absence of selective pressure. A map of the most frequently generated recombinant forms along the env gene is being determined. In parallel, we are characterizing the recombinant proteins generated in these assays in order to characterise the early steps of generation of the recombinant forms found in the epidemics. These researches are aimed at improving the understanding of the complex interplay between virus evolution and adaptation to its host. In the long run, the ability of divergent isolates to lead to the formation of functional chimeric viruses and the analysis of the molecular bases for the failure of non-functional chimeric viruses will highlight structural constraints that could be help in the design of molecules with potential antiviral activity.

Keywords: Signal transduction, NF-kappaB, kinase, NEMO/IKKgamma Retrovirus, HIV. Recombination

Activity Reports 2005 - Institut Pasteur

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