The NEMO Team (PI: Fabrice Agou)


The research group is focused on the study of the molecular mechanisms by which the IKK complex activates the NF-kB signal transduction pathway

Since 2002, we study the protein NEMO (NF-kBEkB signal transmission pathway is known for long to participate in the inflammatory cellular response, and to bacterial (LPS) or viral (TAX) agents. More recently, the NF-kB pathway has also been shown to participate in cancer progression through  inhibition of apoptosis. NEMO plays a pivotal role as a regulator of the so-called IKK complex, a central regulatory multiprotein complex (Fig. 1) (see Hayden and Ghosh., 2008; Vallabhapurapu and Karin, 2009; Israël, 2010, for reviews). In addition, mutations in NEMO are the cause of rare human diseases (see Fig. 2), including Ectodermal Dysplasia Anhidrotic with Immuno Deficiency (Doffinger et al.), Incontinentia Pigmenti (Smahi et al., 2000) and Mendelian Susceptibility to Mycobacterial Disease (Picard et al., 2006).ssentialModulator). The NF-
 

Figure 1:
The canonical NF-kB signaling pathway



 
Major results and objectives

We have shown that NEMO forms several dynamic multiprotein complexes in the cell with the two kinases IKKα and IKKβ (7). The regulatory region of NEMO (Fig. 2) integrates the signals leading to regulation of the IKK complex. This region encompasses, within the C-terminal half of the protein, a coiled-coil domain (CC2-LZ) responsible for the association of the protein in dimers, and a « zinc finger » domain (ZF) at its extreme C-terminus. The solution structure of the ZF domain was determined by NMR, and a model of the complex of this domain with Ubiquitin was established (5, 6) (Fig. 3).

 
 
 
Figure 2: The functional domains of NEMO: CC, coiled-coil; LZ, leucine zipper; HLX, helical domain; NOA, ubiquitin binding site found in NEMO, Optineurin/NRP and ABINs proteins; ZF, zinc finger; UBD, ubiquitin binding domain; NOAZ, ubiquitin binding bipartite (NOA and ZF) domain. Binding sites for the RIP, vFLIP, TANK and CYLD proteins are indicated. Some NEMO mutations causing human pathologies are indicated: red, Incontinentia Pigmenti (IP); blue, anhidrotic ectodermal dysplasia with immunodefiency (EDA-ID); green, Mendelian susceptibility to mycobacterial diseases (MSMD).


Using the directed evolution method of the « Ribosome display », we have isolated proteins from the ankyrin family binding with high affinity to the CC2-LZ domain of NEMO (8). These so called DARPINs (Designed Ankyrin Repeats Proteins) allowed crystallization of a dimeric CC2-LZ domain and its structure was solved by X-ray diffraction at 2,9 A (3) (Fig. 4).
 
 
Figure 3: Model of the NEMO ZF-ubiquitin complex: Docking model proposed for the NEMO ZF-ubiquitin complex (average structure), with the ZF in blue and ubiquitin in grey. The zinc ligand is depicted as a fuchsia sphere.

 

Both the NOA region within the CC2-LZ domain and the ZF domain are ubiquitin binding domains. The high affinity binding which is specific to poly-Ubs K63 requires the presence of both domains forming a new bipartite binding site (4) (see Fig. 2).

We designed synthetic peptides able to block the activation of the NF-kB pathway by extracellular stimuli. These peptides either inhibit NEMO oligomerization (13) or interfer with its poly-ubiquitin binding activity (Chiaravalli et al., in preparation). They specifically kill several primary cancer cell types of the myeloid lineage (9). An important R&D project in association with a pharma company is under way, with the aim to isolate drugable compounds mimicking the action of these peptides.

In parallel, we study the in vitro properties of mutants of the C-terminal part of NEMO, and in particular of its ZF domain, which cause rare human genetic diseases (Courtois et al., 2001) with the aim to understand the molecular mechanisms of the defects (5, 6, 12).

 
Figure 4: Structure of the NEMO CC2-LZ domain in complex with 1D5 DARPin: Side view of the complex in ribbons diagram. NEMO helices (chains A and B) are colored as defined on the vertical bar to the left, i.e. CC2 (blue), stammer (pale green), NOA (red), LZ (turquoise). His-tag residues (aa 240-250) are colored in gray. Chains C and D of the 1D5 DARPin are colored in green (See Ref. 3 for details).
 

References cited
  • Courtois, G., Smahi, A. & Israel, A. (2001). NEMO/IKK gamma: linking NF-kappa B to human disease. Trends Mol. Med. 7, 427-430.
  • Doffinger, R., Smahi, A., Bessia, C., Geissmann, F., Feinberg, J., Durandy, A., Bodemer, C., Kenwrick, S., Dupuis-Girod, S., Blanche, S., et al. (2001). X-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired NF-kappaB signaling. Nat. Genet. 27, 277-285.
  • Hayden, M. S. & Ghosh, S. (2008). Shared principles in NF-kappaB signaling. Cell 132, 344-362.
  • Israel, A. (2010). The IKK complex, a central regulator of NF-kappaB activation. Cold Spring Harb Perspect Biol 2, a000158.
  • Picard, C., Casanova, J. L. & Abel, L. (2006). Mendelian traits that confer predisposition or resistance to specific infections in humans. Curr Opin Immunol 18, 383-390.
  • Smahi, A., Courtois, G., Vabres, P., Yamaoka, S., Heuertz, S., Munnich, A., Israel, A., Heiss, N.S., Klauck, S.M., Kioschis, P., et al. (2000). Genomic rearrangement in NEMO impairs NF-kappaB activation and is a cause of incontinentia pigmenti. The International Incontinentia Pigmenti (IP) Consortium. Nature 405, 466-472. and http: www.incontinentiapigmenti.fr)
  • Vallabhapurapu, S. & Karin, M. (2009). Regulation and function of NF-kappaB transcription factors in the immune system. Annu Rev Immunol 27, 693-733.

Main current Research Projects
  • Structural basis for the recognition of different poly-ubiquitin linkage by the bipartite ubiquitin-binding domain of NEMO (NOAZ).
  • Determination of the IKK complex structure.
  • Study of the recruitment of the IKK complex at the cell membrane in response to extra-cellular stimuli.
  • Development of new peptides or compounds with anti-cancer activity targeting NEMO and inhibiting the NF-κB pathway.
  • Characterization of the molecular defect of new pathological mutants of NEMO.

Technical know-how: Biochemistry, Cell biology, Modelisation
  • Molecular biology (cloning, site directed mutagenesis…).
  • Cellular biology (transfection, transduction, immunofluorescence, western blot, IP…).
  • Confocal microscopy, fluorescence microscopy (FRET, BiFC…).
  • Protein purification.
  • Biophysical techniques (sedimentation velocity and equilibrium, CD, DLS, ITC, Biacore, Stopped flow, fluorescence polarization, HTRF, gel filtration combined with LALS …).
  • Directed evolution (Ribosome display).
  • In-silico modeling.
Advanced methods in crystallogenesis
 

Team members



Fabrice AGOU
PI, Chef de Laboratoire, Institut Pasteur
Phone 01 44 38 95 69



Elisabeth FONTAN
Chargée de Recherche, Institut Pasteur
Phone: 01 45 68 83 80


Keïs NABHANE
Etudiant en Master 2
Phone: 01 40 61 38 10