Unit: Biologie moléculaire de l'expression génique

Director: Israël Alain

The laboratory is involved in the study of two signalisation pathways characterized by inducible proteolysis events that lead to the nuclear translocation of transcription factors. The first project deals with the rel/NF-κB family of transcription factors, whose activity is controled by cytoplasmic retention through inhibitory molecules (IκB's) which get degraded in response to multiple signals. In collaboration with the group of Michel Veron we have identified peptides which interfere with the oligomerization of the NEMO protein, the central component of the NF-kB cascade, and specifically inhibit this signaling pathway.

A second project deals with the Notch receptor which transmits its signal following a series of three proteolytic steps which are followed by the nuclear translocation of the intracellular part of the receptor, which then behaves as a transcriptional co-activator. We have pursued 2 directions of research: we have first identified a new step of the activation cascade, which involves monoubiquitination of the receptor following the second cleavage, allowing the third one to take place. We have then shown that the intracellular region of the Notch ligands interacts with proteins that associate with the cytoskeleton, and therefore regulate the motility of the cells which express these ligands.

Structure of the NEMO protein and identification of peptides which inhibit its oligomerization (G. Courtois, R. Weil, T. Lopez ; collab with M Veron and F Agou)

In collaboration with Fabrice Agou in the lab of Michel Veron, we have studied the determinants of the oligomerization of the NEMO protein. NEMO is the regulatory component of the IKK complex, which also includes two kinases, IKKα and IKKβ, and its oligomerization has been suggested to represent a critical event in the activation of this complex. The self association of NEMO involves the C-terminal half of the polypeptide chains containing two putative coiled-coil motifs, a CC2 and a LZ leucine zipper, a proline rich region and a ZF zinc-finger motif. We have shown that the minimal oligomerization domain of NEMO can be restricted to the CC2-LZ segment and we demonstrated that both CC2 and LZ subdomains are necessary to restore the LPS-dependent activation of the NF-κB pathway in a NEMO deficient cell line. We confirmed the association of the oligomerization domain in a trimer and we demonstrated that both the CC2 and LZ subdomains are necessary for trimer formation. We also showed that upon mixing, isolated CC2 and LZ peptides associate to form a stable hetero-hexamer. We therefore proposed a structural model for the organisation of the oligomerization domain of activated NEMO in which three C-terminal domains associate into a pseudo-hexamer forming a six helix bundle.

To search for drugs inhibiting NF-κB activation, we rationally designed cell-permeable peptides corresponding to the CC2 and LZ subdomains that mimic the contact areas between NEMO subunits. The peptides were tagged with the Antennapedia/Penetratin motif and delivered to cells prior to stimulation with lipopolysaccharide. We showed that both peptides inhibit NF-κB activation with an IC50 in the μm range. These molecules were also shown to be able to induce cell death in human retinoblastoma Y79 cells, which require constitutive NF-κB activity for survival. These results provide the "proof of concept" for a new and promising strategy for the inhibition of the NF-κB pathway activation through targeting the oligomerization state of the NEMO protein.

Role of the NF-κB pathway in the differentiation of NK cells (S. Mémet, D. Ndiaye; collaboration Jim di Santo)

While perturbations in NF-κB activity impact strongly on B- and T-cell development, little is known about the role for NF-κB in natural killer (NK) cell differentiation. We analyzed the cell-intrinsic effects of deficiencies in 2 IκB members (IκBα and IκBε) on NK cell differentiation. Neither IκBα nor IκBε deficiency had major effects on NK cell generation, while their combined absence led to NF-κB hyperactivation, resulting in reduced NK cell numbers, incomplete NK cell maturation, and defective interferon gamma production. Complementary analysis of transgenic mice expressing an NF-κB-responsive reporter gene showed increased NF-κB activity at the stage of NK cell development corresponding to the partial block observed in IκBαxIκBε-deficient mice. These results define a critical window in NK cell development in which NF-κB levels must be tightly controlled.

The Notch signaling cascade (F. Logeat, C. Brou, N. Gupta, E. Six, A. Olry, P. Chastagner, D. Ndiaye)

We have proposed a model according to which the activation of the Notch receptor involves a series of three proteolytic steps. The first cleavage is due to a furin-like convertase, and is required for surface expression of a functional Notch receptor. A second cleavage step takes place in the extracellular region of the receptor and involves the membrane metalloprotease TACE. The third and last cleavage is due to an activity called γ-secretase and results in the liberation of the intracellular region of the receptor, which is then transported to the nucleus where it behaves as a transcriptional coactivator.

One of our projects involved identifying the requirements for the γ-secretase cleavage step : we demonstrated that this cleavage requires a new modification of the Notch receptor, monoubiquitination, as well as clathrin-dependent  endocytosis. These steps are probably required for the transport of Notch to the site where γ-secretase cleavage takes place. Presenilin, the catalytic component of the γ-secretase complex, preferentially interacts with the monoubiquitinated form of ΔE. We identified the lysine residue targeted by the monoubiquitination event and confirmed its importance for activation of the Notch receptor by its ligand Delta1.

Our second project deals with a potential cell-autonomous role for the Notch ligands. Using peptide affinity chromatography followed by mass spectrometry we have identified Dlg1 as a partner of Delta1 C-terminal region. Dlg1 is a human homologue of the Drosophila Discs large tumor suppressor, a member of the MAGUK (Membrane-Associated Guanylate Kinase) family of molecular scaffolds. Moreover we showed that deletion of a canonical C-terminal PDZ binding motif (ATEV) in Delta1 abrogated this interaction. Delta4 also interacted with Dlg1, while Jagged1, another Notch ligand, did not. In HeLa cells, transfected Delta1 triggered the accumulation of endogenous Dlg1 at sites of cell-cell contact. Expression of Delta1 also reduced the motility of 3T3 cells. Finally, deletion of the ATEV motif totally abolished these effects, but did not interfere with the ability of Delta1 to induce Notch signaling and T cell differentiation in coculture experiments. These results point to a new, probably cell-autonomous function of Delta1, which is independent of its activity as a Notch ligand.

Keywords: Signalisation, phosphorylation, proteolysis, ubiquitination, NF-?B, Notch

Activity Reports 2004 - Institut Pasteur

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