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 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. We have identified a series of pathologies caused by mutations in a gene encoding a component of this signaling pathway (the NEMO protein). In addition we have identified a new deubiquitinase which is involved in the NF-κB cascade, and whose mutation leads to a human pathology called cylindromatosis. On the other hand we have set up a system which allows us to dissect the various steps of the mechanisms of NF--κB activation by the TCR. In parallel we have generated a series of transgenic mice where one or more of the IκB molecules have been inactivated, as well as mice where NF-κB activity has been specifically inhibited in certain regions of the brain.
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 mechanism which involves a modification of the receptor following the second cleavage, allowing the third one to take place. Then we have shown that, in a manner reminiscent of the behavior of Notch, its ligand undergo two successive cleavage events, on by a metalloprotease, and the second one by a γ-secretase activity. However, in contrast to the Notch situation, these two cleavages seem to be constitutive.
Genetic and biochemical analysis of the NF-κB signaling pathway (G. Courtois, R. Weil, R. Blaise, C. Lobry, T. Lopez)
In collaboration with the group of JL Casanova (hopital Necker), we had shown that hypomorphic mutations in the gene encoding NEMO, the structural/regulatory subunit of the IKK complex that plays a central role in the NF-κB signaling cascade, lead to a series of symptoms in male patients that associate a defect in innate immunity to a syndrom called EDA (anhydrotic ectodermal dysplasia). A new male patient has been identified who presents with similar symptoms, except that he exhibits an additional deficiency in memory T cells. The analysis of this patient revealed that he carries a mutation in one of the phosphoacceptor Serine residues of IκBα, therefore preventing degradation of this inhibitor and resulting in a partial inactivation of the NF-κB pathway. In parallel we have undertaken a detailed study of NF-κB activation through the TCR. We first demonstrated that the IKK kinase complex is recruited to the immunological synapse and can be coprecipitated with the TCR after T-cell activation. Using ZAP-70-deficient T cells expressing an hybrid molecule between the SH2 domain of ZAP-70 and NEMO, we showed that targetting of NEMO to the immunological synapse was sufficient to selectively restore NF-κB activation in response to TCR ligation. Finally, we demonstrated that the simple targetting of NEMO to the membrane of T cells was sufficient to induce NF-κB activation. This model will allow us to dissect the mechanisms of TCR-induced NF-κB activation and to identify the NEMO-dependent and NEMO-independent events in this cascade.
In vivo analysis of NF-κB activity (S. Mémet, D. Ndiaye)
The generation of mice devoid of both IκBα et IκBε has shown that the phenotype of these double KO mice is more severe than each individual KO : they die at birth and exhibit an increased NF-κB activity, correlated with an increased expression of some of its target genes. The analysis of lymphoid cells at E18.5 shows a deficiency in mature B cells, caused by a massive apoptosis of progenitor cells. Reconstitution experiments in host mice have revealed an unexpected role of NF-κB in the migration of B and T cells towards secondary lymphoid organs. These results confirm the central role played by NF-κB in homeostasis and survival of lymphocytes, and suggest that a decrease or an increase in NF-κB activity can lead to similar types of symptoms.
In parallel we have generated a mouse where the expression of the IκBα inhibitor can be inducibly expressed in the forebrain, and in particular in the hippocampus. This study has allowed us to confirm for the first time in vivo the anti-apoptotic function of NF-κB in neurons.
Finally an ex vivo study of cerebellar granule cells allowed us to characterize the signaling pathways responsible for the basal NF-κB activity observed in these cells. We found that opening of calcium channels at the plasma membrane and at intracellular stores are indispensible for the basal NF-κB activity. We demonstrated further that three cellular sensors of the cytosolic Ca2+ levels, calmodulin, protein kinases C, and the p21ras/PI3K/Akt pathway are simultaneously involved in the steps linking the Ca2+ second messenger to NF-κB activity.
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, which takes place in the extracellular region of the receptor and is required for ligand-induced activation, has been characterized. The responsible enzyme has been purified and identified as TACE, a membrane metalloprotease already known to be involved in TNF shedding. 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.
We have investigated the third proteolytic cleavage which is caused by the activity. We first demonstrated that this cleavage event requires a modification of its substrate, the product of the second cleavage, which is probably required for its transport to the site where γ-secretase cleavage takes place. On the other hand we have set up a genetic screen which should allow us to isolate cells which are devoid of γ-secretase activity : this approach should lead us to the identification of new components of the complex responsible for this activity, or of new regulators of this activity.
Finally we have shown that in a manner reminiscent of the behavior of Notch, one of its ligands, Delta1, undergoes two successive cleavage events, one by a metalloprotease of the ADAM family, and the second one by a γ-secretase activity. We have also shown that the first cleavage was a prerequisite for the second. However, in contrast to the Notch situation, these two cleavages seem to be constitutive. The identification of the ADAM cleavage site has allowed us to design a non-cleavable form of Delta1. In addition, we have shown that the γ-secretase cleavage liberates the intracellular part of the Delta1 molecule, which partially localizes to the nucleus. The possible role this fragment of Delta1 plays in the nucleus is currently being investigated.
Keywords: Signalisation, phosphorylation, proteolysis, ubiquitination, NF-kB, Notch