Group 1 : Notch team (PI: Christel Brou)


Research summary
Development and maintenance of organs and tissues requires constant interaction and communication between cells. Notch pathway allows for communication between adjacent cells and can elicit many downstream responses, including cell-fate specification, progenitor cell maintenance, boundary formation, proliferation and apoptosis. This pathway is used at many steps during development and adulthood, and the precise outcome of the Notch signal is highly sensitive to the cellular context. Haploinsufficiency or gain-of-function of Notch, or Notch-related genes, are responsible for various defects in mammals (1): developmental diseases, including aortic valve disease, Alagille syndrome, and familial forms of cardiomyopathy, or late-onset syndromes, like CADASIL (Cerebral Autosomal Dominant Artheriopathy with Subcortical Infarcts and Leukoencephalopathy), increasing number of cancers (skin, intestine, head and neck squamous cell carcinoma) or leukemia (more than 50% of T-cell acute lymphoblastic leukaemias T-ALL, 15% of chronic lymphocytic leukaemia, myeloid leukaemia). Depending on the tissue, Notch appears to behave as an oncogene or a tumor suppressor. Therefore it is crucial to better understand the molecular mechanisms of Notch signaling and regulation, in order to eventually identify druggable factors or steps.

 

Figure 1 : Notch receptors (left) and their ligands (right). The extracellular domain of Notch exhibits EGF-like repeats, involved in ligand binding, whereas the intracellular part contains ankyrin-like repeats, a PEST domain and several nuclear localization signals. Notch ligands of theDSL family (for Delta and Serrate from Drosophila and Lag-2 from C. elegans; there are 3 Delta and 2 Serrate/Jagged genes in mammals) are also type I transmembrane proteins, characterized by an N terminal DSL domain accounting for receptor binding, and a poorly conserved and structured intracellular domain. dNotch and Delta/Serrate (top) are from Drosophila, the other are mammalian homologs.
The Notch receptor (see figure 1) is expressed as a transmembrane heterodimeric receptor at the cell surface. Notch activation relies on the direct contact between adjacent cells, since Notch ligands are also transmembrane proteins. Following ligand binding, Notch undergoes two consecutive cleavages that allow the release of Notch intracellular domain and its translocation into the nucleus where it acts as a transcriptional activator (figure 2). These enzymatic cleavages of Notch, first by an ADAM metalloprotease (ADAM10 or 17), and second by g-secretase activity (the same enzyme that is affected in Alzheimer’s disease), occur during trafficking of the receptor into the cell (3, 4). This trafficking is regulated by post-translational modifications such as ubiquitinations/deubiquitinations (figure 3) and requires various cofactors and regulators. In addition the proper delivery of Notch cofactors is highly controlled, and their exact function deserves to be further studied.

 

Figure 2: An overview of Notch signaling. Notch is depicted as two orange triangles representing the heterodimer, Delta ligand is in green. 1. In the absence of activation, Notch is internalized and degraded in the lysosomes. 2. Delta ligand is internalized and recycled back to the membrane in a modified form, which has acquired a strong affinity for the receptor. 3. Upon ligand binding, Notch extracellular domain is transendocytosed into the ligand-sending cell, and the remaining receptor undergoes two successive proteolytic cleavages by ADAM metalloproteases (ADAM10 in most cases, otherwise it may be ADAM17/TACE (2)) then by the γ-secretase complex, which acts inside the transmembrane domain and allows the release of the intracellular part of the receptor (NIC). The latter translocates to the nucleus where, together with CSL (for human CBF1, Drosophila Su(H) and C. elegans Lag-1) and Mastermind, it reconstitutes a target-specific transcriptional activator. EE: early endosome; MVB: multivesicular body; RE: recycling endosome; U: ubiquitin.

 
 
 
Among the mechanisms that control the strength of Notch signal, our work focus on the maintenance of an active receptor at the plasma membrane, on the production of an active ligand, or on the regulation of signal transduction after activation. We are particularly interested in the trafficking and post-translational modifications undergone by Notch receptor and ligands.
 
Figure 3: Ubiquitination of a substrate consists in the covalent attachment of the ubiquitin polypeptide to an internal lysine residue of the substrate by members of the large family of E3 ubiquitin ligases that act in concert with E2-conjugating enzymes.Ubiquitination is counteracted by deubiquitination, ie removing of the ubiquitin chain by specific deubiquitinating enzymes called deubiquitinases (or DUBs). This removal can promote or prevent carrying out of the process initiated by ubiquitination (5).

 
Major recent results
We have already highlighted the importance of ubiquitination in the Notch activation process (6) as well as in the regulation of non-activated receptor by identifying the roles of an E3 ubiquitin ligase (7, 8). We have also described two novel deubiquitinases regulating Notch activation and Notch quantity at the cell surface respectively (9, 10). However these steps require additional specific enzymes and cofactors that we are studying now (11).
 
Main current Research Projects
1) Functional characterization of two new families of regulators acting in Notch signaling: Ndfips and Arrestins.
2) Identification of the deubiquitinating enzymes targeting Notch ligands, and regulating ligand quantity and/or activity.
3) Role and mechanism of action of the E3 ubiquitin ligase Deltex in Notch signaling
4) Cell autonomous functions of Notch ligands
 
References cited
1.           Louvi A & Artavanis-Tsakonas S (2012) Notch and disease: A growing field. Semin Cell Dev Biol.
2.           Brou C, et al. (2000) A novel proteolytic cleavage involved in Notch signaling: The role of the disintegrin-metalloprotease TACE. Mol Cell 5(2):207-216.
3.           Bray SJ (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7(9):678-689.
4.           Brou C (2009) Intracellular trafficking of Notch receptors and ligands. Exp Cell Res 315:1549-1555.
5.           Komander D, Clague MJ, & Urbé S (2009) Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol 10:550-563.
6.           Gupta-Rossi N, et al. (2004) Monoubiquitination and endocytosis direct {gamma}-secretase cleavage of activated Notch receptor. J Cell Biol 166(1):73-83.
7.           Chastagner P, Israel A, & Brou C (2006) Itch/AIP4 mediates Deltex degradation through the formation of K29-linked polyubiquitin chains. EMBO Rep 7(11):1147-1153.
8.           Chastagner P, Israël A, & Brou C (2008) AIP4/Itch regulates Notch receptor degradation in the absence of ligand. PLoS ONE 3(7):e2735.
9.           Moretti J, et al. (2010) The translation initiation factor 3f (eIF3f) exhibits a deubiquitinase activity regulating Notch activation. PLoS Biol 8(11):e1000545.
10.         Moretti J, et al. (2012) The Ubiquitin-specific Protease 12 (USP12) Is a Negative Regulator of Notch Signaling Acting on Notch Receptor Trafficking toward Degradation. J Biol Chem 287(35):29429-29441.
11.         Dalton HE, et al. (2011) Drosophila Ndfip is a novel regulator of Notch signaling. Cell Death Differ 18(7):1150-1160.
 
 
 
Publications of the team (2012-2000)
·       Moretti, J, Chastagner, P, Liang, C-C, Cohn, M, Israël, A, Brou, C. (2012) The Ubiquitin Specific Protease 12 (USP12) is a negative regulator of Notch signaling acting on receptor trafficking towards degradation. J. Biol. Chem., 287, 29429-29441.
·       Dalton, HE., Denton, D., Foot, NJ., Ho, K., Mills, K., Brou, C., and Kumar, S. (2011) Drosophila Ndfip is a novel regulator of Notch signaling. Cell Death and Differentiation 18, 1150–1160
·       Moretti, J., Chastagner, P., Gastaldello, S., Heuss, SF., Dirac, AM., Bernards, R., Masucci, MA., Israël, A., and Brou, C. (2010) The translation initiation factor 3f (eIF3f) exhibits a deubiquitinase activity regulating Notch activation. PLoS Biol 8(11): e1000545.
·       Brou, C. (2009) Intracellular trafficking of Notch receptors and ligands. Exp. Cell Res. 315, 1549-1555.
·       Blaise, R., Mahjoub, M., Salvat, C., Barbe, M., Brou, C., Corvol, MT., Savouret, JF., Rannou, F., Berenbaum, F., and Bausero, P. (2009) Involvement of the Notch pathway in the regulation of MMP13 and the dedifferentiation of articular chondrocytes. Arthritis and Rheumatism 60, 428-439.
·       Chastagner, P., Israël, A., and Brou, C. (2008) AIP4/Itch Regulates Notch Receptor Degradation in the Absence of Ligand. PLoS ONE 3, e2735.
·       Clement, N., Gueguen, M., Glorian, M., Blaise, R., Andreani, M., Brou, C., Bausero, P., and Limon, I. (2007) Notch3 and IL-1beta exert opposing effects on a vascular smooth muscle cell inflammatory pathway in which NF-kappaB drives crosstalk. J. Cell Sci. 120, 3352-3361.
·       Chastagner, P., Israel, A., and Brou, C. (2006) Itch/AIP4 mediates Deltex degradation through the formation of K29-linked polyubiquitin chains. EMBO Rep. 11, 1147-1153.
·       Brou, C., and Logeat, F. (2006) Endocytose et voie de signalisation Notch. Médecine/Sciences 22, 685-688.
·       Olry, A., Chastagner, P., Israël, A., and Brou, C. (2005) Generation and characterization of mutant cell lines defective in g-secretase processing of Notch and Amyloid Precursor Protein. J. Biol. Chem. 280, 28564-28571.
·       Six, E., Ndiaye, D., Sauer, G., Laâbi, Y., Athman, R., Cumano, A., Brou, C., Israël, A., and Logeat, F. (2004) The Notch ligand Delta 1 recruits Dlg1 at cell-cell contacts and regulates cell migration. J. Biol. Chem. 279, 55818-55826.
·       Gupta-Rossi, N., Six, E., LeBail, O., Logeat, F., Chastagner, P., Olry, A., Israël, A., and Brou, C. (2004) Monoubiquitination and endocytosis direct g-secretase cleavage of activated Notch receptor. J. Cell Biol. 166, 73-83. (Faculty of 1000 article factor of 13)
·       Six, E., Ndiaye, D., Laâbi, Y., Brou, C., Gupta-Rossi, N., Israël, A., and Logeat, F. (2003) The Notch ligand Delta 1 is sequentially cleaved by an ADAM protease and by g-secretase. Proc. Natl. Acad. Sci. USA 100, 7638-7643.
·       Gupta-Rossi, N., LeBail, O., Brou, C., Logeat, F., and Israël, A. (2002) Control of Notch activity by the ubiquitin-proteasome pathway. Notch from neurodevelopment to neurodegeneration: keeping the fate, research and persectives in Alzheimer's disease. Springer-Verlaged.
·       Brou, C., Israël, A. (2001) Presenilins. Current Biology 11, R543.
·       Gupta-Rossi, N., LeBail, O., Gonen, H., Brou, C., Logeat, F., Six, E., Ciechanover, A., and Israël, A. (2001) Functional interaction between SEL-10, a F-box protein, and the nuclear form of activated Notch1 receptor. J. Biol. Chem. 276, 34371-34378.
·       Brou, C., Logeat, F., Gupta, N., Bessia, C., LeBail, O., Doedens, J.R., Cumano, A., Roux, P., Black, R., and Israël, A. (2000) A novel proteolytic cleavage involved in Notch signaling: role of the disintegrin-metalloprotase TACE. Mol. Cell 5, 207-216.
 
 
Team members


Christel BROU
PI, Chef de Laboratoire, Institut Pasteur
Phone 01 40 61 30 41


 

Patricia Chastagner
Ingénieure Institut Pasteur
Phone 01 40 61 30 41
 



Loredana PUCA
PhD student
Phone 01 40 61 30 41
 
 
 
We are looking for students and post-docs!