Construcción de las representaciones

Figure 1.9 illustrates the core Notch signalling pathway that was deciphered during the 1990s. Notch receptors are constitutively cleaved in the Golgi apparatus by a furin-like convertase prior to transport to the plasma membrane. This post- translational modification is essential for formation of hetero-dimeric active Notch receptors in mammals (Logeat et al., 1998), although this modification is not

Figure 1.8 The structure of Notch and its ligands, Delta and Serrate (Jagged). A prototypical Notch gene encodes a single trans-membrane receptor whose extra- cellular domain consists of an array of conserved epidermal growth factor (EGF) repeats. Up to 36 EGF repeats can be present, of which EGF repeat 11 and 12 are sufficient for ligand interaction. Three juxta-membrane cysteine-rich Lin-12-Notch (LN) repeats modulate interactions between the extra-cellular and intra-cellular domains of the receptor. A hetero-dimerization domain non-covalently binds the membrane-tethered and extra-cellular regions of the receptor. The intra-cellular domain consists of seven ankyrin repeats (acting as structural motifs) flanked by two nuclear localisation signals, a transactivation domain (TAD) and a proline, glutamine, serine, threonine-rich (PEST) region. Delta and Serrate are each composed of a DSL region that interacts with the Notch receptor, and several EGF repeats, with Serrate/ Jagged also containing a juxta-membrane cysteine-rich region. (PM= Plasma

Figure 1.9 Schematic representation of the Notch signalling pathway.A representation of the post-translation modifications, the protein-protein interactions, protein degradation and the transcriptional activation events that occur during Notch signalling and which serve to control cell division, differentiation and death. See section 1.4.1 of the text for description (Schematic taken from Cell Signaling®)

necessary in flies (Kidd and Lieber, 2002). Notch receptors can be modified further by the Fringe class of proteins (Johnston et al., 1997; Irvine, 1999; Bruckner et al., 2000; Visan et al., 2006). Fringe proteins are glycosyl transferases that add sugar residues to sites on the extracellular EGF repeats of the Notch receptor that have been fucosylated by O-Fucosyl transferase (Okajima and Irvine, 2002). This modification increases affinity of Notch receptors for Delta ligands, but reduces Notch-Serrate interactions (Haines and Irvine, 2003). Once at the plasma membrane, mature Notch receptors can bind Delta and Serrate (DSL) ligands on opposing cells, this interaction results in two cleavage events on the Notch receptor (Kopan and Goate, 2000). Firstly a S2 cleavage, mediated by the metalloprotease TACE, releases the extra-cellular domain of the Notch receptor (Brou et al., 2000). This proteolysis enables the γ-secretase enzyme complex to perform a presenillin mediated S3 cleavage, liberating the intracellular domain of the Notch receptor (Notch-ICD) (Schroeter et al., 1998; Taniguchi et al., 2002). Notch-ICD then translocates to the nucleus where it binds to the CSL transcription factor switching it from a transcriptional repressor to a transcriptional activator (Jarriault et al., 1995). This altered gene expression directs the cell towards a specified cell fate.

The simplicity of the canonical Notch signalling pathway described above does not accurately reflect the actual processes that occur in the cells relaying the signal. Notch signalling is much more complex and regulation occurs at many levels (Kadesch, 2004; Bray, 2006). In recent years this complexity has been exposed and novel mechanisms have been discovered that further our understanding of how the Notch signalling pathway works. For example, recent research has shown Notch interaction with its DSL ligand switches the Notch receptor from an auto-inhibited

conformation, to a state that permits metalloprotease mediated S2 cleavage (Gordon et al., 2007). Despite such advances, many questions remain unanswered as to how the Notch signalling pathway elicits a cell response, and how signal transduction is regulated. A significant gap in our knowledge of how the Notch signalling pathway works is in the activity and regulation of the DSL ligands. Endocytosis of the extracellular remains of the Notch receptor, bound to its DSL ligand, is mediated by Neuralized (Neur) and Mind bomb (Mib) (Le Borgne et al., 2005a; Chitnis, 2006). Both are E3 ubiquitin ligases that share few structural similarities, yet can perform the same function (Le Borgne et al., 2005b). A mechanism for how these two proteins promote Notch signalling has yet to be defined. One suggestion is that Neur and Mib activate DSL ligands through partial degradation (Bray, 2006). Neur- and Mib-mediated ubiquitination of DSL ligands makes them targets for adaptor proteins such as Epsin (Wang and Struhl, 2004; Wang and Struhl, 2005). Such adaptor proteins promote endocytosis, which may be necessary for activation of DSL ligands or may occur after binding of the Notch receptor to its ligand, thus promoting Notch signalling as endocytosis permits S2 cleavage of the Notch receptor. The exact mechanism is yet to be defined, but ubiquitination of DSL ligands is necessary for Notch-ICD generation (Koo et al., 2005). Additionally, modulation of the Notch signalling pathway also occurs through ligand and Notch trafficking, ligand processing, CSL regulation, epigenetic regulators, ligand localisation, and Wnt signalling (see reviews Bray, 2006; Fiuza and Arias, 2007), with more levels of regulation likely to be discovered in the future. Notch signalling can also be activated and its signal processed independently of the core pathway illustrated in Figure 1.9. Calcium ions bind to EGF repeats on the Notch receptor, inhibiting its activity. In tissue-culture cells where calcium ions are depleted from the extra-cellular space,

there is potent ligand-independent activation of Notch receptors, leading to Notch signalling (Rand et al., 2000; Raya et al., 2004). CSL-independent Notch signalling is also known to occur, and is dependent on GSK3β (Brennan et al., 1997; Brennan et al., 1999b). This and other findings suggest a functional connection between the Notch and Wnt signalling pathways exists within cells (discussed further in section 1.4.3). In summary, interpretation of the molecular control of Notch signalling is much more complex than originally perceived as many underlying regulatory mechanisms in this pathway remain unknown.