REGLAMENTO INTERNACIONAL DE FIELD TRIAL DE OTOÑO O DE CAZA PRACTICA

The Notch pathway is activated by inter-cellular cross-talk between a signal-sending cell and a signal-receiving cell, resulting from initiation of signalling in the Golgi Apparatus with eventual translation of Notch to the nucleus (Figure 1.15). The EGF domains of the NECD receptors undergo glycosylation by fucosyltransferases, including POFUT1 (protein o-fucosyltransferase), subsequently followed by Fringe modification in the Golgi complex, where specific Fringes (Manic Fringe [Mfng], Lunatic Fringe [Lfng] and Radical Fringe [Rfng]) elongate the o-fucose chains, modifying the Notch receptors and allowing them to bind their ligands.

Figure 1.15. The Notch pathway

(1) Notch signalling is initiated in the Golgi apparatus where the receptor undergoes proteolytic

cleavage at its S1 site, facilitated by furin proteases. (2) It is then transported to the cell surface membrane, where its extracellular domain binds with Notch ligands from the adjacent cell. (3) The Notch receptor is then proteolytically cleaved by ADAM metalloproteases, leading to endocytosis of the ECD into the ligand-expressing cell, followed by the release of the NICD with a hitched membrane. (4) NICD undergoes cleavage by gamma-secretase, leading to an active NICD. (5) Activated NICD translocates to the nucleus, interacting with CSL, forming a complex with MAML, RBPJ. (6) The active complex releases canonical Notch targets HES and HEY. Lack of NICD binding leads to CSL interacting with co-repressor. Adapted from (Suresh and Irvine, 2015).

The NECD undergoes its first proteolytic cleavage (S1) in the Golgi complex, where the LNR is cleaved by furin-like proteases and a heterodimeric receptor with the NECD is

formed in the HD (Lake et al., 2009, Gordon et al., 2009) (Figure 1.15). The Notch ligands are translated in the ER and travel to the cell surface through the Golgi complex. Following Fringe modification, the Notch receptor transfers to the cell surface where it interacts with the ligand, and trans-endocytosis is activated into the signal-sending cell. Trans-endocytosis is triggered by E3 ubiquitin ligases, Neuralized (Neu), recognising delta-ligands or Mindbomb (Mib), recognising jagged ligands, which are necessary for ligand endocytosis (Yamamoto et al., 2014). This process creates a conformational change in the Notch receptor and reveals its S2 cleavage site, normally masked by NRR, which is cleaved by ADAM proteases. This allows Notch extracellular truncation (NEXT) to enter the active site where S3, within the TMD, is sequentially cleaved by γ-secretase (Jorissen and De Strooper, 2010). This cleavage releases the NICD, which translocates into the nucleus and forms a complex with the DNA-binding transcription factor CSL (RBP-jk in mammals, Su(H) in Drosophila) and coactivator mastermind (MAML1,

MAML2, MAML3 in mammals, Mam in Drosophila) through its domains, RAM and ANK

(Borggrefe and Liefke, 2012, Tanigaki and Honjo, 2010). Downstream canonical transcription factors, including Hairy/enhancer of split (HES and HEY) genes are then activated (Kopan and Ilagan, 2009, Liu et al., 2013, van Tetering and Vooijs, 2011, Yamamoto et al., 2014) (Figure 1.15). NICD absence results in the binding of CSL to its analogous sequence, leading to a recruitment of transcription corepressors, including Hairless, CtBP (C-terminal Binding Protein), and Groucho. These corepressors negatively regulate Notch target gene expression.

Notch receptors are initially cleaved during exocytosis at site 1 (S1), regulating signal activity (Figure 1.15). Ubiquitination of the ligand takes place in the ICD and is controlled by E3 ubiquitin ligases including Mindbomb and Neuralized (Kopan and Ilagan, 2009). Lateral patterning during Notch signalling depends on positive (lateral induction) or negative (lateral inhibition) feedback.

1.4.2.1 Notch glycosylation

Notch modification by glycosyltransferases takes place in the Golgi complex where glycans are added to the EGF domains of NECD. The addition of glycans can either reduce or potentiate Ligand-mediated Notch signalling, depending on which EGF domain is being modified (Taylor et al., 2014). Table 1.6 highlights the glycosyltransferases that modify the ECD of Notch1.

Table 1.6. List of glycosyltransferases that modify Notch ECD Glycosyltransferase

(drosophila/ mammals)

Function EGF modification

Ofut1/Pofut1 O-fucosyltransferase/ Chaperon activity

Adds fucose to EGF 11-12

Fringe/

Lunatic,Manic, Radical Fringe

N-acetylglucosaminyl- transferase

Adds GlcNAc to 3’-OH groups of O-fucose EGF 11-12

Rumi (Ogut1)/ Poglut1

O-glycosyltransferase Add glucose to EGF 16-20

Shams/ (GXYLT)1, (GXYLT)2

O-xylosyltransferase Adds first xylose to xylose on O-glucose

*EGF, epidermal growth factor; GlcNAc, N-acetylglucosamine. Adapted from (Rana and Haltiwanger, 2011)

Notch O-fucosylation

Notch modification involves addition of O-fucose or O-glucose to the EGF repeats within the extracellular domain of the receptor. Enzymes involved in this process include POFUT1 (Protein O-Fucosyltransferase 1), which are located and function within the Golgi complex (Figure 1.15, Table 1.6). O-glucose addition is carried out by the enzyme O-glucosyltransferase, POGLUT, encoded by Rumi, and finally, O-GlcNAc, a type of O- glycosylation occurring on hydroxyl amino acids of the EGF repeat, is added by Fringe. (Rana and Haltiwanger, 2011). O-fucosylation can occur on EGF12 as well as other EGF repeats, which may play a vital role in Notch activation (Ge and Stanley, 2008). Amongst the 36 EGF repeats of the Notch receptors, 20 of them have O-fucosylation sites, thus Fringe modifications can take place in any of these residues.

The Fringe proteins (β3-N-acetylglucosaminyltransferases)

Fringes are β3-N-acetylglucosaminyltransferases that transfer a GlcNAc to O-fucose residues in the Golgi complex, and play a crucial role in activating the Notch pathway through specific ligand binding (Okajima et al., 2003, Moloney et al., 2000, Taylor et al., 2014, Bruckner et al., 2000). There are three Fringe proteins that mediate Notch signalling; Lunatic (LFNG), Manic (MFNG) and Radical Fringe (RFNG), all of which elongate the O-fucose chain of the Notch transmembranes (Table 1.6, Table 1.7).

Table 1.7. Fringe-mediated glycosylation of Notch 1

Fringe proteins

Notch 1 EGF domains

EGF6 EGF8 EGF12 EGF26 EGF36

Lfng - Jag1 + Dll1 +Dll1, +Jag1 -Dll1, -Jag1 - Jag1

Mfng - Jag1 + Dll1 + Dll1, +Jag1 -Dll1, -Jag1 - Jag1 Rfng NM (+Jag1, +Dll1) + Dll1 + Dll1 +Dll1, +Jag1 NM (+Jag1, + Dll1)

*NM, not modified by Fringe; +/-, ligand binds/ligand does not bind. Adapted from (Kakuda and Haltiwanger, 2017)