Driever Notch-Signalling

Receptor:

in Humans:

• NOTCH1-4: with SS (secretion signal), EGF repeats (domain for ligand binding / selectivity)

  NICD: nuclear localization signals, Ankyrin repeats (Interaction domain), TAD tansactivation domain, PEST- Protein Stability

  -> can be posttranslationally modified -> different ligand affinity, interactions, targeting

C.elegans: Lin-12, GLP1

Drosophila: NOTCH

Ligands:

• JAGGED [1-2]

• DELTA-like 1, 3, 4

Drosophila: Delta, serrate

C.elegans: Apx-1, Lag-2, DSL-1

No NICD: Transcription Co-Repressors bound to CSL & recruit HDAC to suppress transcription

Notch signaling: The intracellular domain of the NOTCH receptor (NICD) gets cleaved off -> Complex with CSL, MAML (Stability) -> Displace Corepressors & HDAC -> CBP recruitment(activates gene expression), Acetylation of Histones -> Gene Expression

NICD = Notch intracellular domain

Gene activation by CSL and CBP transcription activation and Stability by MAML (=Mastermind like) binding

Epigenetic modifications by recruiting Histone acyltransferases -> promote a more open and transcriptionally active chromatin state that can be stabilized to remain open

Positive feedback loop -> induction of its own signaling components

Synthesis of Notch receptor in the ER -> PTM and processing (Glycosylation [O-fructosylation] and cleavages in the Golgi) -> transmembrane proteine needs to be targeted to the membrane and anchored there

-> then ligand binding

(1) Notch translated in rough ER & glycosylated by glycosyltransferases (O-fut1, Rumi)

(2) Notch traffics to Golgi -> S1 cleavage by Furin-like proteases, Elongation O-fucose chains on EGF repeats by Fringe

(3) Notch trafficks to cell surface

(4) Notch ligand Delta, Serrante translated in ER & traffic to Cell surface through Golgi, Endocytosis & Recycling of Ligands towards Receptor

(5) Ligand & Receptor interact

Ubiquitination of the ligand by NEUR or MIB -> that triggers a pulling force (by clathrin mediated endocytosis) on the receptor where this triggers a conformational change -> ADAM10 cleavage S2 at ECM -> y-SEC cleavage S3 at IM -> nuclear import of the elevated NOTCH signal domain -> transcriptional activation, epigenetic mod, feedback loop

(6) Ligand-Receptor complex endocytosed into signaling cell, E3 Ubiquitin ligases (Neur, Mib) & endocytotic proteins (Dynamin) essential for trans-Endocytosis. Pulling force pulls Notch -> Conformational change reveals S2 cleavage site

(7) S2 cleavage by ADAM proteases to generate NEXT

(8)NEXT S3 Cleavage by g-secretase complex to release NICD

(10) NICD nuclear translocation

(11) NICD & CSL & MAM on target DNA - Corepressors are replaced by Coactivators -> Gene expression on. No NICD: CSL recruits Co-repressors -> gene expression off

Similar to Neur it ubiquitinates the Notch ligand (delta) upon receptor binding and by that it marks it for endocytosis -> pulling force -> cleavage -> signaling in receptor cell

because it is important to generate the pull force that triggers the cleavge and by the signal activation in the receptor cell

that pull force is generated by MiB / Neur Ubiqui of ligand -> endocytosis -> clathrin and Dynamin (ring to squeeze of clathrin coated pits) mediated force

also if no pull force the ligand / rezeptor is internalized, degraded and recycled (transendocytosis)

Epsin/Clathrin - mediated endocytosis of the ligand exerts force on Notch

Pulling force opens the NRR (negative regulatory Region) of the Notch receptor -> cleavage and nuclear translocation

NRR needs to be pulled to free the cleavage site S2 for the ADAM10

by Lateral inhibition

all can potentially become the sorted one but chance / other signaling / fate / statictic leads to one cell expressing more Delta than others and by that it activates Notch in the surrounding cells and they have neurogenin inhibited only in the Delta expressing cell inhibition will stop and NeuroD leads to in this case neuron fate

-> asymmetric cell division = one cell inherits different amount of Notch regulators e.g. Numb = localized determinants

First of all Numb can be asymmetrically distributed in cell division leading to different cell fates that is because Numb controls Notch trafficking in the cell:

Numb inhibits Notch siganling by internalizing Notch rezeptors (speed up internalization) but also inhibit recycling of the Notch rezeptors

-> internalization by bulk endocytosis and not the force promoting clathrin mediated endocytosis

E.g. segmentation clock

Over 3 tiers:

Bottom tier: single cell oscillators

Middle: local synchronisation

Upper: global control of slow and arrest (Notch put in vesicles)

Notch signaling maintains neural stem cells

For example at the levels of Notch PTMs (Ub, phosphorylation, hydroxylation, acetylation) on the intracellular domain (NICD) the signals from different pathways can act directly on the domains of the Notch receptor: e.g. a modification of the PEST domain affects stability, on the NLS domain can inhibit nuclear translocation, on the transactivation domain can change recruitment of other components or on Ankyrin domain can alter Protein - protein interaction e.g. masking

-> accumulation of GMP and undifferentiated cells without the right signals no right differentiation / spezialisation

active Notch gives rise to mitotically active fr5+ SC -> self renewal / proliferation maintanance

Linage disicion Notch promotes Absorptive cell fate and inhibits other fates through TF Math1

1. because of its role in SC niches it plays a role in tumor formation / initiation

2. because its role and interaction with cytoskeleton it plays a role in tomour invasion and metastasis

3. Intra and extravastation

4. tumor neovascularisation -> Notch promotes angiogenesis in tumors

• Notch internalization and cleavage

• Notch ICD translocation and the inhibition through Numb (Numb inactive in cancer cells Numb stabalizes TP53 so it can act as TF and Numb inhibits Notch and HH signaling)

• Transcriptional activation by inhibiting the Histone acetylase or the recruiting components

• Transcription itself or the transcribed mRNAs …

-> mAbs Notch ligands or Notch decoys inhibit Delta like (or Jagged

-> mAbs to Notch rezeptors

-> y-Sec or ADAM10 inhibitors

-> MAML1 inhibitors to disrubt transcription activation complex