Notch signaling. Ramray Bhat 6/09/2017

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1 Notch signaling Ramray Bhat 6/09/2017

2 Lecture 1 introduction, signaling fundamentals, receptor ligand structure, cleavage Lecture 2 Introduction to non canonical signaling, Notch signaling in development: paradigms

3 Salient aspects of Notch signaling 1. Juxtacrine 2. No cascading 3. Receptor and Ligand TM Type 1 4. Delta (DLL) and Serrate (Jagged) (Notch 1-4, DLL1-3, Jagged1-2) 5. 3 cleavage events 6. Nuclear recruitment of NICD changes function of CBF1/Su(H)/Lag-1 or CSL TC. Kopan et al., CSHLP,.2012

4 S1 cleavage 1. Notch cleaved into an NECD and TMIC in S1 cleavage. 2. Prior to cleavage in transgolgi, Notch subjected to glycosylation. 3. Non covalent linkage in trans Golgi. 4. Unprocessed full length Notch does not reach membrane van Tetering and Vooijs, Curr. Mol. Med., 2011

5 S1 cleavage 1. Cleavage occurs through furin/furin-like convertase (PACE, RXRR motif) 2. Inhibiting furin/rxrr site mutation abrogates receptor processing/ receptor presentation. 3. Notch 2 unaffected by removal of furin loop 4. Cripto-1 enhances Notch heterodimerization and Ligand-dependent signaling. van Tetering and Vooijs, Curr. Mol. Med., 2011

6 S2 cleavage 1. S2 site cleavage occurs through ADAM (A Disintegrin And Metalloproteinase) 2. Membrane bound zinc-dependent MPs: ADAM10 (Kuzbanian) or ADAM17/TACE(TNFalpha converting enzyme) 3. Upstream of signal processing. Loss of function phenotype resembles and is severe to Notch mutation. Other substrates involved including Eph- Ephrin 4. Can be rescued by NICD overexpression van Tetering and Vooijs, Curr. Mol. Med., 2011

7 S2 cleavage 1. Notch ligands can also be cleaved by ADAMs/Kuz. Interestingly ADAMdn phenotype can be rescued by DLL overexpression suggesting other sheddases might work. 2. Debate on extent of ligand activation on S2 cleavage: overexpression of ADAM leads to increased signal processing 3. Mechanistic models of ADAM-dependent S2 cleavage: pulling force model, pull and cut model. 4. Pulling force model requires S1 based heterodimerization and ligand internalization. But formaldehyde fixed ligand shows this not to be true. What is the pull and cut model? van Tetering and Vooijs, Curr. Mol. Med., 2011

8 Notch structure and modular organization NRR: negative regulatory regions consist of 3LNR (Lin12 Notch repeats) and HD domain: heterodimerization (proteolysis resistant/autoinhibited state) Ligand independent action requires LNRA and B deletion or HD activating mutations Gordon et al., JCS,.2008

9 S3 cleavage 1. S3 mediated by an aspartyl protease presenilin 2. Presenilin part of a multiprotein complex γ- secretase (along with nicastrin, APH1 and PEN- 2) (aspartyl protease) 3. NICD has 2 NLS domains 4. CSL which is otherwise associated with Hairless/Insensitive/SMRT (corepressors) and HDACs. 5. Upon NICD binding, HDACs are released and so are repressors. On the other hand transcriptional activators such as Mastermind are recruited along with HATs. van Tetering and Vooijs, Curr. Mol. Med., 2011

10 Notch structure and modular organization Gordon et al., JCS,.2008

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12 Notch activation 1. LNR (Cys ) rich domains cover the HD which has sites for S2 2. Deletion of LNR domains leads to ligand independent activation. 3. HD unraveling to enter the deep active site of TACE

13 Regulating the preference of Notch for its ligands Tetrasaccharide modified Notch prefers DLL to Jagged

14 Glycobiological regulation of Notch POGLUT1 (Rumi) adds glucose which results in proper folding and signaling Extension by xylosyl transferase results in impaired signaling through negative regulation of surface expression.