CellSig11 - 31 Flashcards
How was notch identified?
Forward genetic screens in Drosophila - notch wing phenotype - by Thomas Hunt Morgan - 1866-1945
How many notch receptors does Drosophila have?
1
How many delta ligands do Drosophila have?
2 - delta and serrate
How many delta ligands do vertebrates have?
At least 9 members of this family
How many notch receptors do humans have?
4 (Notch 1-4)
What are delta ligands collectively known as?
DSL family
Characterise delta/notch structures and function
1TM domains, with many EC EGF repeats, juxtacrine in function, notch also has many IC ankyrin repeats
What is important about EC EGF repeats?
Made up of repeated cysteines in regular intervals that form sulphide bridges, adding structure
What regulates EGF cysteine pairing?
Protein disulphide isomerase in the ER
Outline the delta-notch pathway
Notch activation > Notch-intra translocates to the nucleus > activation of CSL > transcription
What constitutes CSL?
CBF1/SuH/LAG1
What is furin’s role in notch activation?
Cuts the receptor to produce a heterodimer, S1, which then implants into the PM
What is furin?
Serine protease - controls first cleavage of notch
How is notch cleaved for a second time?
Ligand dependent, TACE cuts off EC domain, but notch remains in membrane because TM domain still attached
What is TACE?
Metalloprotease
What is presenilin?
Gamma-secretase - controls third cleavage, and release of notch from the PM
What releases notch from the PM?
Presenilin
Outline notch degradation
E1 - ubiquitin activating enzyme > E2 - ubiquitin conjugating enzyme > E3 ubiquitin ligase (notch specific) > proteasome
Characterise Su(dx)/Itch
3 major domains - WW binds notch, HECT mediates ubiquitination, C2 localises to the PM
Characterise Sel-10
Recognises Notch-intra, but lacks ubiquitin ligase activity, so recruits one via its F box
Characterise Neur/Mib
Binds delta, RING domain ligases, required to activate endocytic pathway
What domains are present in notch-intra?
NLS - nuclear localisation sequence, RAM and ANK domains - protein/protein interaction domains, activation domain - transcriptional activator
Outline transcriptional activity without Notch present
CBF1 anchors HDAC via SMRT and SKIP near the promoter, reducing DNA acetylation
Outline transcriptional activity with Notch present
Notch-intra displaces SMRT and HDAC and acts as a transcriptional activator
How is Alzheimers related to Notch activation?
Similar mechanism - amyloid precursor protein (APP) cleaved by alpha or beta-secretase, then by presenilin in one of two places (40 or 42 a/a). The 42 a/a version is a major component of the amyloid plaques
How was the first human notch cloned?
From T-cell acute lymphoblastic leukemia patients - Truncated Allele of Notch (TAN1)
What is TAN1?
Translocation of TCR-beta gene fused to Notch-intra
How does TAN1 cause leukemia?
TCR-beta strongly expressed in immature T-cells, therefore Notch is made consituently active
What does CADSIL stand for?
Cerebral AD ateriopathy with subcortical infarcts and leukoencephalopathy
What is CADSIL?
Recurrent strokes leading to dementia, due to build of granular material in smooth muscle of blood vessels
What causes CADSIL?
Mis-sense mutations in Notch3 - usually the first 5 EGF repeats
