Lecture 10

Question 1

Notch signalling is used when a cell has to make a linear fate decision, T or F

Answer 1

F – notch signalling is used when a cell needs to make a binary fate decision

Question 2

Due to their specificity, a single ubiquitin ligase acts in the notch pathway, T or F

Answer 2

F – multiple ubiquitin ligase act upon the notch pathway

Question 3

Explain the significance of the notch signalling pathway in leukaemia

Answer 3

Human notch was first cloned from patients with T-cell acute lymphoblastic leukaemia. These patients has a truncation mutation in notch as a result of a chromosome translocation which lead to the fusion of the TCR-? gene and the intracellular domain of notch. TCR-? is constitutively expressed in immature T-cells resulting in the constituent activity of notch by maintaining T-cells in a stem cell state. This proliferation contributes to tumour formation.

Question 4

Whose is credited with discovering the notch gene by their forward genetic screens in Drosophila

Answer 4

Thomas Hunt-Morgan

Question 5

Before its insertion into the membrane, the notch receptor is already partially cleaved, describe how this occurs

Answer 5

Furin, a serine protease cleaves the notch receptor in the golgi. This results in a weakening of the connection between intracellular and transmembrane/extracellular domains

Question 6

What enzymes regulates the pairing of cysteine residues in the endoplasmic reticulum

Answer 6

Disulphide isomerase

Question 7

The Notch receptor also has internal repeats contained in the intracellular domain, what are these referred to as

Answer 7

Ankryin repeats

Question 8

What is the role of the segmentation clock in patterning the mesoderm

Answer 8

The segmentation clock determines when and where somites bud off from the axial mesoderm

Question 9

What are the vertebrate homologues of delta/serrate

Answer 9

Vertebrates have at least 9 different homologues that all belong to the delta or jagged family – they are collective members of the delta-serrate ligand (DSL) family

Question 10

Explain the roles of Su(deltex) and Sel-10 in the degradation of notch

Answer 10

The WW domain of Su(deltex) binds to the notch receptor via its C2 domain which localises it to the membrane by binding to phospholipids. The HECT domain then mediates ubiquitination of the notch receptor. Sel-10 recognises phosphorylated notchintra in the nucleus but doesn’t contain a ubiquitin ligase domain. Instead, Sel-10 recruits a ubiquitin ligase via its F-box domain to unbiquitinate notchintra.

Question 11

What can be said about the degradation of notch and how is this significant in its signalling pathway

Answer 11

Notch is rapidly degraded by proteasomes which acts as a regulation mechanism for its activity

Question 12

Which 76 amino acid peptide is added to proteins in order to mark them for degradation

Answer 12

Ubiquitin

Question 13

What type of signalling is used by notch-delta

Answer 13

Juxtacrine

Question 14

Why is it that despite being a relatively simple organism Drosophila still have two different genes the code of notch ligands

Answer 14

As Drosophila has evolved this gene for a notch ligand has been duplicated due to its importance in early patterning

Question 15

What is the significance of the notch signalling pathway in Alzheimer’s disease

Answer 15

Amyloid precursor protein (APP) looks very similar in structure to the notch receptor. APP is also cleaved by presenilin after cleavage by an ? or ? secretase in a similar way to how furin and TACE cleave notch. The ?-secretase products are cut by presenilin in one of two positions in its transmembrane domain which results in the production of an extracellular peptide of either 40 or 42 amino acids in length.

Question 16

Describe the pattern of mutations in jagged1 that lead to alagille syndrome

Answer 16

Many of these missense and non-sense mutations occurs within the cysteine repeats contained in the EGF repeats of the Jagged1 protein. This causes improper folding of the protein and may influence binding of the jagged1 ligand to the notch receptor

Question 17

What is the role of jagged1 in notch signalling

Answer 17

Jagged1 is a delta homologue and a ligand that binds to notch receptors

Question 18

Alagille syndrome is an autosomal dominant mutation associated with mutations in jagged1. What are the symptoms of this condition

Answer 18

Chronic liver disease in children but also heart disease, mental retardation and skeletal abnormalities

Question 19

Give an overview of the notch-delta pathway

Answer 19

Delta binds to the notch receptor and activates it. When notch is activated the intracellular domain, Notchintra is cleaved off and translocates from the membrane to the nucleus. Here Notchintra interacts with CSL which shifts CSL from being a transcriptional repressor to a transcriptional activator via Co-A. This leads to the upregulation of notch target genes

Question 20

What are the similarities between delta and notch proteins

Answer 20

Both are single pass transmembrane proteins with many EGF repeats in the extracellular domain

Question 21

What is significant about the jagged1 being a gene involved in early development

Answer 21

Unlike most other developmental genes, jagged1 is haploinsufficient

Question 22

Explain the paradoxical role of ubiquitination and Neur/Mib in activation of the notch pathway

Answer 22

Neur/Mib binds to delta and the RING domain acts as a ubiquitin ligase. As well as degradation, ubiquitination can also mediate endocytosis. Once delta has bound to Notch an enzyme cleaves extracellular Notch domain which is still bound to the delta ligand. Ubiquitination of delta causes endocytosis which brings the Delta-Notch complex into the signalling cell. This pulls the extracellular domain of the Notch receptor in as it is endocytosed into the other cell with the delta ligand. This causes the release of Notchintra which can translocate to the nucleus of the target cell where is can act to upregulate target genes

Question 23

Unlike most genes involved in early development, notch is haploinsufficient, T or F

Answer 23

T

Question 24

Explain the final step of notch receptor activation

Answer 24

In the final stage presenilin, a ?-secretase cuts within the transmembrane domain and releases the intracellular domain of the receptor, notchintra which then translocates to the nucleus where is can interact with CSL to upregulate notch target genes

Question 25

Describe in detail what happens upon ligand binding to the notch receptor

Answer 25

Ligand binding to the partially cleaved notch receptor results in the activity of a second protease known as metalloprotease (TACE) cleaving off the extracellular domain, leaving the transmembrane and extracellular domains still inserted into the membrane. This is referred to as the ligand dependant step

Question 26

What is the significance of these extracellular peptides produced by presenilin in Alzheimer’s pathology

Answer 26

The 42 amino acid peptide produced by presenilin is a major component of the amyloid plaques found in Alzheimer’s brains. Mutations in human presenilins account for most early onset autosomal-dominant cases of Alzheimer’s as a result of increase 42 amino acid peptide being produced.

Question 27

How often intermediate filaments a new somite formed in chick embryos

Answer 27

Every 90mins

Question 28

Explain the cysteine connection in diseases that affect notch signalling

Answer 28

The clustered mutations all affect codons for cysteines within the EGF repeats. These would lead to an unpaired cysteine and may alter the overall structure of Notch3

Question 29

What is the significance of the EGF repeats in the extracellular domains of delta and notch

Answer 29

This commonality mediates receptor-ligand recognition. Extracellular protein-protein interactions are often mediate by domains that contain a repeated amino acid sequence. These EGF repeats are made up of 30-40 amino acids and contain repeated 6 cysteines in regular intervals which form sulphide bridges with cysteines in the other EGF containing protein.

Question 30

There are three different types of enzymes that are involved in marking a protein for degradation, E1, E2 and E3. Explain the different roles of these enzymes in mediating the degradation of proteins such as notch

Answer 30

E1 is known as ubiquitin activating enzyme and along with E2, ubiquitin conjugating enzyme is expressed in all cells. These enzymes are referred to as housekeeping enzymes and are non-specific to the proteins which the mark for degradation. E3, known as ubiquitin ligase enzymes are specific for individual proteins or set of proteins and are responsible for adding the ubiquitin peptide.

Question 31

Recall some of the target genes upregulated by Co-A and CSL

Answer 31

Suppressor of Hairless, LAG1 and CBF1

Question 32

What are the two notch receptor ligands found in Drosophila

Answer 32

Delta, Serrate