Signalling through regulated proteolysis

Question 1

What are the 4 types of signalling

Answer 1

1. Notch signalling
2. Wnt signalling
3. hedgehog signalling
4. NFkB signalling

Question 2

Describe stem cells

Answer 2

1. Stem cells reproduce themselves (self-renewal).
2. Stem cells generate transit-amplifying progeny destined to differentiate into functional cell types.
3. Stem cells persist for a long time (whole life).
4. Stem cell behaviour is regulated by the immediate environment (the niche).
5. Each daughter produced when a stem cell divides can either remain a stem cell or go on to become terminally differentiated.

Question 3

How is stem cell behaviour regulated by the immediate environment

Answer 3

1. Contact dependent- neighbouring cells
2. Soluble factors- growth factors from further away
3. Binding of stem cell to ECM proteins

Question 4

What are transit amplifying cells.

Answer 4

1. In many cases, the daughter that opts for terminal differentiation undergoes additional cell divisions before terminal differentiation is completed

Question 5

What is the Notch signalling pathway

Answer 5

1. contact dependent

Question 6

What are the 4 Notch receptors in mammals

Answer 6

1. NOTCH1,
2. NOTCH2,
3. NOTCH3,
4. NOTCH4

Question 7

What are the 5 ligands that notch receptors interact with

Answer 7

5 ligands of the Delta-Serrate-Lag2 (DSL) family

1. jagged 1 (JAG1),
2. JAG2,
3. delta-like 1 (DLL1)
4. DLL3
5. DLL4

Question 8

What is basics of notch signalling

Answer 8

1. Notch itself is a cell-surface receptor that transduces short-range signals by interacting with transmembrane ligands such as Delta (termed Delta-like in humans) and Serrate (termed Jagged in humans) on neighboring cells

Question 9

Describe the three proteolytic cleavage events involved in notch signalling

Answer 9

1. Cleavage at site (S)1 occurs in the trans Golgi network by a protease called furin during maturation of the receptor and transport to the cell surface.
2. First cleavage generates the mature heterodimeric Notch receptor that is then displayed on the cell surface.
3. The binding to Delta on a neighboring cell triggers the next two proteolytic steps
4. Receptor-ligand binding induces cleavage at S2 mediated by ADAM10 and ADAM17 metalloproteinases.
5. This leads to S3 cleavage by γ-secretase which releases the Notch intra-cellular domain (NICD) from the plasma membrane.
6. The NICD (notch tail) then translocates to the nucleus, where it binds to a transcriptional repressor protein called CSL, which is converted into a transcriptional activator.

Question 10

How does notch signaling determine cell fate

Answer 10

1. induction of lineage specification and commitment in multipotent progenitor cells
2. maintenance of stem cell compartments by inhibition of differentiation
3. initiation and progression of differentiation in stem and progenitor cells.

Question 11

Give an example of how notch signalling affects induction of lineage specification and commitment in multipotent progenitor cells

Answer 11

1. e.g. hematopoiesis
2. active notch signaling helps direct differentiation to T cells
3. at same time inhibits differentiation into b lymphocytes

Question 12

Give an example of how notch signalling affects the maintenance of stem cell compartments by inhibition of differentiation

Answer 12

1. Intestinal epithelium
2. Active notch signaling positively controls stem cells self-renewal
3. At same time inhibits stem cell pool from undergoing terminal differentiation

Question 13

Give an example of how notch signalling affects the initiation and progression of differentiation in stem and progenitor cells.

Answer 13

1. Epidermis

2. Stem or progenitor cells require active notch signaling to undergo terminal differentiation

Question 14

How does lateral inhibition controls neural cell fate in Drosophila neuroectoderm

Answer 14

1. When individual cells in the epithelium begin to develop as neural cells, they signal to their neighbors not to do the same.
2. This inhibitory, contact-dependent signaling is mediated by the ligand Delta, which appears on the surface of the future neural cell and binds to Notch receptor proteins on the neighboring cells.
3. In many tissues, all the cells in a cluster initially express both Delta and Notch, and a competition occurs, with one cell emerging as winner, expressing Delta strongly and inhibiting its neighbors from doing likewise.
4. In other cases, additional factors interact with Delta or Notch to make some cells susceptible to the lateral inhibition signal and others unresponsive to it.

Question 15

How does notch signalling controls terminal differentiation in the epidermis

Answer 15

1. Epidermis is outermost tissue layer of skin
2. Outermost cell layer is stratum corneum
3. Form physical and chemical barrier against mic orgs
4. Prevents extensive water loss
5. Constantly renewed by stem cells - Located in basal cell layer
6. Proliferation only takes place in basal layer
7. Stem cells give rise to transiently amplifying cells
8. Undergo cell division before terminal differentiation
9. Once they decide to undergo terminal differentiation, they detach from basement membrane and are sorted and pushed up into spinous layer
10. This pushes all the cells up towards the surface of skin
11. Undergo biochemical changes which makes them into dead cells that make up stratum corneum

Question 16

How is notch signalling is a tumour suppressive pathway

Answer 16

1. ~50-85% of human cSCCs (Cutaneous Squamous Cell Carcinoma) harbour mutations in Notch1 and Notch2, many of which are inactivating
2. ~5-10% of human cSCCs harbour mutations in Notch3.
3. Epidermis is very exposed to environmental insults
4. Oncogenic mutations happen often
5. Lead to uncontrolled proliferation of stem cell pool
6. Normal epidermis cells notch signals keep mutated stem cells in check
7. Loss of function mutations in Notch then tumours are allowed to develop

Question 17

What is the Wnt signalling pathway

Answer 17

1. paracrine signalling

Question 18

What are Wnt proteins and how do they act

Answer 18

1. Wnt proteins are secreted signal molecules.
2. Wnts act by regulating the proteolysis of a multifunctional protein called β-catenin.
3. In the absence of a Wnt signal, cytoplasmic β-catenin is rapidly degraded by the β-catenin destruction complex.

Question 19

What are the two cellular pools β-catenin exists in

Answer 19

1. one is located at cell-cell junctions.

2. the other is located in the cytoplasm.

Question 20

What happens to β-catenin in the absence of a Wnt signal

Answer 20

1. In the absence of a Wnt signal, β-catenin that is not bound to cell–cell adherens junctions interacts with a degradation complex containing APC, axin, GSK3, and CK1.
2. In this complex, β-catenin is phosphorylated by CK1 and then by GSK3, triggering its ubiquitylation and degradation in proteasomes.
3. Wnt-responsive genes are kept inactive by the Groucho co-repressor protein bound to the transcription regulator LEF1/TCF.

Question 21

What happens to β-catenin in the presence of a Wnt signal

Answer 21

1. Wnt binding to Frizzled and LRP clusters the two co-receptors together, and the cytosolic tail of LRP is phosphorylated by GSK3 and then by CK1.
2. Axin binds to the phosphorylated LRP and is inactivated and/or degraded, resulting in disassembly of the degradation complex.
3. The phosphorylation of β-catenin is thereby prevented, and unphosphorylated β-catenin accumulates and translocates to the nucleus, where it binds to LEF1/TCF
4. It displaces the co-repressor Groucho, and acts as a coactivator to stimulate the transcription of Wnt target genes.
5. The scaffold protein Dishevelled is required for the signaling pathway to operate; it binds to Frizzled and becomes phosphorylated , but its precise role is unknown.

Question 22

What are mutations in Wnt signalling are associated with

Answer 22

1. colorectal cancer
2. 5-10% colon cancers contain activating mutations in beta-catenin- oncogene
3. > 80 % inactivating mutations in Apc- tumour repressor

Question 23

What is importance of APC

Answer 23

1. One of the scaffolding proteins so without APC, the beta catenin complex falls apart- so results in inactive Wnt pathway

Question 24

Give a summary of Wnt signalling

Answer 24

1. Wnt signalling is a paracrine signalling pathway.
2. Wnt proteins act by regulating the proteolysis of a multifunctional protein called β-catenin.
3. Wnt target genes are involved in controlling cell proliferation.
4. Hyperactivation of Wnt signaling can contribute to cancer development

Question 25

Give brief overview of hedgehog signalling

Answer 25

1. In the absence of a HH signal, a latent transcription regulator called Cubitus interruptus (Ci in drosophila, Gli in mammals) is ubiquitylated and proteolytically cleaved.
2. However, Ci is not completely degraded, but processed into a smaller fragment which accumulates in the nucleus where it acts as a transcriptional repressor.
3. HH signalling blocks the proteolytic processing of Ci, thereby changing it to a transcriptional activator.

Question 26

What happens in the absence of hedgehog

Answer 26

1. In the absence of Hedgehog, most Patched is in intracellular vesicles, where it keeps Smoothened inactive and sequestered.
2. The Ci protein is bound in a cytosolic protein degradation complex, which includes the protein kinase Fused and the scaffold protein Costal2.
3. Costal2 recruits three other protein kinases (PKA, GSK3, and CK1), which phosphorylate Ci.
4. Phosphorylated Ci is ubiquitylated and then cleaved in proteasomes to form a transcriptional repressor, which accumulates in the nucleus to help keep Hedgehog target genes inactive.

Question 27

What happens in the presence of hedgehog

Answer 27

1. Hedgehog binding to iHog and Patched removes the inhibition of Smoothened by Patched.
2. Smoothened is phosphorylated by PKA and CK1 and translocates to the plasma membrane, where it recruits the complex containing Fused, Costal2, and Ci.
3. Smoothened inhibits CI proteolysis
4. Costal2 releases unprocessed Ci, which accumulates in the nucleus and activates the transcription of Hedgehog target genes.

Question 28

How does BCC (basal cell carcinoma) develop

Answer 28

1. BCC develops by hyperactivated HH signalling
2. Hyperactivation can also lead to uncontrolled proliferation of cells
3. Basal cell carcinoma
4. Develops from stem cells in epidermis
5. Driven by hyperactivation of HH signalling

Question 29

What do mutations in HH signaling pathway lead to

Answer 29

1. Mutations in HH signaling pathway lead to Gorlin syndrome

Question 30

What is Gorlin syndrome

Answer 30

1. Gorlin syndrome affects about 1 in 31,000 people.
2. Between 70 to 80% of people with Gorlin syndrome have someone else in their family with it and have inherited a genetic mutation from one of their parents.
3. Gorlin syndrome is also called naevoid basal cell carcinoma syndrome (NBCCS), because about 90% of people with the syndrome develop multiple basal cell carcinomas (BCC) of the skin.
4. People who have Gorlin syndrome can also have a number of different medical conditions including:
   a) cysts on the jaw that usually develop during the teens
   b) changes in the bones – rib abnormalities
   c) medulloblastoma (5% of children with Gorlin’s syndrome)
   d) benign tumours of the ovary (ovarian fibroma)
   e) benign tumours of the heart (cardiac fibroma)
5. Most affected patients carry mutations in PTCH1, and mutations in PTCH2 have also been reported.

Question 31

Give a summary of hedgehog signalling

Answer 31

1. HH signalling is a paracrine signalling pathway.
2. HH signalling acts by blocking the proteolytic processing of Ci/Gli, thereby changing it to a transcriptional activator.
3. Hyperactivation of HH signalling underlies basal cell carcinoma development.

Question 32

What are NFkB proteins

Answer 32

1. NFκB proteins are latent transcription regulators that are central to inflammatory and innate immune responses.

Question 33

Describe basics of NFkB signalling pathway

Answer 33

1. Various cell surface receptors activate NFκB signalling.
2. Toll-like receptors recognize pathogens and activate this pathway to trigger innate immune responses.
3. the receptors for the cytokines TNFα and IL1 also activate this pathway to induce inflammation.
4. Activation of the pathway triggers a multiprotein ubiquitylation and phosphorylation cascade that releases NFκB from an inhibitory protein complex.
5. NFκB can now enter into the nucleus and act as a transcription factor to drive gene expression.

Question 34

Describe the NFkB pathway more complexly

Answer 34

1. Both TNFα and its receptors are trimers.
2. The binding of TNFα causes a rearrangement of the clustered cytosolic tails of the receptors, which now recruit various signaling proteins, resulting in the activation of a protein kinase that phosphorylates and activates IκB kinase kinase (IKK).
3. IKK is a heterotrimer composed of two kinase subunits (IKKα and IKKβ) and a regulatory subunit called NEMO.
4. IKKβ then phosphorylates IκB on two serines, which marks the protein for ubiquitylation and degradation in proteasomes.
5. The released NFκB translocates into the nucleus, where, in collaboration with coactivator proteins, it stimulates the transcription of its target genes.

Question 35

Give summary of NFkB signalling

Answer 35

1. NFκB signalling is a paracrine signalling pathway.
2. NFκB proteins are latent transcription regulators that are central to inflammatory and innate immune responses.
3. Activation of the NFκB pathway triggers a multiprotein ubiquitylation and phosphorylation cascade that releases NFκB from an inhibitory protein complex, allowing it to act as a transcription factor.