Signalling Pathways

Question 1

1. give the structure of the delta ligand (2 components)
2. give the stucture of the notch receptor (3 components)
3. what are EGF repeats? what do they do?

Answer 1

1. Extracellular domain comprised of EGF repeats which mediates the interraction with the notch receptor

singal transmembrane domain

2. extracellular EGF repeats

single TM domain

Intracellular ankryn repeats.

3. made up of repeated cysteine residues at regular intervals. the cysteines are paired, which mediates the formation of disulphide bridges

Question 2

Name 3 ubiquitin E3 ligases, the molecule that each binds to, and the structure of each.

Answer 2

1. Su(dx)
   - WW domain which binds to notch
   - Hect domain which mediates ubiquitination

C2 domain which localises the protein to the cell membrane

2. Sel10
   - binds to notch
   - lacks a hect/ring domain thus doesnt have its own ubiquitn ligase activity. A hect/ring domain is recruited by an F-box domain
3. Neur/mib
   - binds to delta
   - ring domain mediates ubiquitination

Question 3

1. Name 3 proteins that are involved in receptor cleavage, where they cleave, and when they cleave.
2. Why is the Neur/Mib E3 ubiqutin ligase required for pathway activation?

Answer 3

1. Furin - cuts the TM domain when protein is in golgi. 2 parts are held together by a cystine bridge
2. presenillin - cuts cysteine bridge within TM domain to allow NICD to translocate to nucleus
3. TACE - cuts the EC domain following ligand binding.

Question 4

1. What is NICD?
2. Name the 4 domains of NICD and what they do.

Answer 4

1. The intracellular domain of notch
2. NLS domain - nuclear localisation

RAM domain - protein protein interractions

Ankryn repeats - protein protein interractions

Question 5

1. Describe the notch signalling pathway
2. How does the NICD promote transcription of certain genes?

Answer 5

1. delta binds to and activates notch

the NICD translocates to the nucleus

in the nucleus, the NICD interracts with the CSL complex transforming it from a Co-R to a Co-A

2. CSL is bound to HDAC via SMRT and SKIP. NICD displaces SMRT thus HDAC, thus DNA is no linger deacetlyated and the transcriptional machinery can gain access to gene.

Question 6

1. How is notch signalling implicated in Alzheimers?
2. How is notch signalling implicated in T-cell acute lymphoblastic leukaemia?
3. How is notch signalling implicated in Alagille syndrome?
4. How is notch signalling implicated in CADSIL?

Answer 6

1. Amyloid plaques formed by misfolded amyloid precursor protein

APP is cleaved by presinellin following cleavage by alpha or beta secretase

beta secretase cleaves APP in one of 2 positions, resulting in an extracellular peptide that is either 40 or 42 aa long.

the 42 aa product is the major component of amyloid plaques

mutations in gamma presenellin result in more of the 42 aa protein being formed and accounts for most of the early onset autosomal dominant form of the disease.

2. trunkated allele of notch (TAN-1) is formed by the translocation of TRC-beta on chromosome 7 and NICD on chromosome 9.

TCR-beta is expressed in T cells. Because the extracellular domain of notch is lost, notch 1 is constituently active in T cells

notch activation keeps cells in an immature state. this type of leukaemia is made up of immature T cells.

3. chronic liver disease, heart disease, retardation and skeletal abnormalities

autosomal dominant mutations in jagged 1 (a delta ligand)

notch pathway is not haplosufficient; pathway is sensitive to gene dosage

4. recurrent strokes leading to progressive dementia

caused by the build up of granular material in blood vessel smooth muscle

mis-sense mutations in notch 3 result in an unpaired cysteine. because a cysteine is unpaired, the formation of sulphide bridges is disrupted resulting in misfolding

misfolded notch 3 forms the granular material in the smooth muscle.

Question 7

1. In which organisms are wnt genes found?
2. Why do vertebrates have more wnt genes?

Answer 7

1. ALL metazoans
2. genome duplications

Question 8

1. what is the wnt ligand produced as? What is cleaved?
2. what modification takes place? how many times does it occur? what residues are modified? what enzyme performs these modifications?
3. which protein targets the ligand to the plasma membrane?
4. due to its hydrophobicity, how may wnt ligands diffuse?
5. What do HSPG do?

Answer 8

1. produced as a precursor. the signalling sequence that targets it to the secretory pathway is cleaved.
2. palmitoylation. twice, on serine 209 and cysteine 77. performed by porcupine
3. wntless.
4. may be loaded onto lipoproteins or form multimers which hide hydrophobic regions
5. aid diffusion of the ligand away from cells

Question 9

1. which 2 proteins form the wnt receptor?
2. which protein inhibits wnt signalling by interfering with the receptor and how?

Answer 9

1. arrow and frizzled.

Wnt binds to the cysteine rich domains of frizzled and to arrow

2. dickkopf

it couples arrow to kremen, which promotes arrow’s internalisation.

Question 10

1. name the 4 components of the destruction complex
2. what molecule acts downstream of wnt?
3. What happens in the absence of wnt?
4. What happens when wnt is present?

Answer 10

1. APC (beta catenin binding protein)

Axin (scaffold protein)

GSK3B (kinase)

CKIa (kinase)

2. beta-catenin
3. beta catenin is phosphorylated by CKIa at a serine residue, which primes the phosphorylation by GSK3B at serine and threonine residues

the phoshphorylation creates binding sites for slimb, thus slimb becomes associated with the destruction complex

slimb is an E3 ubiquitin ligase, thus ubiquitinates beta catenin, which is consequently degraded

due to the degradation, the nuclear concentration of beta catenin remains low. As a result, groucho is bound to DNA. Groucho activates histone deacetlyases.

4. dishevelled is recruited to frizzled and is phosphorylated

dishevelled binds to and inactivates axin, leading to the dissociation of the destruction complex.

beta catenin is no longer degraded, thus its cytoplasmic and nuclear concentrations increase.

beta catenin displaces groucho and activates transcription by recruiting CBP (histone acetylase) and BRG-1 (which is involved in chromatin remodelling)

Question 11

1. what are the roles of Wnt in drosophila? (2)
2. What are the roles of wnt in c.elegans? (2)
3. what are the roles of wnt in zebrafish? (2)

Answer 11

1. segmentation

expression of the D/V boundary of the wing is required for wing outgrowth

2, regulation of the Qr and Qi neuroblasts

signalling in Qi activates the Mab5 gene which induces posterior migration

3. formation of the organiser

ventral/posterior fates.

Question 12

1. give 3 examples non-canonical wnt signalling
2. what evidence suggests that wnt signalling is involved in the maintenance of intestinal stem cells?

Answer 12

1. planar cell polarity and convergent extension movements; axon guidance (RYK and Ror2 are activated by Wnt which induces axon repulsion); negative feedback (DKK1, DKK4 and axin 2 expression)
2. wnts are expressed below the crypt and nuclear beta catenin increases upon decent into the crypt. Stem cells are also found within the crypt.

Question 13

1. how is wnt signalling implicated in cancer?
2. how is wnt signalling implicated in bone disease?

Answer 13

1. ectopic wnt signalling caused by a loss of APC can lead to cancer.

famililal adenomatous polyposis caused by heterozygous APC loss. it causes a large no of intestinal polyps caused by sporadic loss of the remaining wild type copy of APC. these polyps can accumulate other mutations, which cause them to become malignant.

rare stabilising beta catenin mutations can lead to colon cancer

loss of function mutations in axin can cause hepatocellular carcinoma.

2. tetra-amelia syndrome is characterised by the absence of all 4 limbs. it is caused by autosomal recessive LOF mutations in wnt 3.

Question 14

1. name 3 vertebrate homologues of Hh
2. what is the vertebrate homologue of skinny Hh
3. what is the vertebrate homologue of dispatched?
4. name 3 vertebrate homologues of Patched
5. Name 3 vertebrate homologues of Ihog and Boi

Answer 14

1. Desert Hh, Sonic Hh, Indian Hh
2. Hh acetyl transferase
3. dispatched and scube
4. Ptc1, Ptc2 and Hhip
5. CDO, BOC and Gas1

Question 15

1. name the stages in the formation of the Hh ligand (4)
2. which molecules are required for the release of Hh? (2) How?
3. How are HSPGs involved?

Answer 15

1. a N-terminal sequence that targets it to the secretory pathway is cleaved

a c-terminal sequence is removed by autoproteolysis

the N-terminal sequence undergoes palmitoylation performed by skinny Hh

the new c-terminus is coupled to a choleterol molecule

2. dispatched and scube. they load Hh onto lipoproteins
3. long distance diffusion.

Question 16

1. what action does Patched empose on Smoothened?
2. what action does Hh empose on Patched?
3. What occurs to Smo when Hh is bound? Name 3 tightly coupled changes that occur?
4. Describe the cillia model of ptc and smo location.
5. What is ptc thought to act like? Give 3 pieces of supporting evidence.
6. How does Hhip regulate Hh signalling?
7. How do IHog/Boi/CDO/BOC/Gas1 regulate Hh signalling?

Answer 16

1. inhibition
2. inhibition
3. the inhibition from ptc is lifted. Ptc is internalised and degraded so that smo can be trafficked to the surface
4. in the absense of Hh, Ptc is localised to the cillia. Smo is excluded from this region.

When Hh is bound, ptc is removed from the cillia, allowing smo to accumulate there.

5. like a pump, pumping out molecules that activates smo and pumping in molecules that inhibit smo.
   - it is homologous to RND permeases that confer multi-drug resistance to prokaryotes by pumping out toxins/drugs
   - it is homologous to NPC1 which can move molecules across membranes
   - Ptc like proteins in c. elegans are involved in the efflux of lipids and lipid modified molecules that activate smo.
6. it down regulates Hh signalling by mopping up free Hh and preventing it from binding to Ptc.
7. they promote Hh signalling by acting as co-receptors that promote the binding of Hh to Ptc

Question 17

1. what is complex 1 composed of?
2. What is complex 2 composed of?
3. What happens in the absence of Hh?
4. What happesn when the concentration of Hh is low?
5. What happens when the concentration of Hh is high?
6. name the stages of Ci phosphorylation
7. Name the 3 vertebrate homologues of Ci

Answer 17

1. Ci, cos2 and fused
2. SuFu and Ci
3. Smo interracts with Cos2, a scaffold for complex 1.

when it is bound to smo, PKA, GSK3B and CKI phophorylate Ci

Phosphorylated Ci binds to slimb, which promotes the processing of Ci into Ci^R (N-terminal fragment)

Ci^R retains its zinc finger domain, but lacks the transcriptional activation domain

Ci is also retained by complex 2 which keeps Ci in the cytosol

4. complex 1 dissociates, thus Ci is not phosphorylated

Ci^R is not produced thus active repression is lost

5. smo is phosphorylated by complex 1 kinases

complex 1 dissociates and preferrentially associates with complex 2

the kinase activity of Fu counterracts the activity of SuFu.

Full length Ci translocates to the nucleus as Ci^A

Ci^A interracts with CREB and activates transcription

6. PKA firstly phosphorylates Ci, which primes it for phosphorylation by GSK3 and CKI
7. Gli1, Gli2 and Gli3

Question 18

1. Name 3 negative feedback effects of Hh signalling
2. Name a positive feedback effect of Hh signalling

Answer 18

1. Ptc1 transcription; Hhip upregulation; downregulation of CDO/BOC/Gas1
2. induction of Gli1

Question 19

1. name a role of Hh signalling in Drosophila
2. name 2 roles of Hh signalling in vertebrates

Answer 19

1. segment polarity
2. neural development (DV axis)

limb bud patterning

Question 20

1. what can a loss of Hh signalling in development lead to? (3)
2. Sheep that ingest what substance give birth to lambs with these phenotypes?

Answer 20

1. holoprosencephaly, syndactyly and cyclopamine polydactyly
2. corn lillies that contain cyclopamine have polydactyly

Question 21

1. Which components of the Hh pathway are Tumour supressors (i.e. LOF mutations result in cancer?) (2)
2. Which components of the Hh pathway are proto-oncogenes (i.e. GOF mutations result in cancer?) (1)
3. Which component is mutated in Gorlin Syndrome/Nevoid Basal Cell Carcinoma?
4. How does Hh signalling maintain cancers/act as a growth factor?
5. What is GDC0499, and what are the results of its clinical trials?

Answer 21

1. Ptc1 & SuFu
2. Smo
3. LOF mutn in Ptc. Sporadic loss of the functional copy is common, resulting in a large number of skin cancers.
4. Paracrine signalling maintains cancer in an undifferentiated state
5. Smo inhibitor. Initial results were good. After a few months, the cancers returned as a mutant form of Smo confered resistance to the inhibitor.

Question 22

1. name the 2 branches of the TGF-β family
2. what is the role of TGF-β signalling in the maintenance of homeostasis?
3. give 2 roles of TGF-β signalling in development

Answer 22

1. BMP/GDF branch

TGF-β/Activin/Nodal branch

2. maintains the correct balance of proliferation and cell death by promoting apoptosis and inhibiting proliferation
3. activin signalling induces dorsal mesoder

BMP signalling induces epidermal ectoderm

Question 23

1. what type of mutations in TGF-β are frequently found in cancer patients?
2. some cancers also show what? What is this and what does it indicate?
3. what type of mutations are less dangerous than the mutations indicated in Q1 and why?

Answer 23

1. hypomorphic mutations
2. microsatellite instability: microsatellites are repeated sequences of DNA which cause problems in DNA replication and they increase or decrease in length. Unstable microsatellites are thought to affect gene expression and indicates problems in DNA repair machinery.
3. mutations that completely abolish TGF-β signalling
   - TGF-β signalling is used later on in cancer development by promoting an epithelial to mesenchymal transition and angiogenes (which are required for metastasis and cancer survival)
   - TGF-β signalling prevents transformation, as it stops cell cycle at the G1 phase to stop proliferation and induce differentiation, and promote apoptosis while the tumour is still benign.
   - autocrine activity of TGF-β1 enhances tumour progression, leading to poor prognosis.

Question 24

1. What is the TGF-β ligand produced as?
2. how is the ligand modified?
3. What does release of TGF-β entail?

Answer 24

1. a precursor
2. it is cleaved into TGF-β dimers and LAP (which is held together by disulphide bridges)

LAP is tethered to the ECM by LTBP

3. Cleavage of the LAP/LTBP complex by plasmin and calpain proteases and the binding of thrombospondin to LAP.

Question 25

1. Describe the stages in receptor activation 2. What are lefty and antivin and how do they act 3. What is BAMBI and how does it act?

Answer 25

1. the TGF-β dimer binds to the type II receptor the type II receptor recruits and phosphorylates the type I receptor the type I receptor phosphorylates downstream proteins 2. decoy ligands; they lack a alpha-helix loop that allows for dimerisation of the ligand, thus the type I receptor can't be recruited 3. decoy receptor; it resembles the type II receptors but lacks the intracellular domain so the type I receptor can't be phosphorylated.

Question 26

1. Which proteins transduce the signal? 2. which types of these proteins are used by the BMP branch? 3. which types of these proteins are used by the TGF-β branch? 4. what is the name of these proteins that are receptor regulated? What are they anchored to the cell membrane by when the pathway is inactive? 5. what do the Q4 proteins dimerise with when they are phosphorylated? 6. what does the Q5 complex do?

Answer 26

1. Smads 2. 1, 5, 8 3. 2, 3 4. R-smads; anchored by SARA 5. smad-4 6. translocates to the nucleus where it can activate or repress transcription by binding to histone acetyltransferases or deacetylases

Question 27

1. What do I-smads do? 2. Which Smads are I-smads 3. How do they work? 4. Which domains do they lack (compared to R-smads) that gives them this function?

Answer 27

1. antagonise signalling 2. 6 & 7 3. bind to type I receptors and activated R-smads to block their activity 4. MH1 (DNA binding) domain and P target (target for phosphorylation)

Question 28

1. how many RTK genes are there in the human genome? 2. what is the role of RTKs? 3. what do receptors do upon activation. Name one exception to this and why. 4. Give the basic structure of RTKs

Answer 28

1. 58 2. phosphorylation 3. dimerisation of monomers; insulin, as it is already dimerised. 4. short single TM domain; cytoplasmic domain with tyrosine kinase activity; extracellular ligand binding domain which is variable between ligands.

Question 29

1. how do receptors become activated? 2. What fundamental process follows receptor activation? 3. what does the process mentioned in question 3 result in?

Answer 29

1. ligand binding induces dimerisation of receptors which induces a conformational change causing them to become activated. 2. transautophosphorylation, whereby the kinase domain phosphorylates tyrosine residues on the kinase domain of the neighbouring monomer. 3. increased kinase activity, receptor stablility and the creation of docking sites for downstream proteins.

Question 30

1. which domain mediates the docking of proteins to phosphorylated tyrosines? 2. give 4 examples of proteins that dock on phosphorylated tyrosines and their roles.

Answer 30

1. SH2 domains 2. PI-3 kinase (phosphorylates lipid) PLC gamma (activates IP₃ pathway) GTPase activating protein (activates RAS/MAP pathway) c-Cbl (catalyses ubiquitination of the receptor - negative feedback)

Question 31

1. which proteins activate Ras? 2. Which proteins inactivate Ras? 3. how does RTK activation lead to Ras Activation? 4. Describe the MAP kinase pathway.

Answer 31

1. GEFs 2. GAPs 3. GRB2 binds to active RTK and to Sos. Sos is a GEF, thus catalyses the dissociation of GDP from Ras so that GTP can become associated. 4. Ras activates MAP-KKK. MAP-KKK phosphorylates/activates MAP-KK. MAP-KK phosphorylates a specific serine and threonine residue on MAP-K. MAP-K phosphorylates downstream proteins such as TFs, gene regulatory proteins and other kinases.

Question 32

1. How many families of RTKs are there? 2. how do FGFs act? 3. how many FGF receptors are there for _all_ ligands? What mechanism creates some variability? 4. Which 3 domains do all receptors have? What do each of these domains do?

Answer 32

1. 22 2. mainly in a paracrine manner, but also in an intracrine or endocrine manner 3. 4. Alternative mRNA splicing 4. D1 (inhibits ligand binding to regulate receptor activation) D2 and D3 (ligand binding domains)

Question 33

1. which ECM component do FGF receptors and ligands bind to to facilitate ligand binding 2. how does the formation of these complexes do so? 3. In which developmental strucures is FGF8 expressed (5)? In which particular structure is an FGF gradient important 4. what is FGF3R a regulator of? GOF mutations in this receptor lead to what?

Answer 33

1. HSPGs 2. membrane bound HSPGs recruit ligands at the surface via their GAG chains. the complex also favours the high affinity binding between the ligand and the receptor 3. somites, forming limb buds, eye, midbrain/hindbrain boundary and brachial arches. the FGF8 gradient is involved in the determination front of somites 4. regulator of bone growth. Achondroplasia.

Question 34

1. What are TLRs a class of? 2. LOF mutations in TLRs in flies lead to what? GOF mutations in TLRs in flies lead to what? 3. describe the events that lead to the discovery of TLR4 being the receptor for LPS

Answer 34

1. pattern recognition receptors (innate immune system) 2. increased fungal infections; increased anti-fungal proteins 3. some mice were resistant to LPS injections. The genes that confered this resistance were mapped to chromosome 4 (LPS locus). Screening of the LPS locus using positional cloning lead to the identification of TLR4, which makes a complex with CD14 to bind to LPS. Mice that were resistant hand a point mutation in TLR4 or lacked the TLR4 gene entirely.

Question 35

Give the location, and what is recognised by the following... 1. TLR4 2. TLR1 3. TLR6 4. TLR5 5. TLR3 6. TLR7/8 7. TLR9

Answer 35

1. cell surface; recognises LPS (component of gram negative bacteria) 2. cell surface; forms a dimer with TLR2 to recognise triacylated lipoproteins (gram negative bacteria) 3. cell surface; forms a dimer with TLR2 to recognise diacylated lipoproteins (gram positive bacteria) 4. cell surface; recognises flagella 5. endosomal; recognises dsRNA 6. endosomal; recognises microbial ssRNA 7. endosomal; recognises CpG DNA

Question 36

1. What are PAMPs? 2. What are DAMPs? 3. Give the basic structure of TLRs

Answer 36

1. pathogen associated molecular patterns. they are molecular patterns found on pathogens but not usually host cells 2. disease associated molecular patterns. they are endogenous molecules created to alert the host to tissue injury. they may be intracellular molecules released by necrosis or activated following injury or ECM fragments released/upregulated in response to injury. 3. Extracellular domain consisting of leucine repeats that forms a horseshoe structure. TIR/intracellular domain consisting of highly conserved amino acids. 3 conserved boxes are critical for signalling as they interract with adaptor molecules.

Question 37

1. how are specific immunological responses tailored to infecting microbes? 2. which domains of TLRs interract with the same domains of adaptor proteins to initiate the signalling cascade?

Answer 37

1. different microbes activate different TLRs. Different TLRs recruit different adaptor proteins thus activate different signalling cascades 2. TIR domains

Question 38

1. which TLRs use the MyD88 pathway 2. which protein recruits MyD88 to the TLR 3. what does the MyD88 pathway activate thus induce? 4. describe the MyD88 pathway

Answer 38

1. _all_ except TLR3 2. TIRAP 3. NFκB and MAP-K thus inducing cytokine production 4. MyD88's death domain interracts with the death domain of IRAKs IRAKs phosphorylate TRAF6 TRAF6 is a ubiquitin ligase, and creates an unconjugated free polyubiquitin chain the creation of the chain leads to the phosphorylation of NFκB, which induces the expression of cytokines.

Question 39

1. Which TLRs use the TRIF pathway? 2. which protein recruits TRIF to the TLR? 3. what does the TRIF pathway activate thus induce? 4. Describe the TRIF pathway.

Answer 39

1. TLR3 and 4 2. TRAM 3. activates IRF3 and NFκB thus inducing the production of cytokines and type 1 interferons 4. TRIF associates with TRAF3 and TRAF6. TRAF6 activates NFκB TRAF3 undergoes autoubiquitination and acts as a ubiquitin ligase, thus triggering the phosphorylation of IRF3 IRF3 induces the expression of Type 1 interferons.

Question 40

**_Sepsis_** 1. how can the production of pro-inflammatory cytokines lead to oedema and potentially capillary leak syndrome? 2. why is capillary leak syndrome dangerous? 3. Which mutation was found exclusively in patients with septic shock and confered an increased incidence of gram negative infections? 4. What is Eritotan and what are its results in clinical trials? **_Respiratory Syncitial Virus_** 5. why can people be infected more than once? 6. which TLRs detect the virus and by which components? 7. which TLR mutations are over represented in infants under 12 months hospitalised for RSV bronchiolitis?

Answer 40

1. it causes damage to endothelial walls which increases vascular permeability 2. it causes dangerous hypotension, hypoalbumenaemia and haemoconcentration, which can lead to organ failure by hypoxia 3. asp299gly. 4. interferes with TLR4/LPS complex formation. it has been well tolerated in patients with sepsis and confers a significant reduction in mortality 5. adaptive immunity decreases over time 6. TLR4 (fusion protein on the surface); TLR3 (dsRNA); TLR7 (ssRNA) 7. Asp299Gly and Thy399IIe