L5 - Hh and Wnt Flashcards
What organism were Hh and Wnt disocvered in
Flies
Hh and Wnt are what type of genes
Segment polarity genes
Describe a Hh mutant, therefore what is the gene required for
Has no naked cuticle and is covered in a lawn of dendticles
Hh is therefore required for the formation of the naked cuticle
During segment patterening what do Hh and Wnt do for each other
So what can be said about Hh and Wnt mutant phenotypes
Maintain each others expression
Since loss of either Hh or Wnt will also lead to loss of expression of the other one then phenotypes will be similar
Describe the discovery of wnt in mice and flies
Discovered as wingless Wg in flies
Discovered as Int1 in mice
Wnt was an amalgamation of the names
How many Wnts and Hhs do vertebrates have
Many orthologues
What is significant regarding Hh in c.elegans
They do not have Hh present
Describe the translation of the Hh signal
Translated as three regions
1) N terminal signal sequence - targets to sec pathway 2) C terminal autoproteolytic domain - that cleaves itself
3) Middle protein
What is found at the N term end of the Hh when first translated
Signal sequence which targets the protein to the secretory pathways
What is found at the C term end of Hh when first translated
Autoproteolytic domain
What occurs once the signal sequence has been cleaved and the C terminal autoproteolytic domain has been cleaved
(2 modifications)
Cholesterol mod at C’
Palmitoylation at N’
What is the effect of palmitate and cholesterol
Both are strongly hydrophobic so make the Hh hydrophobic
What two proteins important for long range singalling of Hh
Dispatched and scube
How many TMD in dispatched
12
What is the proposed function of dispatched and scube
May help load Hh into lipoprotein particles or cytonemes
What is required for the long range transmission of the hh
HSPGS
What does HSPG stand for
Heparan sulphate proteoglycans
When is the signal sequence of wnt cleaved
Once the protein has entered the secretory pathway
What are the two modification of Wnt signal
Palmitoylation of cys77
Palmitioleic acid mod of ser209
Where is palmitiylation of Wnt addeded
cycy77
Where is palmitoleic acid added to Wnt
ser209
What is the effect of the two modifications added to Wnt
Make wnt insoluble in water
WHat is Wntless involved in
Getting wnt to the cell membrane
What may be involved in the transmission of the Wnt signal
Lipoprotein particles and cytonemes
What is required for the long range transmission of the wnt signal
HSPGs
What is the structure of wntless
7 pass transmembrane protein
What is a cytoneme
Long cellular protrusion used to touch other cells causing them the change thier behaviour - signalling molecule accumulates at the top
What does Hh bind to
Ptc
Ptc strucutre
12 TMD
Similar to the dispatched gene
How does ptc act without hh bound
In a negative way by continuosuly inhibiting smoothened
WHat is the strucutre of Smo
7 TMD
Is there a stoichiometric relationship between ptc and smo
What does this mean?
Doesnt work one to one
Single ptc molecule inhibits many smo by regulating the subcellular location and stability of smo (targets smo to a compartement where it is degraded)
What is the effect on smo and ptc when Hh binds to ptc
Both ptc and smo internalised and degraded
So now smo can be trafficked to the surface
THREE MAIN CHANGES OCCUR TO SMO
Relocation
Accumulation
Phosphorylation
In mammals what is thought to serve as the focal point for Hh signalling
Cilia
Describe localisation of Ptc1 in absence of Hh
What is the effect of this on smo
Ptc1 localised to cilium so smo is excluded from this area
What occurs to the localisation of Ptc1 when Hh binding
What is the effect of this on smo
Ptc1 removed from cilium
Allows smo to accumulate in the cilum and initiate signalling
How was it seen that cilium is involved in Hh signalling
Mutations in mice which disrupt cilia formation Hh signalling is also inhibited
What is the effect on ci when Hh not present
There are two complexes which keep it out of the nuceleus
What are the two complexes which keep ci out of the nucleus
One with supressor of fused gene (SUFU) binds ci
One contains costal2 and a fused serine threonine kinase
How many kinases in the costal2 complex
Name them
What effect do they have on Ci
3
Casein kinase I, protein kinase A, glycogen synthase kinase 3B
Shorten Ci via slimb - involving ubquitination
What is the effect of the shortened version of Ci
Acts as a transcriptional repressor molecule - CiR
Hh target genes are actively repressed
When HH active
What occurs to the three kinases
What effect does this have on ci
Interaction with the three kinases and Ci is blocked so a full length Ci is released and will actively promote transcription of target genes
When HH active
What occurs to SUFU
Phosphrylation of SUFU by fused proteins and promotes the formati`on of the active form of Ci
How does Hh act negatively on its own pathway
Repression of patched - reduces and limits the level of activation
How does Hh act positively on its own pathway
Gli1 is induced and this is unable to be proetolysed into a repressor
How does Hh play a role in wing patterning
In th drosophila imaginal disc Hh expressed posteriorly
It then diffuses into the anterior causing the expression of Dpp
How is Shh involved in the patterning of the neural tube
Shh from notochord induces Shh exp in FP
Shh from FP then diffuses throughout the neural tube and a morphogen gdt is formed
Neural fate dep on how much Shh seen and for how long
How is Hh involved in the AP patterning of the limb
Shh expressed in the ZPA
Confers posterior identity to the developing limb
What are the other examples of Hh signalling
Brain dev Branching in lung Prostate Teeth Tounge
What is holoprosencephaly
Loss of the ventral brain structure
Results in fused eues and cyclopic embryoys
What is cyclopamine
An inhibitor of Smo
What is syn/polydactylyl caused by
Mis regulation of the hedgehog pathway
What mutations can cause cancer by causing over activity of the Hh pathway
What are these genes
Loss of func in Ptc1 or SUFU
Tumour supressor genes
What mutations can cause cancer by causing inhibition of the Hh pathway
Activating mutations in Smo
Smo is a protooncogene
What is gorlin syndrome
Large numbers of basal cell carcinoma due to patient being heterozygous for Ptc
Mistakes can lead to the loss of the functional ptc gene so this cell would then form a BCC
What may be used to treat cancer caused by overactive Hh
What was the issue with this
Smo inhibitors - GFC-0049
Starts well but then tumour comes back due to resistance acquired by a mutation in Smo
What is the crucial transcription factor for Wnt signalling
B-catening
Describe what happens to B-catenin when Wnt not activated
B-catenin is bound and destoryed by a desturction complex
Where does wnt derive its name from
Amalgam of wingless Drosophila gene and Int vertebrate proto-oncogene
Int-1 is a proto-oncogene, what causes its activation
Integration of the mouse mammary gland tumour virus
Wingless (wg) mutants initially discovered produced wingless but viable flies, T or F
T
Explain how wg and hh maintain each other’s expression in an auto-regulatory loop
Wg maintains hh by controlling the expression of engrailed (en), a transcription factor that regulates hh expression. Hh then in-turn maintains and directly upregulates wg
What is significant about hh and wg knockout mutants
They exhibit the same phenotype – larvae with a lawn of denticles
Unlike similar developmental signalling pathways, wnt expression is highly conserved throughout Kingdom Animalia, T or F
T – even found in sponges
Why is it that vertebrates have more wnt genes
Due to genome duplication throughout evolution
The wnt protein is produce by a cleavage event that separates its signalling sequence from the initially translated protein, T or F
T
What is the role of porcupine in the early modification of wnt
Porcupine is an acyl transferase that adds palmitoleic acid modifications to a serine residue at point 209 in the wnt3a structure
What is the hypothesised role of wntless in wnt signalling and what is its basic structure
Wntless is a 7 transmembrane domain protein potentially required for the transport of wnt to the plasma membrane and its subsequent release/presentation to target cells
What is the effect of palmitoylation and palmitoleic acid modification of the wnt protein
Addition of these hydrophobic groups makes wnt insoluble in water
What components of the extracellular matrix are involved in mediating the diffusion of wnts away from the sending cell
Heparan sulphate proteoglycans (HSPGs)
What is significant about the fact that most of the effects of Wnt signalling can be elicited by a membrane bound form of the protein in Drosophila
It suggests that wnts act as juxtacrine signalling molecules or that they don’t diffuse far and act on adjacent cells in Drosophila
Recall the two main receptors involved in reception and transmission of wnt signalling in Drosophila
Frizzled and Arrow
What is the name of the nuclear factor in Drosophila that is induced as a result of wnt signalling and its corresponding vertebrate homologue
Armadillo (vertebrate homolog – ?-catenin)
What are the names of the arrow receptor homologues found in humans
LRP5 and 6
Both the frizzled genes and arrow/LRP5&6 act in combination as receptors for wnt signalling molecules, T or F
T
Describe the structure of the frizzled receptor and how it interacts with wnts
7 transmembrane domain protein. Wnt binds to the cysteine-rich domain (CRD) in the N-terminus of the Fz protein
Describe the structure of the LRP5&6/Arrow receptor for wnt
Single pass transmembrane protein
What happens when wnt binds to LRP5/6/Arrow and the Fz receptors
These two receptors come together to form an active wnt signalling complex
Which important extracellular wnt inhibitor is overexpressed in order to downregulate wnt signalling in experiments
Dickkopf1 (Dkk)
How does Dkk act to downregulate wnt signalling
Dkk is coupled to Kremen. Activation of Dkk by wnt binding promotes the internalisation of the LRP receptors
Describe the composition of the degradation complex involved in wnt signalling
Consists of the scaffold protein axin bound to APC, GSK3?, CK1? and slimb
Describe what happens in the absence of wnt signalling
Also bound to the degradation complex via an interaction with APC is ?-catenin. In the absence of wnt signalling ?-catenin is phosphorylated by CK1? and then by GSK3?. This poly-phosphorylated ?-catenin is then recognised by the slimb protein which ubiquitinates the ?-catenin marking it for degradation by the proteasome system. With low levels/absence of ?-catenin T cell factor (TCF) transcription factors are bound to the promoter regions of wnt target genes. Also bound to these TCFs is a transcriptional repressor known as groucho. Groucho inhibits the transcription of wnt target genes
Describe what happens in the presence of wnt signalling
Wnt binds to its Fz and arrow/LRP 5&6 receptors in the membrane. These receptors come together and form an active complex which recruits the dishevelled protein to the complex. Dishevelled is then phosphorylated and as a result may bind to axin in the intracellular destruction complex. Arrow/LRP is then also phosphorylated this time by GSK3? and the receptor recruits axin also. Binding of the destruction complex to the receptor complex displaces the slimb protein. With slimb lost the destruction complex is inactivated. ?-catenin then accumulates inside the cell due to it not being ubiquitinated and marked for degradation by slimb. It then translocates to the nucleus of the receiving cell and displaces groucho from the TCF DNA binding proteins. In combination with additional downstream transcriptional activators this leads to the transcription and expression of wnt target genes.
Explain how ?-catenin degradation is achieved by the destruction complex
?-catenin is phosphorylated by CK1? first, which primes it phosphorylation by GSK3?. Phosphorylation by both kinases is required for ?-catenin recognition by an E3 Ubiquitin ligase complex (which contains b-TrCP/Slimb) and subsequent degradation by the proteasome. The serine/threonine phosphates and surrounding amino acid sequence in ?-catenin as a result of phosphorylation forms an optimal binding site for b-TrCP/Slimb. ?-TrCP/Slimb binds only after GSK3 phosphorylates the 3rd and 4th phosphorylation sites
Where does CK1? phosphorylation occur within the ?-catenin/armadillo structure
Sites within the N-terminal tail
The S/TXXXS/T(P) is the ideal site for GSK?, what does this mean
A serine or threonine residue followed by 3 residues of any identity and then another serine or threonine that has been phosphorylated by CK1?
What is the name of the vertebrate homologue of slimb
?-TrCP
Describe the structure of the SCF E3 ubiquitin ligase complex involved in ?-catenin/armadillo degradation
The Skp1-Cullin-F-box E3 ubiquitin ligase complex consists of the ring finger protein Roc1 which binds to an E2 ligase, the scaffold protein cul1 and skp1
Explain the role of SCF in ?-catenin/armadillo degradation
The F-box protein interacts with Skp1 via its F-box. The F-box of also interacts with the substrate via the WD40 domain that interacts specifically with phosphorylated targets
How does the absence of Wnt signalling lead to no expression of wnt target genes
Without ?-catenin binding to TCF, groucho remains bound. The transcriptional repression by groucho is mediate by its recruitment of histone deacetylases thought to make DNA refractive to transcriptional activation
How therefore does wnt signalling lead to expression of downstream target genes
In the nucleus increases levels of ?-catenin displace groucho from the TCF complex. Displacement of groucho leads to the recruitment of histone acetylase CBP/p300 and another transcriptional activator called BRG-1. These lead to transcription of wnt target genes
Explain how interactions between TCF/?-catenin and chromatin could also be mediated by legless (Bcl9) and pygopus genes
Mutations in these genes result in wingless-like phenotypes in Drosophila. Both genes also promote wnt signalling in mammalian cell cultures.
Wnt signalling components are also involved in directing planar cell polarity and convergent extension, T or F
T
Explain the negative feedback mechanism of wnt signalling
Dickkopf1 (Dkk) activation by wnt binding promotes the internalisation of the LRP receptors. This decreases further wnt signalling activation and has important homeostatic roles.
Give examples of planar cell polarity events caused by wnt signalling
Wnt signalling aligns all the hairs in the skin in a certain direction
Explain how defective wnt signalling can cause cancer, particularly in the gut
Patients who are heterozygotes for APC loss of function mutations suffer from familial adenomatous polyposis. This is where sporadic loss of the other functional wild type APC allele in the gut results in activation of the wnt signalling in such cells. This causes hyperproliferation and culminates in the formation of polyps which may accumulate further mutations and cause colon cancer.
The APC gene is a proto-oncogene, T or F
F – it’s a tumour suppressor gene (loss of function results in tumorigenesis)
Give an example of another disease phenotype caused by mutation(s) in wnt signalling
Tetra-amelia is a disease where the infant is born without limbs. This is caused by a mutation in wnt3
