Lecture 19 Flashcards
Drosophila larva
- Denticles present across anterior half of each segment
- Posterior half smooth
- In mutant, posterior segment failed to develop, covered in denticles
How many hedgehog proteins in Drosophila?
Single hedgehog (Hh) in Drosophila
Hedgehog proteins in vertebrates?
Sonic (Shh)
Indian (Ihh)
Desert (Dhh)
What are hedgehog proteins and how are they modified?
- Secreted signalling proteins
- Modified by cleavage and addition of cholesterol
Explain hedgehog processing
Inactive precursor - Signal peptide 0-82 - Secretory pathway
20kDa N-terminus is signalling molecule
25kDa C-terminus catalyses cleavage - modifies N-terminus with cholesterol
Cleavage occurs between glycine-257 and cysteine-258
- Sulphur on cysteine attacks peptide bonds - converts into thioester
- Hydroxyl group on cholesterol forms ester bond - completes cleavage
- Palmitoyl fatty acid added to N-terminus - Hh hydrophobic at both ends
- Cholesterol embeds Hh in cell membrane
Explain why hh is tethered to the membrane
- Restrict activity to immediately adjacent cells
Parasegment boundary
- Boundary between stripes is parasegment boundary
- Tethered hedgehog induces diffusible wingless in adjacent cell
What occurs when Hh N-domain lacks cholesterol?
Freely diffusible
Switches on more wingless in more cells
Explain dispatched Hh from membrane
- Dispatches from membrane, not via cleavage - cholesterol remains
- Other partners required to aid diffusion
Explain Patched (Ptc)
Hh receptor
- 12-pass transmembrane domain
- Usually inhibits Hh response
- KO of patched activates Hh response
- Could involve smoothened (Smo)
Hedgehog signalling in Drosophila
- Absence of Shh, Ptc acts to sequester Smo in membrane bound vesicles -> degraded
- Ci/Cos2/Fu complex binds microtubules through Cos 2
Ci phosphorylated by PKA
Slimb recognises Ci-P -> Ci targeted to the proteasome release 75kDa Ci75 fragment
Binds target genes, represses transcription
What occurs in the Drosophila hedgehog pathway in presence of Hh
- Ptc unable to sequester Smo
- Cos2/Fu recruited by Smo - disassociate from microtubules
- Ci released from complex - can’t be PKA phosphorylated
- Full length Ci binds CBP - activates expression
Explain induction of neural fates by Shh
- Nervous system forms ectoderm - overlying notochord
- Folds to make neural tube in neurulation
-Notochord secretes Shh
- Shh induces floor plate
- Floor plate secretes Shh
- Motorneurons specified at high Shh
- V2 interneurons at medium Shh
- V1 interneurons at low Shh
Hh signalling and cancer
Components in Hh pathways can mutate
Stem cell proliferation in basal layer of skin stimulated by Hh
Excess Hh - Over-proliferation
- Ptc1 inhibits signalling - tumour supressor
- High frequency of BCC in ptc-/ptc+
Possibility of anti-cancer drugs - inhibitors of Hh
Who are at high risk of basal cell carcinoma, and why?
People with Gorlin’s syndrome - mutation in Ptc
- BCC not that serious - treated with surgery
- Cancers shrink when treated with small molecule inhibitors of Hh
Structure of Wnt proteins
Vertebrates have multiple Wnt’s between 35 and 45 kDa
N-terminal signal peptide
Cysteine important - forms intra-chain disulphide bonds
Wnt modified with hydrophobic palmitoleane on serine
Addition carried out by porcupine
Wnt signalling pathway in absence of Wnt
- Largely elucidated in Drosophila by looking at mutants
- Lrp and Frizzled used
- GSK3/APC/Axin phosphorylate beta catenin
- Targeted for degradation by Slimb
- Beta-catenin fully degraded
- TCF binds DNA and represses targets
Wnt signalling pathway in presence of Wnt
Wnt binds Fizzled and Lrp
Phosphorylation of Lrp by conformational structure change
High affinity for Axin sequestering it and disassociation from complex
Degradation of beta-catenin disrupted - No phosphorylation by GSK3
Translocates to nucleus and binds TCF -> repressor to activator
What does Nieuwkoop centre do?
Instruct dorsal mesoderm and organizer
Wnt essential to estanlishing centre
Adds beta catenin mRNA to ventral side of Vegetal region -> second Nieuwkoop centre and duplicated axis of development
What occurs when Wnt is over-expressed
Cancer
What can cause colon cancer and melanoma
Mutations preventing beta catanin degradation
Wnt signalling with cancer
Wnt expressed by MC’s in crypt important for ISC proliferation
APC mediates destruction of beta-catenin
Excess Wnt signalling
LOH of APC in patient with null allele -> No beta catenin degradation -> Large numbers of precancerous polyps in colon
- Mutations in Beta-catenin that prevent APC degradation
