Lecture 12 Flashcards
Which family are the vertebrate homologues of the Ci transcription factor
Gli family proteins
Phosphatases are involved in both hedgehog and wnt signalling pathways, T or F
F – kinases are involved in both
What three cancers are directly caused by ectopic hedgehog signalling
Basal cell carcinoma, medullablastoma and rhabdomyosarcoma
Excess activation of smoothened caused by an insensitivity to ptc1 inhibition can cause cancer, thus what type of gene is smo
Proto-oncogene
What is the significance of the non-canonical hedgehog signalling in adipocytes/myocytes in tumorigenesis
Aerobic glycolysis is a feature of many tumour cells and is known as the Warburg effect
What was seen in hh mutant Drosophila embryos
Defects in segmentation. The segments contained a lawn of denticles and no naked cuticles
What is Niemann-Pick Disease and how is this related to ptc
NP disease is caused by a mutation in the niemann pick protein C1 and is a lipid storage disorder that leads to the accumulation of harmful quantities of lipids in the visceral organs and brain. Ptc contains regions homologous to the structure of NPC indicating its potentially similar roles
Explain the hypothesised action of Ptc on small molecules in order to exerts its inhibitory effect on Smo
Ptc either pumps a small inhibitory molecule into cells or a small excitatory molecule out. Or Ptc may pump a small molecule into or out of the cells that effects Smo trafficking or localisation in the membrane
Explain the signal transduction mechanism at high levels of hedgehog ligand
Hedgehog acts through both the CKI/GSK3?/PKA and Cos2/Ci/Fused/SuFu complex by leading to phosphorylation of full-length CiA and/or SuFu. This prevents partial degradation of CiA and allows its translocation to the nucleus in its activator form. Thus, resulting in the promotion of hedgehog target gene transcription.
Both wnt and hh signalling use scaffold proteins, recall the proteins involved in each signalling pathway
Wnt signalling – uses axin. Hedgehog signalling – uses costal2
Give an example of a positive feedback mechanism involved in hedgehog signalling
Gli1 is a downstream target gene of hedgehog signalling. Gli1 was is a constitutive activator of hedgehog gene expression leading to a feedforward response and subsequent increase in hedgehog signalling
What happens when hedgehog binds to its ptc receptor
Leads to an inhibition of the inhibitory action of the ptc receptor
What type of protein is slimb referred to as
F box domain containing protein
What type of gene is hedgehog
Segment polarity gene
In absence of the ligands in both wnt and hedgehog signalling there is transcriptional repression. Which transcription factors mediate this in each pathway
Transcriptional repression is mediate by groucho/TCF in wnt signalling whereas in hedgehog signalling this is carried out by Ci-75/CiR
Discuss the genetic conservation of the hh signalling pathway
Hh signalling is relatively conserved amongst metazoans and kingdom Animalia however not to the extent that wnt signalling is. For example C.elegans lacks hh signalling mechanisms
Inactivation of ptc1 or SuFu can cause cancers, what type of genes are these
Tumour suppressor genes
A single ptc receptor inhibits a single smo receptor, T or F
F – a single patched molecule can inhibit the activity of several smo receptors
What is the significance of the N-terminal signal sequence of the hedgehog ligand when first synthesised
It targets the protein to the secretory pathway
What are the two effects of ptc1 induction by hedgehog
Suppression of the intracellular signalling within the cell and restricting further diffusion of the hedgehog ligand by receptor binding. The latter acts to steepen the gradient of hedgehog expression
Explain the negative feedback mechanism mediated by hedgehog interacting protein
Hedgehog binding signalling leads to hedgehog interacting protein induction. This acts to limit diffusion of the ligand and the level of subsequent signalling by the downregulation of hedgehog signalling stimulators CDO, BOC and GAS1
Cleavage of the N-terminal part of the hedgehog protein is coupled to a cholesterol molecule addition. This occurs as well as palmitoylation. What are the combined effects of these modifications and the effects on the signalling pathway
Addition of these groups makes the hedgehog protein very hydrophobic. This renders the molecule insoluble in water and acts to target its localisation to the membrane. In addition, it makes hedgehog unable to leave the membrane and hence restricts it to signalling only to neighbouring cells
Explain how cilia are involved in mammalian hedgehog signalling
In the absence of the hedgehog ligand, ptc1 is localised to the primary cilium of the cell and smo is excluded from this region. Hedgehog binding to ptc causes its removal from the cilium which allows smo to accumulate there and initiate signalling
By explaining its homology to other similar proteins, describe to evidence for ptc action as a pump
Ptc has homology to RND permeases which pump out toxins and are involved in multi-drug resistance. Ptc also has homology with NPC1 which can transport some molecules across membranes as well as move cholesterol containing vesicles.
What is unusual about antagonists of the canonical hedgehog signalling pathway
They were found to be agonists of the non-canonical hedgehog signalling pathway
What accounts for the increase in hedgehog genes seen in vertebrates compared to invertebrates
Vertebrates contain more homologues of the hedgehog family genes due to genome duplication throughout evolution to account for the importance of the pathway in patterning
How have mutations in cilia identified their role in hedgehog signalling
Mutations that disrupt cilia formation were found to impair hedgehog signalling too
Explain the hedgehog signalling negative feedback loop mediated by ptc/ptc1
One of the downstream targets of hedgehog signalling is the ptc protein. This acts to inhibit subsequent hedgehog intracellular signalling and limits the level of activation of the pathway by reducing the range of movement of the hedgehog signal
Hh and wg are said to be dependent on each other, what does this mean
These genes require each other to be expressed. Loss of either gene will lead to the loss of the others expression. Thus mutations in either gene will give rise to similar phenotypes
Which degradation machinery protein(s) are involved in both wnt and hedgehog signalling
Slimb/TCRP-?
How many transmembrane domains does patched have
12
Explain the mechanism by which full-length Ci (CiA) is partially degraded by an intracellular protease in the absence of hedgehog signalling
PKA initially phosphorylates CiA which primes it for subsequent phosphorylation by GSK3? and CKI. These multiple phosphorylations create a binding site for Slimb protease binding. Slimb binds and partially degrades CiA by removing the activator domain.
Explain the effects of ptc on smo both when the hedgehog ligand is absent and present
Patched regulates the subcellular localisation and stability of smoothened. In the absence of HH, Ptc keeps Smo from getting to the cell surface by causing its trafficking to a compartment where its degraded. When Ptc binds to HH, they both get internalized and degraded. This allows and Smo trafficking to the cell surface
Describe a non-tumour related disease associated with abhorrent hedgehog signalling and what causes it
Cyclopia and holoprosencephaly are caused by a lack of or inhibition of hedgehog signalling in the brain. Sonic hedgehog is required to pattern the ventral midline of the embryo. This diseased is characterised by a failure of the forebrain to separate into two hemispheres and results in fused features, such as eyes etc.
Both wnt and hedgehog signalling molecules are modified by addition of hydrophobic moieties, T or F
T
Gain of function mutations in hedgehog cause cancers, T or F
T
Gorlin syndrome or Nevoid Basal Cell Carcinoma is a disease state caused by problems with hedgehog signalling that leads to massive numbers of BCCs. Explain how this disease is caused and leads to systemic tumour formation
Patients with Gorlin syndrome are heterozygous for a loss of function mutation in ptc1. Hence sporadic inactivation of the other, functional, copy of the patched1 gene leads to tumorigenesis
Describe the role of sonic hedgehog signalling in patterning of the limb
Sonic hedgehog is expressed in the zone of polarising activity in the posterior limb bud. It is responsible for patterning posterior regions of the limb
What proteins are required for hedgehog release from the signalling cell and its long-range diffusion
Dispatched, Scube and other HSPGs
Recall some of the vertebrate homologues of hedgehog
Sonic hedgehog, Indian hedgehog and desert hedgehog
What is the main different in transcription factor degradation reactions that occur in wnt and hh signalling
Ci is only partially degraded, whereas ?-catenin is fully degraded
Ptc is involved in triggering stem cell differentiation, T or F
F – it maintains stem cells in the skin via proliferation
What happens in the hedgehog signalling transduction pathway at low concentrations of the ligand
At low concentrations of hedgehog, the PKA/GSK3?/CKI complex dissociates from the complex containing Ci, fused and cos2. As a result, active repression by CiR is lost
Explain the hedgehog signal transduction pathway in the absence of the ligand
In the absence of the ligand the activator Ci transcription factor is kept out of the nucleus by the combined action of two complexes. The first complex contains costal2 (Cos2) which is a kinesin like molecule that acts as a scaffold protein, and fused, a serine-threonine kinase. The other complex contains the Ci and the suppressor of fused (SuFu) gene. The Cos2-fused complex binds to the smo receptor and causes three other genes to act on Ci, casein kinase I, protein kinase A and glycogen synthase kinase 3?. This kinase complex processes the Ci transcription factor by phosphorylation and creates binding sites for the slimb protease machinery. Slimb protease binds and partially degrades the Ci transcription factor from its full-length activator form, CiA to its repressor form, CiR. CiR then translocates to the nucleus and represses hedgehog target gene expression
What is the result of mutations in sites within the Ci transcription factor that are usually phosphorylated by the CKI/PKA/GSK3?
Reduction in phosphorylation of Ci and processing. This reduces Slimb binding and may lead to increase hedgehog signalling
What similarities are seen in the frizzled and smoothened receptors involved in wnt and hedgehog signalling
They are both 7 transmembrane domain proteins where the heteromeric G-proteins don’t play a central role in signalling
Explain the role of hedgehog signalling in wing patterning
Hedgehog signalling is responsible for anterior-posterior patterning in the wing imaginal disc. Hedgehog is secreted from cells in the posterior wing imaginal disc and diffuses anteriorly to induce the expression of decapentaplegic. At the boundary between the presence and absence of hedgehog, a signalling centre forms which patterns the wing.
What happens to the hedgehog ligand once it reaches the membrane
The N-terminal signal sequence is cleaved off by an autoproteolytic cleavage catalysed by the C-terminus of the protein
Explain the role of non-canonical hedgehog signalling in adipocyte and myocyte cellular metabolism
Activation of smo by decrease inhibition by ptc leads to the stimulation of aerobic glycolysis mechanisms in the target cells. This results in a partial metabolism of glucose and leads to the production of lactate which leaves the cells and acidifies the extracellular environment
What is the name of the complex to which slimb belongs
SCF ubiquitin E3 ligase complex
Recall some of the hedgehog receptors in vertebrates
Patched 1 and 2, Hedgehog interacting protein, Smoothened, CDO, Brother of CDO (BOC)
What was the result of ectopic sonic hedgehog signalling in the anterior limb bud, either by ZPA cell grafting or implantation of sonic hedgehog soaked beads
Mirror image duplication of limb structures
Whose saturation mutagenesis experiments lead to the discovery of the hedgehog signalling pathway
Nusslein-Volhard and Wieschaus
Explain the role of hedgehog signalling in neural development
Sonic hedgehog is secreted from the ventral floor plate and notochord and is responsible for patterning of the neural tube. Once released sonic hedgehog diffuses into the neural tube and patterns cells within it. Depending on the concentration and duration of hedgehog exposure by neural progenitors determines their subsequent fate, this is mostly related to their position
What enzyme carries out the autoproteolytic cleavage of the N-terminus of the hedgehog protein
Hedgehog acetyltransferase in vertebrates and skinny hedgehog in Drosophila
Explain how smo inhibitors have been looked at as potential cancer treatments
GDC-0449 is an antagonist of the hedgehog signalling pathway that inhibits hedgehog signalling and continuous abhorrent proliferation in tumour cells
Explain how hh and wg interact
Hh directly upregulates wg. Wg then controls the expression of the transcription factor, engrailed (en) which in turn then regulates hh expression
What is significant about the three different vertebrate homologues of Ci
Gli1 cannot be cleave by slimb due to missing one phosphorylation site. This means that it isn’t recognised by the degradation machinery and hence is always a transcriptional activator. Gli2 and 3 however can be transcriptional activators or repressors
What is the normal action of ptc in the absence of signalling
Patched constitutively inhibits smoothened in the absence of the hedgehog ligand
Currently smo inhibitors fail after long-term use, why is this
The tumours pick up additional mutations that render the smo inhibitors ineffective due to tumour resistance
