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In the notch pathway, ___ and Jagged are ___.
Delta, ligands
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Notch is composed of ___.
- NECD - Notch extracellular domain
- transmembrane domain
- NID - Notch intracellular domain
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___ cleaves the NECD from the ___.
ADAM, TM-NICD
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What is ADAM?
A desintegrin and metalloprotease
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The NICD makes its way to the ___ and generally results in ___.
- nucleus
- transcription, cell division, differentiation
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Hh are ___, a ___.
- ligands
- family of secreted proteins
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Hh generally functions in ___.
development
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In adult cells Hh can lead to ___.
cancer
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Hh homologues in vertebrates include ___.
sonic, desert, Indian Hh
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What is the receptor for Hh?
Patch
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What are the receptors for Hh in mammals?
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Besides Patch and Hip, what else is needed for Hh pathway activation?
Smo
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The ultimate target of Hh in the fruitfly is ___, a ___.
- cubitus interruptus (Ci)
- transcription factor
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What vertebrate homologue most closely resembles Drosophila Hh?
Desert
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How do Shh, Dhh, and Ihh differ?
Typically by potency - Shh>Ihh>Dhh
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What role in develoment does Hh play?
cellular proliferation, growth, and axon path finding
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What are examples of human developmental disorders from Hh?
- Holoprosencephaly
- Greig cephalopolysyndactyly syndrome
- Pallister-Hall Syndrome
- Gorlin's syndrome
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What are some cancers triggered by malfunctioning Hh?
- Basal cell carcinoma
- Rhabomyosarcoma
- Medulloblastoma
- Small cell lung cncer
- Pancreatic cancer
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What are some components of the Hh pathway?
- Patch(Ptc) - membrane receptor
- Smoothened (Smo) - intermembrane protein
- Intracellular Hh Signaling complex (HSC)
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Describe Patch.
- Membrane receptor which activates Smo when bound to Hh.
- After binding, Ptc levels decrease as a result of endocytosis
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In vertebrates, Ptc does not have ___, so it needs ___.
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Describe Smoothened.
Intermembrane protein that when activated relays signals to HSC
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In vertebrates, Smo is always ___.
at the cell membrane
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Describe HSC.
- Coastal 2 (Co2) - kinesin-related protein
- Fused (Fu) - Ser/Threo kinase
- Supressor of fused (Su/Fu)
- Cubitus Interruptus (Ci)
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What are three Ci homologs as activators in mammals?
Gli1, Gli2, Gli3
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Since Gli acts only as an activator, it does not get ___.
truncated
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What happens to Ci when there is no Hh?
HSC truncates Ci which becomes a repressor
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What happens when Hh binds to Ptc?
Production of Ci which becomes an activator
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In the Drosophila wing imaginal disc, Ci is truncated where?
In all but cell fate 1 nearest the posterior
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In the Drosophila wing imaginal disc, Hh concentration results in activation where?
In all but cell fate 5 (nearest the Anterior) which results in repressor.
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In the Drosophila wing imaginal disc, how does Hh concentration affect expression?
[Hh] is proportional to activation
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What is involved in Hh processing?
- Autocleavage
- Binding of cholesterol to C end
- Addition of palmitate to amino terminus
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Describe the binding of Hh to cholesterol.
- Critical for target cell intake
- Critical for signal transduction after Hh binds to Ptc
- If binding is inhibited, Hh doesn't work
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In the absence of Hh, Ptc ___.
blocks the phosphorylation and stability of Smo.
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What type of receptor does Smoothened have?
G protein coupled receptor
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When there is no Ptc, ___.
Smo is found in endosomal vesicles
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Upon Hh binding to Ptc, ___.
Smo is released and localizes to the cell membrane
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Smo multimers may be required for ___.
high level signaling
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With Hh present, Ci__ is processed to ___ or ___.
- 155
- Weak activator - Ci^act
- Strong activator - Ci*
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If no Hh is present, Ci__ is formed.
75
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How is Ci phosphorylated?
- PKA
- Glycogen synthase Kinase 3-beta
- Casein Kinase 1-gamma
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Upon phosphorylation, Ci is ___ by ___, a ___.
- ubiquitinated
- Slimb (supernumerary limbs)
- proteosome for cleavage
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Describe Coastal 2 (Cos2)
- Possibly a microtubule-motor
- Interacts with Smo
- Responsible for moving Smo
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How do Cos2 and Smo interact?
- Cos2 binds Fu to Smo
- Their interaction is critical for hh pathway signalling
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Where does Cos2 move Smo?
- cell membrane upon Hh pathway activation.
- Intracellular vessicles in the absence of Hh ligand
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Describe Fused (Fu)
- Has kinase activity, i.e. might phosphorylate Cos2 and Su(Fu) upon Hh pathway activation.
- Binds to Cos2 and Su(Fu) via carboxy terminus domain
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Describe Suppressor of Fused
- May be antagonistic to Fu
- No a.a. homology to known proteins
- Binds Fu and Ci, but not Cos2
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What evidence is there that Su(Fu) and Fu might be antagonistic?
Su(Fu)- and Fu- flies yield a wt phenotype
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What are possible functions of Su(Fu)?
- May inhibit Ci activation
- Nuclear translocation of Ci
- Transcriptional regulation in vertebrates
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Describe Hh pathway at no/low [Hh].
- Ptc on cell membrane repressing/sequestering Smo at vesicle with HSC-A inactive.
- HSC-R on MT picking up vesicle leading to truncated Ci^75 by Su(Fu).
- Ci^75 to nucleus as repressor
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Describe Hh pathway at medium [Hh].
- Hh binds Ptc at cell membrane permitting Smo/HSC-A to go to cell membrane
- HSC-R not at MT
- HSC-A allows Ci^act into Nucleus for low activation with possible low amount of Ci^75.
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Describe Hh pathway at high [Hh].
- Hh sequesters Ptc to vesicle permitting Smo Multimer/HSC-A to go to cell membrane
- Fu phosphorylates Cos2/SuFu
- SuFu leaves HSC
- Cos2 drives Ci to Smo
- HSC-R not present/inactivated.
- HSC-A allows Ci* to be processed untruncated into Nucleus by SuFu for high activation
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Research shows that Hh signalling malfuction is responsible for:
- Formation of tumors
- Survival of tumors
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In mammals, absence of Hh leads to ___.
- Gli forms MT-attached complex with Fu and SuFu
- Gli remains in cytoplasm
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In mammals, if Hh is present, ___.
- Hh binds to Ptc
- Smo is activated (no longer supressed)
- Processing of Gli is activated
- Gli is translocated to nucleus
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In mammals, the negative feedback regulators in the Hh pathway are ___.
Ptch, Hip, Gli
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What genes are for cell proliferation?
- Cyclins D1 and D2 (mammalian) -> mitosis
- cMyc
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What are three target proteins?
- Cyclin B - Mitotic P Factor (MPF) - nuclear translocation
- P21 inhibition - tumor supression
- PDGF pathway activation (MAPK) - cell division
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What are two types of genetic problems w.r.t. cancer?
- LOF for tumor supression - Ptc and SuFu
- Overexpression of oncogenes - Shh and Smo
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LOF of SuFu leads to ___.
medulloblastoma
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LOF of Ptc1 leads to ___.
cell nevus carcinoma
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Heterozygous Ptc mice ___.
develop tumors
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Blocking Smo blocks ___.
binding of Hh
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What should be inhibited for cancer treatment?
Smo, Gli
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What are inhibitors of Smo?
- Cyclopamine - binds to Smo, but difficult to synthesize and toxic
- KAAD - modified cyclopamine - less toxic
-
How do you inhibit Gli?
- Forskolin - PKA activator - used in different pathways as well
- RNAi - Not feasible for treatment
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