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BMS 109 > Dev 2 > Flashcards

Dev 2 Flashcards

1
Q

Wingless and …. maintain each others expression.How?

A

Hedgehog.
Hh> Wg>Eng>Hh
Hedgehog directly controls wingless expression, but wingless controls Engrailed, which then regulates hedgehog

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2
Q

Wingless and Hedgehog are what genes?

A

Segment polarity

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3
Q

Loss of function of wnt/wingless?

A

Segment polarity genes, so disrupt segment characteristics.

Loss of naked cuticle, covered in denticles.

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4
Q

Is the wnt translated in the mature form?

A

No. It is transcribed with a signalling sequence which is cleaved off.

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5
Q

How is Wnt modified after post-translation?

A

Porcupine Acyl transferase modifies it by Palmitoylation, and the addition of a Palmitoleic acid on ser209 in wnt 3a.

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6
Q

……… is required for transport of Wnt to the plasma membrane. (and maybe release/presentation)

A

Wntless- 7pass transmembrane protein.

think without the PM would be Wntless

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7
Q

What are required for diffusion of Wnt away from the plasma membrane? Why?

A

Due to the hydrophobicity of the palmitolylation Additions the Wnt is water insoluble so for diffusion requires the formation into multimers or loaded into a lipoprotein.

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8
Q

What is Palmitolylation?

A

The covalent attatchment of fattyacids

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9
Q

What is HSPG?

A

Heparan sulfate Proteoglycan in the EXC matrix required for diffusion away.

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10
Q

Wnt binds to what receptor?

A

Frizzled 1-4, which brings together this and Arrow/LRP 5/6 (in humans)

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11
Q

Frizzled structure?

A

7pass transmembrane spanning protein, Wnt binds to the cysteine rich domain (CRD) of the N terminal extension.

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12
Q

LRP/arrow structure?

A

Single pass transmembrane spanning protein, stands for LDL receptor-related protein

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13
Q

What is an extracellular inhibitor of the Wnt pathway? How? (lecture one)

A

Dickkopf-1 (dickhead) Dkk1
Binds to LRP/arrow and Kremen promoting internalisation of LRP, so cannot fuse with Fz.
Used in Wnt loss of function experiments.

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14
Q

In the canonical wnt pathway, what happens if there is no Wnt to Beta-catenin?

A

Without Wnt B-catenin phosphorylated by Ck1alpha and GSK3beta in a distruction complex.
This is recognised by Slimb causing B-catenin to be ubiquitinated and therefore degraded by proteosmones.
This then leaves the destruction complex empty to degrade other B-catenins.

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15
Q

HSPG are not required in the drosophila, suggesting?

A

Most effects of Wnt signaling can be elicited by a membrane bound form of the protein. This suggests that juxtacrine signaling mediated via cell-cell contact is sufficient in many cases.

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16
Q

In the absence of beta-catenin, what happens to the wnt genes?

A

In the absense of B-catenin, Groucho stays bound to the TCF on the promoter and represses transcription.
Groucho recruits histole deacetylases making the DNA tightly packed and hard to access. (heterochromatin)

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17
Q

What happens to Beta-catenin levels when Wnt is present?

A

Arrow and Fz are brought together and dishevelled protein is phosphorylated, which may bind to axin in the destruction complex and inactivate it. GSK3 also phosphorylates arrow/LRP and axin is recruited to the receptor.
Slimb is then lost from the complex, so B-catenin is not ubiquitinated or degraded.
Therefore B-catenin accumulates, and is translocated to the nucleus, (some stay bound to the destruction complex so others cant be degraded.)

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18
Q

If Wnt is present and beta-catenin accumulates what happens to wnt genes?

A

the B-catenin displaces the repressor groucho from the TCF/LEF family binding protein on the promoter and the wnt genes are transcribed.
Histone acetylases and chromatin remodelling factors make the DNA accessible. (euchromatin)

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19
Q

How is B-catenin phosphorylated?

A

By Ck1 alpha first on the N-terminal , which primes for the three phosphorylations by GSK3 beta. The 4 sites are required for recognition by an E3 ubiqutin ligase complex (slimb)
The serine/threonine phosphates and surrounding amino acid sequence forms an optimal binding site for Slimb.

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20
Q

How does slimb interract with Beta-catenin?

A

Slimb has an F-box which binds to the scaffold proteins e.g. Skp1 of the SCF E3 ligase. The WD40 repeats of the F-box recognises the Phosphorylated sections of beta-catenin, and bind to these also.

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21
Q

Structure of the SCF e3 ligase complex? (and other info) Conufused.

A

The SCF complex consists of the ring finger protein Roc1 binding E2 ligase, a scaffold protein cul1 and skp1. The latter binds the F box of beta TRCP/Slimb. F-box proteins target phosphorylated substrates specifically, leading to addition of Ub to them, and subsequent processing by the proteasome. The WD40 repeats of beta-TrCP interacts with specific substrates, including beta-catenin/armadillo. It also targets Ci from the hedgehog pathway

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22
Q

How is the DNA adapted for translation of Wnt genes when Beta-catenin is present?

A

Histone acetyl transferases recruited and other chromatin remodelling factors such as BRG1.
Euchromatin.
In Drosophila Pygo and Lgs co-factors for transcription- mutations cause wingless phenotype.

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23
Q

What are the negative feedback regulators induced by wnt signalling?

A

Dickkopf-1 and 4, Axin-2
Certain wnts (wnt5a) can inhibit canonical
wnt signaling via ror2
(research more?)

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24
Q

What is the most common wnt pathway?

A

Canonical

others Planar cell polarity/convergent extension

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25
Q

Wnt and convergent extension movements?

A

Non-canonical wnt: in zebrafish wnt11 and wnt5 mutants show clear defects in convergent extension movements.

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26
Q

Wnt and axon guidance?

A

Repulsion by RYK receptor tyrosine kinase

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27
Q

3 examples of planar cell polarity Wnt signalling?

A

Hairs on the arm all facing the same way. Cells have polarity.
Mirror image hexagonal tesselation arund the D/v line of the ommatidium of the drosophila eye.
Convergent extension in the zebrafish, mutant in Vangl has problems. This is a planar cell polarity protein.

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28
Q

Examples of Canonical Wnt in Drosophila?

A

Segment polarity: Drosophila- without wg loss of naked cuticle- sets up segments and parasegments.
Wingbuds: Expressed at d/v boundary of laminal disc in drosophila, mutant= wingless

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29
Q

Examples of Canonical Wnt in vetebrates?

A

Anterior/posterior axis: E.g. zebrafish highest posterior giving different fates etc e.g. overexpression too anteriorly (LiCl experiment) gives loss of eye development.
Orgniser: early beta-catenin along with Nodal sets apart the Nieuwkoop centre, which signals to the organiser through Wnt II.

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30
Q

Wnt in c.elegans?

A

In C. elegans another important model system wnt signaling can be studied by analysing the migration of two neuroblasts: the QRd and QLd cells.
Wnt signaling in the QLd cell induces a homeobox gene mab5 which induces posterior migration. Thus in the absence of the signal both cell move anteriorly whereas ectopic activation of wnt leads to both cells migrating posteriorly.

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31
Q

Wnt in intestinal stem cells?

A

Wnt maintains the gut stem cell population int he villi Crypts which are used to constantly renew gut cells.
If downregulate Wnt signalling by increasng Dkk (or -/- TCF4)= loss of stem cells as differentiate .

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32
Q

Wnt signalling and cancer?

A

Loss of APC tumor supressor gene (truncating events that inactivate) e.g. causes Familial adenomatous polyposis.
(dominant but throughout life one somatic cell mutation + the germ cell =cancer)
APC negatively regulates (inhibits) B-catenin, which when levels increase too high results in overproliferation of the tissue from the stem cells.
This can cause multiple polyps in colon and rectum which can go on to form cancer..
90% of rectum cancer causes by gain of Wnt (loss APC). Also in other cancers e.g breast.

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33
Q

Axin loss of function?

A

Axin l.o.f.-hepatocellular carcinoma
loss of axin in the destruction complex= b-catenin accumulating= increased wnt signalling, so stem cell proliferation increases.

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34
Q

beta- catenin regulates stem cells how?

A

Maintains stem cells, so gain of wnt -= benign tumours etc.

35
Q

LRP5/ Frizzled mutation?

A

LRP5-point mutant (frizzled) G.O.F
insensitive to Dkk-> not internalised ever, so increase in wnt signalling -> increased bone density.
Loss of function: decreased density= osteoporosis.

36
Q

Axin 2 loss of function?

A

Axin2- loss= B-catenin increases, so wnt signalling increases.
severe tooth agenesis (oligodontia)
-multiple missing teeth,
also pre-disposition for colon cancer.

37
Q

2 broad functions of Hedgehog pathway?

A

Involved in patterning and development of organs, involved in the regulation of stem cells and the initiation of cancer

38
Q

What type of gene is hedgehog?

A

It is a segment polarity gene.

39
Q

Hedgehog knockout?

A

Loss of naked cuticle, so too many denticles, as it and wingless with engrailed are in a feedback loop.

40
Q

Vertebrate hedgehog homologues?

A

Hh -> sonic hedgehog, indian and desert

41
Q

How is the hedgehog post translational modifications?

A

Transcribed with the N-terminal signalling sequence in tact, which enables it to be secreted.
This is removed.
C-terminal is cleaved-autoproteolytic cleavage
Cholesterol is added to the new C terminus
Palmitoyl group is added to the N-terminus.

42
Q

What enzyme causes palmitoylation of Hedgehog? Drosophila? Vertebrae?

A

Skinny hedgehog Ski n drosophila

Hhat in vertebrates

43
Q

What is the impact of the hedgehog ligand being hydrophobic? How is this overcome?

A

It needs help inn order to diffuse away or would be stuck to the cell membrane.
Dispatched/Scube is required for release.
Multimers/lipoproteins can hide the hydrophobic regions and with help from HSPG can diffuse away a long distance.

44
Q

What is the receptor to hedgehog?

A

Ptc in drosophila, and in vertebrates Patched 1 and 2

45
Q

WHat makes the hedgehog ligand hydrophobic?

A

Cholesterol and palmitate modifications.

46
Q

Ptc is constituitively …… what? What happens if the ligand binds?

A

Inhibiting the Smo protein.

If hedgehog ligand binds to Ptc its internalised, causing an inhibition of the inhibition so activates smo.

47
Q

Hedgehog binding tranduction pathway?

A

Binds to ptc to inhibit it, which causing the disinhibition of Smo, so Smo is activated. Smo binds to the membrane and accumulates .This inhibit the proteolytic cleavage of the Ci protein, which enables it to translocate into the nucleus and bind to the hedgehog genes to activate.

48
Q

How does Ptc inhibit Smo?

A

It is thought that Patched is involved in the translocation of Smo to a compartment where it gets degraded. When ptch isnt present three events can happen to smo: Relocation, accumulation and phosphorylation.
Evidence suggests that Ptc could also be used as a translocator (e.g. C elegans, cholesterol in Niemann Pick) so could pump Smo inhibiting small molecules such as Cyclopamine.

49
Q

In vertebrates where are ptc1 and Smo found?

A

On cilla, Ptc accumulates if no smo and vice versa.

50
Q

Evidence that hedgehog signalling takes place on cillia?

A

Mouse with a mutation that disrupts the formation of cillia causes the hedgehog singalling to be impaired.

51
Q

Ptc has a homology to what?

A

A prokaryote RND permease which confers drug resistance.

52
Q

Ptc is also related to what membrane protein disease?

A

Niemann Pick Protein C1- involved in transporting cholesterol vesicles along microtubules and across the plasma membrane. Implicated in people with Nieumann Pick disease, which causes accumulation of lipids in organs.

53
Q

Ptc in C.elegans?

A

No hedgehog signalling but Ptc is used for cell membrane remodelling

54
Q

Smo is inhibited by..?

A

Cyclopamine

55
Q

What is the effect of hedgehog interracting protein?

A

In vertebrates. Acts to most likely mop up excess hedgehog, so keeping signals down.

56
Q

CDO/BDO or vertebrate Gas1 function?

A

promote the binding of Hh to Ptc.

57
Q

Structure of Smoothened?

A

Smoothened is a 7 pass transmembrane protein

58
Q

Current model of Hh impact on Ci?

A

No Hh, ci bound in a complex,

59
Q

Ci or Gli?

A

Ci is the drosophila, and Gli the vertebrate (vertebrates more complex so longer name)

60
Q

If no hedgehog what happens to Ci?

A

Complex of costal2 (acts as a scaffold protein) and fused (serine threonine kinase) bound to Smo.
Ci is bound to SuFu- Supressor of Fused.
GSK3 B, PKA and CK1 in another complex that are recruited by the complex on Smo.
This is recognised by Slimb, which partially breaks Ci down into CiR, the active repressor of the gene.
(Cig- when the hedgehogs gf gets involved who worked at GSK for 3yrs, has a Pink KA and CK1 bra then his cig habit is repressed by the want to be slim)

61
Q

Low Hedgehog impact on Ci?

A

Smo partially activated, CK1, GSK3, PKA complex dissociates. Ci is not broken down into the repressor, but also no activation.

62
Q

High Hedgehog impact on Ci?

A

Smo activated, fused phosphorylates SuFu (suppressor of fused) its thought so Ci is released from the complex at the full length- CiAct.

63
Q

How is Ci made into an active form?

A

PKA phosphorylates Ci first, priming phosphorylation by GSK3 and CK1.
Mutation of any of these sites reduces processing to cleaved Ci, and reduces Slimb binding

64
Q

What is slimb’s structure?

A

Slimb is an F box protein, a component of the SCF ubiquitin E3 ligase complex

65
Q

When Ci is cleaved what length to? from?

A

Ci-155 to Ci-75

66
Q

Vertebrate homologues of Ci? Difference between them?

A

Gli 1,2,3
Gli1 does not get cleaved to a shorter repressor form and acts only as a transcriptional activator, whereas Gli 2 and 3 can work as both activators and repressors.

67
Q

How does Slimb repress Ci?

A

Slimb binds to non-optimal binding sites on Ci and partially proteolyzes it, leaving only the N terminus in tact- ciR

68
Q

How does Shh negatively regulate its own concentration?

A

Induction of Patched1 in vertebrates by hedgehog will:
-Act to supress the level of signal inside the particular cell
-The increased amount of Patched1 is used to bind extracellular sonic hedgehog and restrict diffusion.
-Stimulators of the signal (CDO/BOC and GAS1) are downregulated by Shh
This steepens the gradient

69
Q

2 Examples of Non-Canonical Hh/Shh signallign?

A
  1. Smo activates RhoA GTPase in endothelial cells.
  2. Smoothened can boost Ca2+ and activation of AMPK in myocytes and adipocytes. This causes an increase in aerobic respiration in mitochondria, incomplete metabolism of glucose , producing lactose. Can be seen in the cell by the acidification of the ECM. (warburg effect)
70
Q

Warburg effect and Hedgehog signalling?

A

Aerobic respiration is often a feature of tumour cells, known as the Warburg effect. hedgehog non-canonical signalling can cause Smo to increase Ca and activate AMPK, which causes a shift towards aerobic respiration.

71
Q

Why could patients taking Shh inhibitors lose weight and have muscle cramps?

A

Some inhibitors of the Canonical pathway of Hh are activators of the non-canonical. E.g. Smo causing activation of AMPK, causing increased glucose uptake into muscle cells and fatty acid oxidation. Also increased Ca, increasing aerobic respiration in mitochondia of muscle cells, thus using more energy up.

72
Q

How do Hh and Wg regulate each other?

A

Hh -> Wg-> Eng->Hh

73
Q

Hh in the wing patterning?

A

In the Drosophila Hh is expressed in the posterior and diffuses into the anterior. This induces expression of decapentaplegic (Bmp, tgf like gene), patterning the wing.

74
Q

Shh in the neural tube?

A

Concentration of Shh seen determines the neuron cell, highest in the notocord and floorplate. Dorsal/ventral patterning.

75
Q

Shh in the limb bud?

A

Shh in the zone of polarising activity ZPA. conferring a posterior identity. Leads to certain HOX genes activation.
E.g. if transplant ectopically on other side get bilateral symmetry of the fingers in chick.

76
Q

Hh and disease?

A

Holoprosencephaly- loss of Hh-forebrain fails to develop in two hemispheres. (LOOK UP NOTES LAST TERM)
e.g. lambs that eat cyclopamine (inhibitor) can cause cyclopedia.
Polydactyl (increased Shh in ZPA) and syndactyly (webbed feet)

77
Q

Shh in developement summary?

A

Limb bud-ZPA posterior
Neural tube- neurons- highest NC and floorplate
wing patterning- posterior diffuses to A- patterning
brain development- splits procencephalon (forebrain)
Lung- branching.
etc

78
Q

Gain of Hh?

A

Cancer.
E.g. Basal cell carcinonoma. (most common cancer-skin)
Stem cell proliferation.
Gain in Smo- Proto-oncogene, or loss in Ptc tumour supressor

79
Q

Inactivation of Patched1 or SuFu? disease?

A

rhabdomyosarcoma- cancer of soft tissues, often muscles.
Loss in Ptc- tumour supressor
Medulloblastoma (ptc loss)

80
Q

inherited Hh mutation disease?

A

Nevoid basal-cell carcinoma syndrome/ Gorlin syndrome, is an inherited medical condition involving defects within multiple body systems such as the skin, nervous system, eyes, endocrine system, and bones. only 10% of people with don’t develop BCC. Mutation in Patched gene (loss) and then a spontaneous somatic cell mutation will cause -/-.

81
Q

How can Hh cause cancer without causing mutations in the pathway proteins?

A

Hh can act as a growth factor e.g. Small cell lung carcinoma, Pancreatic, prostate.
Supports tumour growth.

82
Q

Smo inhibitors use?

A

Cancer treatment?
e.g. GDC-0449. Initially a very good result, to reduce cancerous tumours on the spinal cord, however they aquired a resistance by mutating Smo and grew back.

83
Q

Similarities of Hh and Wnt?

A
  • 2 hydrophobic modifications to each.
  • Depend on HSPG and TM proteins for diffusion. And multimers/lipoproteins.
  • Frizzled and Smo both 7TM proteins
  • Scaffold proteins: Axin, Costal2
  • Both proteolyzed: Ci partially proteolyzed, B-catenin fully degraded.
  • Both have a repressor in absence of signal; CiR or Groucho.
  • Both receptors act via a kinase to cause gene expression.

BMS 109 (20 decks)

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