Autophagy

Question 1

Describe the autophage pathway

Answer 1

PI3K –> AKt –| TSC1/2 –| Rheb –> mTOR1 –| autophagy

Question 2

how many forms of mTOR are there

Answer 2

2

Question 3

When does autophagy occur

Answer 3

Starvation
Cellular Stresss
Developmental and structural Remodelling
Removed damaged cytoplasmic components

Question 4

What 2 things can activate AMPK

Answer 4

Metformin

Resveratenol (grape skins)

Question 5

What does ULK1 phosphorylate in the initiation of autophage

Answer 5

Components of the PI3KC3 complex

Question 6

What occurs in the nucleation stage of autophagy

Answer 6

vpo34 or PI3KC3 complex phosphorylates PI3P –> PI3P is alipid that trafficks proteins to the isolation membrane
Beclin-1 is a key scaffold protein of the isolation membrane

Question 7

How is LC3 alteredt

Answer 7

It has a lipid attached to it via atg3 and atg7 –> these lipids are eseential and knockout prevents the formations of autophagosomes.

LC311 is a crucial part of the autphagosome membrane

Question 8

How does mTOR inhibit autophage

Answer 8

inhibits phosphorylations in the initiation complex

Question 9

How does AMPK trigger autophagy

Answer 9

1) Phosphorylaiton of ULK1
2) Phosphoryation of Raptor ( A component of mTOR)
3) Phosphorylates TSC2 –> this causes inhibition of mTOR

Question 10

How is ULK1 normally activated

Answer 10

Starvation or nutrient deprivation –> mTOR moves away from ULK1 –> unmasks ULK1 kinase activity –> FIP200 phosphorylated and Atg proteins are recruited to the autophagosome site.

Question 11

What phosphorylations are inhibitor on ULK1

Answer 11

Phosphorylations by mTOR and on Atg13

Question 12

Roles of autophagy in cancer

Answer 12

1) Can be a cell survival mechanism
2) Method of caspase-independent cell death
3) Arsenic trioxide can cause a selective autophagosome degradtion of oncogenic fusion proteins e.g. bcr-abl and Pml-Rar

Question 13

What do we want to do autophagy in cancer

Answer 13

Some cancers are addicited to autophagy i.e. Ras driven cancer –> hence inhibitoin of autophagy can increase the sensitivity of tumour cells to cytotoxic drugs

Question 14

Name an autophagy inhibitor

Answer 14

Gossypol

Question 15

Did gossypol have good effects

Answer 15

1) Caused pro -survival autophagy in breast and cervical hela cancer cells
BUT cause autophagic death in glioma cells!

Question 16

What are the 3 criteria to prove a cell dies from autophage

Answer 16

1) Cell dies without apoptosis
2) iIncrease in autophagic flux
3 ) autophagy inhibited with autophagy inhibitors

Question 17

DISCUSS THE BH3 MIMETIC AND AUTOPHAGY DRUG USED IN OVARRIAN CANCER CELLS

Answer 17

Obatoclax (A BH3 mimetic) accumulated in lysosomes during treatment and alkalinised them and inhibited the end stage of autophagy hence stopped sucesful treatment

Question 18

Name inhibitors of mTOR

Answer 18

Rapamycin! / Everolimus

Question 19

what can phosphorylate Beclin-1

Answer 19

DAPK

Question 20

What p53 target can promote autophagosome formations

Answer 20

DRAM

Question 21

Name some autophagy inhibitors

Answer 21

1) Hydroxychloroquine –> Disrupts lysosomal functional2
2) Lucanthone –> traditionally used to treat Schistosomiasis –> also disrupts lysosomal function and inhibit autophage –> made breast cancer cells 10x more potent
3) Lys 05 –> a water soluble compound that can accumulate ready in the lysosome!

Question 22

What therapy has been shown to induce apoptosis and necrosis

Answer 22

Photodynamic therapy!

Question 23

What are the molecules called that can induce Lysosomal Membrane Permeabilisation

Answer 23

Lusosomotropic molecules e.g. Hydrochloroquine !!

Most of these molecules are weak bases that become trapped in the lysosome and increases its leakiness

Question 24

Why do lysosomotropic molecules have great potential

Answer 24

Because they dont rely on other pathways!

Question 25

What protein can inhibit LMP

Answer 25

HSP70 and this protien is overexpressed in many cancers!!