Cell Death Lecture Sep 24

Question 1

If the DNA damage is too severe for the cell to handle, p53 will turn on expression of a gene other than the Ckis. What is it and what does it do?

Answer 1

p53 will turn on expression of Bax.

Bax is the trigger for cell death

Question 2

What are the three main forms of cell death?

Answer 2

apoptosis (orderly, regulate, no cell lysis)

necrosis (includes cell lysis and the associated immune response)

Autophagy (development of autophagosomes within the cell).

Question 3

Why can autophagy be considered more of a cell survival response instead of a cell death response?

Answer 3

The autophagosomes develop in an attempt to destroy the damaged part of the cell so that new parts can be made.

Cell death only occurs if this attempt fails and the autophagosomes increase in number and degrade the cell.

Question 4

What are the two pathways of apoptosis?

Answer 4

the extrinsic pathway involving the TNF receptors along the plasma membrane

and the intrinsic pathway involving the mitochondrial release of Cyt C

Question 5

What are the steps of the extrinsic pathway of apoptosis?

Answer 5

1. the TNF receptors bind their ligands (TNF, Fas-L, TRAIL, etc.)
2. THe bound receptors will timerize
3. The trimerization allows the receptors to act as a scaffold for the binding of adapter proteins along the death domain.
4. The adapter proteins will bind procaspase 8, which is an initiator caspase.

Note: the combo fo the receptor, adapters, and procaspase 8 make up the DISC.

5. The procaspase has proteolytic capabilities which are always on to some extent. So when the procaspases are bound, one of them will eventually break down the end ot its neighbor, thus activating it. The activated neighbor will then do the same fo all the other caspases and so forth, activating them all.
6. The activated caspase 8 of the DISC will then activate the proteolytic capabilities of the effector caspases (executional caspases) C3, C6, C7.
7. These have a number of functions that will all result in the destruction of the genome and the inability of the cell to replicate its DNA.

Question 6

In the extrinsic apoptosis pathway, which caspases are the initiator caspases?

Which are the effector caspases?

Answer 6

caspase 8 is the initiator

C3, 6, and 7 are the effector caspases

Question 7

What are the three things the effector cascpases will do in the cell?

Answer 7

1. They will destroy the enzymes used for DNA repair, like PARP
2. They will activate nucleases to chew up the DNA further and prevent it from being repaired
3. They will phosphorylate lamins to promote the condensation of the chromatids and degradation of the nuclear lamin

Question 8

Describe the steps of the intrinsic pathway of apoptosis.

Answer 8

1. DNA damage and/or inapprorpriately increased C-myc will activate p53.
2. p53 will increase the expression of Bax
3. Within the mitochondrial membrane, Bax will bind to itself to form a pore.
4. A number of things will be able to leave the mitochondrial matrix trhoguh this pore, the most important of which is Cyt C.
5. Cyt C in the cytosol will bind to APAF-1.
6. The Cyt c/APAF-1 complex will pull in procascpase 9. Binding of C9 results in the formaiton of the flower-like apoptosome.
7. THe proteolytic capabilities of the caspase 9 on the apoptosome will activate the effector caspases C3, C6, and C7, which will then carry out their roles as they did in the extrinsic pathway

Question 9

What other mitochondrial membrane protein will inhibit the Bax pore formation?

Answer 9

Bcl2

Bcl2 is in the same protein family as Bax, and can therefore bind to Bax and inhibit pore formation.

So it’s really the balance of Bcl2 and Bax within the membrane that determines whether a cell will undergo apoptosis through this pathway or not:

Relatively high Bax = pore formation

Relatively hihg Bcl2 = no pore formation

Question 10

What will mutations in Bcl2 result in?

Answer 10

Mutated Bcl2 will be hyperacive, therefore, Bax will almost never be able to form a pore and apoptosis will not occur. This results in cancer

Question 11

What two things will stimulate Bax expression?

Answer 11

Bax expression is turned on by p53

p53 is activated by DNA damage and by inappropriately high C-myc levels

Question 12

What molecule links the extrinsic and the intrinsic pathways of apoptosis together?

Answer 12

There is a third protein from the Bax Bcl2 family which is called Bid

Bid is a target for caspase 8 in the extrinsic pathway. Caspase 8 will cleave it to form tBid.

tBid cooperates with Bax to promote pore formation and apoptosis

This is known as the mitochondrial amplification loop

Question 13

What are the three proteins that inhibit Bcl2 and thus indirectly promote Bax pore formation?

Answer 13

BAD

NOXA

PUMA

Question 14

How does the binding of survival factors work to oppose apoptosis?

Answer 14

Binding of survival factors to their receptors will result in two things:

1. increase transcription of the Bcl2 gene, so you get more Bcl2 to inhibit Bax pore formation
2. activation of PI3Ks, which will phosphorylate and activate AKT. AKT inhibits NOXA, PUMA, and BAD so that they are not there to inhibit Bcl2.

Question 15

What are two examples of survival factors?

Answer 15

insulin or growth factor

Question 16

What are the three main types of autophagy?

Answer 16

microautophagy, macroautophagy, and chaperone-mediated autophagy

Question 17

In what brand of autophagy are atuophagosomes formed?

Answer 17

macroautophagy

Question 18

What two main proteins regulate autophagy (macroautophagy)?

Answer 18

mTOR (inhibits autophagy)

Beclin-1 (form scaffold on which proteins assemble to make the autophagosomes)

Question 19

Describe the mTOR pathway in autophagy?

Answer 19

1. Nutrient and growth factor availability activate the PI3K/Akt/mTOR pathway
   - class 1 PI3K kinases are active
   - maintain activation of AKT
   - AKT phosphprylates mTOR
2. This promotes cell survival and growth, inhibiting cell death and autophagy
3. However…..if energy is low, AMPK activity increases
4. AMPK inhibits AKT, so mTOR does not become activated, and autophagy is allowed to occur

(ER stress can also directly inhibit mTOR)

Question 20

What pathway activates mTOR?

Answer 20

appropriate nutrients>

PI3K active>

AKT active

> phsophorylates and acti ates mTOR

Inhibits apopotsis

Question 21

Describe Beclin-1 signalling in autophagy

Answer 21

1. Under healthy conditions, Beclin 1 is inhibited by Bcl2
2. WHen AMP is high (low energy), the JNK pathway is activated
3. JNK will inhibit Bcl2
4. the inhibition of Bcl2 means that Beclin can do it’s thing
5. Beclin is activated by class 3 PI3K
6. Beclin will promote the formation of the autophagosome

Question 22

What two branches of regulation lead to the formation of autophagosomes?

Answer 22

1. The JNK pathway in response to low energy (high AMP). This activates Beclin 1

THe AMPK pathway in response to low energy and DNA damage. This inhibits mTOR

Question 23

How does the JNK patway activated Beclin1?

Answer 23

Becline-1 is held in the inactive form by Bcl2 (no surprises there).

JNK will phosphorylate Bcl2, to result in the dissociation of Bcl2 from Beclin-1.

Question 24

How does an autophagosome form?

Answer 24

1. A double membrane structure (derived from the ER) engults the old parts of the cell it wants to get rid of
2. The autophagosome docks with a lysosome.
3. The contents are broken down using lysosomal hydrolytic enzymes

Beclin-1 and PI3K class 3, and ATG proteins are instrumental in this process