Apoptosis

Question 1

When is a cell dead?

Answer 1

1. Cell has lost integrity of the plasma membrane – defined by vital dyes in vitro
2. Cell including its nucleus has undergone complete fragmentation -> discrete bodies
3. Its corpse or fragments have been engulfed by an adjacent cell in vivo.

Question 2

Caspase independent pathway

Answer 2

1. AIF is located in intermembrane space of mitochondria – damage causes release of AIF –> cytoplasm
2. Translocate into nucleus -> binds to DNA and destroys it.
   Nerve cells usually use this method

Question 3

Intrinsic pathway steps

Answer 3

1. When DNA damage cannot be repaired this is detected by ATM, ds breaks and ATR
2. Serine/threonine kinases add phosphate to checkpoint kinase 1 and checkpoint kinase 2 to activate them.
3. P53 is activated and transactivates genes NOXA, PUMA, pro-apoptotic proteins BH3 only proteins.
4. Levels in cytoplasm are elevated
5. Bind and sequester anti apoptotic proteins BCL-2/BCL-XL
6. Promotes oligomerisation of pro-apoptotic proteins BAK and BAX
7. Aggregates and form a pore – allow release of cytochrome C from inner mitochondrial space
8. Cytochrome C binds to apoptotic protease activation factor (APAF-1) to form apoptosome.
9. Apoptotic protease = caspase. Apoptosome activate caspase 9 –> active caspase 9 complex
10. Organelles and DNA are targeted cell-death

Question 4

Activation of caspases in initiator pathway

Answer 4

1. The initiator pro-caspase 9 binds to centre of apoptosomes via CARD domain
2. Proteolytic cleavage occurs between the pro-domain and large subunit – additional cleavage between large and small subunits
3. Large and small subunits are produced, bind together to form an active caspase 9 – need 2 of each
4. Activate effector caspases via cleavage
5. Amplification of signal (+ feedback loop) recruit more caspases – enough force to dismantle the cell.

Question 5

Steps in initiating extrinsic pathways - Fas receptor

Answer 5

1. Fas ligand binds to receptor –> activation of FADD –> association with another protein that has a DED (death effector domain) = FADD associated death domain adaptor protein (FADD adaptor protein)
2. Dimerisation –> binding of pro-caspase 8 or pro-caspase 10
3. Cleavages –> large/small subunits –> active caspases

Question 6

DISC in extrinsic pathway steps

Answer 6

Pro caspases 8/10 mounted on FADD = DISC

1. Large and small subunits cleaved off and combine with other units –> (2 of each) active caspases
2. Large and small subunits of the same initiator caspase (no chimeras)
3. Proteolytic cleavage occurs between pro-domain and large subunit
4. Additional cleavage between large and small subunits
5. • feedback reaction and amplification of signal and activation of effector/execution caspases

Question 7

What is c-Flip

Answer 7

procaspase 8 analogue that is able to compete with initiator pro-caspases 8 with binding to FADD = cell doesn’t die

Question 8

The cross talk between extrinsic and intrinsic apoptosis

Answer 8

1. Activation of caspase 8 –> activation of intrinsic mitochondrial pathway –> cleaves BH3 only protein BID –> truncated BID (tBID)
2. tBID can permeabilise the mitochondrion –> MOMP

BID is a substrate of Caspase 8

Question 9

Poly (ADP ribose) polymerase (PARP)

Answer 9

Inactivated by caspase

Found in nucleus and detect and signals ssDNA breaks

Question 10

Apoptosis and cancer - IAPs, what does it do

Answer 10

XIAP - inhibit Caspase 3, 7 and 9

SMAC can bind XIAP through n-terminal IAP binding motif - prevent inhibition of Caspase –> remove apoptosis blockade