10: Apoptosis

Question 1

Difference between necrosis and apoptosis?

Answer 1

Apoptosis= PROGRAMMED cell death, controlled disassembly of cellular contents without disruption, NO inflammatory response

Necrosis = UNREGULATED cell death associated with trauma, cellular disruption and INFLAMMATORY response

Question 2

What is necrosis?

Answer 2

Plasma membrane becomes permeable
Cell swelling/rupture
Release of proteases -> autodigestion/dissolution of cell
Localised inflammation

Question 3

Mechanism of apoptosis?

Answer 3

Latent phase - Death pathways activated, but cell is mrophologically the same

Execution phase - cell shrinks, loss of plasma membrane asymmetry, phosphatidylserene appears in outer leaflet, chromatin/nuclear condensation, DNA fragmentation, fragmentation into membrane-enclosed apoptotic bodies

Question 4

What happnes in apoptosis?

Answer 4

Latent phase - Death pathways activated, but cell is mrophologically the same

Execution phase - Loss of microvilli/cell junctions, cell shrinks, loss of plasma membrane asymmetry, phosphatidylserene appears in outer leaflet, chromatin/nuclear condensation, DNA fragmentation, fragmentation into membrane-enclosed apoptotic bodies

Plasma membrane remains INTACT so NO INFLAMMATION

Question 5

What are caspases?

Answer 5

The ‘executioners’
Activated by proteolysis
Cascade of activation

Question 6

What are the types of caspases?

Answer 6

Initiator caspases - CARD + DED domains provide homotypic protein-protein interactions

Effector caspases

Question 7

Describe the synthesis of caspases

Answer 7

Synthesised as zymogens - PROCASPASES
Inactive domains are cleaved
Forms active hetero-tetramer (2 large, 2 small chains)

Question 8

Describe the caspase cascade

Answer 8

Initiator caspases trigger apoptosis by cleaving + activating other caspases

Effector caspases carry out apoptosis

Question 9

How do effector caspases carry out apoptosis?

Answer 9

1. Cleave and INACTIVATE proteins

2. Activate/release enzymes by direct cleavage or cleavage of inhibitory molecules.

Question 10

Mechanisms of caspase activation?

Answer 10

Death by design - Receptor mediated pathways (intrinsic)

Death by default - Mitochondrial (intrinsic) death pathway

Question 11

What receptors are involved in receptor mediated apoptosis

Answer 11

Death receptors

Trimerise, unlike TK receptors which dimerise

Question 12

What adaptor proteins are involved in receptor mediated apoptosis?

Answer 12

FADD = activation (1 DED + 1DD domain)
FLIP = inhibition (2 DED domains)

Question 13

How does signalling work?

Answer 13

Fas (receptor) is trimerised by Fas-L (on lymphocytes)
Recruitment of FADD by DD domain on Fas (intracellularly)
Recruitment of procaspase 8 by DED domain on FADD

Need at least 2 procaspases to form active tetramer

End result: forms DISC (Death inducing signalling complex)

Question 14

What inhibits death receptor activation?

Answer 14

FLIP competes with procaspases for binding to receptor tails/FADD
Interferes with trans-cleavage
CANT MAKE TETRAMER

Question 15

What is the intrinsic pathway of apoptosis?

Answer 15

Regulated by Mitochondria
Loss of mitochondrial potential
Release of cytochrome c
Release of other apoptosis factors
Formation of APOPTOSOME complex

Question 16

What can trigger the intrinsic pathway?

Answer 16

Cellular stresses

e.g. lack of/overstimulation of growth factors, DNA damage, ROS

Question 17

Describe the structure of the apoptosome

Answer 17

‘Wheel of death’
Composed of Apaf-1, cytochrome c, ATP, procaspase 9
Apaf-1 have a CARD domain and WD-40 domains
Cyt c binds to WD-40 domains
Each Apaf-1 can bind a procaspase 9

Question 18

How is receptor mediated + mitochondrial apoptosis linked?

Answer 18

Caspase 8 cleaves Bid which enhances release of mitochondrial proteins, thus activating the mitochondrial pathway

Question 19

List anti-apoptotic proteins

Answer 19

Bcl-2
Bcl-xL

These work in the mitochondria

Question 20

List pro-apoptotic proteins

Answer 20

Bid
Bad
Bax
Bak

These move from cytoplasm to mitochondria

Question 21

Which domain in the Bcl-2 protein family allows dimerisation? Why is this important?

Answer 21

BH3

Dimerisation means you can concentrate a signal, whether it is anti or pro-apoptotic

Question 22

What is the PI3-kinase signalling pathway

Answer 22

Promotes cell survival and proliferation
PI3 kinase is a LIPID kinase (not protein)
Activates protein kinase PKB/Akt which is ANTI-apoptotic

Question 23

How does PKB/Akt block apoptosis and induce cell survival?

Answer 23

Phosphorylates and inactivates Bad + caspase 9

Inactivates FOXO transcription factors (FOXOs promote expression of pro-apoptotic genes)

Question 24

Explain the mechanism by which apoptosis occurs via Bcl-2 family proteins and PKB/Akt in the cell

Answer 24

1. Cell survival: Bcl-2/Bcl-xL are bound to Bax/Bak via BH3 domains. Bad is phosphorylated and inactive by PKB/Akt
2. In the absence of GFs, Bad dephosphorylated and RELEASED from PKB/Akt
3. Bad displaces and frees Bax/Bak from Bcl-2/Bcl-xL
4. Bax/Bak forms tetramer, creating a large pore in the mitochondrial membrane
5. Cytochrome c released via the pore and activates apoptosis

Question 25

What does PTEN do?

Answer 25

counteracts PI3-K signalling
Reverses the phosphorylation of lipid
(TRIphosphate -> BIphosphate)

Question 26

What do Inhibitors of Apoptosis Proteins do?

Answer 26

Extrinsic pathway:
Bind to procaspases and prevent activation
Bind to caspases and inihibit activity

Question 27

What are the cytoprotectie/anti-apoptotic pathways?

Answer 27

Intrinsic pathway: Bcl-2/Bcl-xL
Extrinsic pathway: FLIPs, IAPs
Growth Factor pathways: PI3-K, PKB/Ak