Apoptosis

Question 1

What is necrosis?

Answer 1

Unregulated cell death associated w/trauma, cellular disruption and an inflammatory response

Question 2

What is apoptosis?

Answer 2

Regulated cell death
Controlled disassembly of cellular contents without disruption
No inflammatory response

Question 3

What is the difference between necrosis and apoptosis?

Answer 3

Apoptosis vs. necrosis
Regulated - unregulated
No cellular disruption - disruption
No inflammatory response - inflammatory response

Question 4

What cellular mechanisms execute the apoptotic response?

Answer 4

1. Caspases - executioners
2. Initiating death programme
   a) death receptors (extrinsic)
   b) mitochondria (intrinsic)
3. Bcl-2 family - regulators
4. Stopping death programme

Question 5

How can the Bcl-2 family of proteins modulate apoptosis?

Answer 5

• Can be pro-apoptotic or anti-apoptotic
• Common feature = BH3 protein-protein interaction domains that mediate binding btwn different members of family
• Anti-apoptotic members (Bcl-2, Bcl-xL) bind to pro-apoptotic members (Bax, Bak) in mitochondrial membrane via their BH3 domains, holding them in an inactive state
• When this interaction is disrupted by a “BH3-only” family member (e.g. Bad), pro-apoptotic proteins assemble into a pore in mitochondrial membrane
• This allows cyt c release and triggering of apoptosome
• Bad is normally prevented from activating apoptosis by phosphorylation by protein kinase Akt/PKB which is itself under GF control via PI3’kinase
• Another pro-apoptotic family member Bid is cleaved and activated by active caspase8 and engages mitochondrial pathway when receptor-mediated pathway has been triggered

Question 6

What are caspases?

Answer 6

Cysteine-dependent aspartate-directed proteases

Initiator or effector

Question 7

How are caspases synthesised and activated?

Answer 7

Synthesised as essentially inactive precursors - procaspases
Become cleaved (proteolysis) by themselves or other caspases into light subunit + heavy subunit
Subunits tetramerise into L2S2 active tetramer
Initiator caspases activate effector caspases causing a cascade of activation

Question 8

How does the structure of initiator and effector caspases differ?

Answer 8

Initiator:
Contain N-terminal CARD (caspase recruitment domain) or DED (death effector domain) for homotypic protein-protein interactions

Effector:
No protein-protein interaction domains

Question 9

What does the cascade of activation set off by caspases allow?

Answer 9

Amplification
Divergent responses
Regulation

Question 10

How do effector caspases carry out the apoptotic programme?

Answer 10

Cleave and inactivate proteins or complexes (e.g. nuclear lamins –> nuclear breakdown)

Activate enzymes )e.g. protein kinases, nucleases) by direct cleavage, or by cleavage of inhibitory molecules

Question 11

What are the mechanisms of caspase activation?

Answer 11

1. Death by design = receptor-mediated (extrinsic) pathways

2. Death by default - mitochondrial (intrinsic) death pathway

Question 12

How do death receptors regulate apoptosis?

Answer 12

1. Death receptors (e.g. Fas) bind secreted or membrane-bound ligands (e.g. Fas-L) causing their trimerisation
2. Promotes binding on cytoplasmic side of CM of adaptor proteins (e.g. FADD) and initiator procaspase8 to form a Death-Inducing Signalling Complex (DISC)
3. Interactions mediated by protein-protein interaction domains: Death Domain (DD) or Death Effector Domain (DED)
4. Proximity of the procaspase8 molecules induces cross-cleavage and activation, followed by cleavage and activation of effector procaspase3 and triggering of apoptotic programme

Question 13

How do mitochondria regulate apoptosis?

Answer 13

1. Cellular stresses (e.g. lack of/overstimulation by GFs, DNA damage (via p53)) cause loss of mitochondrial membrane potential
2. This causes release of cytochrome c resulting in formation of heptameric apoptosome complex (Apaf-1, cyt c, initiator procaspase9 via the CARD domains on Apaf-1 and procaspase9)
3. Proximity of multiple procaspase9 induces cross-cleavage, production of active caspase9
4. Caspase9 activates effector caspases (e.g. 3).

Question 14

What are the phases of apoptosis?

Answer 14

Latent phase

Execution phase

Question 15

What happens during the latent phase of apoptosis?

Answer 15

Death pathways activated, but cells appear the same

Question 16

What happens during the execution phase of apoptosis?

Answer 16

Loss of microvilli and IC junctions
Cell shrinkage
Loss of plasma membrane symmetry
Chromatin and nuclear condensation
DNA fragmentation
Formation of membrane blebs
Fragmentation into membrane-enclosed apoptotic bodies
Plasma membrane remains intact - no inflammation

Question 17

Why do cells undergo apoptosis?

Answer 17

To get rid of:

1. Harmful cells (e.g. cells w/viral infection, DNA damage)
2. Developmentally defective cells (e.g. B cells expressing antibodies against self antigens)
3. Excess/unnecessary cells (embyronic development, e.g. brain to eliminate excess neurons; liver regeneration; sculpting of digits and organs)
4. Obsolete cells (e.g. mammary epithelium at end of lactation)
5. Exploitation - chemotherapeutic killing of cells

Question 18

Why do cells with high ATP levels tend to die via apoptosis and cells with low levels via necrosis?

Answer 18

Assembly of apoptosome requires ATP