Lecture 7: Apoptosis

Question 1

What are meant by external signals?

Answer 1

1. Activate via “death receptors”
2. Detect presence of EC death signals, in response, ignite the intrinsic apoptotic machinery
3. Death receptors activate apoptosis independent of p53
4. 30 members total:
   a. cysteine rich EC domains
   b. cytoplasmic death domain (DD)

Question 2

How do “death receptors” function?

Answer 2

1. FasL: binds 3 Fas molecules
2. Clustering of death domains
3. Adapter protein FADD (Mort1) binds via DD = DISC
4. FADD also contains DED, which binds to analogous domain in pro-caspase 8 (FLICE, MACH)
5. DED = CARD
6. Pro-caspase 8 drives own activation via self-cleavage
7. Caspase 8 activates down-stream effector caspases (eg caspase 3) committing cell to apoptosis

Question 3

What is meant by DD

Answer 3

death domain

Question 4

What is meant by FADD

Answer 4

Fas associated death domain

Question 5

What is meant by DISC

Answer 5

Death inducing signalling complex

Question 6

What is meant by DED

Answer 6

Death effector domain

Question 7

What is meant by CARD

Answer 7

Caspase recruitment domain

Question 8

What are some details about death receptors

Answer 8

1. membrane bound FasL = 39kDA
2. Proteolytically cleaved by MMPs
3. Soluble form (26kDa) sFasL
4. Both forms can self-associate and trimerise after it can bind with receptor Fas

Question 9

What are the inhibitors of Fas?

Answer 9

1. FAP-1 (Fas-associated phosphatase-1) inhibits Fas export to cell surface
2. SODD silencer of TNRF1 death domain. Prevents trimerization
3. FLIP inhibits FLICE (=caspase 8)

Question 10

What is the signalling cascade of death receptors?

Answer 10

1. TNF prod mainly by activated macrophages and T-cells in response to infection
2. TNFR1: activation of TFs, pro-inflammatory and immunomodulatory genes
3. Adapter protein TRADD (TNFR associated DD) binds via DD
4. FADD or RIP bind to TRADD via DD
5. FADD activation leads to apoptosis if other signals blocked = context dependent
6. TNFR1-TRADD-FADD-Caspase 8 = DISC

Question 11

What are other triggers of apoptosis

Answer 11

DNA damage
Metabolite deprivation
Physical damage
Heat shock
Hypoxia

Question 12

What are the two types of apoptosis via caspases

Answer 12

Caspase-dependent
Caspase-independent

Question 13

What is the apoptosome?

Question 14

What is the process of apoptosis via cytochrome c?

Answer 14

1. Pro-apoptotic signals triggers mitochondria to release cytochrome c and Smac/DIABLO
2. Cytochrome c and
   Apaf-1 hybridise (using dATP)
3. Procaspase 9 (initiator procaspase) binds and it enters the apoptosome
4. Executioner procaspases (3, 6, & 7) become caspases, cleaving death substrates (ICAD, Lamin, Vimentin, Actin).
5. This can be inhibited by IAPs (Smac/Diablo derivatives)

Question 15

What are caspases?

Answer 15

1. Cysteine aspartyl-specific proteases
2. absolute specificity for aspartic acid in the P-1 position [x/x/x/asp/y/]
3. cascade = disassembly of cell
4. synthesised as inactive pro-enzyme (zymogen), activated by apoptosis trigger (proteolytic cleavage), & subsequent formation of a dimer

Question 16

What proteins protect cells from apoptosis?

Answer 16

1. I^CAD: inhibitor of nuclease CAD responsible for DNA fragmentation
2. Poly-adenosyl ribose polymerase (DNA Repair)
3. Lamin (disintegration of nucleus and chromatin condensation)
4. Fodrin (critical for cell membrane structure)
5. Cell cycle regulatory proteins
6. Bcl-2 proteins

Question 17

How do viruses inhibit apoptosis?

Answer 17

IAPs (inhibitors of apoptosis)

1. highly regulated
2. can mark caspases for ubiquitination and degradation, OR bind directly and inhibit activity
   a. XIAP
   b. cIAP1
   c. cIAP2
   d. NAIP
   e. Survivin (caspase
   3,7,9)
3. IAPs: contain 1-3 BIR (baculoviral IAP repeat) domains can contain CARD, RING, and UBC domains
4. IAPs inhibited by:
   a. Smac/Diablo
   b. HtrA2/Omi

Question 18

What is the Bcl-2 family

Answer 18

All posses conserved motif called “Bcl-2 homology domains” (BH1-4)

Question 19

What are the two types of Bcl-2’s

Answer 19

1. anti-apoptosis (8)
   a. Bcl02
   b. Bcl-xL
   c. Bcl-w
2. Pro-apoptosis (18)
   a. Bax
   b. Bak
   c. Bok
   d. Bik
   e. Bad

Question 20

Where is Bcl-2 found and what is their role

Answer 20

Outer mitochondrial, ER, and nuclear membranes

Bid (cytosolic) cleaved by DR activated caspase 3

Role: proection from free radicals, cytochrome c, and Ca2+ release?

Question 21

What do Bcl family members do

Answer 21

Can heterodimerize: titrate functions. Ratio (relative concentrations) important: “Death-life rheostat”

Some anti-apoptotic members can directly inhibit caspases

Post translational modifications also important (serine phosphorylation)

Question 22

What is the role of Ca2+?

Answer 22

1. enters cell and mt upon trigger
2. Triggers:
   a. calcineurin -
   dephosphorylates Bax
   (activated)
   b. Catabolic enzymes -
   calpain (degrades
   fodrin), nuclear
   scaffold protease
   c. Transglutaminase - X
   links proteins,
   stabilizes apoptotic
   bodies
3. affects intracellular structures as it interacts with cytoskeletal elements
4. movement of phosphotidyl serine

Question 23

What is the role of endonuclease?

Answer 23

Formation of larger DNA fragments (300kb + 50kb) prior to oligonucleosomal fragments

Chromatin topology important as associated proteins are cleaved by caspases; DNA exposed to nuclease

Nucleases:
1. CAD
2. EndoG

Question 24

What is CAD?

Answer 24

“Caspase-activated deoxyribonuclease”

Present in inactive complex until caspase inactivation of inhibitor I^CAD

Question 25

How are apoptotic bodies disposed of?

Answer 25

Phagocytosis
1. RECOGNITION
a. Trigger causes
exteriorization of
phosphatidylserine
(PS) and receptor PSR
interact
2. SIGNALLING
a. PS > Cdc42-Rac, Gaz
Mer, ICAM-3-CD14?
3. Full clearance:
a. inflammation results if
defective
b. if incomplete - risk of
autoimmune and
cancer, also problem if
cell contain parasite
c. defective in lupus
erythmatosus