Lecture 7: Apoptosis Flashcards

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1
Q

What are meant by external signals?

A
  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)
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2
Q

How do “death receptors” function?

A
  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
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3
Q

What is meant by DD

A

death domain

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4
Q

What is meant by FADD

A

Fas associated death domain

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5
Q

What is meant by DISC

A

Death inducing signalling complex

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6
Q

What is meant by DED

A

Death effector domain

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7
Q

What is meant by CARD

A

Caspase recruitment domain

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8
Q

What are some details about death receptors

A
  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
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9
Q

What are the inhibitors of Fas?

A
  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)
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10
Q

What is the signalling cascade of death receptors?

A
  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
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11
Q

What are other triggers of apoptosis

A

DNA damage
Metabolite deprivation
Physical damage
Heat shock
Hypoxia

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12
Q

What are the two types of apoptosis via caspases

A

Caspase-dependent
Caspase-independent

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13
Q

What is the apoptosome?

A
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14
Q

What is the process of apoptosis via cytochrome c?

A
  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)
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15
Q

What are caspases?

A
  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
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16
Q

What proteins protect cells from apoptosis?

A
  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
17
Q

How do viruses inhibit apoptosis?

A

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
18
Q

What is the Bcl-2 family

A

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

19
Q

What are the two types of Bcl-2’s

A
  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
20
Q

Where is Bcl-2 found and what is their role

A

Outer mitochondrial, ER, and nuclear membranes

Bid (cytosolic) cleaved by DR activated caspase 3

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

21
Q

What do Bcl family members do

A

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)

22
Q

What is the role of Ca2+?

A
  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
23
Q

What is the role of endonuclease?

A

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

24
Q

What is CAD?

A

“Caspase-activated deoxyribonuclease”

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

25
Q

How are apoptotic bodies disposed of?

A

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