Lecture 15 - Apoptosis

Question 1

Roles of apoptosis?

Answer 1

1. Embryogenesis.
2. Cytotoxic T cells.
3. Tissue homeostasis.
4. Outer lens of the eye.

Question 2

Necrosis?

Answer 2

Cell death induced by damage/injury. Inflammatory response.

Question 3

Key regulatory family of apoptosis?

Answer 3

Bcl-2 family of proteins. They can be pro- or anti-apoptotic. Activators, repressors and derepressors.

Question 4

What domain do Bcl-2 proteins possess? How many of these domains do they possess?

Answer 4

Bck-2 homology (BH) domain. Can have 1 to 4 of these domains.

1. If they have 4 domains = ANTI.
2. 1, 2, 3 of these domains = PRO.

Question 5

The balance of pro/anti- apoptotic proteins controls whether apoptosis occurs.

Answer 5

• Bim = activator of apoptosis. If bound to the anti-apoptotic protein Bcl-2 then it is inhibited.
• However, Bim can be swapped with Bad, a derepressor.
• Bim is now liberated and can activate apoptosis.
• Activates Bcl-2 proteins and Bax which are TM proteins of the mitochondria = they release CytC.

Question 6

CytC initiates a signalling cascade mediated by what?

Answer 6

Caspases.

Question 7

Three classes of caspases?

Answer 7

1. Activators/ initiators.
2. Executioners / effectors.
3. Inflammatory (not involved in apoptosis).

Question 8

How are caspases activated?

Answer 8

Activated by dimerisation or cleavage. Activators are activated through dimerisation, which then cleave the effectors, and this then activates the effectors. Effectors cleave cellular substrates.

Question 9

How does CytC recruit caspases and initiate apoptosis?

Answer 9

• CytC binds APAF = Apoptotic Protease Activating Factor-1.
• APAFs have CARDs = CAspase Recruitment Domains. They associate with each other to form the apoptosome.
• Apoptosome recruits procaspase-9. Dimerisation of procaspase-9.
• Apoptosome cleaves execuitioner caspases: caspase 3, 6, 7. They cleave substrates and begin apoptosis.

Question 10

So which one is the activator caspase?

Answer 10

Procaspase-9.

Question 11

Executioner/effector caspases?

Answer 11

Caspases 3, 6, 7.

Question 12

Key target of executioner caspases?

Answer 12

ICAD = Inhibitor of Caspase-Activated DNase. Cleaved and CAD is released which begins to fragment DNA.

Question 13

Other examples of executioner caspase targets?

Answer 13

Nucealr (e.g. lamins) and cytoskeletal (e.g. actin) proteins.

Question 14

How do TFs prevent the activation of apoptosis?

Answer 14

Binding of TFs can activate downstream signals that promote differentiation and survival, e.g. PI-3 kinase and Akt signalling.

1. Activating Akt causes Bad to be phosphorylated.
2. Therefore it cannot bind to the anti-apoptotic Bcl-2 protein. Bcl-2 protein binds the apoptotic activator Bim and prevents apoptosis.

This is intrinsic apoptosis.

Question 15

Intrinsic vs extrinsic apoptosis?

Answer 15

Intrinsic = normally involves the release of CytC. 
Extrinsic = signal from extracellular environment.

Question 16

What are death receptors?

Answer 16

Cell surface receptors that initiate apoptosis following ligand binding?

Question 17

Give two examples of death receptors and their respective ligands.

Answer 17

Fas and FasL; TNF-R1 and TNF-alpha.

Question 18

Mechanism of action?

Answer 18

1. FasL binds Fas death receptor.
2. Fas undergoes a conformational change that activates procaspase-8.
3. Procaspase-8 interacts with the adaptor protein FADD (Fas-Associated Death Domain). The DDs (death domains) of Fas and FADD interact.
4. FADD has a DED domain; binds procaspase-8 via its DED domain.
5. DED = Death Effector Domain.
6. Forms DISC = Death Inducing Signal Complex.
7. . Procaspase-8 cleaves the executioner capsases, activated.

This is extrinsic apoptosis.

Question 19

The extrinsic pathway can activate the intrinsic pathway. How?

Answer 19

Procaspase-8. Can cleave bid, a pro-apoptotic Bcl-2 family member. Generates tBid. tBid binds to the mitochondrial membrane and releases CytC = intrinsic pathway.

Question 20

Fas in influenza defence.

Answer 20

Infected cells will present virus peptides on MHC class I. Bind to cytotoxic T cells via a TcR. On the T cell there are FasL molecules, which bind to Fas on the target cell and induce apoptosis.

Question 21

How does HIV affect the expression of FasL in CD4 T cells?

Answer 21

All CD4+ cells express Fas. HIV-infected cells will also express a high level of FasL. Non-infected CD4 cells will bind to overexpressed FasL on the infected cell, inducing apoptosis of the infected T cell.