Apoptosis II

Describe how cytochrome c activates caspase 9.

1. Bax/Bak oligomer forms pore in OMM, releasing cytochrome c and Smac/Diablo.

2. Cytochrome c binds Apaf1, which then leads to procaspase 9 cleavage and activation during the formation of the apoptosome. Activated caspase 9 = initiator caspase.

3. Initiator caspase cleaves and activates executioner/effector procaspases 3/6/7.

4. Active executioner/effector caspases cleave substrates.

What are caspases, and what are the two functional classes?

Capases are proteases that used conserved cysteine residues in their active site to cleave proteins after aspartate residues.

• Initiator caspases: Caspase-8, caspase-9

  • When active, can cleave effector procaspases to activate them

• Effector caspases: Caspase-3, caspase-6, caspase-7

How do IAPs (inhibitors of apoptotic proteins) negatively regulate/inhibit caspases?

1. Simply bind to procaspases to inhibit them – BIR domain on IAPs bind and inhibit procaspases

2. Could act as E3 ubiquitin ligases to target procaspases for proteasomal degradation – RING domain on IAPs interact with ubiquitin machinery

Thus, IAPs protect against unintended procaspase activation.

Remember, during intrinsic pathway activation, Smac/Diablo is released alongside cytochrome c, which inhibits IAPs so that apoptosis can occur (procaspases not subject to proteasomal degradation).

What are the 5 types of death receptors (DR) involved in the extrinsic pathway? What are the advantages to death receptors?

FAS, DR3, DR4, DR5, TNFR1

FAS receptor: Expressed in many cells (ie. activated T and B lymphocytes, macrophages, liver, spleen, lung, testis, brain, ovaries, intestines, heart).

• However, FasL (Fas ligand) expression is tightly regulated. Primarily induced in T/B lymphocytes, macrophages, and NK cells.

Advantage: Clear unneeded lymphocytes post-infection. Lymphocytes that are repeatedly stimulated by antigens upregulate both FAS and FasL to promote their own and each other’s demise (ie. autocrine and paracrine apoptosis). The same goes for cytotoxic T cells and NK cells.

Describe the steps of the general extrinsic/death receptor/”instructed to die” pathway.

1. FasL (from another cell) binds Fas.

2. Fas-FasL recruits FADD protein via homotypic interactions between death domains in both Fas and FADD.

3. DEDs in FADD recruit initiator procaspase 8 or 10, which activate downstream effector procaspases. FADD acts as a scaffold to bring two initiator procaspases in close enough proximity for their auto-activation – important, as it bypasses the need for cytochrome c to be released.

What is the DISC complex?

Death-inducing signaling complex: Composed of death receptor + FADD + initiator caspase → this entire complex allows activation of effector caspases, triggering the caspase cascade.

Given the intrinsic and extrinsic pathways. What are the potential strategies for restoring apoptosis in cancer cells?

1. Inhibiting pro-survival Bcl-2 proteins using BH3 only protein mimetics (ie. Venetoclax)

2. Inhibiting IAPs using Smac/Diablo mimetics (ie. IAP inhibitors)

3. Indirect way: Inhibit the PI3K pathway and hence Akt activity so that Bad (a BH3 only protein) can drive apoptosis.

4. Identify death receptor ligands for receptors preferentially expressed on tumor cells