L11: apoptosis

Question 1

What are the targets of active caspases?

Answer 1

Targets of active caspases
- Cleavage occurs at aspartate residue in sequence
- Why is this important?
o Selective cleavage of downstream targets
o Effects the ‘execution’ phase of apoptosis in a controlled, albeit irreversible manner (why irreversible?)

Question 2

What are the caspase substrates and their function?

Answer 2

• Other cleavage-activated hydrolases (or inhibitors of hydrolases), e.g. calpain (selective protease), caspase-activated DNAse (helps breakdown chromatin)
• Activation of other enzymes that facilitate breakdown, e.g. activation of Rho-kinase (signals cell contraction –> blebs)
• Targets that initiate engulfment (last stage of apoptosis)

Question 3

What is the mechanism of engulfment during apoptosis?

Answer 3

Engulfment
- Caspase activates proteins that pump out chemoattractants to bring phagocytes in towards the apoptotic cell
- Also expose ‘eat-me’ signals, so that the phagocytes know to target the apoptotic blebs for degradation
- One famous ‘eat-me’ signal is phosphatidyl-serine, normally on the inside of the cell, but when cells are dying becomes flipped to the outside

Question 4

How does a cell know why and how to commit apoptosis?

Answer 4

• Apoptosis is mainly broken down into two pathways: extrinsic and intrinsic, both have similar downstream apparatus
• extrinsic pathways tend to be signals from outside the cell that act on cell surface receptor, causes clustering of caspases together
• the other pathway (intrinsic) is various stimuli that cause mitochondrion to become depolarised and pump out or allow passive release of the proteins, which forms complexes in the cytosol such as cytochrome C, which activates caspases

Question 5

What triggers apoptosis?

Answer 5

• Self-elimination of pre-malignant cells (tumour suppression)
  o Oncogenic stress, e.g. Ras or c-myc activation
  o Stress associated with rapid cell growth e.g. hypoxia, protein fold stress
  o DNA damage (precursor of mutation)
  o Replication stress in dividing cells
  o Anti-tumour adaptive immune response, e.g. ‘killer’ T-cells
  o Loss of pro-survival growth factor signals (tell cell it is in correct niche)
• External agents – drugs, radiation etc

Question 6

What are BH3 domain proteins?

Answer 6

• Involved in architecturally forming this pore in the mitochondria to trigger apoptosis
• two main constituents of the pore are Bax and Bak proteins
• anti-apoptotic Bcl-2 family proteins block the formation of a pore needed for apoptosis

Question 7

What is used to trigger apoptosis therapeutically?

Answer 7

• Targeting cancer cells directly:
  o DNA damaging agents (chemotherapy, radiotherapy)
  o Small molecule agents (p53 reactivators, IKK inhibitors)
  o Biologics, e.g. TRAIL ligand (death receptor agonist)
• Indirect
  o Reactivation of immune response (immunotherapy)
  o Starve cells of blood (anti-angiogenic therapy)
  o Targeting niche (growth-factor blocking)

Question 8

How can you detect apoptosis?

Answer 8

• Cellular morphology (e.g. blebbing)
• Nuclear morphology (breakdown of nucleus)
• Molecular analyses:
  o Flow cytometry (analyses molecular profile of every single cell, Annexin V stains PS in membrane)
  o Enzyme assays (determining caspases activation in cells)
  o Immunoblotting (antibodies against cleaved caspase substrates)
  o Immunostaining

Question 9

What is Bortezomib?

Answer 9

Case study – Bortezomib
- Multiple myeloma is a disease of B-cell (plasma cell) lineage
- These are antibody producing cells
- In multiple myeloma these proliferate out of control
- Currently incurable, but drugs can keep in remission

Proteasome is a molecular target of Bortezomib
- Binds and blocks proteasome from degrading substrates
- Bortezomib inhibits myeloma xenograft growth, either alone or together with other chemo drugs