Apoptosis

Why is apoptosis important?

• Homeostasis balances cell division and cell death. Problems with division → tumours; problems with death → organ failure.

• Apoptosis is a natural suicide process, also essential in development:

–    Tadpole tail is shed by apoptosis.

–    Webbed digits in early embryos are sculpted by apoptosis.

–    Many neurons die in the embryo to give the correct ratio of neurons to target cells.

What is the difference between apoptosis and necrosis?

Necrosis is caused by trauma or injury, ruptures the membrane and is associated with inflammation; apoptosis is regulated.

What are the steps of apoptosis?

• Specification: the cell to die is chosen.

• Killing: the death programme is triggered.

• Execution: the "get rid of the body" step (clearance).

What are the hallmarks of apoptosis?

• Chromatin condensation, nuclear fragmentation, membrane blebbing, cell fragmentation.

• DNA ladder: genomic DNA is digested in ~150 bp steps because nucleosome-wrapped DNA is protected (seen by gel electrophoresis).

• Phosphatidylserine (PS) is normally on the inner leaflet of the plasma membrane; in apoptosis polarity is lost and PS is spread equally between both leaflets (visualised with a dye).

Why was C. elegans used to study apoptosis?

• The worm can live without apoptosis, so it can be studied.

• It is transparent, so apoptosis can be watched live under the microscope.

• Invariant somatic lineage: always 1090 cells made, 131 die → 959 somatic cells, of which 302 are neurons.

How was the apoptotic pathway discovered in C. elegans?

Genetic screens found CED-3 and CED-4 regulating apoptosis. Switching genes off revealed the pathway:

• Inhibit CED-4 → no CED-3 → no apoptosis → excess neurons.

• Inhibit CED-9 → CED-4 overproduced → CED-3 overactivated → excess apoptosis → too few neurons. So CED-9 is anti-apoptotic.

• Inhibit EGL-1 → CED-9 active → blocks CED-4 → CED-3 not activated → no apoptosis → excess neurons. So EGL-1 inhibits CED-9.

Worm pathway: EGL-1 ⊣ CED-9 ⊣ CED-4 → CED-3 → apoptosis

What are caspases?

• Caspase = Cysteine-ASPartic acid protease; cleaves after an Asp residue; substrate specific. They are the executioners of apoptosis in humans.

• Made as inactive pro-caspases, activated by cleavage (one trigger: many caspases in the same area).

• Organised as a cascade: initiators at the top, effectors at the base. A few initiators activate many effectors, so rapid amplification makes apoptosis fast.

How is the apoptosome formed?

• Apoptotic peptidase activating factor 1 (APAF-1) is inactive (closed) before apoptosis.

• Cytochrome c is released from mitochondria.

• Cytochrome c + dATP make APAF-1 open and recruit several procaspase-9.

• This forms the apoptosome, which is very processive and activates procaspase-3 (an executioner).

What are the roles of BCL-2 family proteins?

• The family has three groups: anti-apoptotic (BCL-2, BCL-XL, MCL-1), pore-forming effectors (BAX, BAK) and BH3-only proteins (BAD, BID, BIM, PUMA, NOXA).

• BCL-2 governs mitochondrial outer membrane permeabilisation (MOMP): it stops cytochrome c release and so blocks apoptosome formation.

• BAX/BAK oligomerise in the outer membrane to cause MOMP (effectors, not inhibitors). BH3-only proteins are stress sensors: they neutralise BCL-2-type proteins or directly activate BAX/BAK. These essentially act against BCL-2.

How does the extrinsic pathway cause apoptosis?

• A killer lymphocyte with Fas ligand binds the Fas (death) receptor on the target cell.

• Adaptors and inactive procaspases are recruited; caspase-8 oligomerises and activates.

• These cleave executioner caspases, which then cleave structual proteins such as tubulin (cell structure).

How does the intrinsic pathway cause apoptosis?

• Triggered by cytochrome c release, which activates the apoptosome → more caspases → the cell is digested from the inside.

• Activators: UV, genotoxic stress, caspases. Release may be due to mitochondrial matrix swelling or through open channels.

• The two pathways are interlinked: the extrinsic pathway eventually leads to cytochrome c release and the apoptosome.

What is p53 (TP53)?

p53 is a TF and one of the most effective tumour suppressors: it promotes cell-cycle arrest, DNA repair and apoptosis.

How is p53 regulated?

• p53 forms a tetramer and is constitutively degraded: MDM2 (a ubiquitin ligase) ubiquitinates it and the proteasome destroys it.

• Oncogene activation inactivates MDM2 → p53 is no longer ubiquitinated → stabilised.

• DNA damage or chemotherapy → p53 is phosphorylated (by ATM, CHK1, ERK) and acetylated (by CBP or PCAF). It translocates to the nucleus and its transcriptional activity increases.

What are the outputs of p53?

• Cell-cycle arrest (blocks proliferation of pre-cancerous cells).

• DNA repair, e.g. by inducing GADD45.

• Apoptosis if damage is beyond repair: targets include FAS receptor, BAX, p53AIP, so p53 stimulates both extrinsic and intrinsic pathways, and blocks BCL-2 by a mechanism that is still unclear.

• Logic: damage → arrest → repaired = cycle resumes; not repaired = apoptosis. Escaping apoptosis can lead to a tumour.

How is p53 targeted in cancer?

Therapy: p53 is a prime target for anti-cancer drugs; the level matters: low p53 stops the cell cycle (suppresses tumour development), high p53 induces apoptosis (better for eliminating tumour cells).