Apoptosis intro Flashcards
Why is programmed cell death important to the normal development ofan organism?
- In moulding structures and tissue of the body.
- It is also used in the immune system to prevent self-immunity, such as in the clonal deletion of autoreactive T-cells.
- It can also occur during cell stress, for example when DNA damage is too severe to repair.
- Important in clearing neutrophils that have ingested bacteria during immune reactions.
There are 2 main pathways for initiation of apoptosis.
What are they?
- Extinsic Pathway
- Intrinsic Pathway
Describe the extrinsic pathway
- A set of specialized death signals are received by death receptors on the cell surface.
- All the receptors have death domains
- Ligand binding induces the formation of a death-inducing signalling complex (DISC)
- Adaptor proteins (e.g. Fas-associated death domain-FADD) are recruited
- These recruit procaspase-8 and activate it by proteolytic cleavage.
The extrinsic pathway can involve cell-cell interactions or…
ligand-cell interactions
mTNF-alpha is a membrane embedded form of the cytokine
How do cytotoxic T clls induce apoptosis in target cells?
Draw a diagram to show which ligands on the T cells interact with the receptors on the target cell
Cytotoxic T cells use the cell-cell mechanism to induce apoptosis in target cells, such as infected or cancerous cells
TRUE or FALSE:
Several soluble ligands of death receptors exist
TRUE
mTNF-alpha is a membrane embedded form of which cytokine?
How can the intrinsic pathway be activated?
- The intrinsic pathway can be activated by many varied stresses and treatments (including oxidative stress)
- All signals seem to converge on the mitochondria and cause mitochondrial outer membrane permeabilization (MOMP)
All signals seem to converge on the mitochondria and cause mitochondrial outer membrane permeabilization (MOMP):
What are the 2 models for how thiss happens?
- Pore formation by pro-apoptotic proteins Bax and Bak
- Induction of the mitochondrial permeability transition (MPT) by formation of a permeability transition pore (PTP)
Explain the events in the Intrinsic Pathway
- MOMP allows release of mitochondrial proteins, e.g. cytochrome c (from the intermembrane space)
- Cytochrome c interacts with & polymerizes Apaf-1 (apoptotic protease-activating factor-1).
- Procaspase-9 joins the complex to form the apoptosome, and is activated.
- Caspase-9 catalyses the activation of the executioner caspase cascade by proteolytic cleavages.
- Other digestive proteins are also released, e.g. endonuclease G.
Draw a diagram to show the activation of apoptosis via the extrinsic pathway
Draw a diagram to show the activation of apoptosis via the intrinsic pathway
Caspases: Cysteine Aspartate Proteases
Describe their functions
- Caspases are the responsible for cleavage of key proteins seen at onset of apoptosis.
- Caspases mediate disassembly of cells via proteolysis, which is irreversible.
- Inhibition of caspases inhibits apoptosis.
- Post-translational activation correlates with onset of apoptosis
How are caspases synthesised?
- Caspases are synthesised as precursors capable of auto-regulation and activation, in order to prevent inappropriate damage to the cell.
- The reactions are highly specific, which helps to ensure selective protein degradation.
- Cleave after aspartate, XXXXD, where XXXX depends on enzyme (e.g. DEVD for caspase-3)
Describe Caspase structure & Activation
- Caspases are constitutively present
- Cleavage (between the pro-domain and the large domain) of the zymogen activates the enzyme
- The active enzyme can initiate a cascade
- Apoptosis can be initiated in 30 mins
- Effective regulation is essential otherwise unnecessary damage would occur
Show the staining of apoptotic cells
- Figure right. Nuclei stained blue, shows condensation in apoptotic cells (little blue blobs).
- Green stain is Annexin V against phosphatidylserine, showing PS externalization on the surface of the 2 middle cells (apoptotic).
What does apoptosis look like?
- Cyt c in mitochondria (labelled light green) disperses in the cell.
- Phosphatidylserine is externalized on the membrane surface (stained red).
- Cell blebs form on the cells surface.
- DNA (stained blue) is no longer just in the nucleus
Explain the initiation of apoptosis as a result of oxidative damage to DNA
- DNA damage switches on p53, which results in activation of pro-apoptotic proteins Bax and Puma.
- Bax inhibits the anti-apoptotic protein Bcl-2, which normally blocks Cyt c and Smac release from the mitochondria.
- Puma triggers Smac release from mitochondria, which in turn inhibit IAPs. IAPs are protective, as they inhibit caspase-3 and its downstream effects.
- p53 also directly activates the pro-apoptotic factor Apaf-1, which activates caspase-9.
Describe the mechanism of Fas Redox-Dependence
- The death receptor Fas, forms a complex with Apoptosis signal-regulating kinase 1 (ASK1), together with the redox-sensing and antioxidant protein thioredoxin (TRx).
- This has thiol groups (-SH, reduced form) but oxidative stress (hydrogen peroxide, H2O2) oxidizes them to disulfides, which releases Trx.
- This allows Ask-1 to be activated and signal via MAP kinases to phosphorylate and activate c-Jun N-terminal kinases (JNKs) which tend to cause apoptosis.
