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
Explain how caspase-3 cleaves substrates
- The active site in caspase-3 has a cysteine-histidine dyad, Cys163 and His 121.
- The cysteine is nucleophilic and attacks the peptide bond next to Asp in the substrate’s cleavage site.
What are the main Downstream actions and effects of caspases?
DNA Degradation and Nuclear Condensation
How do caspases cause Inactivation of DNA repair and replication enzymes?
•The effector caspase-3 inactivates DNA protein kinase, DNA replication complex C and PARP (Poly-ADP Ribose Polymerase).
The latter is a repair enzyme.
How do caspases cause DNA fragmentation (laddering)?
- Caspase-activated deoxyribonuclease (CAD) is normally held in an inactive complex with ICAD (inhibitor)
- Caspase-3 cleaves ICAD, resulting in CAD activation and internucleosomal strand breaks
How do caspases cause nuclear condensation?
- Nuclear lamina is made of polymers of proteins called lamins that underlie the nuclear membrane.
- Lamins are cleaved by caspase-6, causing the lamina to collapse and chromatin to condense
How do caspases indirectly cause changes in the cell membrane which induce phagocytosis?
- Caspase-3 degrades aminophospholipid translocase, resulting in phosphatidylserine (PS) externalisation (a marker of apoptosis).
- This changes membrane structure and allows blebbing (bubbles on the surface of the cell) to occur.
- The externally exposed PS is recognised by phagocytes and acts as an “eat-me” signal for macrophages, causing phagocytosis of the apoptotic cell.
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
Describe the Balance of Apoptosis vs Cell Survival
- Apoptosis is induced by external signals such as stress or ligands that bind to death receptors.
- It can be prevented by growth factors and other cell survival signals.
- Cell cycle and apoptosis functions are co-ordinately regulated through gene expression.
Which family of proteins controls apoptosis in response to stress stimuli?
- Group I members – e.g. bcl2, bclxl = anti-apoptotic, protect cell from death. Localised in membrane through a hydrophobic C terminal domain.
- Group II members – e.g. PUMA, Bax and Bak = pro-apoptotic activity. Present in cytosol. (See the diagram on the next slide).
- Group III- e.g. Bid, Bad share a common BH3 domain; they are additional regulatory factors leading to apoptosis.
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.
How does the Cellular Redox State Influence Apoptosis?
- Transcription factors for expression of apoptotic (especially p53 and AP-1) are redox sensitive, i.e. active under oxidizing conditions.
- Apoptosis signal-regulating kinase 1 (ASK1) is downstream of the death receptors.
- Death receptor signalling is redox dependent too: ASK-1 is blocked by reduced thioredoxin but not oxidized Trx
What is Thioredoxin (Trx)?
Thioredoxin (Trx) is a small protein containing thiol (-SH) groups that keeps proteins in a reduced state.
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.
Describe the consequences of the failure to induce apoptosis
- Failure to induce apoptosis in response to DNA and cell damage results in cancer.
- Failure to apoptose in the immune system leads to autoimmunity and adenopathy.
- CD95 (FasL) produced by T cells is an autocrine killing signal leading to retention of memory cells only.
Where is apoptosis derimental?
- AIDs involves increased T cell apoptosis as HIV tat sensitizes cells to CD95 mediated apoptosis.
- Experimental models of stroke show enhanced caspase activation.
- Ischemia causes enhanced expression of caspases and apoptosis is suggested to mediate slow cell death around ischemic lesion in stroke.
- In Alzheimer’s disease, beta amyloid and pre-senilins induce apoptosis in vitro. There is evidence of DNA strand breakage in PM AD brains.
- Adenopathy is swelling of the lymph nodes, which can occur in autoimmune lymphoproliferative syndrome (ALPS), when there is a defect in T-cell pro-apoptotic proteins leading to accumulation of auto-reactive T-cells in the lymph nodes.
