10. Apoptosis Flashcards
What is necrosis?
- Unregulated cell death
- Associated with trauma, cellular disruption and an inflammatory response
- Cells burst or membrane permeability increases => loss of contents to environment
What is apoptosis?
- Regulated, controlled disassembly of cellular contents
- Without disruption - no inflammatory response
- Plasma membrane remains intact
What happens during necrosis?
- Trauma (mechanical, bacterial etc.)
- Plasma membrane becomes permeable
- Cell swelling and rupture
- Release of proteases leading to auto-digestion and dissolution of the cell
- Localised inflammation due to attraction of immune (phagocytic) cells
What are the 2 phases of apoptosis and what happens in them?
- Latent phase - death pathways are activated, but cells appear morphologically the same
- Execution phase - orderly activation of specific proteins and kinases
Outline what happens during apoptosis
- Loss of microvilli and intracellular junctions
- Dramatic cell shrinkage
- Loss of plasma asymmetry
- Chromatin and nuclear condensation
- DNA fragmentation
- Formation of membrane blebs
- Fragmentation into membrane-enclosed apoptotic bodies
What does DNA modification in apoptosis involve and how can this be seen in the lab?
- Fragmentation of DNA ladders - seen on agarose gel
* Formation of more ‘ends’, labelled by adding an extra fluorescently-tagged base in a tunel assay (brighter)
What does apoptosis-like PCD involve?
- Some, but not all, features of apoptosis
- Display of phagocytic recognition molecules, even before plasma membrane lysis
- Some inflammatory response
What does necrosis-like PCD involve?
- Variable features of apoptosis before cell lysis
* “Aborted apoptosis”
What are the 4 parts in the mechanism of apoptosis?
- Caspases (“executioners”)
- Initiating death programme - death receptors (extrinsic) + mitochondria (intrinsic)
- Bcl-2 family - regulators
- Stopping death programme
What do caspases have in their active site and what are they activated by?
- Cysteine residue in active site (required for activity)
* Activated by proteolysis - cut proteins just after their aspartate residue
How can caspases be divided into different classes - describe them?
Initiator and effector
Describe initiator caspases
• Initators are the first to be triggered
• Contain specific motifs:
- CARD - caspase recruitment domain
- DED - death effector domain
• Located at the end of their respective caspases to form homotypic protein-protein interactions (DED with DED)
Describe effector caspases
- Don’t contain specific motifs
* Just proteases
Describe the activation of caspases
- Synthesised as inactive pro-caspases (zymogens) - single chain polypeptides
- Have a pro-domain to maintain the inactivated stage
- Proteolysis - cleavage of the pro-domain => formation of the heterodimer
- Folding of 2 large (L) and 2 small (S) chains to form active L2S2 heterotetramer
What are main purposes of the caspase cascades?
- Amplification
- Divergent responses
- Regulation
Once apoptosis is triggered, what do the initiator caspases do to the effector caspases?
Initiator caspases cleave and activate the effector caspases, which then carry out the apoptotic programme
What are the 4 different consequences that can occur in the apoptotic programme (from the clipping of different proteins)?
Loss of function
• Inactivation - monomeric substrate cleaved by caspase
• Disassembly - multimolecular complex, cleaved at many different points e.g. nuclear lamins
Gain of function
• Activation - direct cleavage resulting in free active component, as it will unfold and change conformation
• Release - caspase cleaves a protein that is holding another protein making it inactive (inhibitory molecule) - results in break down of DNA
What do the following caspase mechanisms refer to:
• Death by design
• Death by default
- Design - receptor-mediated (extrinsic) pathways. Particular stimulus outside the cell that tells it to kill itself.
- Default - mitochondrial (intrinsic) pathway. Cell detects something off inside.
What do death receptors on all cells consist of?
- Extracellular cysteine-rich domain
- Single transcellular domain
- Cytoplasmic tail (with a death domain)
When are death receptors activated?
• When they encounter secreted or transmembrane trimeric ligands e.g. TNF-alpha or Fas, (death ligands)
What are the adaptor proteins in receptor-mediated apoptosis and what do they do?
- FADD - positive regulator, required for death pathway to be activated and promotes cell death
- FLIP - negative regulator, inhibits the death pathway and allows it to be regulated
What domains do FADD and FLIP have?
DED = death effector domain DD = death domain
- FADD = DED + DD
- FLIP = DED + DED
What happens to death receptors when activated (in cytotoxic T lymphocytes) and how does it lead to the execution of the death programme?
1) Fas ligand binds to Fas receptor on the cytotoxic T lymphocytes
2) Fas receptor trimerises
3) Receptor brings 3 cytoplasmic DD domains of the positive adapter protein, FADD, together
5) Recruitment of oligomerisation of pro-caspase 8 through its DED, to the FADD DED
6) This binding forms a death-inducing signalling complex (DISC)
7) Disc formation results in the cross-activation of pro-caspase 8 - (at least 3) pro-caspase 8s come into close contact and cleave each other
8) This releases the active initiator caspase 8 tetramer
9) Cleaves effector caspases to execute the death programme
What happens once caspase 8 is activated and has carried out its action?
- FLIP adaptor proteins inhibit the caspase system (contain DED domains on their N-terminus)
- No proteolytic activity
- Competes with pro-caspase 8 to bind to the DED domains of FADD
- Prevents trans-cleavage, blocking the formation of active caspase 8
When caspase 8 is activated, which effector caspases are activated downstream?
Caspase 3 and 7 - these carry out the apoptotic programme
Outline the activation of the intrinsic pathway to the formation of the apoptosome?
- Cellular stresses
- e.g. overstimulation by GFs, DNA damage, reactive oxygen species
- Causes a loss of mitochondrial membrane potential
- Release of cytochrome C and other apoptosis-inducing factors
- Stimulates the formation of an apoptosome complex - equivalent of DISC on death receptors
What does the apoptosome consist of and what does it do?
• APAF-1, cytochrome C, ATP, pro-caspase 9
• APAF-1 = ATPase
- WD-40 repeats at the C-terminus involved in protein-protein interactions
- caspase recruitment domain (CARD) at the other end
• When cytochrome C binds to the WD-40 repeats, it forms a heptamer
- i.e. monomer associates with 6 other units to make a wheel of 7, bound via the CARD domain
• Process requires ATP
- The CARD domains of the apoptosome can interact with the CARD domains on pro-caspase 9 (oligomerisation)
- Close proximity of pro-caspase 9s (that bind to the apoptosome) can cross-cleave and activate each other
- Pro-caspase 3 comes along, dimerises with caspase 9 and is activated by it
- Caspase 3 splits into 2 subunits (large and small) - and 2 of each join to produce an active caspase 3 (tetramer)
Can ATP levels make a difference between death via necrosis or apoptosis?
Yes, as apoptosis is an active process and necrosis doesn’t require energy
What does ‘Bid’ do?
- Links the receptor-mediated (extrinsic) and mitochondrial death (intrinsic) pathways
- Caspase 8 from the receptor-mediate pathway can cleave Bid
- This enhances the release of mitochondrial proteins
What is the difference between the receptor-mediated and mitochondrial death pathway?
The mitochondrial pathway requires ATP
What family of proteins does Bid belong to and what is it characterised by?
Bcl-2 family of apoptosis regulators (Group 3)
Describe the Bcl-2 family
- Characterised by domains in their protein sequence = Bcl-2 homology (BH) domains (1-4)
- Some of them have a transmembrane domain
- Bcl-2 is the found member - group 1
- Group 3 only contains BH3
- BH3 (Bcl-2 homology domain 3) is a dimerisation motif
- Pro-apoptotic - Bax, Bid, Bad, Bak (move between cytosol and mitochondria)
- Anti-apoptotic - Bcl-2, Bcl-xL (localised to mitochondrial membrane)
Describe how growth factors create anti-apoptotic effects?
- Ligand binding causes dimerisation and cross-phosphorylation of tyrosine kinase receptors
- This initiates signal transduction pathways
- Creates docks for adaptor proteins, which can mediated interactions e.g. activating Ras, activation of MAPK/ERK => growth
• Another phosphorylation site on the TK receptors triggers the PI3-kinase pathway
- site on cytoplasmic tail attracts and adaptor p85 (which is with p110, forming PI3 kinase)
• This (p110 part) phosphorylates a lipid (so must be close to the membrane)
• PIP2 => PIP3 phosphorylation [by PI3-K]
• Recognised by adapter subunit pf PKB/Akt (protein kinase B)
• PKB is recruited to the cell membrane and activated
• Results in mitogenic and anti-apoptotic signals
What are the 3 main subunits of PI3-K?
- Targeting subunit
- Adapter subunit
- Catalytic subunit
Why is the activation of PKB/Akt anti-apoptotic?
- Phosphorylates and inactivates Bad
- Phosphorylates and inactivates caspase 9
- Inactivates FOXO transcription factors (FOXOs promote expression of apoptosis-promoting genes)
- Other e.g. stimulates ribosome production and beneficial protein synthesis
How do the pro-apoptotic Bcl-2 family proteins play a part in the apoptosis mechanism?
- When GFs are absent, the PI3-kinase pathway is not activated
- PKB is not activated, so Bad is not phosphorylated/held in an inactive heterodimer with PKB
- Bad is free to go to the mitochondrial membrane and binds through its BH3 domain to the BH3 domains of the anti-apoptotic Bcl-2 family
- This displaces the pro-apoptotic members from inhibition by the anti-apoptotic members
- The pro-apoptotic members can form a pore in the mitochondrial membrane, allowing cytochrome C to escape into the cytosol
- Apoptosis is induced
Where do Bcl-2 and bax dimerise?
They are transmembrane domains that dimerise on the mitochondrial surface
What Bcl-2 members are phosphorylated (inactive) in the cytosol
Bad and Bax
What does PTEN (lipid phosphatase) do?
- Counteracts the production of PKB
* This reduces regulation of cell survival, therefore promotes apoptosis
What do IAPs do?
- Bind to pro-caspases and prevent activation
- Also bind to active caspases and inhibit activity
- This regulates programmed cell death
Which proteins regulate the extrinsic, and intrinsic pathway?
- Extrinsic - FLIP, IAPs
* Intrinsic - Bcl-2, Bcl-xL
Which proto-oncogenes/TSGs are associated with apoptosis?
- Bcl-2 (oncogene - over-expression promotes cancer)
- PKB/Akt (oncogene - over-expression promotes cancer)
- PTEN (tumour suppressor - inactivation promotes cancer)
