10: Apoptosis Flashcards
Difference between necrosis and apoptosis?
Apoptosis= PROGRAMMED cell death, controlled disassembly of cellular contents without disruption, NO inflammatory response
Necrosis = UNREGULATED cell death associated with trauma, cellular disruption and INFLAMMATORY response
What is necrosis?
Plasma membrane becomes permeable
Cell swelling/rupture
Release of proteases -> autodigestion/dissolution of cell
Localised inflammation
Mechanism of apoptosis?
Latent phase - Death pathways activated, but cell is mrophologically the same
Execution phase - cell shrinks, loss of plasma membrane asymmetry, phosphatidylserene appears in outer leaflet, chromatin/nuclear condensation, DNA fragmentation, fragmentation into membrane-enclosed apoptotic bodies
What happnes in apoptosis?
Latent phase - Death pathways activated, but cell is mrophologically the same
Execution phase - Loss of microvilli/cell junctions, cell shrinks, loss of plasma membrane asymmetry, phosphatidylserene appears in outer leaflet, chromatin/nuclear condensation, DNA fragmentation, fragmentation into membrane-enclosed apoptotic bodies
Plasma membrane remains INTACT so NO INFLAMMATION
What are caspases?
The ‘executioners’
Activated by proteolysis
Cascade of activation
What are the types of caspases?
Initiator caspases - CARD + DED domains provide homotypic protein-protein interactions
Effector caspases
Describe the synthesis of caspases
Synthesised as zymogens - PROCASPASES
Inactive domains are cleaved
Forms active hetero-tetramer (2 large, 2 small chains)
Describe the caspase cascade
Initiator caspases trigger apoptosis by cleaving + activating other caspases
Effector caspases carry out apoptosis
How do effector caspases carry out apoptosis?
- Cleave and INACTIVATE proteins
2. Activate/release enzymes by direct cleavage or cleavage of inhibitory molecules.
Mechanisms of caspase activation?
Death by design - Receptor mediated pathways (intrinsic)
Death by default - Mitochondrial (intrinsic) death pathway
What receptors are involved in receptor mediated apoptosis
Death receptors
Trimerise, unlike TK receptors which dimerise
What adaptor proteins are involved in receptor mediated apoptosis?
FADD = activation (1 DED + 1DD domain) FLIP = inhibition (2 DED domains)
How does signalling work?
Fas (receptor) is trimerised by Fas-L (on lymphocytes)
Recruitment of FADD by DD domain on Fas (intracellularly)
Recruitment of procaspase 8 by DED domain on FADD
Need at least 2 procaspases to form active tetramer
End result: forms DISC (Death inducing signalling complex)
What inhibits death receptor activation?
FLIP competes with procaspases for binding to receptor tails/FADD
Interferes with trans-cleavage
CANT MAKE TETRAMER
What is the intrinsic pathway of apoptosis?
Regulated by Mitochondria Loss of mitochondrial potential Release of cytochrome c Release of other apoptosis factors Formation of APOPTOSOME complex
What can trigger the intrinsic pathway?
Cellular stresses
e.g. lack of/overstimulation of growth factors, DNA damage, ROS
Describe the structure of the apoptosome
‘Wheel of death’
Composed of Apaf-1, cytochrome c, ATP, procaspase 9
Apaf-1 have a CARD domain and WD-40 domains
Cyt c binds to WD-40 domains
Each Apaf-1 can bind a procaspase 9
How is receptor mediated + mitochondrial apoptosis linked?
Caspase 8 cleaves Bid which enhances release of mitochondrial proteins, thus activating the mitochondrial pathway
List anti-apoptotic proteins
Bcl-2
Bcl-xL
These work in the mitochondria
List pro-apoptotic proteins
Bid
Bad
Bax
Bak
These move from cytoplasm to mitochondria
Which domain in the Bcl-2 protein family allows dimerisation? Why is this important?
BH3
Dimerisation means you can concentrate a signal, whether it is anti or pro-apoptotic
What is the PI3-kinase signalling pathway
Promotes cell survival and proliferation
PI3 kinase is a LIPID kinase (not protein)
Activates protein kinase PKB/Akt which is ANTI-apoptotic
How does PKB/Akt block apoptosis and induce cell survival?
Phosphorylates and inactivates Bad + caspase 9
Inactivates FOXO transcription factors (FOXOs promote expression of pro-apoptotic genes)
Explain the mechanism by which apoptosis occurs via Bcl-2 family proteins and PKB/Akt in the cell
- Cell survival: Bcl-2/Bcl-xL are bound to Bax/Bak via BH3 domains. Bad is phosphorylated and inactive by PKB/Akt
- In the absence of GFs, Bad dephosphorylated and RELEASED from PKB/Akt
- Bad displaces and frees Bax/Bak from Bcl-2/Bcl-xL
- Bax/Bak forms tetramer, creating a large pore in the mitochondrial membrane
- Cytochrome c released via the pore and activates apoptosis
What does PTEN do?
counteracts PI3-K signalling
Reverses the phosphorylation of lipid
(TRIphosphate -> BIphosphate)
What do Inhibitors of Apoptosis Proteins do?
Extrinsic pathway:
Bind to procaspases and prevent activation
Bind to caspases and inihibit activity
What are the cytoprotectie/anti-apoptotic pathways?
Intrinsic pathway: Bcl-2/Bcl-xL
Extrinsic pathway: FLIPs, IAPs
Growth Factor pathways: PI3-K, PKB/Ak
