Cancer 9 - Apoptosis Flashcards
Why do cells undergo apoptosis (programmed cell death - PCD)
- Harmful cells
- Developmentally defective cells
- Excess/unnecessary cells
- Obsolete cells
- Exploitation
What are the differences between necrosis and apoptosis
Necrosis = unregulated cell death, associated with trauma, cellular disruption and INFLAMMATORY RESPONSE
Apoptosis = regulated cell death, controlled disassembly of cellular contents without disruption, NO INFLAMMATORY RESPONSE
Explain how necrosis happens (localised inflammation)
- Plasma membrane becomes permeable (e.g. trauma)
- Chromatin condenses, fluid rushes in –> cell swells and cellular membrane ruptures
- Release of proteases –> autodigestion and dissolution of cell
- Cell lysis, invasion of phagocytic cells, inflammation
What are the 2 phases of apoptosis and what happens in each phase
- Latent phase - death pathways activated but cell remains morphologically the same
- Execution phase - Loss of microvilli and intercellular junctions
- cell shrinkage
- loss of plasma membrane asymmetry (phosphatydylserine appears in outer leaflet/membrane)
- Chromatin and nuclear condensation
- DNA fragmentation
- Formation of membrane blebs
- fragments enclosed into membrane enclosed apoptotic bodies
Apoptotic bodies are phagocytosed by….?
Macrophages
Why is there no inflammation in apoptosis?
Because the cellular contents is not released so no mediators are attracted
As the cell shrinks in apoptosis, what closes around it? Why
Epithelium closes around it to prevent entry of things such as pathogens via the lumen
How can apoptotic nuclei be detected
- DNA ladders - show fragmentation as DNA is being broken down
- Adding fluorescent tagged base - DNA fragmentation = more ends = brighter nuclei show more fragmentation
Aside from necrosis and apoptosis, what are the other forms of cell death
This is a graded response
- Apoptosis-like PCD = has some but not all features of apoptosis - phagocytic recognition molecules displayed before plasma lysis
- Necrosis-like PCD - variable features of apoptosis before cell lysis - “aborted apoptosis”
What do caspases (Cysteine-dependent Aspartate-directed Proteases) do?
They degrade everything inside the cell - “the executioner”
They are activated by proteolysis - trigger cascade of activation
What are the 2 types of caspases
- Initiator caspases - these are triggered first.
2 domains:
CARD (Caspase Recruitment Domain) (cascades 2, 9)
DED (Death Effector Domain) (cascades 10, 8) - these have homotypic protein-protein interactions, provide scaffolding
- Effector caspases - these do the chopping up (caspases 3,6,7)
How does caspase maturation happen
Zymogens (procaspases) undergo proteolytic cleavage –> removes the pro-domains and it separates to form 2 distinct long (L) and short (S) subunits –> which then forms an active L2S2 hetero-tetramer
Which 3 things do caspase cascades involve
- Amplification
- Divergent responses
- Regulation
How do initiator cascades trigger apoptosis?
By cleaving and activating effector caspases (which carry out the apoptotic programme)
What 4 actions can effector caspases have
(Monomeric substrates)
- Inactivation
- Activation
(Multiprotein complexes)
- Disassembly
- Release
Give an example of a nuclease that is activated by effector caspases
Caspase-Activated-DNase (CAD)
Give an example of a protein/complex being cleaved and inactivated by effector caspases
Nuclear lamins cleaved - leading to nuclear breakdown
What are the 2 mechanisms of caspase activation
- Death by design - receptor mediated (extrinsic) pathways
2. Death by default - mitochondrial (intrinsic) pathway
Describe death receptors
- They are transmembrane, cysteine-rich extracellular domains that are trimerised
What do intracellular death domains attract
Adapter proteins
Name and describe the roles of 2 adapter proteins in receptor-mediated apoptosis
- FADD - has a DED and DD domain - responsible for activating apoptotic path
- FLIP - 2x DED domain - inhibits apoptotic path
DD and DED domains bind to similar domains on other proteins
Explain how signalling via death receptors occurs in the Fas/L
- Fas receptor trimerised - due to FasL on lymphocyte
- FADD (adapter protein) recruited via its DD to the DD of Fas
- Recruitment and oligomerisation of procaspase 8 (via its DED to FADD DED) —> triggers DISC (Death-Inducing Signalling Complex)
How many procaspases are needed to form an active tetramer
2
Which 2 ways can activate procaspase 8
- Transcleavage
- Conformational change upon oligomerisation
(active procaspase is released when procaspase 8 interacts with DED which causes cleavage)
What are the two types of FLIP?
Small and long FLIP - they have the same homology in the DED domain as the procaspases but they have no proteolytic activity - so the FLIPs must compete with the procaspase to inhibit activation of caspase 8
Essentially FLIP inhibits procaspase 8 activity by binding onto the DED domain of the Fas/FADD receptor tail - no proteolytic compartment so no transcleavage
Describe the intrinsic pathway of apoptosis (mitochondrial regulation)
- Loss of mitochondrial membrane potential due to cellular stress
- Release of Cytochrome C and other apoptosis inducing factors into cytoplasm
- Formation of the apoptosome complex
What are the constituents of the apoptosome complex
Apoptotic activating factor-1 (APAF-1), procaspase 9, ATP, Cytochrome C
Describe the role(s) of Apaf-1 in the apoptosome
Apaf-1 is a scaffolding protein to organise the apoptosome
- Has CARD domains at the centre of the wheel to recruit caspases
- Has WD-40 repeats at C terminal - which binds to cytochrome c
When stimulated, the apoptosome is a ……..meric structure
Heptameric
How are the caspases involved in the apoptosome
Procaspase 9 binds (docks) onto the CARD, and forms a dimer with caspase 9 in the middle of the wheel - potentially 7 procaspase 9s can bind as well (heptameric)
Oligomerization brings many procaspase 9s together - which results in cleavage and activation –> release of active caspase 9 tetramer –> initiating the caspase cascade —> which triggers apoptosis
What is an essential requirement for the apoptosome
ATP
What may determine whether cell death is by necrosis or apoptosis
Energy levels in the cell
How may the extrinsic and intrinsic apoptotic pathway be linked
The extrinsic pathway, upon activation of caspase 8, activates Bid proteins which stimulates the intrinsic (mitochondrial) pathway by stimulating release of mitochondrial proteins into cytoplasm.
Caspase 8 and apoptosome then go onto activate caspase 3
Which family of proteins modulates apoptosis?
Bcl-2
some have Transmembrane domains
How many groups of Bcl-2 proteins are there?
3
Group 1
Group 2
Group 3 - contains BH3 only
(BH3 = dimerisation motif)
Which are the anti-apoptotic proteins
Bcl-2, Bcl-xL (hooked on mitochondria)
What are the pro-apoptotic proteins
Bid, Bad, Bax, Bak (move between cytosol and mitochondria)
Group 2 and 3
Which 2 pathways does activation of the PI3 receptor (Growth factor receptor) cause
PI3 kinase stimulated by growth factors (e.g. EGF, insulin)
- ERK pathway (growth)
2. PI3 kinase pathway (survival, proliferation)
What is PI3 kinase and what is it involved in
It is a lipid kinase (2 subunits - P110 and P85)
It is involved in growth control and cell survival
Which anti-apoptotic protein kinase does PI3 kinase activate
PKB/Akt (via PIP3)
PI3 kinase converts PIP2 –> PIP3
What is P13 kinase blocked by?
What effect does this have
Blocked by PTEN
Reduces cell survival
4 ways in which PKB/Akt induces cell survival by blocking apoptosis
(intrinsic pathway)
- Phosphorylates and inactivates Bad
- Phosphorylates and inactivates caspase 9
- Inactivates FOXO Tfs (FOXOs promote expression of pro-apoptotic genes)
- Other, e.g. stimulates ribosome production and protein synthesis
Explain how apoptosis is regulated by BH3 dimerisation
- Usually (GF present), Bad is inactive and phosphorylated in cytosol
- Bcl-xL/2 on mitochondria and form inactive dimers via BH3
GFs absent
- Bad dephosphorylated and released
- Displaces Bcl-2xL dimer
- Bax/Bak form a pore in the mitochondrial membrane, out of which cytochrome c leaks out
- Apoptosis triggered
What is PTEN?
Lipid phosphatase
Means that we can alter lipid levels in the cell to control apoptosis/survival
Which proteins can regulate extrinsic apoptosis
Inhibitor of Apoptosis Proteins (IAPs)
- They bind to procaspases and prevent activation
- bind to active caspases and inhibit their activity
Name anti-apoptotic pathways for intrinsic pathway
Bcl-2, Bcl-xL
Name anti-apoptotic pathways for the extrinsic pathway
FLIP, IAPS
Name 2 therapeutic uses of apoptosis
- Kill harmful (oncogenic) cells - e.g. virally infected cells, cells w DNA damage
- Chemotherapeutic killing of tumour cells (e.g. dexamethasone stimulates DNA cleavage)
