Cancer 10: Apoptosis Flashcards
What are the 4 main functions of apoptosis ?
- Harmful cells
- developmentally defective cells
- remove excess / unnecessary cells
- obsolete cells (e.g maxillary epithelium at end of lactation)
- exploitation (e.g chemotherapy)
what is the difference btw necrosis vs apoptosis
necrosis = unregulated cell death associated with trauma, cellular disruption and an inflammatory response
Apoptosis = regulated cell death; controlled disassembly of cellular contents without disruption; no inflammatory response
What happens in necrosis?
- Plasma membrane becomes permeable
- Cells swell and cellular membrane ruptures
- Chromatin condense
- Release of proteases leading to autodigestion and dissolution of the cell
- cells lyse
- Localised inflammation occurs
What are the 2 phases of apoptosis?
- latent phase
- execution phase
What happens in the latent phase of apoptosis?
- death pathways are activated,
but cells= morphologically the same
What happens in the execution phase of apoptosis?
- Loss of microvilli and intercellular junctions
- Cell shrinkage –> then epithelium closes around
- Loss of plasma membrane asymmetry (phosphatidylserine lipid appears in outer leaflet)
- Chromatin and nuclear condensation
- DNA fragmentation occurs
- Formation of membrane blebs
- Fragmentation of membrane-enclosed apoptotic bodies
Is there inflammation involved in apoptosis?
no
What happens to the apoptic body ?
- it is phagocytosised by surrounding cells
e. g macrophages
how can DNA modification help detect apoptosis?
a) DNA LADDERS
you can detect apoptosis by DNA degradation –> (DNA fragmentation)
- separate DNA by size
at first: DNA = too big cant move don’t down gel
- later: DNA ladder fragments visible
b) TUNEL assay
-
What are 2 Other types of cell death (except necrosis + apoptosis)
(PCD = programmed) - shows spectrum/graded response
- Apoptosis-like PCD = shows some, but not all, features of apoptosis. Display of phagocytic recognition molecules before plasma membrane lysis
- Necrosis-like PCD = shows variable features of apoptosis before cell lysis; “Aborted apoptosis”
What are 4 mechanisms of apoptotic cell death?
- The executioners – Caspases
- Initiating the death programme
- via Death receptors OR Mitochondria - The Bcl-2 family
- Stopping the death programme
What are caspases (in apoptosis) ?
what are the 2 types of caspases?
caspases
- -> triggers apoptosis
- -> activated by proteolysis
- -> initiates cascade of activation
there are 2 types of caspases:
a) Initiator caspases – first triggered (2, 9, 10, 8)
has 2 domains:
–> CARD = (CAspase Recruitment Domain) localises caspases at specific sites in cell
–> DED (Death Effector Domain) specific for caspases 8/10
CARD + DED ==> targeting subunits - protein-protein interactions (adapters), provide scaffolding
b) Effector caspases = 3, 6, 7 (P20/10 motifs)
Describe the maturation of the Caspase
- They are made as zymogens (pro-caspases) - inactive
- -> proteolytic cleavage of 2 procaspases to remove respective pro-domains releases the long + short subunits
- Next, folding of 2 large + 2 small chains –> forms active L2S2 hetero-tetramer
Describe the caspase cascades
caspase cascades
- allows amplification,
- allows divergent responses
- allows regulation
– caspase 8 + 9 –> initiator caspases that trigger apoptosis by cleaving and activating
- others = effector caspases
carry out apoptotic programme
How do effector caspases execute the apoptotic programme?
2 methods
a) they cleave and inactivate proteins or complexes -that would normally stop apoptosis (e.g lamin cleavages –> nuclear breakdown)
b) They activate enzymes by direct cleavage, or
cleavage of inhibitory molecules
What are the 2 mechanisms of caspase activation?
- Death by design
– Receptor-mediated (extrinsic) pathways
- ligand binds to death receptors –> which activates the death domain in the cytoplasmic domain of the receptor –> DED of procaspase binds to DD –> forms (DISC) –> activates procaspase –> activation
- Receptors have Transmemb, cysteine-rich extracellular domains
- Intracellular death domains –> attract adapter proteins
FADD = activating adaptor proteins
FLIP = inhibitory adaptor proteins
–> tightly controlled
Death by default – Mitochondrial (intrinsic) death pathway:
- exposure to cellular stress
- Loss of mitochondrial membrane potential (ΔΨ)
- Release of cytochrome c
- Release of other apoptosis-
inducing factors
- Formation of the apoptosome complex (Caspase 9, Apaf1)
What are the 3 ways of signalling through death receptors ?
e.g Fas/Fas-Ligand
- Receptor (Fas) trimerisation by ligand (Fas-L on lymphocyte)
- Recruitment of adapter protein (FADD) through its DD to DD of Fas
- Recruitment and oligomerisation of procaspase 8 through its DED to FADD DED –> Death-Inducing Signalling Complex (DISC)
–>
How does initiator procapsase 8 oligomerisation results in cleavage and activation ?
- Procaspase 8 interacts with DED –> cleaves procaspase –> active tetramer released
- Some initiator procaspases have (some) intrinsic low catalytic activity – oligomerisation allows transcleavage
- Other caspases = activated by conformational change on oligomerisation
- Need at least 2 procaspases to form active tetramer
How does FLIP inhibit procaspase 8 activation?
- FLIP competes for binding to receptor tails / FADD via DED domains
–> and Incorporates into receptor-procaspase complexes and interferes with transcleavage
–> because it cant form tetramer
CARD = docking site for bringing lots of caspases together
-
Apoptosis requires / doesn’t require energy
Necrosis requires / doesn’t require energy
apoptosome requires ATP
Necrosis doesn’t require energy
Principal mechanisms of apoptosis: Bid links receptor and mitochondrial death pathways
apoptosome feeds into caspase 3 –> causes proteolysis and cell death
caspar 8 can cleave protein BID –> promotes and inhales proteolysis ad cell death
(requires ATP)
What are the main modulators of apoptosis?
- Anti-apoptotic proteins = Bcl-2 and Bcl-xL, (hooked on mitochondria)
- Pro-apoptotic proteins = Bid, Bad, Bax, Bak (move between Cytosol and Mitochondria)
How is PI3’-Kinase signalling pathway involved in growth + cell survival?
o PIP3 activates protein kinase PKB/Akt which is anti-apoptotic + Phosphate-dependent kinase (PDK) –> induces cell survival
- -> PKB/Akt acts by:
1. 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 protein synthesis
