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
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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
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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
Describe the Regulation of apoptosis by Bcl-2 proteins via BH3 heterodimerisation:
Bcl-xL/2 localised on mitochondria + form inactive dimers w/ other Bcl-2 family
- In cytosol, have inactive phosphorylated Bad
- If remove GFs, Bad dephosphorylated –> released from docking proteins + becomes active –> moves to mitochondria –> displace Bcl-2/xL
- Bax/bak form pore in mitochondrial membrane –> release cytochrome C –> pro-apoptotic
PTEN (lipid phosphatase) counteracts PI3’-K signalling
????
how does inhibitor of Apoptosis Proteins (IAPs) regulate Programmed cell death?
Extrinsic pathway
- they Bind to procaspases –> prevent activation
- can also Bind to active caspases –> inhibit activity
- Bcl-2, Bcl-xL regulate intrinsic /extrinsic pathway
- FLIP, IAPs regulate intrinsic / extrinsic pathway
- GF pathways via PI3’-K and PKB/Akt –> promote cell survival / death
- Bcl-2, Bcl-xL regulate intrinsic pathway
- FLIP, IAPs regulate extrinsic pathway
- GF pathways via PI3’-K and PKB/Akt –> promote cell survival
What are some therapeutic uses of programmed cell death?
Harmful (oncogenic) cells (e.g. cells with viral infection, DNA damage)
Chemotherapeutic killing of tumour cells, e.g. Dexamethasone stimulates DNA cleavage
how many apaf 1 monomers join the apoptosome?
a) how is the monomer become an apoptosome?
7
a) monomoer –> signal – heptomer –> recruit caspases –>form apoptosome
apoptosome requires/ doesn’t require ATP
apoptosome requires ATP
note: BH3 = allows dimerisation of the motifs
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