Apoptosis Flashcards
(39 cards)
What cells do we need to remove?
- harmful cells (viral infection/DNA damage)
- developmentally defective cells
- excess/unnecessary cells
- obsolete organs (e.g. mammary epithelium at the end of lactation)
- exploitation (chemotherapeutic killing of cells)
What is necrosis?
Unregulated cell death associated with trauma, cellular disruption and an INFLAMMATORY RESPONSE
What is apoptosis?
programmed cell death
Regulated, controlled disassembly of cellular contents without disruption – NO INFLAMMATORY RESPONSE
active process (requires energy)
What happens during necrosis?
- plasma membrane becomes permeable
- cell swelling and rupture of cellular membranes
- release of proteases leading to auto-digestion and dissolution of the cell (unregulated action
- localised inflammation occur due to the attraction of immune cells
What are the phases of apoptosis?
Latent phase – death pathways are activated, but cells appear morphologically the same.
Execution phase – an orderly activation of specific proteins and kinases:
- Loss of microvilli and intercellular junctions
- Dramatic cell shrinkage
- Loss of plasma membrane asymmetry
- (Phosphatidylserine lipid appears in outer leaflet)
- Chromatin and nuclear condensation
- DNA fragmentation
- Formation of membrane blebs
- Fragmentation into membrane-enclosed apoptotic bodies
Why is the no inflammation in apoptosis?
Up to the blebbing, the plasma membrane remains intact.
Because the contents aren’t released, there is no inflammation
How is DNA modifies in apoptosis?
Because the nucleus gets condensed and destroyed, whatever is inside the nucleus must be degraded
If you extract DNA from the cell undergoing apoptosis, and run it on agarose gel, there is fragmentation of the DNA.
- Fragmentation of DNA ladders (in agarose gel)
- Formation of more ‘ends’, which are labelled by adding an extra fluorescently-tagged base in a TUNEL assay
What is apoptosis-like PCD?
some, but not all, features of apoptosis. There may be a display of phagocytic recognition molecules, even before plasma membrane lysis
What is necrosis-like PCD?
Variable features of apoptosis before cell lysis – “Aborted apoptosis” (can occur up to a certain point down the process)
Whatare the mechanisms of apoptotic cell death?
- The executioners – Caspases (key enzymes)
- Initiating the death programme: via death receptors (extrinsic) and mitochondria (intrinsic)
- The Bcl-2 family
- Stopping the death programme
How are caspases activated?
- Executioners of apoptosis that have a cysteine residue in their active site (required for activity)
- Activated by proteolysis, and cut proteins just after their aspartate residue
- Cascade of activation – have a recognition motif, which will cleave when interacting with these motifs
What are the classes of caspases?
initiators and effectors
- Initiator caspases are the first to be triggered (2, 9, 10 and 8) - contain specific motifs (e.g. CARD for 2 and 9, DED for 10 and 8)
- Effector caspases (3, 6 and 7) don’t contain these motifs
Describe the maturation of caspases
- synthesised as pro-caspases (zymogens)
- have a pro-domain to maintain the inactivated stage
- Proteolysis results in cleavage of the pro-domain -> formation of the heterodimer.
- Cleavage of the inactive pro-caspase precursor is followed by folding of 2 large and 2 small chains to form an active L2S2 heterotetramer.
- These cleavages are done by the caspases themselves
What is the purpose of the caspase cascade?
Amplification, Divergent responses, Regulation
Describe the caspase cascade
apoptosis is triggered -> the initiator caspases cleave and activate the effector caspases -> allows the commencement of cascades
Initiator caspases trigger apoptosis by cleaving and activating
Once effector caspases are activated, they carry out the apoptotic programme (activate further caspases downstream)
How do effector caspases carry out apoptosis?
- Cleaving and inactivating various proteins and complexes (e.g. nuclear lamins leading nuclear breakdown)
- Activating enzymes by direct cleavage, or cleavage of inhibitor molecules (e.g. protein kinases, nucleases such as Caspase-activated DNAse (CAD))
How are caspases activated?
- Death by design – Receptor-mediated (extrinsic) pathways
- Death by default – Mitochondrial (intrinsic) death pathway
What do death receptors consist of?
- Extracellular cysteine-rich domain
- Single transcellular domain
- Cytoplasmic tail (with a death domain)
What activated death receptors?
only activated when they encounter secreted or transmembrane trimeric ligands (e.g. TNF-alpha or Fas) – these are called death ligands
What do death receptors do?
When activated, receptors activate the caspases
Once activated, they will dimerise/trimerise -> attract adaptor protein
- 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 pro-caspase 8 through its DED to FADD DED -> DISC (Death-Inducing Signalling Complex)
- active caspase 8 is then released, and it cleaves effector caspases to execute the death programme
What are the adaptor proteins in receptor-mediated apoptosis?
FADD: POSITIVE regulator (required for the death pathway to become activated) and promotes cell death.
FLIP: negative regulator (inhibits the death pathway and allows it to be regulated)
FADD = DED + DD FLIP = DED + DED
- DED = Death Effector Domain
- DD = Death Domain
How is initiator caspase?
Once recruited, the trimerised receptors FADD contain DED domains. The pro-caspase 8 (initiator) will interact with the DED domain. There will be 3 pro-caspase 8 molecules
They will be cleaved (trans-cleavage) by one another -> the tetramer can be assembled together to form an ACTIVE CASPASE 8. The trimerised receptor enables close proximity of the pro-caspase, so trans-cleavage can take place -> active initiator caspase.
Initiator pro-caspases bind, via their DED domains, to the DED domains of FADD - three initiator pro-caspase 8s come into close contact, which allows cleavage -> releases the active initiator caspase 8 tetramer
How does FLIP inhibit activation of caspase 8?
FLIP – caspase homology in DED domain, but has no proteolytic activity therefore it competes with pro-caspase
Because of the DED domain of the FLIP inhibitor protein, it will insert itself in-between the pro-caspase, and PREVENT the efficient cleavage -> it blocks the formation of new, active caspase 8
What happens in mitochondrial regulation of apoptosis?
intrinsic pathway whereby cellular stresses (e.g. lack of/overstimulation by growth factors, DNA damage etc.) cause a loss of mitochondrial membrane potential -> release of cytochrome C and other apopotosis-inducing factors -> stimulate the formation of an apoptosome complex
