Apoptosis Flashcards
Define Necrosis.
Unregulated cell death associated with trauma, cellular disruption and an inflammatory response
Define Apoptosis.
Regulated cell death; controlled disassembly of cellular contents without disruption – no inflammatory response
Describe the process of necrosis.
- Plasma membrane becomes permeable
- Cell swelling and rupture of cellular membranes
- Release of proteases leading to autodigestion and dissolution of the cell
- Localised inflammation
What are the two phases of apoptosis?
Latent phase
Execution phase
Describe the latent phase of apoptosis
Death pathways are activated, but cells appear morphologically the same
Describe the Execution phase of apoptosis
- Loss of microvilli and intercellular junctions
- 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 enclose apoptotic bodies (these are then taken up by macrophages)
What is an important feature of apoptosis that distinguishes it from necrosis?
Plasma membrane remains intact – no inflammation
How is DNA fragmentation observed in apoptosis?
- Fragmentation of DNA ladders (seen in agarose gel)
- TUNEL assay = DNA fragmentation leads to more “ends” which are labelled by adding an extra fluorescently-tagged base
What other types of cell death are there other than necrosis and apoptosis? Describe the nature of cell death in reality
- Apoptosis-like cell death
- Necrosis-like cell death (sort of like an aborted apoptosis that ends up being necrosis)
Cell death is a GRADED response i.e. shows features of both apoptosis and necrosis
What are caspases?
Cysteine-dependent aspartate-directed proteases that bring about apoptosis
Which caspases are the ‘initiator caspases’? What are they responsible for?
2, 8, 9 and 10
They initiate the apoptosis signal
Which caspases are the ‘effector caspases’? What are they responsible for?
3, 6 and 7
They carry out the mass proteolysis that leads to apoptosis
Describe the structure of effector caspases.
They are single chain polypeptides consisting of a small and large subunit
Describe the structure of initiator caspases.
They also have a large and small subunit found in effector caspases but they also have a targeting subunit (protein-protein interacting domain also called the pro domain)
What are the two types of targeting subunit that initiator caspases can have?
CARD – caspase recruitment domain; e.g. Caspase-2, -9
DED – death effector domain; e.g. Caspase-8, -10
Describe the activation of initiator Caspases
- Initiator Caspases normally exist as inactive procaspase monomers.
- Their activation is initiated by dimerisation, which is facilitated by binding to adaptor proteins via the protein–protein interacting domains.
- Both pro-caspases undergo cleavage by autocatalysis. This leads to removal of the prodomain and cleavage of the linker region between the large and small subunit.
- An active heterotetramer is formed
Describe the activation of effector Caspases. What do they do once they are activated?
- Inappropriate activation of the effector caspases is prevented by their production as inactive procaspase dimers that must be cleaved by initiator caspases.
- This cleavage is between the large and small subunits
- The resulting small and large subunit of each Caspase will associate, resulting in an active heterotetramer.
- Once activated, a single effector caspase can cleave and activate other effector caspases, leading to an accelerated feedback loop of caspase activation.
More specifically, what can effector Capsases go on to do to execute the apoptotic programme?
- Cleave for example nuclear lamins leading to nuclear breakdown
- Activate kinase and nuclease enzymes by direct cleavage, or
cleavage of inhibitory
molecules; e.g. Caspase-Activated DNase (CAD)
What are the two mechanisms of Caspase activation, and hence apoptosis
- Death by design or extrinsic pathway (receptor-mediated)
2. Death by default or intrinsic mitochondrial death pathway
Describe the three domains of the death receptor mediating the extrinsic pathway
- Extracellular cysteine rich domain (N-terminus)
- Transmembrane domain
- Intracellular tail with a Death Domain, DD (C-terminus)
What are the two important adaptor proteins in the death by design pathway and how are they different, in terms of their function and important domains?
- FADD = positive regulator that promotes cell death – has Death Effector Domain (DED) and DD
- FLIP = negative regulator – has DED + DED
Describe signalling of apoptosis through Fas.
- Fas ligand on CTLsbinds to Fas receptor and the Fas receptors undergo trimerisation, which brings the three DDs together
- The trimerised DDs recruit FADD, which binds via its own DD
- FADD then recruits and oligomerises procaspase 8 through the DED of procaspase 8
- Binding of procaspase 8 to FADD forms DISC (death-induced signalling complex)
- DISC formation results in cross-activation of procaspase 8
- Active caspase 8 is released, which then activates effector caspases
Describe the importance of oligomerisation in this pathway.
Some initiator caspases have intrinsic low catalytic activity
Oligomerisation brings them close enough together to allow transcleavage (auto-catalytic cleavage)
Also, at least 2 procaspases are required to form an active caspase
Describe how FLIP acts as an inhibitor of apoptosis
FLIP is evolutionarily related to caspases but has lost its catalytic activity
It has two DED domains and can compete with procaspase 8 to bind to the receptor tails or FADD via its DED domains
It can incorporate into receptor-procaspase complexes and interfere with transcleavage
