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
As an overview, describe death by default.
Cellular stresses (e.g. lack of or overstimulation by growth factors, DNA damage, ROS) causes a change in mitochondrial membrane potential
=> Release of cytochrome C and other apoptosis-
inducing factors from the mitochondrion
=> Formation of the apoptosome complex
What does the apoptosome consist of?
APAF-1 (apoptotic activating factor 1)
Cytochrome C
ATP
Procaspase 9
Describe the domains found within APAF-1.
CARD domain
ATPase domain
WD-40 repeats (protein-protein interactions)
How does the APAF-1, Cyt c and ATP form the apoptosome
The cytochrome C released from the mitochondria bind to the WD-40 repeats of APAF-1 and causing formation of a heptamer (x7 APAF-1); this requires ATP
- The 7 CARD domains are in the middle, each of which can interact with a CARD domain of procaspase 9
- Seven procaspase 9 bind via their CARD domains to the APAF-1 heptamer and their close contact allows them to cross-cleave each other to generate activate caspase 9
What pro-apoptotic protein links the death by default and death by design pathways? Explain how it works.
Bid Caspase 8 (generated by the death by design pathway) cleaves Bid, which travels to the mitochondrion and promotes the release of cytochrome C – thus triggering the mitochondrial death pathway
How can energy levels of a cell show whether a cell is going through apoptosis or necrosis?
Apoptosis requires ATP whereas necrosis does not
State the important family of proteins that act as intrinsic modulators of apoptosis?
Bcl-2 family
There are three main groups of Bcl-2 proteins. What is common to all three groups?
BH3 domain – this is a dimerisation motif, which allows members of the family to form dimers with each other
What are the anti-apoptotic Bcl-2 proteins and where are they found?
Bcl-2
Bcl-xL
They are found localised on the mitochondrial membrane
What are the pro-apoptotic Bcl-2 proteins and where are they found?
Bid Bad Bax Bak These are found in the cytoplasm and in the mitochondrial membrane
Other than Ras signalling, what other pathway does growth factor binding to growth factor receptors activate?
PI3-kinase pathway
What type of molecule is PI3-K?
Lipid kinase (so NOT a protein kinase); = Phosphatidylinositol 3’-kinase
What are the main subunits of PI3-K?
Adaptor subunit
Targeting subunit
Catalytic subunit
What is the main action of PI3-K?
PI3-K catalyses the convertion PIP2 to PIP3
What effect does this action have that leads to inhibition of apoptosis?
PIP3 is recognised by the adaptor subunit of Protein Kinase B (PKB/Akt)
This allows PKB to move to the cell membrane where it becomes activated
PKB phosphorylates and inactivates Bad
Describe the arrangement of the anti-apoptotic and pro-apoptotic proteins when growth factor signalling and the PI3-K pathway is active.
This means PI3-K can produce PIP3 – so PKB/Akt is activated meaning that Bad is phosphorylated and inactivated
Bad is held in an inactive heterodimer with 14-3-3
On the mitochondrial membrane, Bak and Bax are held in inactive heterodimers with Bcl-2 and Bcl-xL
Describe how loss of growth factor signalling can lead to apoptosis.
This means loss of activation of the PI3K pathway so less PIP3 produced so less activation of PKB/Akt
Bad gets dephosphorylated and dissociated from its inactive heterodimer
Bad then moves to the mitochondrial membrane and binds to the anti-apoptotic proteins (Bcl-2 and Bcl-xL) via its BH3 domain
This displaces Bax and Bak from their inactive heterodimers
So Bax and Bak then form a pore in the mitochondrial membrane allowing the release of cytochrome C from the mitochondrion – this leads to apoptosis
Summarise the effects of PKB/Akt in promoting cell survival.
Phosphorylates and inactivated Bad
Phosphorylates and inactivates caspase 9
Inactivates FOXO transcription factors (FOXOs promote the expression of apoptosis-promoting genes)
Name two extrinsic regulators of apoptosis and describe their actions.
PTEN
- Lipid phosphatase
- Counteracts the activation of PKB
- Reduces cell survival and promotes apoptosis
IAPs (Inhibitor of Apoptosis proteins)
- Binds to procaspases and prevents their activation
- Can bind to activate caspases and inhibit their activity
Are the following tumour suppressor genes or oncogenes?
a. Bcl-2
b. PTEN
c. PKB/Akt
a. Bcl-2
Oncogene – increased activation would mean reduced likelihood of apoptosis (cancers are anti-apoptotic)
b. PTEN
Tumour suppressor gene –inactivation will mean reduced likelihood of apoptosis
c. PKB/Akt
Oncogene
