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.
Cells and organelles swell Chromatin condenses Membrane is comprimised causing fluid to rush in Proteases are released leading to dissolution and autodigestion of the cell Cell lysis There is localised inflammation - phagocytic cell infiltration
What are the two phases of apoptosis? Describe them.
Latent phase Death pathways are activated, but cells appear morphologically the same Execution phase: Loss of microvilli and intercellular junctions Cell shrinkage - adjacent epithelial cells close around gap Chromatin and nuclear condensation DNA fragmentation Formation of membrane blebs Fragmentation into membrane enclose apoptotic bodies these fragemtnts are phagocytosed by neighbouring cells and roving macrophages
What is an important feature of apoptosis that distinguishes itfrom necrosis?
Plasma membrane remains intact – no inflammation
What DNA modification is seen during apoptosis?
Fragmentation of DNA ladders (seen in agarose gel) Formation of more ends, which are labelled by adding an extra fluorescently-labelled tag in a TUNEL assay
What other types of cell death are there other than necrosis and apoptosis?
Apoptosis-like PCD: Similar to apoptosis but display of phagocytic recognition molecules before plasma membrane lysis, therefore induces inflammation Necrosis-like PCD (sort of like an aborted apoptosis that ends up being necrosis) NOTE: cell death is GRADED
What are caspases?
Cysteine-dependent aspartate-directed proteases They are the executioners of apoptosis They are activated by being proteolytically cleaved
Which caspases are effector caspases?
3, 6 and 7
Which caspases are initiator caspases?
2, 8, 9 and 10
Describe the structure of effector caspases.
They are single chain polypeptides consisting of a small and large subunit The subunits are released by proteolytic cleavage
Describe the structure of initiator caspases.
They have the same two subunits found in effector caspases but they also have a targeting subunit (protein-protein interacting domain)
What are the two types of targeting subunit that initiator caspases can have? Which Initatior caspases belong to each?
CARD – caspase recruitment domain caspases 2&9 DED – death effector domain caspases 8&10
How are active caspases formed?
Cleavage of inactive procaspases Initiator is cleaved at its pro domain (DED or CARD) and also at the juction of its large and small segments Effector is cleaved into its large and small segment Then folding of 2 large and 2 small chains occurs to form an active L2S2 heterotetramer
What are the two mechanisms of apoptosis
Death by design (receptor-mediated) Death by default (mitochondrial (intrinsic) death pathway)
Describe the structure of death receptors.
Cysteine-rich extracellular domain Transmembrane domain Intracellular tail with a death domain (DD)
What are the two important adaptor proteins in the death by design pathway and how are they different?
FADD – positive regulator that promotes cell death – DED + DD domains FLIP – negative regulator – DED + DED domains
Describe signalling of apoptosis through Fas.
- Death ligand(e.g. Fas ligand) binds to death receptor(e.g. Fas receptor (on CTLs))
- The Fas receptors undergo trimerisation which brings the three DD domains of the Fas receptor together
- Recruitment of adapter protein FADD through binding of the Fas receptor DD domain to the FADD DD domains
- FADD then recruits and oligomerises procaspase 8 through the DED domains of the FADD and the DED domains of procaspase 8
- Binding of procaspase 8 to FADD forms DISC (death-induced signalling complex)
- DISC formation results in cross-activation of procaspase 8 as the initiator procaspase 8s cleave eachother
- Active initiator caspase 8 tetramer is released from the receptor, which then activates effector caspases
Describe the important of oligomerisation in this pathway.
Some initiator caspases have intrinsic low catalytic activity Oligomerisation brings them close enough together to allow transcleavage 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 DED domains of FADD It can incorporate into receptor-procaspase complexes and interfere with transcleavage
As an overview, describe death by default.
Cellular stress causes a change in mitochondrial membrane potential This leads to release of cytochrome C from the mitochondrion This stimulates 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)
Explain fully, how death by default leads to caspase activation.
The cytochrome C released from the mitochondria bind to the WD-40 repeats of APAF-1 These dimers can form a heptamer structure (apoptosome) using ATP It has 7 CARD domains in the middle, which can interact with CARD domains of procaspase 9 Seven procaspase 9 bind via their CARD domains to the apoptosome and their close contact allows them to cross-cleave each other Release as an active caspase 9 tetramer
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 energy whereas necrosis does not
What is an important family of proteins that act as intrinsic modulators of apoptosis?
- Bcl-2 family
- some are pro apoptotic and some are anti apoptotic
What is common to all three groups of BCL-2 family proteins?
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 mitochondria
Other than Ras signalling, what other pathway does growth factor binding to growth factor receptors activate?
PI3-kinase
What type of molecule is PI3-K?
Lipid 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 converts PIP2 to PIP3
Describe the arrangement of the anti-apoptotic and pro-apoptotic proteins when growth factor signalling and the PI3-K pathway is active.
- PI3-K can produce PIP3 – so PKB/Akt is activated
- This means Bad is phosphorylated and inactivated
- Bad is held in an inactive heterodimer with 14-3-3 in the cytosol
- On the mitochondrial membrane, Bak and Bax are held in inactive heterodimers with Bcl-2 and Bcl-xL via their BH3 domains
- CELL SURVIVAL
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
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
What kind of cells does apoptosis occur in?
- Harmful cells (e.g. cells with viral infection, DNA damage).
- Developmentally defective cells (e.g. B lymphocytes expressing antibodies against self antigens).
- Excess / unnecessary cells: (embryonic development: brain to eliminate excess neurons; liver regeneration; sculpting of digits and organs).
- Obsolete cells (e.g. mammary epithelium at the end of lactation).
- Exploitation - Chemotherapeutic killing of cells.
What mechanisms do effector caspases use to execute the apoptotic program?
Loss of function: 1) Cleave a protein 2) disassemble a complex Gain of function: 3) Direct cleavage 4) Cleavage of inhibitory molecules
What effect does the main action of PI3-K 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
- phosphorylates and inactivates caspase 9
- PKB also inactivates FOXO transcription factors
Whatdo FOXO transcription factors do?
FOXOs promote expression of apoptosis-promoting genes
