Apoptosis Flashcards
Define Necrosis.
Unregulated cell death associated with trauma, cellular disruption + 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 more permeable: the cell swells + membrane ruptures
Proteases are released leading to dissolution + autodigestion of the cell
There is localised inflammation
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 + intercellular junctions
Cell shrinkage
Loss of plasma membrane asymmetry
Chromatin + nuclear condensation
DNA fragmentation
Formation of membrane blebs
Fragmentation into membrane enclosed apoptotic bodies (digested by macrophages)
What is an important feature of apoptosis that distinguishes it from necrosis?
Plasma membrane remains intact so there is no inflammation
What DNA modification is seen during apoptosis?
Fragmentation of DNA ladders (seen in agar gel)
Formation of more “ends”, which are labelled by adding an extra fluorescently-labelled bases in a TUNEL assay
What other types of cell death are there other than necrosis and apoptosis?
Cell death is GRADED:
Apoptosis-like cell death (display phagocytic recognition molecules before membrane lysis)
Necrosis-like cell death (features of apoptosis before lysis, like an aborted apoptosis)
What are caspases?
Cysteine-dependent aspartate-directed proteases
Executioners of apoptosis
Activated by cleavage (Proteolysis)
Which caspases are effector caspases?
3, 6 + 7
Which caspases are initiator caspases?
2, 9, 8 + 10
Describe the structure of effector caspases.
Single chain polypeptides consisting of a small (p10)+ large (p20) subunit
Subunits are released by proteolytic cleavage
Describe the structure of initiator caspases.
They have the same 2 subunits found in effector caspases but they also have a targeting subunit (for homotypic protein-protein interactions)
What are the two types of targeting subunit that initiator caspases can have?
CARD: caspase recruitment domain (Caspase 2 + 9)
DED: death effector domain (Caspase 8 + 10)
How are active caspases formed?
Cleavage of inactive procaspases is followed by the folding of 2 large + 2 small chains to form an active L2S2 heterotetramer
What are the two mechanisms of apoptosis
Death by design (receptor-mediated, extrinsic)
Death by default (mitochondrial (intrinsic))
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)
FLIP: negative regulator (DED + DED)
Describe signalling of apoptosis through Fas.
Fas ligand (on CTLs) binds to Fas receptor
Fas receptors undergo trimerisation, which brings the 3 DDs together
Trimerised DDs recruit FADD, which binds via its own DD
FADD recruits + 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 the death by design pathway
Some initiator caspases have intrinsic low catalytic activity
Oligomerisation brings them close enough together to allow transcleavage
Thus, at least 2 procaspases are required to form an active caspase
Describe how FLIP acts as an inhibitor of apoptosis
FLIP has 2 DED domains but no proteolytic activity so can compete with procaspase 8 to bind to the DED domains of FADD
It can incorporate into receptor-procaspase complexes + interfere with transcleavage
As an overview, describe death by default.
Cellular stress causes change in mitochondrial membrane potential
Mitochondrion releases cytochrome C + other apoptotic inducing factors
This stimulates formation of the heptameric 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 (for protein-protein interactions)
Explain fully, how death by default leads to caspase activation.
Cytochrome C released from mitochondria binds to WD-40 repeats of APAF-1 + leading to formation of a heptamer structure (apoptosome)
This requires ATP
It has 7 central CARD domains, which can interact with CARD domains of procaspase 9
7 procaspase 9’s bind via their CARD domains to the apoptosome
Their close contact allows them to cross-cleave each other to generate active caspase 9, which initiates a caspase cascade, resulting in apoptosis
What pro-apoptotic protein links the death by default and death by design pathways? Explain how it works.
Bid Caspase 8 (generated by death by design pathway) cleaves Bid, which travels to the mitochondrion + promotes release of cytochrome C, thus triggering the mitochondrial death pathway
What may determine whether a cell undergoes apoptosis or necrosis?
Apoptosis requires energy (ATP) whereas necrosis does not
What is an 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: 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
Found localised on the mitochondrial membrane
What are the pro-apoptotic Bcl-2 proteins and where are they found?
Bid Bad Bax Bak Found in the cytoplasm + in the mitochondrial membrane
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 domain (binds to tyrosine phosphorylated receptor) Kinase domain
What is the main action of PI3-K?
PI3-K converts PIP2 to PIP3
What effect does conversion of PIP2 to PIP3 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
Has mitogenic + anti-apoptotic signal
Describe the arrangement of the anti-apoptotic and pro-apoptotic proteins when growth factor signalling and the PI3-K pathway is active.
PKB/Akt is activated meaning Bad is phosphorylated + inactivated (held in an inactive heterodimer with 14-3-3 protein)
On the mitochondrial membrane, Bak + Bax are held in inactive heterodimers with Bcl-xL + Bcl-2
Describe how loss of growth factor signalling can lead to apoptosis.
Loss of activation of the PI3K pathway so less PIP3, so less activation of PKB/Akt
Bad gets dephosphorylated + dissociated from its inactive heterodimer
Bad moves to the mitochondrial membrane + binds to the anti-apoptotic proteins (Bcl-2 + Bcl-xL) via its BH3 domain
This displaces Bax + Bak from their inactive heterodimers
Bax + Bak form a pore in the mitochondrial membrane allowing 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 + promotes apoptosis
IAPs (Inhibitor of Apoptosis proteins):
Binds to procaspases + prevents their activation
Can bind to activate caspases + inhibit their activity
Are the following tumour suppressor genes or oncogenes?
a. Bcl-2
b. PTEN
c. PKB/Akt
Bcl-2
Oncogene: increased activation would mean reduced likelihood of apoptosis (cancers are anti-apoptotic)
PTEN
Tumour suppressor gene: inactivation will mean reduced likelihood of apoptosis
PKB/Akt
Oncogene
What cells would require programmed cell death?
Harmful cells (damaged DNA/ Infected) Developmentally defective cells (B cells expressing antibodies against self) Excess cells (sculpting of digits + organs) Obsolete cells (mammary epithelial at end of lactation)
What are caspase cascades? What do they result in?
Initiator caspases trigger apoptosis by cleaving + activating effector caspases, which carry out the apoptotic programme
Cascades generate amplification of signal, divergent responses + points for regulation
What actions can caspases have?
Cleave + inactivate proteins/ complexes e.g. nuclear lamins for nuclear breakdown
Activate enzymes e.g. protein kinases by direct cleavage or cleavage of inhibitory molecules
Summarise the effects of PKB/Akt in promoting cell survival.
Phosphorylates + inactivates Bad + caspase 9
Inactivates FOXO transcription factors (which promote expression of apoptosis promoting genes)
Stimulates ribosome production + protein synthesis
List the cytoprotective anti-apoptotic pathways
Bcl-2, Bcl-xL (intrinsic pathway)
FLIP, IAPs (extrinsic pathway)
Growth factor pathways via PI3’-K + PKB/Akt
