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.
The plasma membrane becomes more permeable – the cell swells and the membrane ruptures
Proteases are released leading to dissolution and autodigestion of thecell 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 and intercellular junctions
Cell shrinkage
Loss of plasma membrane asymmetry
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 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 cell death
Necrosis-like cell death (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 cleavage (proteolysis cascade)
Which caspases are effector caspases?
3, 6 and 7 - carry out apoptosis process
Which caspases are initiator caspases?
2, 8, 9 and 10 - trigger apoptosis
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?
CARD – caspase recruitment domain
DED – death effector domain
How are active caspases formed?
Cleavage of inactive procaspases is followed by the folding of 2 large and 2 small chains 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
FLIP – negative regulator – DED + DED - inhibits death pathway and allows regulation
Describe signalling of apoptosis through Fas (extrinsic pathway).
- Fas receptor is upregulated when cells need to apoptose
- Fas ligand (on cytotoxic T-cells) binds to Fas receptor and the Fas receptors undergo trimerisation–> brings the three death domains (DDs) together.
- The trimerised DDs recruit FADD (adaptor protein), which binds via its own DD
- FADD then recruits and oligomerises procaspase 8 (through the DED (death effector domain) of procaspase 8)
- Binding of procaspase 8 to FADD forms DISC (death-induced signalling complex)
- DISC (death inducing signalling complex) formation results in cross-activation of procaspase 8—> caspase 8
- Active caspase 8 is released, 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 (eg lack of GF)
–>causes a change in (loss of) mitochondrial membrane potential
–>This leads to release of cytochrome C from the mitochondrion
–>This stimulates formation of the apoptosome complex
What does the apoptosome (aka wheel of death) 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
—> make it form a heptamer structure (apoptosome)
This requires 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
Their close contact allows them to cross-cleave each other to generate activate caspase 9’s
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
Bid 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 (ATP) whereas necrosis does not
What is an important family of proteins that act as intrinsicmodulators of apoptosis?
Bcl-2 family
There are three main groups of Bcl-2 proteins. What is common toall three groups?
All contain BH3 domain – this is a dimerisation motif, which allows members of the family to form dimers with each other
Some contain Transmembrane (TM) domains
What are two anti-apoptotic Bcl-2 proteins and where are they found?
Bcl-2 and 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 (move between the two)
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 (not protein)
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–> binds protein kinase B/Akt adaptor subunit
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 (which is pro-apoptopic)
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 (PKB/Akt)
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 inactivates 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 activated 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–overexpression–>^cell survival–>cancer
