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 the cell
- There is localised inflammation as immune cells are attracted
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 it from necrosis?
- Plasma membrane remains intact – no inflammation
What DNA modification is seen during apoptosis and by what techniques can you observe these changes?
- 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 - some but not all features of apoptosis. There may be a display of phagocytic recognition molecules, even before cell surface membrane lysis
- 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
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 (one large and one small) subunits found in effector caspases
- … but they also have a targeting subunit (protein-protein interacting domain)
- They have CARD and DED motifs that effector caspases do not have
How do effector caspases actually carry out the apoptotic programme - give 2 example mechanisms?
- Cleave or inactivate various proteins and complexes - e.g. nuclear lamins leading to nuclear breakdown
- Activating enzymes by cleavage or indirect cleavage of inhibitor molecules - e.g. Caspase Activated DNAases which break down the DNA
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?
- Extrinsic - Death by design (receptor-mediated)
- Intrinsic - Death by default (mitochondrial 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
Describe signalling of apoptosis through Fas
- Fas ligand (FasL) binds to Fas receptor (on cytotoxic T lymohocytes) and the Fas receptors undergo trimerisation, which brings the three DDs in the intracellular domain 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 and its own DED
- Binding of procaspase 8 to FADD forms DISC (death-induced signalling complex)
- DISC formation results in cross-activation of procaspase 8 by allowing cleavage
- Active caspase 8 is released and forms an active caspase 8 tetramer, which then activates effector caspases
Describe the importance of oligomerisation in the Fas / FasL pathway
- Some initiator caspases have intrinsic low catalytic activity
- Oligomerisation brings them close enough together to allow transcleavage which allows activation
- Also, at least 2 procaspases are required to form an active caspase tetramer
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 therefore incorporate into receptor-procaspase complexes or in other words in between the procaspases that have bound with FADD and therefore prevent cross-activation of the pro-caspases by interfering with the trans-cleavage
- So you don’t form the active caspase-8 tetramers that would otherwise go on to activate the effector caspases involved in apoptosis
As an overview, describe death by default (intrinsic pathway)
- 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 and briefly state their roles
- CARD domain - caspase recruitment domain - attracts procaspase 9
- ATPase domain - cleaves ATP required to provide energy for binding
- WD-40 repeats (protein-protein interactions) - binding site for cytochrome C
Explain fully, how the intrinsic (death by default) pathway leads to caspase activation and therefore apoptosis
- The cytochrome C released from the mitochondria bind to the WD-40 repeats of APAF-1 and make it form a heptamer structure (apoptosome)
- This requires ATP, and APAF-1 uses its ATPase domain for this
- 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 APAF-1 molecules and their close contact allows them to cross-cleave each other to generate activate caspase 9
- Active caspase 9 can go on to activate effector caspases which ultimately carry out the apoptotic programme
1) What type of caspases are caspase 8 and 9?
2) How do caspase 8 and 9, activated by the death of design and death by default apoptotic pathways result in the actual carrying out of the apoptotic programme?
1)
- Initiator caspases
2)
- They both converge on activating caspase 3 - an effector caspase which is the thing that actually carries out the apoptotic pathway
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
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 promoting cell survival
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
Outline the whole pathway that goes from growth factor binding then via PI3-K and how it has its effects in regards cellular survival and apoptosis
- Growth factors bind receptors
- This causes dimerisation of these receptors
- This dimerisation results in cross-phosphorylation of tyrosine kinase domains within the intracellular parts of the receptor
- This phosphorylation causes morphological change allowing binding of adaptor proteins
- An adaptor binds which then recruits PI3-K
- PIP3-K (kinase) carries out PIP2 → PIP3
- 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 (a member of the BCL-2 anti-apoptotic family) and has many other effects
- Therefore it promotes cell survival and proliferation
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 is allowed to get 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 promotes the expression of apoptosis-promoting genes)
Outline regulation of apoptosis by BCL-2 family proteins via BH3 heterodimerisation, and thus go over again (there’s another flashcard in here about it) how absence of growth factors and the PI3-K pathway leads to apoptosis
- Pro-apoptotic proteins belonging to the BCl-2 family including Bax and Bak are held in their inactive heterodimers by their BH3 domains by the anti-apoptotic proteins belonging to the BCl-2 family including BCl-2 and BCl-xl
- When growth factors are absent, the PIK-3 pathway is inactive
- So PIP3 is not formed
- So PKB is not activated
- This means that Bad cannot be phosphorylated and held in an inactive heterotrimer
- Therefore it is dephosphorylated in the absence of active PKB
- Thus it is released from its heterotrimer
- Now it can form pores in the mitochondrial membrane, which allows cytochrome C to escape into the cytosol and induce apoptosis - cytochrome C apoptosome thing
1) Name two extrinsic regulators of apoptosis (anti-apoptotic) and describe their actions
2) Then mention an intrinsic anti-apoptotic regulator - no need to describe their actions
1)
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
2)
Anything in the BCL-2 family
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
