Apoptosis Flashcards
What is apoptosis also known as?
Programmed cell death
Why do we have programmed cell death?
To remove:
- 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 - this happens during embryonic development e.g:
- Brain to eliminate excess neurons
- Sculpting of digits and organs
- Sculpting of digits prevents webbing of hands (fingers being joined together)
- Liver regeneration
- Apoptotic cells releasing growth signals that stimulates the proliferation of progenitor cells
- Obsolete organs
- e.g. Mammary epithelium at the end of lactation
We can also exploit apoptosis → chemotherapeutic killing of cells
Define apoptosis.
- Regulated cell death
- Controlled disassembly of cellular contents without disruption
- NO inflammatory response
Define necrosis.
- Unregulated cell death
- Associated with:
- Trauma
- Cellular disruption
- Inflammatory response
Describe the process of necrosis.
- Plasma membrane becomes permeable
- Plasma membrane damaged due to trauma so it becomes more permeable
- There is cell swelling and rupture of cellular membranes
- More water can enter the cell due to increased permeability → rupture
- Proteases are released → autodigestion and dissolution of the cell
- Lysosomes rupture → lysosomal enzymes (including proteases) released into cyctoplasm → autodigestion
- Dissolution - i.e. cell dissolves in ECF
- Localised inflammation
- Cellular contents released into ECF
- Once cellular contents ae no longer contained in the cell, they are perceived as DAMPs → triggers inflammatory response
Describe the process of apoptosis.
Apoptosis has two phases:
- Latent phase
- Execution phase
Latent phase
- Death pathways are activated
- BUT cells appear morphologically (structurally) the same
Execution phase
- Loss of microvilli and intercellular junctions
- Cell shrinkage
- Loss of plasma membrane asymmetry
- Phosphatidylserine (phospholipid) is normally in the inner leaflet
- However, during apoptosis it appears in the outer leaflet
- This allows the apoptotic cell to be recognised and removed by phagocytes
- Chromatin and nuclear condensation
- DNA fragmentation
- Formation of membrane blebs
- Bleb = bulge of plasma membrane
- Fragmentation into membrane-enclosed apoptotic bodies
- Therefore, no inflammation
Why is there no inflammation in apoptosis?
Because the plasma membrane remins intact
- This is unlike necrosis where the plamsa membrane ruptures and cellular contents is released into the ECF
-
Once cellular contents ae no longer contained in the cell, they are perceived as DAMPs → triggers inflammatory response
- DAMP = damage associated molecular pattern
How is DNA modified in apoptosis and how can you tell this experimentally?
DNA modificaton - DNA fragmentation
Experimental evidence:
- DNA ladders on agarose gel
- Lots of DNA fragments of different sizes forms a ladder-like appearance during gel electrophoresis
- More DNA ‘ends’
- These are labelled by adding an extra fluorescently-tagged base
- This method for detecting DNA fragmentation is known as a TUNEL assay
What are apoptotic bodies removed by?
Apoptotic bodies are phagocytosed by surrounding cells which can include:
- Non-professional phagocytes - e.g. epithelial cells
- Macrophages
Give 4 types of cell death? Define the last 2.
- Apoptosis (PCD)
- Necrosis
- Apoptosis-like PCD
- Necrosis-like PCD
PCD = programmed cell death
Apoptosis-like PCD
- Some, but not all, features of apoptosis
- Example of a difference:
- In apoptosis-like PCD, you have display of phagocytic recognition molecules before plasma membrane lysis
Necrosis-like PCD
- Variable features of apoptosis before cell lysis
- You don’t get chromatin condensation
-
But you get varying degrees of other apoptosis-like features
- e.g. Externalisation of phosphatidylserine before lysis
- ‘Aborted apoptosis’
- A standard apoptosis programme is initiated
- But is blocked at the level of caspase activation, so takes alternative, caspase-independent routes
REMEMBER:
- There are more than just these 4 types of cell death
- Essentially there is a graded response of cell death
- Cell death can be apoptosis, necrosis, or anywhere in between the two
What are caspases?
Caspases = cysteine-dependent aspartate-directed proteases
Caspases are:
- Executioners of (i.e. execute) apoptosis
- Activated by a proteolysis cascade
How they work:
- They have a cysteine residue in their active site that is required for their activity
- They cleave proteins just after their aspartate residue
NOTE: Residue = monomer
What are the classes of caspases? Desribe each class.
INITIATOR CASPASES
These are caspases: 2, 9, 8, 10
FUNCTION:
- Trigger apoptosis by cleaving and activating the effector caspases
STRUCTURE:
- They have 2 subunits: p20 and p10
- Subunit = separate polypeptide chain
- But p20 and p10 are initially joined (part of the same polypeptide) on the procaspase protein
- The procaspases have an prodomains
-
Caspase 2 and 9: CARD
- Caspase recruitment domain
- Caspase 8 and 10: DED (x2)
- Death effector domain
-
Caspase 2 and 9: CARD
- These pro-domains allow homotypic (within the same type) protein-protein interactions
EFFECTOR CASPASES
These are caspases: 3, 6, 7
FUNCTION:
- Carry out the apoptotic programme
STRUCTURE:
- They have 2 subunits: p20 and p10
How do procaspases mature to become active caspases?
- Caspases activated by proteolytic cleavage to form a large subunit (LS) and small subunit (SS)
- Initiator caspases also have a prodomain which is cleaved and removed
- 2LS + 2SS → active heteroteramer (i.e. active caspase)
EXPLANATION (extra):
Initiator caspases - e.g. caspase 8
- Initiator procaspases have a prodomain (e.g. DED)
- This allows recruitment of other initiator procaspases of the same type → oligomerisation
- e.g. Oligomerisation between procaspase 8 molecules
- Oligomerisation allows the procaspases to cleave each other → transcleavage
- This forms a:
- Large subunit (LS) - p20
- Small subunit (SS) - p10
- Prodomain - removed
- LS and SS from each procaspase (so 2LS and 2SS) fold to form a heterotetramer
Effector caspases - e.g. caspase 3
- Initiator caspases cleave the effector procaspases to form a:
- Large subunit (LS) - p20
- Small subunit (SS) - p10
- LS and SS from each pro-caspase (so 2LS and 2SS) fold to form a heterotetramer
What is the main purpose of the caspase cascade?
- Amplification
- Divergent responses
- Regulation
REMEMBER: Initiator caspases trigger apoptosis by cleaving and activating effector caspases which carry out the apoptotic programme
NOTE:
- Caspase 2 is an initiator caspase as it can undergo transcleavage
- But it can also be cleaved by other caspases which makes it like an effector caspase
How do effector caspases execute the apoptotic programme?
Effector caspases carry out their function by cleavage
They can:
-
Inactivate proteins or complexes
- e.g. Cleavage of nuclear lamins → nuclear breakdown
-
Activate enzymes by:
- Direct cleavage of pro-enzyme (zymogen)**
- Cleavage of inhibitory molecules, releasing the active enzyme
- e.g. Caspase-activated DNase (CAD)
- Types of enzymes activated:
- Protein kinase
- Nuclease → DNA fragmentation (e.g. CAD)
What are the two mechanisms of caspase activation?
Death by design – receptor-mediated (extrinsic) pathways
Death by default – mitochondrial (intrinsic) death pathway
Describe the structure of death receptors and their ligands.
All cells have death receptors on their surface
They have a:
- Extracellular cysteine-rich domain
- Transmembrane domain
- Intracelllular cytoplasmic tail which contains the death domain (DD)
Death receptor ligands:
- Are trimers → receptors trimerise
- Can be:
- Secreted (from other cells)
- Transmembrane - present on the plasma membrane of another cell
In receptor-mediated apoptosis, what do the death receptors interact with?
Death receptors interact with adaptor proteins - there are two:
- FADD
- FLIP
FADD
- Activates apoptosis
- Structure - two main domains:
- DED (death effector domain)
- DD (death domain)
FLIP
- Inhibits apoptosis
- Structure:
- 2 DED domains
- The long form (FLIPL) can also have a caspase-like domain
DD and DED domains bind to similar domains on other proteins
Describe the signalling process through death receptors (extrinsic pathway).
EXAMPLE:
- Death receptor = Fas
- Fas is upregulated on infected cells
- Ligand = Fas ligand (FasL)
- Fas L - trimeric transmembrane protein expressed on lymphocytes
Process:
- Trimeric ligand binds to death receptor → receptor trimerisation
- The receptor then recruits the adaptor protein FADD
- This is done via interactions between the DD domain of the receptors and the DD domain of FADD
- The FADD molecules can then recruit pro-caspase 8 molecules → procaspase 8 oligomerisation
- This is done via interaction between the DED domain of FAD and the DED pro-domain of procaspase 8
- All of this results in formation of DISC (death-inducing signalling complex) - composed of:
- Death receptor
- FADD
- Procaspase 8
- Oligomerisation of procaspase 8 (DISC formation) results in cleavage and activation
Describe how oligomerisation of procaspase 8 results in their activation.
- Oligomerisation of procaspase 8 molecules brings them into close contact
- This allows transcleavage of procaspase 8
- One procaspase in the oligomer cleaves the other
- Cleavage → large and small subunit (gets rid of prodomain)
- Transcleavage can happen because:
- Some initiator procaspases have intrinsic low catalytic activity
- Oligomerisation triggers a conformational change in some initiator caspases, activating them
- Due to transcleavage, you need at least 2 procaspases to form an active tetramer
- Because one procaspase has to cleave the other
How does FLIP inhibit procaspase 8 activation?
- FLIP has 2 DED domains at its N-terminus, same as procaspase 8
- i.e. They are homologous in terms of their DED domains
- This means that FLIP can also interact with FAD via their DED domains
- i.e. Interaction between the DED domain of FAD and the DED domain of FLIP
- This means that FLIP competes with procaspase 8 for binding to FADD
- However, FLIP has no proteolytic activity
- So it incorporates into the receptor-procaspase complexes and prevents procaspase activation by interfering with transcleavage
What does caspase 8 do once activated?
Caspase 8 activates downstream effector caspases (in a proteolytic cleavage cascade)
- The effector caspases carry out the apoptotic programme
Describe the mitochondrial regulation of apoptosis (intrinsic pathway).
- Cellular stresses
- Loss of mitochondrial membrane potential
- Release of cytochrome c and other apoptosis inducing factors from mitochondria
- Formation of the apoptosome complex
NOTE: Each step leads to the next
Give examples of cellular stresses which can lead to loss of the mitochondrial membrane potential.
- Lack of or overstimulation by growth factors
- p53 activation due to DNA damage
- Reactive oxygen species (ROS)
NOTE: Mitochondrial membrane potential refers to the inner membrane
Describe the formation of the apoptosome.
The apoptosome (‘wheel of death’) is made up of:
- APAF-1 (apoptotic activating factor-1)
- Cyctochrome c
- ATP
- Procaspase 9
APAF-1 structure
- CARD domain (at its N-terminus)
- CARD = caspase recruiting domain
- ATPase domain
- WD-40 repeats
- Made up of repeats of a sequence of approx. 40 AAs
- Mediate protein-protein interactions
Apoptosome formation:
- When cytochrome c binds to the WD-40 repeats of APAF-1 , the APAF-1 proteins come together to form a heptamer
- Also requires ATP (binding to the APAF-1)
- The CARD domains of the heptamer can then interact with procaspase 9 molecules via their CARD domains
- So each Apaf-1 in the heptamer can potentially bind a procaspase 9
- Interaction between the APAF-1 heptamer and procaspase 9 molecules → procaspase 9 oligomerisation
- Oligomerisation → transcleavage of procaspase 9 → active tertramer released (2LS + 2SS)
What does caspase 9 do once activated?
Initiate a caspase cascade (proteolytic cleavage cascade)
What determines whether a cell will undergo apoptosis or necrosis?
Energy levels in the cell determines whether it will undergo apoptosis OR necrosis
- Apoptosis requires energy because the apoptosome requires ATP
What is the role of Bid?
- Bid links the receptor-mediated and mitochondrial death pathways
- Caspase 8 from the receptor-mediated pathway cleaves Bid, activating it
- BID enhances release of mitochondrial proteins (including cytochrome c), thus engaging the intrinsic pathway
- The major difference between the intrinsic and extrinsic is that the intrinsic pathway requires ATP
- So, whichever pathway is stimulated initially, the intrinsic pathway is involved in apoptosis
- Therefore, apoptosis is an active process - requires ATP
What are the Bcl-2 family proteins?
These are intrinsic modulators of apoptosis (i.e. modulate the intrinsic pathway)
There are 3 main groups of proteins in the Bsl-2 family
- They all contain a BH3 domain
- BH3 is a dimerisation motif
- Allows proteins in the Bcl-2 family to associate and dimerise with each other
- BH3 is a dimerisation motif
- Some of the proteins contain other domains including a transmembrane domain
- The transmembrane domain attaches these proteins to the outer mitochondrial membrane
- Group 3 is the BH3 only family
- Can have a transmembrane domain but no other BH domains
What two categories are the Bcl-2 family proteins divided into?
Anti-apoptotic
- These include:
- Bcl-2
- Bcl-xL
- They inhibit apoptosis
- They are localised to the mitochondria
- Specifically the outer mitochondrial membrane
Pro-apoptotic
- These include:
- Bid
- Bad
- Bax
- Bak
- They promote apoptosis
- They move between the cytosol and mitochondria
- Specifically the outer mitochondrial membrane
Describe the two pathways which can occur when a growth factor binds to a receptor.
Pathway 1
- Growth factor (ligand) binds to growth factor receptor (tyrosine kinase receptor)
- This leads to receptor dimerisation → cross-phosphorylation → docking of Grb2 (adaptor protein) → activates Ras → ERK cascade → cell growth (and proliferation)
Pathway 2
- Growth factor (ligand) binds to growth factor receptor (tyrosine kinase receptor)
- This leads to receptor dimerisation → cross-phosphorylation → docking of a different protein: PI3-K → activates PI3-K pathway → cell survival (i.e. anti-apoptotic effects) and proliferation
Describe the PI3-K signalling pathway.
PI3-K = phosphatidylinositol 3-kinase
- A lipid kinase (NOT a protein kinase)
- i.e. phosphorylates lipids not proteins
- Involved in:
- Control of cell growth
- Cell survival
Pathway:
- PI3-K binds to the receptor → PI3-K activated
- Once activated PI3-K phosphorylates PIP2 → PIP3
- PIP2 is a phospholipid present in the plasma membrane
- PIP3 activates protein kinase B (PKB)
- PKB is also known as Akt
- PKB is anti-apoptotic
IMPORTANT:
- PI3-K has 2 subunits
- p85 - adaptor
- p110 - kinase (i.e. catalytic)
- PI3-K binds to phosphorylated tyrosines on the tyrosine kinase receptor via its adaptor subunit
- p85 subunit has SH2 domains which allow this
- This activates the kinase subunit which catalyses PIP2 → PIP3
How does PKB/Akt block apoptosis to induce cell survival?
- Phosphorylates and inactivates Bad
- Phosphorylates and inactivates caspase 9
- Inactivates FOXO transcription factors
- FOXOs promote expression of apoptosis-promoting genes
- Promotes the intrinsic and extrinsic pathway
- FOXOs promote expression of apoptosis-promoting genes
- Other - e.g. stimulates ribosome production and protein synthesis
- Impaired ribosome biogenesis → p53-mediated apoptosis
- So stimulating ribosome production prevents this
- Also, in general, you need ribsome production to allow protein synthesis
- And you need sufficient protein synthesis, so the cell has enough resources (e.g. enzymes) to carry out its normal function and survive
Describe how the Bcl-2 family of proteins regulate apoptosis.
Cell survival (picture A)
- The anti-apoptotic proteins (Bcl-2. Bcl-xL) form a heterodimer with the the pro-apoptotic proteins (Bax, Bak)
- Dimerisation via BH3 domains
- Both pro- and anti-apoptotic proteins have BH3 domains
- Heterodimer on outer mitochondrial membrane
- Dimerisation via BH3 domains
-
PKB phosphorylates Bad (pro-apoptotic)
- This results in Bad forming a heterodimer with the protein 14-3-3
- REMEMBER: This requires presence of a growth factor stimulating the PI3-K pathway
- The pro-apoptotic proteins are held inactive in their heterodimers
Apoptosis (picture B)
- Growth factor absent → no PI3-K pathway → PKB does not phosphorylate Bad → Bad is active
- Bad binds to Bcl-2 and Bcl-xL, displacing the pro-apoptotic proteins (Bax, Bak)
- i.e. Bax and Bak released from their heterodimer → active
- Bax and Bak then form a pore in the outer mitochondrial membrane → cytochrome C released from mitochondria
- Bax and Bak oligomerise to form a pore complex
- Once cytochrome C is in the cytosol, it can stimulate the formation of the apoptosome → apoptosis
What is the role of PTEN?
- It’s a lipid kinase which dephosphorylates PIP3 back to PIP2
- This turns off activation of PKB and the PI3-K pathway
- This pathway promotes cell survival and protein synthesis (and proliferation)
- So by turning off this pathway, it promotes apoptosis
- By turning off a pathway that promotes cell survival and proliferation, PTEN acts as a tumour suppressor
- You need this negative regulation to prevent the cell from dividing uncontrollably
What do IAPs do?
IAP = Inhibitor of Apoptosis Proteins
IAPs inhibit (regulate) the extrinsic pathway of apoptosis by:
- Binding to procaspases and preventing activation
- Binding to active caspases and inhibiting their activity
Summarise how the intrinsic and extrinsic pathways of apoptosis are inhibited.
NOTE: Inhibiting the apoptotic pathways is cytoprotective because it protects the cell from death
Intrinsic pathway inhibition:
- Bcl-2
- Bcl-xL
Extrinsic pathway inhibition:
- FLIP
- IAPs
Both pathways:
- Growth factor pathways via PI3’-K and PKB/Akt
State some proto-oncogenes and tumour suppressors associated with apoptosis.
Proto-oncogenes:
- Bcl-2
- PKG/Akt
EXPLANATION (extra):
- These are anti-apoptotic genes → promote cell survival
- So, if these genes mutated, you get cell survival even when apoptosis should occur (e.g. DNA is damaged)
- Cell survival allows cell proliferation, which could lead to tumour formation
Tumour suppressor:
- PTEN
- It inhibits the PI3-K pathway which promotes cell survival and proliferation
- This negative regulation prevents the cell from dividing uncontrollably
State some therapeutic uses of apoptosis.
- Stimulating apoptosis in harmful cells
- e.g. Cells with viral infection or DNA damage
- These cells can be oncogenic (can potentially cause cancer)
- Chemotherapeutic killing of tumour cells
- e.g. Dexamethasone stimulates DNA cleavage
- DNA fragmentation is a key feature of apoptosis
- e.g. Dexamethasone stimulates DNA cleavage
