Apoptosis Flashcards
1
Q
What is apoptosis also known as?
A
Programmed cell death
2
Q
Why do we have programmed cell death?
A
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
3
Q
Define apoptosis.
A
- Regulated cell death
- Controlled disassembly of cellular contents without disruption
- NO inflammatory response
4
Q
Define necrosis.
A
- Unregulated cell death
- Associated with:
- Trauma
- Cellular disruption
- Inflammatory response
5
Q
Describe the process of necrosis.
A
- 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
6
Q
Describe the process of apoptosis.
A
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
7
Q
Why is there no inflammation in apoptosis?
A
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
8
Q
How is DNA modified in apoptosis and how can you tell this experimentally?
A
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
9
Q
What are apoptotic bodies removed by?
A
Apoptotic bodies are phagocytosed by surrounding cells which can include:
- Non-professional phagocytes - e.g. epithelial cells
- Macrophages
10
Q
Give 4 types of cell death? Define the last 2.
A
- 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
11
Q
What are caspases?
A
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
12
Q
What are the classes of caspases? Desribe each class.
A
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
13
Q
How do procaspases mature to become active caspases?
A
- 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
14
Q
What is the main purpose of the caspase cascade?
A
- 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
15
Q
How do effector caspases execute the apoptotic programme?
A
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)
17
Q
Describe the structure of death receptors and their ligands.
A
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
18
Q
In receptor-mediated apoptosis, what do the death receptors interact with?
A
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
19
Q
Describe the signalling process through death receptors (extrinsic pathway).
A
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
20
Q
Describe how oligomerisation of procaspase 8 results in their activation.
A
- 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
21
Q
How does FLIP inhibit procaspase 8 activation?
A
- 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
22
Q
What does caspase 8 do once activated?
A
Caspase 8 activates downstream effector caspases (in a proteolytic cleavage cascade)
- The effector caspases carry out the apoptotic programme
27
Q
What determines whether a cell will undergo apoptosis or necrosis?
A
Energy levels in the cell determines whether it will undergo apoptosis OR necrosis
- Apoptosis requires energy because the apoptosome requires ATP
28
Q
What is the role of Bid?
A
- 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
29
Q
What are the Bcl-2 family proteins?
A
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
31
Q
Describe the two pathways which can occur when a growth factor binds to a receptor.
A
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
32
Q
Describe the PI3-K signalling pathway.
A
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
34
Q
Describe how the Bcl-2 family of proteins regulate apoptosis.
A
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
35
Q
What is the role of PTEN?
A
- 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
