10. Apoptosis Flashcards
1
Q
Why do we need programmed cell death?
A
To remove:
- Hamful cells (e.g. with viral infecion, DNA damage)
- Developmentally defective cells (e.g. B lymphocytes expressing antibodies against self-antigens)
- Excess/unecessary cells:
- Embyronic development: e.g. brain to eliminate excess neurones; liver regeneration; sculpting of digits and organs)
- Obsolete organs (mammary epithelium at the end of lactation)
- Exploitation - chemotherapeutic killing of cells
2
Q
Define necrosis.
A
unregulated cell death associated with trauma, cellular disruption and an INFLAMMATORY RESPONSE
3
Q
Define apoptosis.
A
regulated cell death; controlled disassembly of cellular contents without disruption - NO INFLAMMATORY RESPONSE
4
Q
Describe what happens in necrosis.
A
- The plasma membrane becomes permeable
- There is cell swelling and rupture of cellular membranes
- Proteases are released leading to autodigestion and dissolution of the cell
- Localised inflammation
5
Q
Describe what happens in apoptosis.
A
- 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 (phosphatidylserine lipid appears in outer leaflet)
- Chromatin and nuclear condensation
- DNA fragmentation
- Formation of membrane blebs
- Fragmentation into membrane-enclosed apoptotic bodies
- IMPORTANT FEATURE OF APOPTOSIS: plasma membrane remains INTACT - so there is NO inflammation
- Once the cell have broken down in apoptosis, the apoptotic bodies are taken up by macrophages
-
DNA modification occurs during apoptosis. this leads to:
- Fragmentation of DNA ladders (seen in agarose gel)
- Formation of more ‘ends’, which are laballed by adding an extra fluorescently-tagged base in a TUNEL assay.
6
Q
What are other types of cell death?
A
- Apoptosis-like programmed cell death - has some, but not all features of apoptosis. Display of phagocytic recognition molecules before plasma membrane lysis.
- Necrosis-like programmed cell death - displays variable features of apoptosis before cell lysis - this is like an ‘aborted’ apoptosis that ends up being necrosis
- So cells quite often die of something that is in between necrosis and apoptosis - it is a graded response
7
Q
What are the mechanisms of apoptotic cell death?
A
- The executioners - Caspases
- Initiates the death programme
- Death receptors
- Mitochondria
- The Bcl-2 family
- Stopping the death programme
8
Q
What are the functions of Caspases?
A
- Caspases - Cysteine-dependent aspartate-directed proteases**
- They have cysteine residue in their active site that is required for their activity
- They cut proteins just after their aspartate residue
- They are activated by proteolysis
- They take part in a cascade of activation
9
Q
Describe the classes of caspases.
A
-
Effector Caspases (3, 6 and 7)
- They start as a single chain polypeptide with 2 subunits (large and small)
- The subunits are released by proteolytic cleavage during maturation
-
Initiator Caspases (2, 8, 9 and 10)
- They also have the same 2 subunits that are found in effector caspases
- They also have an extra targeting subunit (protein-protein interacting domain)
- The targeting subunit directs them to a particular location
- CARD - Caspase Recruitment Domain
- DED - Death Effector Domain
10
Q
Describe Caspase maturation.
A
- Procaspases (zymogens) are single chain polypeptides
- To become activated, the procaspases must undergo proteolytic cleavage to form large and small subunits
- NOTE: Initiator caspases must also be cleaved to release the targeting subunit
- These cleavages are done by the caspases themselves
- After the cleavage, you get folding of 2 large and 2 small chains to form L2S2 heterotetramer
11
Q
Describe the caspase cascade.
A
12
Q
What is the function of effector caspases?
A
- Effector Caspases carry out the apoptotic programme in 2 ways:
- Cleaving and inactivating various proteins and complexes (e.g. nuclear lamins leading nuclear breakdown)
- Activating enzymes by direct cleavage, or cleavage of inhibitor molecules (e.g. protein kinases, nucleases such as Caspase-activated DNAse (CAD)
13
Q
Name the mechanisms of caspase activation.
A
- Death by design - receptor-mediated (extrinsic) pathway
- Death by default - mitochondrial (instrinsic) death pathway
14
Q
Describe the death by design pathway of caspase activation.
A
- All cells have death receptors on their surface
- Death receptors consist of:
- Extracellular cysteine-rich domain
- Single transcellular domain
- Cytoplasmic tail (with a death domain)
- These receptors are only activated when they encounter secreted or transmembrane trimeric ligands (e.g. TNF-alpha or Fas) - these are called death ligands
-
Two ADAPTER PROTEINS are very important in this pathway
- FADD - positive regulator (required for the death pathway to become activated) and promotes cell death
- FLIP - negative regulator (inhbits the death pathway and allows it to be regulated)
- Leads to tight control of apoptosis
- FADD and FLIP are different in structure
- FADD = DED + DD
- FLIP = DED + DED
15
Q
Explain signaling through death receptors (e.g. Fas/Fas-ligand)
A
- Fas is a death receptor that is upregulated if apoptosis is required e.g. if a cell is infected by a virus
- The Fas ligand binds the the Fas receptor on the surface of cytotoxic T lymphocytes
- The Fas receptors then undergo trimerisation, which brings the three cytoplasmic DD (death domains) together
- The trimerised death domains recruit the positive adapter protein FADD by its own DD
- The binding of FADD causes recruitment of oligomerisation of procaspase 8 through its DED to the FADD’s DED.
- The binding of procaspase 8 to FADD forms a death-inducing signalling comples (DISC)
- DISC formation results in cross-activation of procaspase 8, whereby they cleave each other within the complex (due to close proximity)
- The activate capase 8 is then released, and it cleaves effector caspases to execute the death programme.
16
Q
A
- Initiator procaspases bind, via their DED domains, to the DED domains of FADD
- This brings three initiator procaspase 8s into close contact, which allows cleavage
- This releases the active initiator caspase 8 tetramer
17
Q
What is the function of FLIP?
A
- Death receptor activation of procaspase 8 is inhibited by FLIP
- FLIP is evolutionarily related to caspases but it has lost its catalytic activity
- It has no proteolytic activity so it can compete with procaspase 8 to bind to the DED domains of FADD
- FLIP competes to bind to the procaspase via the DED domains
18
Q
What is the function of caspase 8?
A
- Caspase 8 activates downstream effector caspases
- The effector caspases go on to carry out the apoptotic programme by activating caspase 3 and 7
19
Q
Describe death by default.
A
- This is the intrinsic pathway whereby cellular stresses (e.g. lack of/overstimulate by growth factors, DNA damage etc.) cause a loss of mitochondrial membrane potential
- This results in the release of cytochrome C and other apoptosis-inducing factors
- These stimulate the formation of an apoptosome complex
20
Q
Describe the apoptosome.
A
- The apoptosome consist of:
- APAF-1 (apoptotic activating factor 1)
- Cytochrome C
- ATP
- Procaspase 9
- At one end, APAF-1 contains a number of repeats that are involved in protein-protein interactions
- There is also an ATPase domain within APAF-1
- At the other end of APAF-1 there is a caspase recruitment domain (CARD), which is also found in some initiator caspases (e.g. caspase 9)
- When cytochrome C binds to the WD-40 repeats on APAF-1, it forms a heptamer (the apoptosome)
- This process also requires ATP
- The CARD domains at the centre of the apoptosome can interact with the CARD domains on procaspase-9 (so even procaspase 9s can bind to the apoptosome)
- The close proximity of the procaspase 9s that bind to the CARD domains of the apoptosome can cross-cleave and activate each othe to produce caspase 9
- The activated caspase 9 is then released, which is able to trigger the caspase cascade, which leads to apoptosis.
21
Q
Describe the principle mechanism of apoptosis.
A
- Bid links the receptor-mediated and mitochondrial death pathways
- When one pathway is triggered, it can trigger the other pathway
- Caspase 8 from the receptor-mediated pathway can cleave Bid, which enhances release of mitochondrial proteins, thus engaging the intrinsic pathway
- The difference between the two mechanisms is that the mitochondrial pathway requires ATP
- Bid promotes the release of cytochrome C from the mitochondrion, which triggers the mitochondrial death pathway
- NOTE: apoptosis is an ACTIVEprocess, which requires energy so the energy levels of a cell may determine whether death is by necrosis (less ATP) or apoptosis (more ATP)
22
Q
What is the function of Bcl-2 family proteins?
A
These are intrinsic modulators of apoptosis
There are THREE main groups of Bcl-2 proteins, all of which contain BH3 domains
Some of the proteins contain other domains including a transmembrane domain
BH3 is a dimerisation motif (for protein-protein interaction) that allows proteins in the Bcl-2 family to associate and dimerise with each other
Members of this family fall into TWO categories:
- Anti-apoptotic proteins - localised to the mitochondrial membrane and INHIBIT apoptosis
- Pro-apoptotic proteins - move between the cytosol and the mitochondrial membrane and they PROMOTE apoptosis
23
Q
Describe the kinase signalling pathway in the cell cycle and apoptosis regulation.
A
- Growth factors may activate TWO growth factor pathways associated with anti-apoptotic effects
- Ligand binding causes dimerisation and cross-phosphorylation of the tyrosine kinase receptors.
- Phosphorylation of the tyrosine kinase receptor initiates signal transduction pathways as well as creating docking sites for adapter proteins (e.g Grb2), which can bind to mediate the protein-protein interactions within the pathways (e.g. activating Ras, which leads to activation of the MAPK/ERK cascase)
- Another phosphorylation site on the tyrosine kinase receptors triggers the PI3-kinase pathway, which is involved in cell survival and has anti-apoptotic effects
- Phosphatidylinositol 3-kinase (PI3-K) is a lipid kinase involved in growth control and cell survival
- It has 3 main subunits:
- Targeting subunit
- Adapter subunit
- Catalytic subunit
- It phosphorylates PIP2 to PIP3, which is then recognised by the adapter subunit of PKB/akt (Protein kinase B)
- PKB is then recruited to the cell membrane and it is activated - it has anti-apoptotic effects
- PKB phosphorylates and INACTIVATES Bad (part of the Bcl-2 family)
- Other pro-apoptotic proteins (such as Bax and Bak) are held in their inactiveheterodimers (by their BH3 domains) to the anti-apoptotic Bcl-2/xL proteins
- As the pro-apoptotic proteins are held in the inactive heterodimers, cell survival and proliferation are promoted
- When growth factors are ABSENT, the PI3-kinase pathway is not activated, so PIP3 is NOT generate and, hence, PKB is NOT recruited to the cell membrane and activated
- This means that Bad can NOT be phosphorylated and held in an inactive heterodimer (with PKB)
- So the Bad is dephosphorylated and released from the heterodimer
- Bad can then go to the mitochondrial membrane, where it can bind through its BH3 domain to the BH3 domains of the anti-apoptotic Bcl-2 family members thus DISPLACING the pro-apoptotic Bcl-2 family members
- Once the pro-apoptotic Bcl-2 family members (e.g. Bax and Bak) are released from inhibition by the anti-apoptotic Bcl-2 family members, they form a pore in the mitochondrial membrane, which allows cytochrome C to escape into the cytosol and induce apoptosis
24
Q
What are the extrinsic regulators of apoptosis?
A
- PTEN is a lipid phosphatase that counteracts the production of PKB, therefore reducing the regulation of cell survival and promoting apoptosis
- IAPs (Inhibitor of Apoptosis Proteins) bind to procaspases and prevent activation
- IAPs also bind to active caspases and inhibit their activity
25
Q
Name the anti-apoptotic pathways.
A
Bcl-2, Bcl-xL = intrinsic pathway
FLIP, IAPs = extrinsic pathway
Growth factor pathways via PI3-kinase and PKB/Akt
26
Q
What are the proto-oncogenes/tumour suppressors associated with apoptosis?
A
Bcl-2 (oncogene - because over-expression of Bcl-2 will promote cancer)
PKB/Akt (oncogene - because over-expression of PKB/Akt will promote cancer)
PTEN (tumour suppressor - because inactivation of this gene promotes cancer)
