Apoptosis 1. Explain the difference between necrosis and apoptosis and describe how they may be differentiated
Apoptosis = programmed cell death (PCD); •
•
Why PCD? 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 – e.g. in embryonic development in the brain to eliminate excess neurones and to sculpt digits and organs Obsolete cells – e.g. mammary epithelium at the end of lactation Chemotherapeutic killing of cells
Necrosis = unregulated cell death associated with trauma, cellular disruption and an inflammatory
response 1.
Plasma membrane becomes
2.
permeable Cell swelling and rupture of cellular membranes
3.
4.
Release of proteases leading to auto-digestion and dissolution of the cell Localised inflammation
Apoptosis (PCD) = regulated death – controlled
1
disassembly of cellular contents and no inflammatory response (e.g. with haematopoietic cells) •
Latent phase of apoptosis: Death pathways activated Cells appear morphologically the same Execution phase of apoptosis: Loss of Microvilli and intercellular junctions Cell shrinkage Loss of plasma membrane asymmetry (phosphatidylserine appears in the outer membrane) Chromatin and nuclear condensation DNA fragmentation Formation of membrane blebs
•
2
3
Fragmentation into membrane enclosed apoptotic bodies Plasma membrane remains intact 1
2
3
After the apoptotic bodies are produced there is phagocytosis by surrounding cells – e.g. macrophages •
2. Discuss whether necrosis and apoptosis are the only forms of cell death There are 4 1. 2.
3. 4.
mechanisms of cell death: (remember 4 deaths and a funeral!) Necrosis – unregulated cell death associated with cellular disruption and an inflammatory response Apoptosis (programmed cell death) – regulated cell death, controlled disassembly of cellular contents, no inflammatory response (e.g. haematopoietic cells) Apoptosis-like PCD – some but not all the features of apoptosis. Display of phagocytic recognition molecules before plasma membrane lysis Necrosis-like PCD – variable features of apoptosis before cell lysis – “aborted apoptosis”
There is evidence of a graded response: Is there a graded response?
Apoptosis
Necrosis
Features of both Programmed cell death
3. Describe the proteolytic caspase cascades which execute the apoptotic response The Mechanisms of Cell Death: • The Executioners – Caspases • Initiation of the Death Programme – Death Receptors, Mitochondria • The Bcl-2 Family The Executioners – The Caspases: • Cysteine-Dependant Aspartate Directed Proteases The executioners of apoptosis Activated by proteolysis Cascade of activation • Other proteases such as calpains and cathepsins may also be involved
There are 3 classes of caspases: 1. Initiator caspases 2. Effector caspases 3. Other caspases •
Caspases have:
CARD – Caspase Recruitment Domain DED – Death Effector Domain • Caspases undergo homotypic protein-protein interactions
Caspase maturation: Pro-caspases (zymogens) are converted into the active enzyme via proteolytic cleavage •
Caspase Cascades: Caspases activate each other in order to provide amplification, a divergent response and regulation • Starts with Caspase 8 • Initiator caspases – trigger apoptosis by cleaving and activating • Effector caspases – carry out the apoptotic programme •
Mechanisms of Caspase Activation: Two main pathways: •
Death by design – receptor mediated
(extrinsic) pathways
•
Death by default – mitochondrial (intrinsic death) path pathway
Death Receptors: • • •
Death receptors are secreted or transmembrane trimeric ligands E.g. TNFR1 – tumour necrosis factor receptor 1 Adapter proteins are needed in receptor-mediated apoptosis
Signalling through death receptors: The death receptor undergoes trimerisation by death ligand Activation of the receptor leads to recruitment of adaptor protein through death domain of receptor Recruitment of adaptor protein causes formation of DISC (death inducing signalling complex) which induces recruitment and oligomerisation of caspase 8 (initiator) The oligomerisation of the initiator caspase leads to cleavage and activation Some initiator pro-caspases have an intrinsic low catalytic activity – oligomerisation allows transcleavage Some pro-caspases are activated by conformational change on oligomerisation Caspase 8 activates downstream effector caspases which execute the apoptotic programme
Signalling through Death Receptors, e.g. Fas/Fas-ligand • Receptor (Fas) trimerization by ligand (Fas-L on lymphocyte)
L
(e.g. Fas)
(Fas-L)
• Recruitment of adapter protein (FADD) through DD DISC
• Recruitment and oligomerization of caspase 8 through DED –> Death-Inducing Signalling Complex (DISC)
Death receptor inhibition: Death receptor activation of caspase 8 is inhibited by FLIP (has caspase 8 homology but no proteolytic activity – acts as an antagonist for the death receptor) •
• •
Competes for binding to receptor tails Incorporates into receptor caspase complexes and interferes with transcleavage FLIP inhibits caspase 8 activation
FLIP • Competes for binding to receptor tails/ FADD • Incorporates into receptor -caspase complexes and interferes with
transcleavage
Effector caspases execute the apoptotic programme: • Effector caspases cleaved in response to caspase cascade initiated by caspase 8 • Cleave and inactivate proteins or complexes (e.g. nuclear laminins leading to nuclear breakdown) • Activate enzymes (including protein kinases, nucleases – e.g. CAD (caspase activated DNase) by direct cleavage of cleavage of inhibitory molecules
4. Discuss how apoptosis may be mediated through death receptors and/or the mitochondria •
For death receptor (extrinsic) mediation of apoptosis see above
Mitochondrial regulation of apoptosis: • • •
Cellular stressors cause loss of mitochondrial membrane potential Release of cytochrome C and other apoptotic inducing factors Formation of the apoptosome complex (Apaf1 + caspase 9)
The apoptosome (the wheel of death): Cytochrome c + Apaf-1 (apoptotic activating factor 1) + ATP + Caspase 9 Each Apaf-1 in the heptameric apoptosome can bind a caspase 9. • Oligomerisation activates caspase 9 which is cleaved, released and initiates a caspase cascade leading to apoptosis • •
The apoptosome : Cytochrome c, Apaf-1 (Apoptotic activating factor -1), ATP, caspase 9
cyt c
Principal mechanisms of apoptosis: • • •
Principal mechanisms of apoptosis: Bid links
Bid links receptor and mitochondrial death pathways Caspase 8receptor cleaves Bidand whichmitochondrial enhances release of mitochondrial proteins death pathways Bid causes release of cytochrome c from mitochondria Caspase 8 cleaves Bid which enhances release of mitochondrial proteins
FasL, TNF
Cellular stresses
ROS
FADD
Caspase 8
_ __ Cytochrome c
Bid
Apoptosome (Apaf1, caspase 9, ATP)
Caspase 3
Proteolysis
Apoptosis requires energy • •
The apoptosome requires energy Energy levels in the cell will determine whether death is by necrosis or apoptosis Apoptosis requires energy The
apoptosome
requires ATP
Energy levels in the cell may determine whether death is by necrosis or apoptosis + ATP Apoptosis
Inhibitor of Apoptosis Proteins (IAP’s) regulate PCD:
- ATP Necrosis
Bind to procaspases and prevent activation Bind to active caspases and inhibit their activity • Mitochondrial leakage also releases an inhibitor of IAP’s (DIABLO) that potentiates cell death • •
5. Discuss how Bcl-2 family proteins may modulate apoptosis • The Bcl-2 family of proteins modulate apoptosis • Anti-apoptotic (mitochondrial): Bcl-2 Bcl-XL • Pro-apoptotic (cytosolic/mitochondrial): Bad Bcl-xS Bax The regulation of apoptosis by Bcl-2 family proteins by heterodimerization (see below): • In A heterodimers are inactive and so Bad is inactive – therefore cell survival • In B the Bad is dephosphorylated and released which releases Bax/Bak from thr Bcl2/-xL pore leading to apoptosis
Cytoprotective Pathways: • • •
Bcl-2, Bcl-xL – the intrinsic pathway FLIP, IAP – the extrinsic pathway Phosphatidylinositol 3-Kinase (PI3-K) and Protein Kinase B (PKB)
PI3-K and PKB:
PI3K and PKB ( Akt) Phosphatidylinositol 4,5-bisphosphate ATP
Phosphoinositide 3’-kinase (PI3K)
Phosphatidylinositol 3,4,5-trisphosphate Protein kinase B (PKB)/ Akt
Cell survival
•
Protein synthesis
The PI3-K pathway is used to produce
PKB
PKB and cell survival: Phosphorylates and inactivates Bad Inactivates FOXO transcription factors (FOXO’s promote expression of apoptosis-promoting genes) Phosphorylates and inactivates caspase 9 Probably has other activities PTEN (lipid phosphatase) inhibits PI3K signalling and so prevents PKB synthesis
•
Proto-oncogenes/tumour suppressors associated with apoptosis: • Bcl-2 • PKB/Akt • PTEN