H7 apoptosis
Apoptosis = regulated cell death
- Caspases = proteases
- Blebbing of membrane
Necrosis = unregulated cell death
- Membrane rupture
7.1 Molecular mechanisms of apoptosis
Caspases:
Cysteine rich aspartate proteases
- Inactive pro-caspases cleaved at aspartate residues active caspase
cuts with cysteine in active site at target aspartate caspase activation
cascade
Extrinsic pathway: mediated by membrane death receptors
Triggered by death factors such as:
Fas ligand (membrane-bound on neighboring cells)
TNF (soluble)
1. Death factors bind to death receptors:
Fas ligand → Fas receptor
TNF → TNF receptor
Binding causes receptor trimerization and a conformational change.
exposes death domains (DDs) from receptor on the cytoplasmic side.
2. Adaptor proteins bind with their DD to the DD of the receptor DD-DD
interaction
FADD (Fas-associated death domain)
TRADD (TNF receptor–associated death domain)
3. Adaptor protein recruit procaspase-8 via their death effector domains
(DEDs).
procaspase-8 comes together dimerization:
Self-cleavage (due to their low basal activity)
Formation of active caspase-8 (an initiator caspase
Death-Inducing Signaling Complex (DISC) = complex of ligand, receptor,
adaptors and procaspase-8
4. caspase cascade:
Caspase 8 Activates executioner caspases (caspase-3, -6, -7)
Executioner caspases cleave key cellular proteins → apoptosis
Cleavage targets of caspases
Nuclear lamins → nuclear shrinkage
Cytoskeletal proteins (actin, intermediate filaments) → cell restructuring
, Kinases → disruption of signaling
Caspase-activated DNase (CAD) → chromatin cleavage
RB tumor suppressor protein, which normally inhibits apoptosis TNF-
induced apoptosis
Apoptosis detection
CAD cuts DNA between nucleosomes → blot: DNA ladder (180 bp
multiples)
TUNEL= TdT enzyme builds fluorescent dUTP into DNA breaka
Inhibition:
c-Flip, a inactive caspase-8 homolog, binds FADD or caspase-8
and prevents caspase-8 recruitment/activation.
The intrinsic pathway: mediated by the mitochondria
Triggered by internal stress signals (eg. DNA damage, oxidative stress).
Bcl-2 family overview
~25 members; all contain BH domains for protein–protein interactions.
o Pro-apoptotic proteins (eg. Bax, Bak) → promote apoptosis by forming
pores.
o Anti-apoptotic proteins (eg. Bcl-2, Mcl-1) → inhibit apoptosis by
binding/sequestering pro-apoptotic molecules.
Special subgroup: BH3-only proteins (eg. Bim, Bid)
o BH3-only activators → activate Bax/Bak.
o BH3-only sensitizers → inhibit anti-apoptotic Bcl-2
proteins.
Apoptosis = regulated cell death
- Caspases = proteases
- Blebbing of membrane
Necrosis = unregulated cell death
- Membrane rupture
7.1 Molecular mechanisms of apoptosis
Caspases:
Cysteine rich aspartate proteases
- Inactive pro-caspases cleaved at aspartate residues active caspase
cuts with cysteine in active site at target aspartate caspase activation
cascade
Extrinsic pathway: mediated by membrane death receptors
Triggered by death factors such as:
Fas ligand (membrane-bound on neighboring cells)
TNF (soluble)
1. Death factors bind to death receptors:
Fas ligand → Fas receptor
TNF → TNF receptor
Binding causes receptor trimerization and a conformational change.
exposes death domains (DDs) from receptor on the cytoplasmic side.
2. Adaptor proteins bind with their DD to the DD of the receptor DD-DD
interaction
FADD (Fas-associated death domain)
TRADD (TNF receptor–associated death domain)
3. Adaptor protein recruit procaspase-8 via their death effector domains
(DEDs).
procaspase-8 comes together dimerization:
Self-cleavage (due to their low basal activity)
Formation of active caspase-8 (an initiator caspase
Death-Inducing Signaling Complex (DISC) = complex of ligand, receptor,
adaptors and procaspase-8
4. caspase cascade:
Caspase 8 Activates executioner caspases (caspase-3, -6, -7)
Executioner caspases cleave key cellular proteins → apoptosis
Cleavage targets of caspases
Nuclear lamins → nuclear shrinkage
Cytoskeletal proteins (actin, intermediate filaments) → cell restructuring
, Kinases → disruption of signaling
Caspase-activated DNase (CAD) → chromatin cleavage
RB tumor suppressor protein, which normally inhibits apoptosis TNF-
induced apoptosis
Apoptosis detection
CAD cuts DNA between nucleosomes → blot: DNA ladder (180 bp
multiples)
TUNEL= TdT enzyme builds fluorescent dUTP into DNA breaka
Inhibition:
c-Flip, a inactive caspase-8 homolog, binds FADD or caspase-8
and prevents caspase-8 recruitment/activation.
The intrinsic pathway: mediated by the mitochondria
Triggered by internal stress signals (eg. DNA damage, oxidative stress).
Bcl-2 family overview
~25 members; all contain BH domains for protein–protein interactions.
o Pro-apoptotic proteins (eg. Bax, Bak) → promote apoptosis by forming
pores.
o Anti-apoptotic proteins (eg. Bcl-2, Mcl-1) → inhibit apoptosis by
binding/sequestering pro-apoptotic molecules.
Special subgroup: BH3-only proteins (eg. Bim, Bid)
o BH3-only activators → activate Bax/Bak.
o BH3-only sensitizers → inhibit anti-apoptotic Bcl-2
proteins.
