BB20023:DNA: make, break & disease 2004-05
APOPTOSIS
There are two kinds of cell death, apoptotic (programmed cell death) and necrotic.
NECROSISAPOPTOSIS
Morphological features
Dr. MV Hejmadi
BB20023:DNA: make, break & disease 2004-05
- loss of membrane integrity
- swelling of cytoplasm & mitochondria
- total cell lysis
- no vesicle formation
- disintegration of organelles
- Membrane blebbing, no loss of integrity
- Aggregation of chromatin at nuclear membrane
- Shrinking of cytoplasm & nuclear condensation
- Fragmentation into smaller bodies
- Formation of apoptotic bodies
- Mitochondria become leaky
Dr. MV Hejmadi
BB20023:DNA: make, break & disease 2004-05
Biochemical features
Dr. MV Hejmadi
BB20023:DNA: make, break & disease 2004-05
- Loss of regulation of ion homeostasis
- No energy requirement
- Random digestion of DNA
- Postlytic DNA fragmentaion
- Tightly regulated process
- Energy dependent
- Non random DNA fragmentation
- Prelytic DNA fragmentation
- Activation of caspase cascade
- Release of various factors into cytoplasm
Dr. MV Hejmadi
BB20023:DNA: make, break & disease 2004-05
Physiological features
Dr. MV Hejmadi
BB20023:DNA: make, break & disease 2004-05
- Affects groups of cells
- Evoked by non physiological disturbances
- Phagocytosis by adjacent cells
- Significant inflammatory response
- Affects individual cells
- Induced by physiological stimuli
- Phagocytosis by macrophages
- No inflammatory response
Dr. MV Hejmadi
BB20023:DNA: make, break & disease 2004-05
Caspases: the central executioners:(cysteinyl aspartate specific proteases)
Dr. MV Hejmadi
BB20023:DNA: make, break & disease 2004-05
These are highly conserved proteases that usually exist as inactive zymogens that are activated to induce cell death. So far, at least 14 homologues have been identified, 11 in humans. Caspases can be broadly divided into either 1) Initiator caspases: Caspases 2, 8,9,10 or 2) Effector caspases: caspase 3,6,7
Structure:Caspases comprise of 3 domains
- a highly variable NH2 domain
- large subunit (~20kD)
- small subunit ( ~10kD)
2 key features:
- variable N domain regulates activation
- all domains derived from proenzyme at cleavage specific sites
Highly specific
- unusual & absolute requirement for cleavage after aspartic acid
- recognition of at least 4 amino acids NH2 terminals to the cleavage site
How do caspases disassemble a cell?It slices, it dices, and that's not all!
Selectively cleave a discrete set of proteins in several ways
- Inactivate inhibitors of apoptosis eg bcl-2, or CAD/ICAD
- Direct disassembly of the cell structures e.g. nuclear lamina.
- Indirect cleaving of proteins involved in cytoskeletal regulation eg. Gelsolin
How are caspases activated? More than one way to skin a cat
Mechanisms of caspase activation include proteolytic cleavage by an upstream caspase (a), induced proximity (b) and holoenzyme formation (c). Proteolytic cleavage by an upstream caspase is straightforward and effective, and is used mostly for activation of downstream, effector caspases. It is probably also used for induction of apoptosis by non-caspase proteases, such as granzyme B. In the second mechanism, recruitment or aggregation of multiple procaspase-8 molecules into close proximity somehow results in cross-activation. The actual process is most probably more sophisticated and more tightly regulated than shown in panel b. In holoenzyme formation, cytochrome c and ATP-dependent oligomerization of Apaf-1 allows recruitment of procaspase-9 into the apoptosome complex. Activation of caspase-9 is mediated by means of conformational change, not proteolysis. Stoichiometry of the apoptosome is not known; it is shown in panel c as a hexamer solely for aesthetic reasons
The roads to ruin:
A) Caenorhabditis elegans:
One of the apoptotic pathways is triggered by internal signals of the CED system
CED-3 & 4 promote apoptosis
CED-9 inhibits apoptosis
Apoptotic stimuli causes CED-9 dissociation by EGL-1 thereby activating CED-3.
B) Mammalian cells: two major apoptotic pathways
1) External signals
- Specific ‘death ligands’
- Loss of contact with surroundings
2) Internal signals
- Irreparable internal damage
- Conflicting signals for cell division
External signal driven by death receptors (DR)
e.g. CD95 (or Fas/Apo)
- Each CD95L trimer binds to 3 CD95 leading to DD clustering.
- FADD ( Fas associated death domain/ Mort 1) binds via its own DD
- FADD effector DD to analogous domain in caspase –8
- Caspase –8 oligomerisation drives activation thru self cleavage
- Caspase –8 then activates downstream effector caspases like caspase –9 (CED-9 homolog)
- Apoptosis initiation
B) Internal signal driven by inactivation of bcl-2, structural & functional homolog of CED-9.
One model for Apaf-1 regulation by the bcl-2 family.
- Bcl-xL binds toApaf-1
- death signal provokes interaction of Bik/ Bax with Bcl-xL,
- Apaf-1 bind to procaspase-9 (in presence of cytochrome c)
- promote dimerisation & activation by autocatalysis.
- Caspase –9 subsequently activates effector caspases leading to apoptosis
References:
1) Science (1998)Vol 281: No 5381; pgs 1298-1326
2) Nature (2000) 12th Oct, Vol 407Insight articles
- The biochemistry of apoptosis by MO Hengartner pp 770 - 776
3) Molecular mechanisms of caspase regulation by S Reidl and Y Shi.
Nature Reviews in Mol & Cell Biology (Nov 2004) Vol 5 pp 897
4) Apoptosis: Molecular mechanismsby Wu, Min; Ding, Han–Fei and Fisher, David E
Dr. MV Hejmadi