Download apoptosis

BB20023:DNA: make, break & disease
2004-05
APOPTOSIS
There are two kinds of cell death, apoptotic (programmed cell death) and necrotic.
NECROSIS
APOPTOSIS
Morphological features
 loss of membrane integrity
 Membrane blebbing, no loss of integrity
 swelling of cytoplasm & mitochondria
 Aggregation of chromatin at nuclear membrane
 total cell lysis
 Shrinking of cytoplasm & nuclear condensation
 no vesicle formation
 Fragmentation into smaller bodies
 disintegration of organelles
 Formation of apoptotic bodies
 Mitochondria become leaky
Biochemical features
 Loss of regulation of ion homeostasis
 Energy dependent
 No energy requirement
 Non random DNA fragmentation
 Random digestion of DNA
 Prelytic DNA fragmentation
 Postlytic DNA fragmentaion
 Activation of caspase cascade
 Tightly regulated process
 Release of various factors into cytoplasm
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Physiological features
Affects groups of cells
 Affects individual cells
Evoked by non physiological disturbances
 Induced by physiological stimuli
Phagocytosis by adjacent cells
 Phagocytosis by macrophages
Significant inflammatory response
 No inflammatory response
Caspases: the central executioners: (cysteinyl aspartate specific proteases)
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
Dr. MV Hejmadi
BB20023:DNA: make, break & disease
2004-05
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 noncaspase 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
Dr. MV Hejmadi
BB20023:DNA: make, break & disease
2004-05
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 407 Insight articles
a. 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 mechanisms by Wu, Min; Ding, Han–Fei and Fisher, David E www.els.net
Dr. MV Hejmadi