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Apoptosis
– mechanisms and role in
cancer therapy
TYPES OF CELL DEATH: Necrotic or apoptotic
APOPTOSIS
External signals
WHEN DOES APOPTOSIS OCCUR?
Normal development e.g. immune system
WHEN DOES APOPTOSIS OCCUR?
Disease states e.g. Alzheimer’s disease
Amyloid plaques in the brain
Caspases – key executioners of apoptosis
(cysteinyl aspartate specific
proteases)
Highly conserved proteases
inactive zymogens
Caspases divided into
Group I
Inflammatory caspases
Caspases 1,4,5,11,12,13,14
Group II
Initiator caspases
Caspases 2,8,9,10
Group III
Effector caspases:
caspases 3,6,7
Caspase structure
Properties of proteases
Irreversible Autocatalytic: triggered by cofactor
binding or inhibitor removal
Proteases can regulate their own
activation
protease inhibitors
specificity
Caspase structure
3 domains
1) highly variable NH2 domain
2) large subunit (p20; ~20kD)
3) small subunit ( p10; ~10kD)
Highly specific
absolute requirement for cleavage after aspartic acid
recognition of at least 4 amino acids NH2 terminals to
the cleavage site
Caspase structure
2 key features:
variable N domain regulates
activation
all domains derived from
proenzyme precursor by
cleavage specific sites
Scheme of procaspase
activation:Cleavage of the
procaspase at the specific
Asp-X bonds leads to the
formation of the mature
caspase, which comprises
the heterotetramer p202–
p102, and the release of
the prodomain.
Structure of caspase-3 heterotetramer
Each heterodimer is formed
by hydrophobic interactions
resulting in the formation
of mostly parallel ß-sheets,
composed of 6 antiparallel
ß-strands.
Two heterodimers fit
together with formation of
a 12-stranded ß-sheet that
is sandwiched by a helices.
N and C termini of the small
and large protease subunits
are indicated
Basic apoptotic machinery
DNA fragmentation,
chromatin condensation,
membrane blebbing,
cell shrinkage &
disassembly into apoptotic bodies
engulfment
Initiator caspases inactivate proteins that protect cells
from apoptosis
Effector caspases are responsible for cellular changes
associated with apoptosis.
How do caspases disassemble a cell?
It slices, it dices!
Selective cleavage of specific proteins
eg bcl-2, or CAD/ICAD
e.g. nuclear lamins
eg. Gelsolin
What triggers apoptosis?
• Growth factor withdrawal
• Specific ‘death ligands
• Loss of contact with surroundings
• Irreparable internal damage
• Conflicting signals for cell division
How are caspases activated?
Proteolytic cleavage
2 key features:
variable N domain regulates
activation
all domains derived from
precursor by cleavage specific
sites
Cleavage of the procaspase at the specific Asp-X bonds
leads to the formation of the mature caspase, which
comprises the heterotetramer p202–p102, and the release
of the prodomain.
How are caspases activated?
Induced proximity
aggregation of multiple procaspase-8 molecules into
close proximity somehow results in cross-activation
How are caspases activated?
Holoenzyme formation
Activation of caspase-9 is mediated by means of
conformational change, not proteolysis
nematode - C.elegans
One of the apoptotic pathways is triggered by internal
signals- CED
CED-3 & 4 promote apoptosis
CED-9 inhibits apoptosis
Apoptotic stimuli causes CED-9 dissociation by EGL-1
thereby activating CED-3.
Caspase signaling in Mammalian systems
IN Lavrik et al The Journal of Clinical Investigation 115(10):2665-72. (October 2005)
Mammalian systems
Mammals
External signals
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
Caspase –8 oligomerisation drives
activation through self cleavage
Caspase –8 then activates downstream
effector caspases like caspase –9 (CED-9
homolog)
Internal signals
BCL-2
TRIGGER
DNA damage
Death receptors
Growth factor
withdrawal
REGULATOR
P53
Bcl-2 family
Cytochrome c
oncogenes
EXECUTIONER
Apaf-1
Caspases
Green and Kroemer The Journal of Clinical Investigation 115(10):2610-17 (October 2005)
References
Chapter 12: Cellular & Mol Biology by Knowles and Selby
AND/OR
Science (1998) Vol 281: No 5381; pgs 1298-1326
AND/OR
J. Clin Invest (10 Oct 2005) 115(10):2665-72
AND/OR
Cancer Biology by RJB King pgs 160-167
AND/OR
NATURE | VOL 407 | 12 OCTOBER 2000 pp770-776
The biochemistry of apoptosis by M.O. Hengartner
Optional
NATURE REVIEWS MOLECULAR CELL BIOLOGY Vol 5 | NOV 2004 | 897
Molecular mechanisms of caspase regulation during apoptosis
Stefan J. Riedl and Yigong Shi
(Only read it if you want to know more about caspase structure)