Transcript integration from proteins to organs: the physiome project

Apoptosis
– mechanisms and role in
cancer therapy
TYPES OF CELL DEATH: Necrotic or apoptotic
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.
APOPTOSIS
WHEN DOES APOPTOSIS OCCUR?
Normal development e.g. oocyte development
© J Yuan, Harvard
Mammalian oocytes (left) undergo apoptosis around birth (right).
Disease states e.g. Alzheimer’s disease
Amyloid plaques in the brain
What triggers apoptosis?
• Growth factor withdrawal
• Specific ‘death ligands
• Loss of contact with surroundings
• Irreparable internal damage
• Conflicting signals for cell division
Caspases – key executioners of apoptosis
(cysteinyl aspartate specific proteases)
Highly conserved proteases
inactive zymogens
Irreversible reaction
Autocatalytic: triggered by cofactor binding or
inhibitor removal
Regulate their own activation
High specificity
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
Caspase structure
3 domains
1) highly variable NH2 domain
2) large subunit (p20; ~20kD)
3) small subunit ( p10; ~10kD)
Key features:
Highly specific: absolute requirement for cleavage after ASPARTATE
Recognition of at least 4 amino acids N-terminal to the cleavage site
Variable N domain regulates activation
All domains derived from precursor by cleavage specific sites
Scheme of procaspase activation
Cleavage of the procaspase at specific D-X bond leads to
activated caspase
Formation of 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
Caspase families
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
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
The roads to ruin
Apoptotic pathways in multicellular organsims
nematode - C.elegans
Out of 1090 cells that form an hermaphrodite
adult, exactly 131 apoptose
apoptosis activation in C. elegans
Cell death abnormal (CED)-4 dimers sequestered by CED-9 on the
outer surface of mitochondria (inactive conformation)
Apoptotic cells produce the egg-laying defective (EGL)-1
Binding of EGL-1 to CED-9 results in release of CED-4 dimer.
Two CED-4 dimers free to form a tetramer and recruit proCED-3
molecules to form the so-called apoptosome.
CED-3 becomes activated and apoptosis is triggered
Getting rid of the evidence!
Dead cells engulfed by ‘friendly neighbouring’ cells
Apoptotic activation in mammalian systems
Internal
and
external
signals
converge
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
TRIGGER
DNA damage
Death receptors
Growth factor
withdrawal
REGULATOR
P53
Bcl-2 family
Cytochrome c
oncogenes
EXECUTIONER
Apaf-1
Caspases
Anti-apoptotic strategies by cancer cells
Blue: decreased levels / activity
Red: decreased levels / activity
Green and Kroemer The Journal of Clinical Investigation 115(10):2610-17 (October 2005)
References
Biology of Cancer by RA Weinberg – Chap 9 pp334-350
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
Optional
Nature Reviews Molecular Cell Biology 7, 97-108 (February 2006)
Developmental apoptosis in C. elegans: a complex
CEDnario by M.O. Hengartner
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)
External signals
Overall structure of ced4/ced9 complex
Each CED-4 molecule comprises four sequential domains—CARD domain
(green), / -fold (blue), helical domain (cyan) and winged-helix domain
(magenta)—and contains a bound ATP molecule and a magnesium ion.
Nieng Yan et al Nature 437, 831-837 (6 October 2005)