1. Apoptosis – mechanisms and role in cancer therapy

2. TYPES OF CELL DEATH: Necrotic or apoptotic

3. APOPTOSIS

4. External signals

5. Normal development e.g. immune system
WHEN DOES APOPTOSIS OCCUR?
Normal development e.g. immune system

6. Disease states e.g. Alzheimer’s disease
WHEN DOES APOPTOSIS OCCUR?
Disease states e.g. Alzheimer’s disease
Amyloid plaques in the brain

7. 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

8. Caspase structure
(A) The caspase family. Three major groups of caspases are presented. Group I: inflammatory caspases; group II: apoptosis initiator caspases; group III: apoptosis effector caspases. The CARD, the DED, and the large (p20) and small (p10) catalytic subunits are indicated. (B) 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. The residues involved in the formation of the active center are shown. (C) The 3D 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 -helices. N and C termini of the small and large protease subunits are indicated.

9. Properties of proteases
Irreversible -
Autocatalytic: triggered by cofactor binding or inhibitor removal
Proteases can regulate their own activation
protease inhibitors
specificity

10. 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

11. 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.

12. 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

13. 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.

14. 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

15. What triggers apoptosis?
Growth factor withdrawal
Specific ‘death ligands
Loss of contact with surroundings
Irreparable internal damage
Conflicting signals for cell division

16. 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.

17. How are caspases activated?
Induced proximity
aggregation of multiple procaspase-8 molecules into close proximity somehow results in cross-activation

18. How are caspases activated?
Holoenzyme formation
Activation of caspase-9 is mediated by means of conformational change, not proteolysis

19. 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.

20. Caspase signaling in Mammalian systems
Extrinsic pathway, DISC formation leads to caspase-8 activation. Two signaling pathways downstream from the receptor were established.
In type I cells (shown in light blue) caspase-8 directly cleaves caspase-3, which starts the death cascade.
In type II cells (shown in light red) an additional amplification loop is required, which involves tBid-mediated cytochrome c release from mitochondria followed by apoptosome formation. Initiation of the intrinsic pathway results in mitochondria-mediated apoptosome formation, followed by caspase-9 and -3 activation, leading to destruction of the cell.
Caspase action can be modulated on several levels.
Activation of caspases at the DISC is inhibited by c-FLIP proteins;
Activation of effector caspases is inhibited by IAPs.
Effector caspases are shown in light green; cellular caspase inhibitors are presented in yellow. The targets for pharmacological modulation are shown with an orange arrow.
IN Lavrik et al The Journal of Clinical Investigation 115(10): (October 2005)

21. Mammalian systems
Mammals

22. 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)
Apoptosis initiation

23. Internal signals

24. BCL-2

25. TRIGGER REGULATOR EXECUTIONER DNA damage Death receptors
Growth factor withdrawal
P53
Bcl-2 family
Cytochrome c
oncogenes
Apaf-1
Caspases

26. Green and Kroemer The Journal of Clinical Investigation 115(10):2610-17 (October 2005)

27. References
Chapter 12: Cellular & Mol Biology by Knowles and Selby AND/OR Science (1998) Vol 281: No 5381; pgs AND/OR J. Clin Invest (10 Oct 2005) 115(10): AND/OR Cancer Biology by RJB King pgs AND/OR
NATURE | VOL 407 | 12 OCTOBER 2000 pp
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)