1. Apoptosis – mechanisms and role in cancer therapy

2. TYPES OF CELL DEATH: Necrotic or apoptotic

3. 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.

4. APOPTOSIS

5. Normal development e.g. oocyte development WHEN DOES APOPTOSIS OCCUR? Mammalian oocytes (left) undergo apoptosis around birth (right). © J Yuan, Harvard Disease states e.g. Alzheimers disease Amyloid plaques in the brain

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

7. 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

8. 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

9. 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

10. 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.

11. 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

12. 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

13. How are caspases activated? Proteolytic cleavage 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. 2 key features: variable N domain regulates activation all domains derived from precursor by cleavage specific sites

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

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

16. The roads to ruin Apoptotic pathways in multicellular organsims

17. nematode - C.elegans Out of 1090 cells that form an hermaphrodite adult, exactly 131 apoptose

18. 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

19. Getting rid of the evidence! Dead cells engulfed by friendly neighbouring cells

20. Apoptotic activation in mammalian systems Internal and external signals converge

21. 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

22. Internal signals

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

24. Anti-apoptotic strategies by cancer cells Blue: decreased levels / activity Red: decreased levels / activity

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

26. 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 References Nature Reviews Molecular Cell Biology 7, 97-108 (February 2006) Developmental apoptosis in C. elegans: a complex CEDnario 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)

27. External signals

28. Overall structure of ced4/ced9 complex Nieng Yan et al Nature 437, 831-837 (6 October 2005) Each CED-4 molecule comprises four sequential domainsCARD domain (green), / -fold (blue), helical domain (cyan) and winged-helix domain (magenta)and contains a bound ATP molecule and a magnesium ion.