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Apoptosis – mechanisms and role in cancer therapy

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.

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Apoptosis – mechanisms and role in cancer therapy

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  1. Apoptosis – mechanisms and role in cancer therapy

  2. TYPES OF CELL DEATH: Necrotic or apoptotic

  3. APOPTOSIS

  4. External signals

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

  6. 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

  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 IN Lavrik et al The Journal of Clinical Investigation 115(10):2665-72. (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 P53 Bcl-2 family Cytochrome c oncogenes Apaf-1 Caspases DNA damage Death receptors Growth factor withdrawal

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

  27. References Chapter 12: Cellular & Mol Biologyby Knowles and SelbyAND/ORScience (1998)Vol 281: No 5381; pgs 1298-1326 AND/ORJ. Clin Invest (10 Oct 2005)115(10):2665-72 AND/ORCancer Biology by RJB King pgs 160-167AND/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)

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