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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)