1. Apoptosis
    programmed cell death by activating an intracellular death program. A genetically regulated process to destroy a cell itself y a serial well defined biochemical and morphological changes
  2. Necrosis
    accidental cell death without order caused by injury, radiation, chemicals or lack of nutrition. These dead cells do not send signals to phagocytes and thus often causes inflammation - necrotic cell typically swell and burst
  3. Ced-1 or Ced-1/-3 mutants to study apoptosis
    • ced-1: mutation prevents engulfment of dead cells, no phagocytosis of apoptotic cells
    • ced-1/ced-3: no apoptotic cells
    • conclusion: ced 3 protein is required for programmed cell death
  4. Growth factors are important for cell survival: experiment with neuron growth (NGF and TrkA)
    NGF and its receptor TrkA: nerve growth factor, needed for pain sensing neurons - Nociceptive neurons; knock out of either results loss of pain sensing
  5. Growth factors are important for cell survival: experiment with neuron growth (Nt-3 or TrkC)
    NT-3 and TrkC: neurotrophin and its receptor. Needed for balancing; propioceptive neurons - innervating muscle spindles, control balancing
  6. Apoptosis (process)
    • dense chromosome condensation occurs along the nuclear periphery
    • cells shrink (most organelles remain intact)
    • chromatin compaction and margination; condensation of cytoplasm
    • cytoskeleton collapse
    • nuclear pyknosis: DNA and nuc fragmentation, break up of nuclear envelope
    • blebbing: generation of bbbles
    • Cell fragmentation: apoptotic bodies formation - memraben boun so that the intracellular consitituents are not released into the extracellular where they might have a deleterious effects on neighborign cells
    • Apoptotic bodies are engulfed by nearby cell of macrophage/phagocytic cells via phagocytosis, need: killer, destraction, engulfment proteins
  7. Caspases
    • usually synthesized in the cell as inactive precursors or pro-caspase, which are usually activated by clevage at aspartic acid (XEXD) by other caspases
    • caspases are proteases
    • All are dependent on cys containing nucleophilic group (QACRG) in active sight - signatre motif. Cys cleave at Asp (D) residue
    • cysteine-sapartic acid protease
  8. Targets of Caspases
    • Nuclear lamins
    • Nucleases (fragment DNA)
    • Mitochondrial protein
    • Cytoskeletal and signaling proteins (actin, gelsolin, PKC, MEKK1)
    • GRASP65, SNAREs
  9. Extrinsic death pathway
    death receptors and DISC assembly (activation from outside of the cell) eg. a killer T cell killing a cancer cell: killer T cell generate killer ligand and trigger apoptosis
  10. Intrinsic death pathway
    • mitochondria, cytochrome c release, apoptosome assembly
    • activation from inside the cell: ER stress, DNA damage caused cell death
  11. Fas dependent killing
    • Extrinsic pathway - from outside
    • Fas ligand on killer lymphocyte/T cell binds to Fas protein (death receptor) on target cell membrane
    • Binding leads to formation of Death inducing signaling complex (DISC) - Fas, Fas ligand, Procaspase-8 form complex (also adaptor protein)
    • DISC leads to cleavage of procaspase-8 - activated caspase 9
    • LEad to caspase-3 mediated apoptosis
    • Loss of Fas ligand leads to lymphoproliferative and autoimmune diasese
    • Loss of Fas/death ligand receptors is associated with cancer
  12. The intrinsic pathway in the absense of trophic factor: caspase activation
    • cytochrome c is released form the mitochondria into the cytosol where it binds to Apaf1 and procaspase 9 (injured mitochondria release cytochrom c)
    • cleaveage of procaspase 9 leads to activated caspase 9
    • caspase 9 acts on procaspase 3 and cleavaging, into caspase 3
  13. Apaf1
    • apoptotic protease activation factor
    • Apaf1 and cyt c bind procaspase 9, form complex
    • complex = apoptosome
  14. apoptosome
    • a complex formed by apaf1, cytochrome, and procaspase 9
    • assembly is essential for caspase activation and intrinsic apoptotic pathway
  15. Bad
    • a soluable pro-apoptotic protein, binds to Bcl2 and Bcl-xl
    • prevents the anti-apoptotic proteins from interaction with bax
  16. Bcl-2 and Bcl-xl
    • anti-apoptotic proteins that are inserted into the mitochondrial membrane
    • in the absense of Bad, Bcl2 and Bcl-xl inhibits Bax for ion influx and cytochrome release
  17. Bax
    • a membrane bound pro-apoptotic protein
    • forms a homo oligomeric channel in the membrane that mediates ion flux
    • leads to release of cytochrom c into cytosol
  18. Presence of trophic factor: inhibition of caspase activation
    • Trophic factor binds to the receptor (cytokine such as TNF)
    • PI-3 kinase is activated and activates PKB pathway
    • PKB pathway leads to phosphorylation of Bad
    • Bad-P no longer binds to Bcl-2/Bcl-xl on the mitochondrial membrane
    • instead, binds to 14-3-3 and stays in the cytosol
    • Bcl-2/Bcl-xl no longer inhibited, acts to inhibit Box - inhibit ion influx and cytochrome release
    • No caspase made
  19. Assembly of the mammalian apoptosome
    • cytochrome c release from mitochondria
    • cytochrome binds Apf-1 monomer - dATP hydrolysis
    • Leads to Apaf-1 oligomerization/nucleotide exchange - formation of Apoptosome
    • procaspase 9 recruitment to Apaf-1; caspase 3 and IAP recruitment to Apaf-1
  20. Activation of Ced-3 in C. elegans
    • Ced-4 dimer (=Apaf1) bidn to Ced-9 (=Bcl-2) - Ced-9 inhibits Ced-4
    • EGL-1 (death signal) binds to Ced-9 and releases Ced-4 dimer
    • Ced4-4 forms tetramer
    • activates Ced3 (=caspase-9) - Ced3-zymogen becomes active Ced-3
    • lead to cell death
    • Ced9 mutants (no functioning Bcl02) all cell undergo apoptosis, can be rescued with mammalian Bcl2
    • No cytochrome c required
  21. IAP
    • Inhibitor of apoptosis
    • IAP overactivation leads to cancer, overinhibition of apoptosis, no cell death
  22. ER-stress
    • overload of misfolded protein or Ca2+ imbalance
    • Caspase activation: Caspase 12 release from ER and activation by Ca2+
    • Loss of mitochondria membrane potential
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