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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
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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
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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
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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
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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
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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
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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
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Targets of Caspases
- Nuclear lamins
- Nucleases (fragment DNA)
- Mitochondrial protein
- Cytoskeletal and signaling proteins (actin, gelsolin, PKC, MEKK1)
- GRASP65, SNAREs
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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
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Intrinsic death pathway
- mitochondria, cytochrome c release, apoptosome assembly
- activation from inside the cell: ER stress, DNA damage caused cell death
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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
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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
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Apaf1
- apoptotic protease activation factor
- Apaf1 and cyt c bind procaspase 9, form complex
- complex = apoptosome
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apoptosome
- a complex formed by apaf1, cytochrome, and procaspase 9
- assembly is essential for caspase activation and intrinsic apoptotic pathway
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Bad
- a soluable pro-apoptotic protein, binds to Bcl2 and Bcl-xl
- prevents the anti-apoptotic proteins from interaction with bax
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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
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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
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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
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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
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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
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IAP
- Inhibitor of apoptosis
- IAP overactivation leads to cancer, overinhibition of apoptosis, no cell death
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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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