Cell Biology: Apoptosis

Cell Biology: Apoptosis

1. Difference between

   • Apoptosis
     • programmed cell death
   • Autophagy
     • Recycles organelles as cellular substrates, sometimes causes cell death using different pathways from apoptosis
   • Autolysis
     • Cessation of active processes in the cell causes release of digestive enzymes by the lysosome into the cytoplasm
     • Usually occurs in injured or dying tissue
     • Necrosis
       • Cell injury resulting in premature cell death

2. Caspase

   • Coded by CED3 gene
   • has activator
     • APAF1
       • Coded by CED4 gene
       • Apoptotic Protease Activating Factor 1
   • Function
     • Cysteine proteases (active Cys residues at catalytic sites)
       • Cleaves substrates specifically at Asp residues
   • Domains
     • CARD
       • CAspase Recruitment Domain
     • Long domain
     • Short domain

3. Initiation

   • Flippase maintains phosphatidyl-serine on inner leaflet of plasma membrane
     • Deactivation of flippase allows P-serine to flip into the outer leaflet
       • Annexin 5 binds to P-serine, tagging it for apoptosis
         • Apoptotic bodies released into extracellular matrix, recognised and eaten by Phagocytes

4. Extrinsic (Receptor) Apoptotic Pathway

   • Mechanism (Using FAS as example)
     • Cytotoxic T-cell has FasL on outer membrane that exists as a trimer and induces trimerisation of FasR on target cell
       • Activated FasR use their Death Domains (DDs) in the cytoplasm to recruit
         • Fas-Associating protein with Death Domain (FADD)
           • FADD acts as adaptor and uses its Death Effector Domain (DED) to recruit
             • Pro-Caspase 8
               • Activated by "Induced Proximity", leading to autocatalytic cleavage (energy not required)
   • Receptors
     • Tumor Necrosis Factor (TNF) receptor
     • FasR
   • Ligands
     • FasL(igand)
       • Member of TNF family
     • TNFα (cytokine)

5. Intrinsic (Mitochondrial) Apoptotic Pathway

   • Mechanism
     • Cytochrome C released, binds to APAF1 inducing conformational change to expose oligomerisation domain for heptamer (7) formation
       • Apoptosome is formed, consisting of 7 APAF1, 7 Caspase 9 and 7 Cyt C with ATP bound to it
         • Caspase 9 (Initiator)
           • In vertebrates
           • Caspase 9 cleaves inactive caspase precursor at 2 sites; betwen large and pro domain and between large and small domain
             • 2 pairs of large and small domains oligomerise to become tetrameric active caspase
               • Caspase 3 and 7 (Effector)
                 • Cleaves subtrate proteins (eg. PARP, ICAD, lamin, cytoskeletal and cell junction proteins)
                   • Cell death
                   • ICAD
                     • Inhibitor of Caspase Activated DNAase
                     • Cleaved by Caspase 3 at Asp residues using its DEVD consensus sequence
           • Initiator pro-caspase different in structure from effector pro-caspases with larger CARD (pro-domain) containing protein-protein interaction motifs
         • Binding of at least 2 caspase 7 allows conformational change to activate each other
       • Cytochrome C
         • Initially bound to outer surface of inner membrane of mitochondria via electrostatic interactions (+ve charged and -ve phospholipid groups)
           • Release is result of
             • MOMP
               • Mitochondrial Outer Membrane Permeabilisation
               • Controlled by
                 • BCL2 Family
                   • Mediators
                     • Either have BH-1,2,3 (eg. Bax and Bak) or BH-3 only (eg. Puma, Bid, Bim, Bad)
                   • Inhibitors
                     • Contain additional BH4 domain

6. Cell Types

   • Type I Cells
     • Apoptosis is solely via mitochondria independent pathway
     • eg. thymocytes (becomes T cells)
   • Type II Cells
     • Mitochondrial death pathway is amplified by Death Receptor Pathway
       • Mechanism
         • where weak activation of caspase 8 cleaves BID (another BH3-only protein) into tBID (truncated)
           • tBID then activates Bax and Bak to allow greater release of Cytochrome C for APAF1 and hence Caspase 3 activation
     • eg. liver cells

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