Chapter 18 Cell Death

  1. types of cell death

    1. growth, development, and maintenance depend on the production of cells, but also their destruction

    2. apoptosis

      1. programmed cell death

      2. Cells will shrink and condense, the cytoskeleton will collapse, the nuclear envelope disassembles, chromatin breaks, and the cell breaks into apoptotic bodies

    3. necrosis

      1. Happens in response to an insult

      2. The cell will swell and burst, spilling all of its contents and causing inflammation

  2. Apoptisis

    1. cell shrinks and chromatin decondenses

    2. membrane blebs

    3. The nucleus collapses, and the DNA starts to get fragmented, and the cell continues blebbing

    4. apoptotic bodies begin to form

    5. apoptotic bodies are cleaned up by immune system cells that can do phagocytosis

      1. macrophages

  3. Autophagy and necrosis

    1. Necrotic cells burst and spill out their cellular contents because the cell is so damaged

      1. Content spill causes inflammation in the tissue and causes neighboring cells to undergo necrosis

  4. Functions of apoptosis

    1. removing damaged cells

    2. tissue architecture 

      1. tissue sculpting - removing webbing between digits from development

    3. T and B cell development

      1. When the cells attack something in their own body, they are eliminated

  5. Caspases 

    1. protein mediators of apoptosis

      1. There is cysteine in the active site

      2. They cleave target proteins at aspartic acid residues

        1. Initiator caspases function to activate executioner caspases (amplification)

        2. Executioner caspases catalyze widespread cleavage events that kill cells

          1. They have over 1000 known targets

  6. Caspase activation during apoptosis

    1. Initiator caspases are monomers in an inactive form

      1. The major initiation caspases are caspase 8 and caspase 9 

    2. When the cell receives an apoptotic signal and adaptor proteins, the signal will cause caspases to dimerize

    3. When in the dimer, the caspases will cut and activate each other

    4. The complex will go find executioners that are already dimer, but inactive unless cut

    5. Executioners are cut and activated by initiators and go to cleave targets and enact the cell death pathway

      1. The 3 major execution caspases are caspase 3, caspase 6, and caspase 7

  7. DNA fragmentation during apoptosis

    1. iCAD is the inhibitor of CAD

      1. CAD - caspase-activated DNA

      2. iCAD - a target of executioner caspases

    2. CAD is a protein that chews up DNA, but it can only do that if the DNA is in the linker region, not DNA wrapped around histones, so CAD fragments DNA in a very even pattern

      1. The smallest fragment CAD can make is 200 base pairs

    3. If you look at DNA undergoing apoptosis, the gel will form a banded pattern

    4. Tunel stain is a fluorescent stain that marks the ends of DNA

  8. Extrinsic pathway

    1. Triggered by extracellular signal proteins binding to death receptors

    2. TNF (tumor necrosis factor) is a family of receptors and ligands

    3. DISC - death-inducing signaling complex

  9. Extrinsic pathway activated by Fas

    1. A killer T cell has a Fas ligand, which is the signal

    2. The Fas ligand binds to the Fas death receptor on the target cell

      1. target cell components are homophilic binders

      2. They have death domains and death effector domains

    3. The death receptor with the death domain recruits FADD, which is an adaptor protein that has death domains and death effector domains

    4. FADD allows for the recruitment of initiator caspase 8, which has the death effector domain 

      1. The initiator caspase is the major caspase for the extrinsic pathway

    5. The complex formed by the receptor, adaptor, and caspase is called DISC

    6. Caspase 8s cut and activate each other, then go to activate executioners

  10. Intrinsic (mitochondrial) pathway

    1. activated from inside the cell as a response to stress

    2. depends on the release of cytochrome c from the mitochondria

    3. can be initiated by the extrinsic pathway

      1. The link is the protein Bid, activated by the extrinsic and activates the intrinsic

  11. intrinsic pathway

    1. Cytochrome c is released into the cytosol from the mitochondria

    2. Cytochrome c finds Apaf1 and binds, changing the conformation of Apaf1

    3. The card domain of Apaf-1 binds to other card domains

      1. 7 cytochrome c’s activate 7 Apaf1’s to form a complex called the apoptosome, which is a heptamer

        1. The nickname for this complex is the wheel of death

    4. The apoptosome recruits 7 initiator caspases (caspase-9s)

    5. caspase 9s bind to the wheel and activate each other, and activate the executioners

  12. Release of cytochrome c

    1. regulation

      1. Bcl2 famil;y

        1. The intrinsic pathway is tightly controlled to prevent apoptosis 

          1. The process is irreversible

        2. The Bcl2 family is the main family that regulates apoptosis, and the family is composed of pro- and anti-apoptotic members

    2. Bcl2 family members

      1. They are all family members because they share homology between domains

        1. anti-apoptotic members

          1. BH4, BH3, BH2, and BH1 domains

          2. Bcl2 and BclXL

        2. pro-apoptotic effector Bcl2 family proteins

          1. BH3, BH2, nad BH1 domains

          2. Bak and Bax

        3. pro-apoptotic BH# only proteins

          1. BH3 domain only

          2. Bad, Bim, Bid, Puma, and Noxa

    3. Role of pro-apoptotic family members Bak and Bax

      1. Bax and Bak make holes in the mitochondrial membrane through oligomeric pores

      2. Bax is normally present in the cytosol, and Bak is always in the membrane

      3. Bcl2 and BclXL are direct inhibitors of Bax and Bak

    4. BH3 only pro-apoptotic members

      1. The largest subclass of the Bcl2 family of proteins

      2. They are the crucial link between apoptotic stimuli and the intrinsic pathway

      3. Different stimuli activate different BH3 members

        1. survival signals block the synthesis and activity of some members

        2. p53 accumulates, causing the transcription of Puma and Noxa

    5. BH3-only proteins promote apoptosis by inhibiting anti-apoptotic members

      1. They are inhibitors of the inhibitors Bak and Bax (so they inhibit Bcl2 and BclXL)

      2. Bid is the link between intrinsic and extrinsic, so the extrinsic pathway can activate the intrinsic pathway if it needs to be amplified

  13. Inhibitors of apoptosis (IAPs)

    1. The cell has multiple robust mechanisms to control apoptosis

    2. IAPs bind to and inhibit activated caspases

      1. XIAP in mammals

    3. Anti-IAPs inhibit IAPs

      1. Fly: Grim, Reaper, and Hid

      2. Mammalian: Smac/Diablo, Omi

  14. Survival signals - extracellular

    1. If cells don’t receive survival signals, they will die by apoptosis

    2. Many survival pathways affect Bcl2 family members

      1. When neurons are first developing, they will extend their axons towards a target cell, and the target cell is secreting survival signals

      2. If a neuron is the first to make it to that target it will get the signals and grow and survive; others will die by apoptosis

  15. Survival pathways effects

    1. Bax and bak

      1. Are pro-apoptotic

      2. So a survival pathway should inhibit them

    2. IAP

      1. Is anti-apoptotic

      2. So a survival pathway should activate it

  16. Pathways acting on Bcl2 members

    1. Pathway acting on Bcl2

      1. survival signal binds receptor

      2. The receptor activates a transcription factor

      3. The transcription factor causes the transcription and translation of Bcl2

      4. Bcl2 blocks apoptosis because it is an anti-apoptotic Bcl2 family member

    2. Pathway acitng on BH3 only members

      1. Signal binds a receptor that is a kinase

      2. activates the Akt kinase

      3. Akt inhibits Bad

      4. Bad releases Bcl2, and Bcl2 blocks apoptosis

    3. inactivation of anti-IAPs

      1. signal binds receptor

      2. activates a kinase cascade

      3. Hid (a protein that inhibits IAPs) is phosphorylated

      4. IAPs block apoptosis

  17. Apoptosis is tidy

    1. The cell breaks into apoptotic bodies

    2. The bodies display the “eat me” signal, which is phosphatidylserine

    3. If phosphatidylserine is expressed on the outside of apoptotic bodies, it is the signal for macrophages to come and swallow the bodies

      1. The macrophage is secreting several signaling molecules as it is eating the apoptotic body

        1. The signaling molecules are TGFbeta, IL10, and PGE2, and they are all anti-inflammatory cytokines

  18. Defects in apoptosis cause disease

    1. too much apoptosis

      1. heart attacks and strokes can stimulate massive levels of apoptosis (and necrosis)

      2. Tissue damage

    2. Too little apoptosis

      1. autoimmune diseases

      2. cancers

        1. Bcl2 was first discovered in B-cell lymphoma