Programmed Cell Death A genetically controlled cell suicide pathway

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Programmed Cell Death A genetically controlled
cell suicide pathway
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video
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The Morphology of Apoptosis

• Cytoplasm shrinks

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Difference Between Apoptosis and Necrosis

• Necrosis (pathological cell death) dying cells
  swell and lyse toxic contents leak out and
  result in inflammatory response.
• Apoptosis (physiological or programmed cell
  death) dying cells shrink, are engulfed and
  degraded by other cells, leave no trace, and
  dont result in harmful outcomes

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Functions of apoptosis

• Sculpt body structures, e.g. hand digit

Produced in excess, e.g. extra neurons are
removed by apoptosis during neurogenesis.
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The Nematode C. elegans As a Model Organism in
the Study of PCD

• A great genetic system
• Completely defined cell lineage
• Study of cell death at a single cell resolution
  in living animals

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The C. elegans Cell Lineage
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B
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Cell Death Can Be Studied at a Single Cell
Resolution
P11
X
X
P11aap
Adapted from Sulston and Horvitz, Dev. Bology 56,
110-150, 1977
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The First Cell Death Mutants Identified in C.
elegans

• In 1976, J. Sulston first described programmed
  cell death in nematodes and reported the first
  cell death mutant (nuc-1), in which DNA in the
  death cells fail to be degraded.

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In 1980, E. Hedgecock isolated two cell death
mutants (ced-1 and ced-2) which are pivotal for
identification of the other cell death genes.
What went wrong with the ced-1 mutant?
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What could go wrong with the ced-1 mutant?

• a) Apoptotic cells fail to die
• b) Normal cells die ectopically
• c) Apoptotic cells fail to be engulfed
• d) Normal cells are ectopically engulfed
• e) Cells undergo necrosis

1. a and b
2. b and c
3. c and d
4. d and e
5. e and a

ced-1
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Phenotypic analysis of ced-1 and ced-2 mutants

1. More cell deaths?
2. Dying cells cannot be removed or engulfed

How to distinguish these two possibilities?
Follow the cell lineage in the mutant animals
What is next?
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Suppressor screens ced-3 and ced-4
What are the functions of ced-3 and ced-4?
H. Ellis and R.H. Horvitz
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What could be the functions of ced-3 and ced-4?

• ced-3 and ced-4 promote cell corpse engulfment
• 2) Inhibitors of cell corpse engulfment
• 3) ced-3 and ced-4 could promote cell deaths
• 4) ced-3 and ced-4 could inhibit cell death
• 5) ced-3 and ced-4 could promote necrosis

1. 1 and 2
2. 2 and 3
3. 3 and 4
4. 4 and 5
5. 5 and 1

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What are the functions of ced-3 and ced-4?

• ced-3 and ced-4 promote cell corpse engulfment?
• Then the mutations must be increase-of-function
• 2) Inhibitors of cell corpse engulfment?
• Then the mutations should be loss-of-function
• 3) ced-3 and ced-4 could promote cell deaths
• Then the mutations should be loss-of-function

How to distinguish 2) and 3)?
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• Lineage analysis suggest
• Many cells that normally die now survive
• ced-3 and ced-4 are involved in cell killing

• How do ced-3 and ced-4 kill the cells?
• Cells die by murder?
• Cells die by suicide?
• cells die by aging?
• Cells die because of injuries?
• Cells die by sickness?

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Cells Die by Suicide Rather Than Murder

• Yuan and Horvitz demonstrated by mosaic analysis
  that ced-3 and ced-4 function in the dying cells
  to kill.
• ced-3 encodes a protein with homology with IL-1b
  converting enzyme (ICE), a cysteine protease.
• ced-4 encodes a protein similar to apoptotic
  protease-activating factor (Apaf-1).

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cps-6
ceh-30
CEM cells
psr-1
wah-1
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Caspases Are Cell Death Executors

• Yuans group using cell culture experiments
  showed ICE and CED-3 can both kill in mammalian
  cells
• CED-3/ICE define a family of cysteine proteases,
  named caspases (aspartate-specific proteases),
  which so far has 16 family members
• A caspase is first synthesized as an inactive
  protease precursor and later activated by
  specific proteolysis at specific aspartate
  residues.

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Caspase Family
caspase
Adapted from Thornberryand Lazebnik, Science 281,
1312-1216, 1998
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Stucture of Caspase-3 (CPP32)
Adapted from Thornberryand Lazebnik, Science 281,
1312-1216, 1998
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Activation of Caspases
1) By self-activation
2) By another cysteine protease or caspase
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Programmed Cell Death
The biochemical basis
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How Are Caspases Activated

• Xiao-dong Wang first set up an in vitro cell-free
  system to study the caspase activation process
• His group found that dATP can trigger caspase-3
  activation in S100 Hela cell extracts.

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Purification of Caspase Activating Factors
S100dATP
Pinkish protein
Apaf-2 (Cytochrome C)
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Apaf-1 Is Similar to CED-4
Adapted from Zou et al. Cell 86 147-157, 1997
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Model for Caspase Activation
Adapted from Zou et al. Cell 86 147-157, 1997
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Discovery of Bcl-2

• In 1986, three groups independently cloned bcl-2
  oncogene. bcl-2 oncogene causes follicular
  lymphoma and is a result of chromosome
  translocation t(1418 that has coupled the
  immunoglobulin heavy chain locus to a chromosome
  18 gene denoted bcl-2
• In 1988, Vaux, Cory, and Adams discovered bcl-2
  oncogene causes cancer by inhibiting lymphocyte
  cell deaths, providing the first evidence that
  cancer can result from inhibition of cell death
• 1990, Stanley Korsmyers group showed Bcl-2
  localized to mitochondria

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C. elegans ced-9 Gene Is a Functional Homologue
of Bcl-2

• A gain-of-function mutation in ced-9 protects
  against all cell deaths in nematodes, while
  loss-of-function mutations cause massive ectopic
  cell deaths
• ced-9 encodes a protein similar to Bcl-2
• Bcl-2 inhibits cell death in nematodes and can
  partially substitute for ced-9
• CED-9 is localized at mitochondria

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Bcl-2/ced-9 Define a Family of Cell Death
Regulators

• Korsmyers group purified a protein, Bax, that
  associates with and modulates the activity of
  Bcl-2. Bax by itself can also cause apoptosis in
  a Bcl-2-independent and caspase-independent
  manner.
• Thompsons group identified a gene, named bcl-x,
  which can be alternatively spliced to generate
  two proteins that have opposite functions in
  apoptosis. The long form (Bcl-xL) inhibits
  apoptosis and the short form (Bcl-xs) cause cell
  death.
• Korsmyers group identified another
  Bcl-2-interacting and death inducing-protein,
  Bid, which only has one Bcl-2 homology domain
  (BH3).
• Subsequently, more Bid-like death-inducing
  proteins were identified, all of which has only
  one BH3 domain. This protein family was called
  BH3-only Bcl-2 subfamily.