APOPTOSIS: Programmed Cell Death APOPTOSIS

APOPTOSIS: Programmed Cell Death
156:201 Principles of Molecular & Cell Biology
October 24, 2003
Reading: Lodish Chapter 22, pp. 924-930
Discussion Group: October 27, 2003
Paper: Sogame N, Kim M, Abrams JM. Drosophila p53 preserves genomic
stability by regulating cell death. PNAS 100: 4697-4701, 2003.
Jackie Bickenbach, PhD
jackie-bickenbach@uiowa.edu
APOPTOSIS
Dept. of Anatomy & Cell Biology
1-457 BSB
DEFINITION: Capacity of a cell to respond to stimuli by initiating pathways that
lead to its death by a characteristic set of reactions.
Organized community of cells with a regulated
balance between cell proliferation and
cell death.
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Cell division is controlled by survival proteins.
This results in an expansion of cells and
tissues.
Cell death is programmed (regulated) by
death proteins.
This results in cell suicide.
A loss of cells is critical in both embryonic
development and in adult tissues:
To decrease or maintain tissues
To get rid of damaged cells
Greek: apo = off
ptosis = a falling
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Apoptosis is not the same as Necrosis
Necrosis
- cells are damaged
- cells swell, then burst
- triggers a destructive immune
response
- acute “messy” response
Apoptosis
- cells die neatly
- cells do not damage neighbors
- programmed, regulated response
- no attraction of destructive immune cells
- apoptotic cells are neatly cleared away
(engulfed by macrophages or by neighboring cells)
Regulated Cell Death During Development
In frog development, an increase in thyroid
hormone induces apoptosis in the
cells in the tadpole tail.
In mouse development, digits are formed
only if cells in the web between each
digit are programmed to die.
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Regulation of the number of nerve cells
Binding of specific neurotrophins
(NGF, BDNF, NT3) to their specific
Trk receptor (receptor tyrosine
kinases) produces survival signals for
a specific class of neurons.
Lodish figure 22-28
suckling 9 hrs. post-suckling
Only nerve cells that
receive a survival signal
live.
Balances number of nerve
cells to number of target
receptors.
Balance of cell
proliferation with
apoptosis in adult
mammary tissues
Ductal epithelial cells proliferate in response to
hormones produced by birth of offspring.
Sucking triggers milk production via oxytocin
hormone produced in hypothalamus.
When suckling stops, TGFb3 (brown stain in B)
accumulates in ducts with undrained milk
- triggers apoptosis (brown cells in C)
- unneeded cells are eliminated
- ducts regress and gland goes back to
resting state
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Apoptosis: Programmed
(Regulated) Cell Death
Apoptosis is characterized by:
Shrinking cells
Chromatin compaction
Degradation of chromosomal DNA
Nuclear fragmentation
Condensation of cytoplasm
Blebbing
Cell fragmentation
Cell surface alterations cause rapid phagocytosis,
which allows recycling of cellular proteins.
Apoptosis can be triggered by internal or external
stimuli.
Apoptosis Movie
Lodish Figure 22-26
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Sigma-Aldrich
There are
multiple
apoptotic
pathways
In all pathways control
of apoptosis is
regulated by a balance
between activation and
inhibition.
It is thought that all cells
have the capacity to
apoptose.
Thus, the critical
determinant of whether
a cell lives or dies may
depend upon its ability
to activate or inhibit
these apoptotic
pathways.
Fas-Fas Ligand Triggers Apoptosis
Fas receptor on a target cell is
activated with the FasL protein.
(Fas is related to TNF receptor,
FasL is related to TNF.
FasL protein is on an activating cell
plasma membrane.
Upon interaction with FasL, Fas
trimerizes.
Fas has a cytoplasmic domain often
called the “death domain”.
The death domain is involved with
protein-protein interactions.
e.g. Fas-FADD-capsase 8
Note: Fas and FasL can be on
different cells or one the same
cell.
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Caspases:
family of cysteine aspartate proteases
Caspases have a catalytic cysteine and cleave
their targets at an asparate.
2 subfamilies:
Caspase 1 involved in inflammation.
Caspases 3, 6-10 involved in apoptosis.
All are synthesized as inactive procaspases.
All, but the initial caspase in a cascade, are
activated by proteolytic cleaving by another
caspase family member. (First caspase in
the cascade is autocleaved)
ÿ Prodomain is cleaved off
ÿ Then caspase is cleaved into small
and large subunits
ÿ Cleaved subunits associate to form
active caspase (likely a tetramer)
Caspase Cascade
Caspase Cascade:
1. Small number of caspases
activated
2. Rapid autocleaving of more
caspases.
3. These initiator caspases also
activate other caspases by
cleaving procaspases
4. These then activate additional
caspases (Effector Caspases)
5. The latter caspases cleave other
cellular proteins
e.g. cellular proteins to release
DNAse, nuclear lamin to
release DNA
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Caspases first discovered in c. elegans
C. elegans has one effector caspase, CED-3.
Humans have 15 caspases, 6 involved in apoptosis (3, 6-10).
Three types of proteins are involved in regulation of apoptosis:
Regulator proteins can either promote or suppress apoptosis.
(CED-9 and Bcl-2 are suppressors)
Adapter proteins coordinate the action of regulator and effector proteins to
control apoptosis by interacting with both types of proteins.
In the absence of trophic factors, they promote activation of effectors
In the presence of trophic factors, they are inhibited by regulators.
Activation of the
Mitochondrial
Caspase Pathway
The caspase pathway moves from the
cell membrane to the mitochondrial
membrane via caspase 8.
Caspase 8 cleaves Bid to release a Cterminal
domain that translocates to
the mitochondria.
Bid is a member of the Bcl2 family and
acts to cause the mitochondria to
release cytochrome c.
Mitochondria are also involved in the
caspase cascade in c.elegans.
Sigma-Aldrich
Lodish
Figure 22-31
vertebrates
c.elegans
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Mitochondria’s Roles in Apoptosis
Pro-apoptotic role
Cells are damaged or stressed.
Mitochondria are induced to release
cytochrome c (electron carrier)
Binds Apaf-1 (adaptor protein)
Aggregates procaspase-9 proteins
They cleave each other
Release active caspase 9
Activates downstream effector
caspases 3,6,7
Anti-apoptotic role
Proteolytic activity of caspase 9 can
be inhibited by IAP proteins.
Proteins that antagonize IAPs may
be released by mitochondria.
Inhibitors of
Apoptosis
Sigma-Aldrich
Apoptosis can be inhibited at stages
catalyzed by the later caspases.
IAP (inhibitor of apoptosis) Family
bind some procaspases
bind some caspases
In order for apoptosis to proceed, IAPs
must be blocked.
When mitochondria release cytochrome
c, they also release a protein
(diablo) that blocks IAPs
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Bcl family members play roles as inhibitors
and as promoters of apoptosis
Bcl family
Death Inhibitors
Bcl-2 and Bcl-X block
cytochrome c release
Promoters of Apoptosis
Bad--inhibits death inhibitors
Bax, Bak, Bim, & Bid stimulate
release of cytochrome c
Caspase Activation vs.Inhibition
Lodish Figure 22-32
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Mechanisms of Ageing and Development 123: 245-260, 2002.
Mutations in
apoptotic genes
may extend life
DAF-2 (c. elegans)
Similar to IFG-I (mammals)
Controls daur larva phase
Animals with DAF-2 mutations
Increase AGE-1 (PI3K)
Increase wt AKT-1 (Akt)
Increase IKK
Results:
• No cell death in the worm
• Worms live twice as long
Is there a switch between apoptosis and
proliferation?
J. Clin., Invest. 109: 437-442, 2002.
FLIP
Diverts molecules activated by
Fas-FasL from caspase cascade
to proliferative pathway
Contains 2 DED (similar to
caspase 8)
Associates with
Raf-1 (ERK) -> proliferation
TRAF-1 (NF-kB) -> differentiation
Thus cells with FLIP resist
caspase -8 apoptosis and might
be stimulated to proliferate
instead.
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Apoptosis Movie
References for Apoptosis
1. Earnshaw et al (1999) “Mammalian Caspases: structure, activation,
substrates and functions during apoptosis” Ann. Rev Biochem. 68, 383.
2. Rhind and Russell (1998) “ Mitotic DNA damage and replication checkpoints
in yeast” Curr. Opin. Cell Biol. 10, 749.
3. Budihardjo, I., et al., Biochemical pathways of caspase activation during
apoptosis. Annu. Rev. Cell Dev. Biol., 15, 269-290 (1999).
4. Adrain, C., and Martin, S.J., The mitochondrial apoptosome: a killer
unleashed by the cytochrome seas. Trends Biochem. Sci., 26, 390-397
(2001).
5. References listed at the end of Lodish Chapter 22.
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