APOPTOSIS

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Immunity in Health and Disease (BS963) 2009-
2010 Dr Minnie OFarrell
APOPTOSIS
Apoptosis in model systems. Molecular mechanisms
of apoptosis in mammalian cells, mitochondrial
pathway (intrinsic) and cell death receptor
pathway (extrinsic). bcl-2 gene family.
Caspases. Apoptosis in the development of the
immune system and in pathologies.
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General text books/monographs Alberts et al. The
Molecular Biology of the Cell 5th ed Lodish et
al. Molecular Cell Biology 6th ed. Weinberg The
Biology of Cancer
Normal white blood cell Apoptotic white
blood cell
Lodish 6th Fig 1-19
During apoptosis (programmed cell death) cells
bleb and eventually break apart without releasing
contents.
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Alberts et al. Fig 18-3 Apoptosis during the
metamorphosis of a tadpole into a frog. The cells
in the tadpole tail are induced to undergo
apoptosis stimulated by the increases in thyroid
hormone that occurs during metamorphosis
The nematode Caenorhabditis elegans has also been
a very important model system for studying
apoptosis in development.
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Types of cells that undergo apoptosis
Apoptosis in the embryonic mouse paw. Apoptosis
occurs between 12.5 to 14.5 days in embryogenesis
Weinberg Fig 9.19 Apoptosis and normal
morphogenesis. TUNEL assay (immunodetection)
detects breaks in DNA as cells undergo apoptosis
Cell Biology Pollard and Earnshaw
1 day
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Apoptosis is also important in the development of
the nervous system
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Two lymphocytes
Staurosporin-treated HeLa cells
Healthy Apoptotic
Apoptosis
Apoptosis
Weinberg Fig 9.18 Different parts of the
apoptotic programme. Markers for the process.
Fragmentation of DNA
Apoptotic cell
Pyknotic nuclear fragments
Phagocytosis of apoptotic bodies
Fragmentation of Golgi bodies
Phosphorylation of Histone 2B
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Comparison between two forms of cell death,
apoptosis and necrosis
Apoptosis
Necrosis
Apoptosis is a highly regulated process requiring
gene expression.
Weinberg Table 9.3 Apoptosis v necrosis
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Apoptosis is a very clean process. Phagocytic
macrophage removing remains of apoptotic
cell. Also a highly regulated process involving
cell-cell interaction and signalling.
Cell and Molecular Biology Karp
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This slide and the next Two useful tables
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Regulators of apoptosis
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An important model system in which the molecular
basis of apoptosis has been studied
Lodish et al. 6th Fig 1.25
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Caenorhabditis elegans Nomarski interference
microscopy Lodish et al. 5th Fig 22.8
Lineage of all somatic cells, from fertilized egg
to mature worm has been traced. 1030 cells
generated but 131 cells die
C. elegans Cell fate data
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Genetic screens Ced-3, Ced-4 and Egl1 required
for cell deaths Ced-9 represses cell death
programme
ced-1 mutant (defective in engulfment so dead
cells visible (refractile) and can identify
apoptosed cells (yellow arrows)
ced-1- ced-3 double mutant. There are no
refractile cells in these double mutants
indicating that no cell deaths occurred (yellow
arrowheads)
Mutations in the ced-3 gene block apoptosis
Lodish et al.6th Fig 21.36
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Lodish et al. 6th Figure 21.37 Evolutionary
conservation of apoptotic pathways
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There are two major pathways of
apoptosis intrinsic pathway extrinsic pathway
Maniati et al. 2008 Fig 1 The molecular basis of
apoptosis in mammals. Simplified overview
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A family of apoptosis proteins has been
discovered in mammalian cells . The first member
of the Bcl-2 family was identified during a study
of B cell lymphoma. The oncogenic version is
formed through a reciprocal chromosomal
translocation in which parts of the chromosome 14
and chromosome 18 are exchanged. The translocated
bcl-2 gene is now under the control of an active
immunoglobulin promoter that drives high levels
of constitutive expression.
Bcl-2 is pro-survival (anti-apoptotic) and is
homologous to CED-9 in Caenorhabditis
elegans. Quite early in the study of Bcl-2 it
was found to localise to the outer membrane of
the mitochondria. We now know that there are at
least 24 Bcl-2-related proteins, 6 are
anti-apoptotic and 18 are pro-apoptotic. Lets
examine these in a little more detail.
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Introducing the BCL-2 family of proteins
important in apoptotic pathways
anti-apoptotic
Weinberg Fig 9.25 Bcl-2 and related proteins part
A
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The structure of many of these proteins has been
determined and interactions between them
investigated.
Weinberg Fig 9.25 Bcl-2 and related proteins
The anti-apoptotic protein, BCL-XL , is
inhibited by binding of the pro-apoptotic BH3
only protein (orange) in the groove between BH1
and BH3
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BH3 only protein binding specificity for BCL-2
homologues
BIM and PUMA bind to all BCL-2 family members
tested by contrast NOXA only binds to A1 and
MCL1. These binding specificities recapitulate
the ability of these proteins to activate
apoptosis e.g. BIM et al can induce apoptosis
alone whereas a combination of NOXA and BAD is
required.
Youle and Strasser (2008) The BCL-2 protein
family opposing activities that mediate cell
death. Nature Reviews Molecular Cell Biology, 9,
47-59
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Conformational changes in BCL-2 family members
during apoptosis. BAX undergoes extensive
conformational changes during the mitochondrial
translocation process. The protein changes from
a soluble cytoplasmic protein in healthy cells to
one that appears to have at least 3 helices
inserted in the mitochondrial membrane in
apoptotic cells.
Youle and Strasser (2008) The BCL-2 protein
family opposing activities that mediate cell
death. Nature Reviews Molecular Cell Biology, 9,
47-59
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Alberts et al. Fig 18-7 Release of cytochrome c
from mitochondria during apoptosis
GFP Green Fluorescent protein
Control UV-treated to induce apoptosis
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To summarize.. BCL-2 family of proteins have
opposing apoptotic activities 1st set (e.g.
Bcl-2 itself) inhibit apoptosis. 2nd set (e.g.
BAX) promotes apoptosis. 3rd set (e.g. the BH3
only proteins) bind and regulate the
anti-apoptotic BCL-2 proteins to promote
apoptosis.
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Extrinsic or death receptor pathway
Maniati et al. 2008 Fig 1 The molecular basis of
apoptosis. Simplified overview
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Death receptors
Weinberg Fig 9.31 The extrinsic apoptotic pathway
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Opferman (2008) Fig 1 Death receptor- mediated
apoptosis
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Maniati et al. 2008 Fig 1 The molecular basis of
apoptosis. Simplified overview
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Weinberg Fig 9.29 The intrinsic apoptotic pathway
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Heptamer ( 7 chains) The blue helices in the
middle bind and activate procaspase 9 to caspase
9 Caspase 9 activates procaspases 3, 6 and 7
Weinberg Fig 9.28 The APOPTOSOME is assembled in
the cytoplasm when cytochrome c is released from
the mitochondria and binds to Apaf-1
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Weinberg Fig 9.32 Convergence of intrinsic and
extrinsic apoptotic pathways
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Caspases are proteolytic enzymes activated by
both extrinsic and intrinsic pathways
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Caspase family, 12-13 members Two
classes Initiators Effectors All caspases
have a similar domain structure Not all
mammalian caspases participate in apoptosis. For
example Caspases 1, 4, 5, and 12 are activated
during innate immune responses and are involved
in the regulation of the inflammatory reponse
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Effector caspases (such as caspase-3, -6 and -7
in mammals) function to breakdown cell structures
through cleavage of specific substrates. Actin
cytoskeleton Lamins Golgi Translation apparatus
Taylor et al. (2008)
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Cory and Adams (2002) Nature Reviews Cancer, 2,
647-656
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Phagocytic macrophage removing remains of
apoptotic cell. This is a highly regulated
process involving recognition and signalling
between the apoptotic cell and macrophage.
Cell and Molecular Biology Karp
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Stages of engulfment of apoptotic cells can be
divided into 4 stages
Binding Recognition Phagocytosis Internali
zation
Kinchen and Ravichandran (2007) Fig1
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Lauber et al.(2004) Fig 2 Lack of dont eat
me signals on the surface of apoptotic cells
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Taylor et al. (2008)
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Lauber et al.(2004) Fig 1 The engulfment synapse
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Lauber et al. (2004) Fig 6 The three steps of
apoptotic cell removal.
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Alberts et al. Fig 25-13 Mechanisms of
immunological tolerance to self antigens
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25-64
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Alberts Fig 25-47 Two strategies by which
effector cytotoxic T cells kill their target cells
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Following are three supplementary slides for
overview and glossary of terms.
A useful diagramatic overview with a short
review. Willis et al. (2003) J. Cell Science,
116, 4053-4057
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Cory and Adams (2002) Nature Reviews Cancer, 2,
647-656
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Cory and Adams (2002) Nature Reviews Cancer, 2,
647-656
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