apoptosis

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Immunity in Health and Disease (BS963) 2009- 2010
Dr Minnie O’Farrell
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.
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.
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.
Types of cells that
undergo apoptosis
Weinberg Fig 9.19 Apoptosis
and normal morphogenesis.
TUNEL assay
(immunodetection) detects
breaks in DNA as cells undergo
apoptosis
Apoptosis in the
embryonic mouse
paw.
Apoptosis occurs
between 12.5 to 14.5
days in
embryogenesis
Cell Biology
Pollard and Earnshaw
+ 1 day
Apoptosis is also important in the development of the nervous system
Two lymphocytes
Healthy
Staurosporin-treated HeLa cells
Apoptosis
Apoptosis
Apoptotic
Weinberg Fig 9.18 Different parts of the
apoptotic programme. Markers for the
process.
Fragmentation of DNA
Pyknotic
nuclear
fragments
Apoptotic
cell
Phagocytosis of apoptotic bodies
Fragmentation of Golgi bodies
Phosphorylation of Histone 2B
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
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
This slide and the next……… Two useful tables
Regulators of apoptosis
An important model system in which the molecular basis of
apoptosis has been studied
Lodish et al. 6th Fig 1.25
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
Genetic screens:
Ced-3, Ced-4 and
Egl1 required for
cell deaths
Ced-9 represses
cell death
programme
Mutations in the
ced-3 gene
block apoptosis
Lodish et al.6th
Fig 21.36
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)
Lodish et al. 6th Figure 21.37 Evolutionary conservation of apoptotic pathways
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
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 antiapoptotic and 18 are pro-apoptotic.
Lets examine these in a little more detail.
Introducing the BCL-2 family of proteins important in apoptotic pathways
= anti-apoptotic
Weinberg Fig 9.25 Bcl-2 and related proteins part A
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
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
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
Alberts et al. Fig 18-7 Release of
cytochrome c from mitochondria
during apoptosis
GFP = Green Fluorescent protein
Control
UV-treated to induce
apoptosis
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 antiapoptotic BCL-2 proteins to promote apoptosis.
Extrinsic or death receptor pathway
Maniati et al. 2008 Fig 1 The molecular basis of apoptosis. Simplified overview
Death receptors
Weinberg Fig 9.31 The extrinsic
apoptotic pathway
Opferman (2008) Fig 1 Death receptormediated apoptosis
Maniati et al. 2008 Fig 1 The molecular basis of apoptosis. Simplified overview
Weinberg Fig 9.29
The intrinsic
apoptotic pathway
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
Weinberg Fig 9.32
Convergence of
intrinsic and
extrinsic apoptotic
pathways
Caspases are proteolytic enzymes activated by both extrinsic
and intrinsic pathways
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
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)
Cory and Adams
(2002) Nature Reviews
Cancer, 2, 647-656
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
Stages of
engulfment of
apoptotic cells can
be divided into 4
stages
Binding
Recognition
Phagocytosis
Internalization
Kinchen and
Ravichandran (2007) Fig1
Lauber et al.(2004) Fig 2 Lack of “don’t eat me” signals on the surface of
apoptotic cells
Taylor et al.
(2008)
Lauber et al.(2004) Fig 1 The engulfment synapse
Lauber et al. (2004) Fig 6 The three steps of apoptotic cell removal.
Alberts et al. Fig 25-13 Mechanisms of immunological tolerance to self antigens
25-64
Alberts Fig 25-47 Two strategies by which effector cytotoxic T cells kill their target cells
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
Cory and Adams
(2002) Nature Reviews
Cancer, 2, 647-656
Cory and Adams
(2002) Nature Reviews
Cancer, 2, 647-656
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