Transcript active

Biology of Disease CH0576
Aspects of Apoptosis
CH0576/RHY
Apoptosis
• Apoptosis, or ‘programmed cell death’
is a term used to describe how cells
die under both pathological and
physiological conditions.
• It is an active process, unlike necrosis.
• It is a genetically controlled process,
unlike necrosis.
CH0576/RHY
Genes & apoptosis
• One of the most important characteristics
of apoptosis is that it is under genetic
control.
• In the early 1980’s it was demonstrated
that inhibitors of RNA and protein
synthesis such as actinomycin D and
cycloheximide also inhibited apoptosis.
• The implication being that specific genes
needed to be switched on as the cell
prepared to undergo apoptosis.
Genes & apoptosis
• The genes regulating apoptosis
rapidly gained the trivial name of
‘suicide genes’ or ‘death genes’.
• Since the early 80’s a number of
suicide genes and anti-suicide genes
have been identified.
• A number of these apoptosis
regulating genes turned out to be
oncogenes e.g. bcl-2, p53 and c-myc.
Genes & apoptosis
• Bcl-2 was the first gene identified as
having a role in the regulation of apoptosis.
• Cells which expressed high levels of this
gene were more resistant to the induction
of apoptosis.
• Cells transfected with this gene gained
this resistance.
• Expression was associated with follicular
cell lymphoma.
Genes & apoptosis
• The implication was that the cancer may be
due more to a lack of cell death rather
than an excessive proliferation.
• It was suggested that cells which failed to
die at the end of their allotted lifespan,
live on and contribute to tumour
development.
• Expression of this gene confers an
increased resistance to apoptosis induction
by several cytotoxic drugs.
Genes & Apoptosis
• Hence bcl-2, by inhibiting cell death not
only contributes to tumour development
but also makes the tumour cells more
difficult to kill with conventional antitumour drugs.
• Genes within the ‘pro-death’ camp include
p53 and c-myc.
• p53 is a gene which is found to be mutated
in the majority of human cancers.
Apoptosis:
Physiological examples
• A number of clear situations exist, in which
apoptosis is fundamentally important, in
the development and survival of the
organism.
– Limb development in embryology
– Potentially autoreactive thymocytes, or cells
bearing inappropriate receptors, fail to develop,
they undergo apoptosis, being destroyed before
populating the secondary lymphoid organs.
– Involution of the thymus with age.
CH0576/RHY
Apoptosis:
Pathological examples
• In pathological states apoptosis can be
induced by:-
– Cytotoxic T-cells ***
– Natural Killer cells (NK cells)
– By the action of cell free cytokines such as
tumour necrosis factor (TNF)
– Apoptosis plays an important role in a range of
autoimmune diseases, viral infections, and in
some tumour cell death.
– Mutations in various elements of the apoptotic
pathways have recently been linked with
specific disease states
CH0576/RHY
Mechanisms of apoptosis
Activation of the Caspase System
• Work with the nematode worm C.elegans
revealed that an enzyme activity, ced-3,
was essential for apoptosis to occur.
• Various enzymes present in mammalian
systems, which are homologous to ced-3,
have been described.
• The first such enzyme was termed ICE.
CH0576/RHY
Caspase cascade
• Several such enzymes have now been found and
described as ICE-like enzymes, more recently
they are described as CASPASES.
• These are a series of cysteine protease
enzymes which become activated within the cell
undergoing apoptosis.
• The activated caspases can cause the cleavage
of poly(ADP-ribose) polymerase, or PARP, a
crucial enzyme in DNA repair.
CH0576/RHY
Mechanism of apoptosis:
• Hence DNA repair mechanisms become
disabled, leading to the activation of a
range of apoptotic endonucleases, which
initiate the fragmentation of DNA.
• A wide range of cellular proteins are
broken down by the activated caspase
enzymes.
• The question is: How is the caspase enzyme
cascade activated initially?
CH0576/RHY
Caspase Classes
• Caspases are grouped into two classes
based on similarities in their primary
structures.
• Class I Caspases possess long N-terminal
prodomains.
• Classs II Caspases possess either short
prodomains or lack prodomains.
• Activation of the Class II molecules (e.g.
Caspases-3,6, and 7) seem to need
proteolytic processing by class I caspases
CH0576/RHY
Caspase classes
• Class II caspases are the downstream
caspases that mediate the proteolysis of a
range of cellular proteins, in a cell destined
to die.
• The activation of the class I caspases
(upstream) is fundamental in cell death
commitment.
• Recent work suggests how these class I
caspases are activated.
CH0576/RHY
Molecule-Molecule
Interactions
• Interaction between various components of
the death complexes are crucial in apoptotic
signalling.
• Three types of protein-protein interaction
domains are present in apoptotic molecules.
• DDs - death domains.
• DEDs - death-effector domains.
• CARDs - caspase recruitment domains.
CH0576/RHY
Molecule-Molecule
Interactions
• DDs are common in:-
– the upstream components of the apoptotic
pathway, such as the death receptors (e.g. Fas,
TNFR and DR3)
– molecules that are recruited to these death
receptors (e.g. FADD, TRADD and RIP) - these
molecules are cytoplasmic ‘adaptor’ molecules.
• DEDs and CARDs are responsible for
recruiting class I caspases to specific death
complexes through adaptor molecules.
CH0576/RHY
Molecule-Molecule
Interactions
• The interaction between the death domains of
the death receptors and those of the adaptor
molecules require the initial activation of the
death receptor by a suitable ligand.
• The binding of the ligand to the receptor
causes a conformational change in the
intracytoplasmic death domain and allows
combination with a similar domain in the
adaptor molecules.
CH0576/RHY
Activation of Caspase
cascade:
• Activators or inducers of apoptosis
interact with receptors present on the cell
surface.
• These receptors are members of the
TNF/NGF receptor family.
• The two key receptors are Fas, also known
as APO-1 or CD95, and TNF receptor.
• Both these receptors possess an
intracytoplasmic domain associated with
them termed a ‘death domain’.
CH0576/RHY
Caspase activation
• The binding of a suitable ligand to the specific
receptor on the cell surface causes a signal
transduction to the death domain and the
activation of a pro-caspase activity.
• Fas receptors are closely associated with a procaspase 8 activity, and TNF receptors
associated with a pro-caspase 2 activity.
• This activation triggers a sequential cascade
within the cell.
CH0576/RHY
Caspase activation: (2001)
• Activation Induced Cell Death.
– Current Opinion in Immunology, 2001, 13:3:
356-362
• Oligomerization of Fas by FasL induces the
recruitment of the adaptor molecule FADD
by their mutual DDs.
• The opposite end of FADD contains DEDs
which allows the recruitment of either procaspase 8 or FLIP (an inhibitory molecule)
CH0576/RHY
Caspase Activation
CH0576/RHY
Caspase activation
• Active caspase-8 promotes the cleavage of
various downstream (class II) caspases
including caspase-3,6 and 7.
• These are smaller ‘effector’ caspases
which lack the amino-terminal domains such
as DDs, DEDs or CARDs.
• They function to degrade various cellular
components such as nuclear lamins, and the
cytoskeletal proteins fodrin and gelsolin.
CH0576/RHY
Caspase activation
• The inhibitor of caspase-activated DNAase
(ICAD) is also cleaved.
 Activation of CAD to degrade DNA.
• Caspase-8 can also cleave Bid, a pro-apoptotic
member of the Bcl-2 family of molecules, to
form an active truncated Bid (tBid).
• This molecule complexes with and inhibits
Bcl-2, in the outer mitochondrial membrane.
CH0576/RHY
Caspase activation
• This initiates the mitochondrial death
sequence.
• Disruption of the outer mitochondrial
membrane allows the leakage of two crucial
mediators of apoptosis: cytochrome-c and
Smac (synonym DIABLO).
• Cytochrome-c complexes with Apaf-1 and
recruits caspase-9, forming the apoptosome.
• Caspase -9 becomes activated.
CH0576/RHY
Caspase activation: (2001)
• Active caspase-9 can cleave and activate
procaspase-3 to its active form which can go
on to activate further caspases (e.g. 6 and 7)
and causes the breakdown of several
cytoskeletal proteins and the inactivation of
the DNAase inhibitor, ICAD.
• Smac inactivates the naturally occurring
inhibitor of apoptosis (IAP)
 Resulting in the further activation of the
downstream caspases.
CH0576/RHY
Killing by Cytotoxic T-cells:
• In addition to the activation of the caspase
system through the death domains of Fas
and TNFR, the process of granule mediated
apoptosis takes part in both innate
immunity, via NK cells, and also adaptive
defence mechanisms, via CTL’s against:• Intracellular pathogens (e.g. viruses)
• Tumours
• Non-self cells.
CH0576/RHY
Granule mediated apoptosis
• This is a calcium dependent mechanism
induced by the actions of a pore froming
protein called PERFORIN and a family of
granule associated proteases called
GRANZYMES.
• The granules of cytotoxic T cells and
their constituent proteins are synthesized
about 24 - 48 hours after stimulation via
the T cell receptor.
CH0576/RHY
Granule mediated apoptosis
• Perforin and granzymes are stored
within cytoplasmic cytotoxic granules.
• Following recognition of a specific target
by the CTL a tight junction is formed
between the effector and target cell.
• When purified granules from CTLs are
added to suitable targets in vitro the
cells are destroyed by the creation of
pores within the lipid bilayer.
CH0576/RHY
Granule mediated apoptosis
• The pores consist of polymers of perforin.
• Perforin, in the presence of calcium ions,
spontaneously polymerises and forms a
cylindrical structure.
• The outer surface of the cylinder is lipophilic,
and the inner surface of the structure is
hydrophilic.
• The inner diameter of the cylinder being about
16nms.
CH0576/RHY
Granule mediated apoptosis
• The structure inserts itself into the target
membrane.
• The pore allows the passage of water and
salts into the cell and causes a rapid
destruction of the target by a lytic event.
• In vitro there is no evidence of the nuclear
DNA fragmentation which is a feature
characteristic of apoptosis.
CH0576/RHY
Granule mediated apoptosis
• It is suggested that this lytic method of killing
is only seen at artificially high levels of
perforin.
• It is probably not a true reflection of the role
of perforin in apoptosis initiated by CTLs in
vivo.
• It is more likely that sub-lytic levels of
perforin are generated in vivo, serving as a
means of entry of granzymes into the target
cell.
CH0576/RHY
Granule mediated apoptosis
• Recent evidence suggests that there are a
number of means by which granzymes enter
the target cell in order to initiate
apoptosis.
• Granzymes are not themselves directly
responsible for DNA fragmentation, they
are proteases and not nucleases.
• Granzymes A  G have been identified,
the major granzymes in terms of apoptosis
are GrA and GrB.
CH0576/RHY
Granzymes:role in apoptosis
• Granzyme B in an aspase, i.e. it cleaves
proteins after an aspartic acid residue.
• The pro-caspases are generally activated by
cleavage at specific aspartic acid residues.
• GrB is capable of activating most of the
caspases (3,6,7,8,9,and 10).
• Evidence suggests that in addition to
activation of caspases, GrB can activate ‘death
substrates’ directly.
CH0576/RHY
Granzymes:role in apoptosis
• In experiments performed using
purified GrB the enzyme cleaved
nuclear substrates including DNA-PK
and NuMA.
• The cleavage sites were different to
those acted upon by the caspases.
CH0576/RHY
Death Receptors
• The well known death receptors, Fas and
TNFR1 trigger apoptosis by recognition of
their specific ligands.
• A recent finding is that there are several
homologues of Fas and TNFR1.
• Death Receptor (DR) 3,4,5 and 6 all function
as receptors which signal apoptosis.
• Decoy Receptors (DcR) 1, 2 & 3 act as decoys
– they compete with DRs for ligand binding.
CH0576/RHY
Death Ligands
• Research using ‘gene knockout’ studies in
the murine system has allowed some of the
links between death receptors and the
cell’s apoptotic mechanism to be elucidated.
• Studies suggest that FasL is critical for
the activation-induced, or instructive
apoptosis of T -cells.
– Individuals with mutations in the genes which
encode Fas or FasL accumulate enormous
numbers of lymphocytes.
– There is massive, lethal lymphadenopathy.
CH0576/RHY
Death Ligands
• The research and clinical findings suggest
that the main biological role for FasL is to
initiate instructive apoptosis and deletion
of peripheral lymphocytes.
• TNF or TNFR ‘knockout’ mice show an 
susceptibility to microbial infection and a 
inflammatory response, when challenged
with bacterial endotoxins:
– main biological role for TNF is the induction of
inflammatory response and stress response
genes.
CH0576/RHY
Death Ligands
• Through bioinformatics database screening
two groups of workers found another death
ligand - Apo2 ligand or TRAIL
– TNF-related apoptosis-inducing ligand.
• Apo2L’s closest sequence homologue is FasL
• Research indicates that in vitro both these
ligands potentially induce apoptosis of
tumour cells.
• Apo2L mRNA is expressed in many tissues.
CH0576/RHY
Death Ligands
• Transcript levels  in T cells following
their stimulation with mitogens such as
PHA.
• Resting peripheral T cells are resistant to
the induction of apoptosis by Apo2L, but
IL-2 stimulated T cells acquire sensitivity
to this ligand.
•  this ligand plays a role in peripheral
deletion of lymphocytes.
CH0576/RHY
Death Ligands
• Apo2L may also contribute to the instructive
apoptosis of virally infected cells.
• T-cells from HIV infected patients are more
susceptible to this ligand than are uninfected
cells.
• Apo2L also seems to be involved in tumour
cell killing mechanisms.
CH0576/RHY
Death Receptors
• The receptors in the TNFR family all
possess several cysteine rich domains
(CRD’s) in their NH2 terminal region.
• The family of receptors is broadly divided
into two subgroups on the basis of their
cytoplasmic amino acid sequences.
• The subgroups either possess or lack a so
called ‘death domain’
• This death domain links the receptor to
the caspase cascade.
CH0576/RHY
Death Receptors
• Receptors bearing the ‘death domain’ (DDs)
include:–
–
–
–
–
–
–
TNFR1
Fas
DR3 (also called Apo3 plus other synonyms)
DR4
DR5 (also called TRAIL-R2, TRICK2, KILLER)
DR6
The death domains of these molecules either
connect the receptor to the caspases, which
induce apoptosis or to kinase cascades, capable
of activating genes.
CH0576/RHY
Decoy Receptors
• The other subgroup of TNFR family, lacking a
functional ‘death domain’ act as inhibitors as
opposed to transducers of signalling.
• They are referred to as the Decoy Receptors.
• This subgroup includes DcR1 and DcR2 - both
cell surface expressed molecules.
• It also includes DcR3 and osteoprotegerin
(OPG) - both of which are secreted, soluble
proteins.
CH0576/RHY
Decoy Receptors
• DcR3 acts as a decoy for FasL and has
been shown to have a high expression in
several tumours.
• It is thought to play a role in tumour
immune evasion.
• DcR1 and DcR2 both act as decoys for
Apo2L, inhibiting its activation of DR4 and
DR5 - see explanatory diagram.
CH0576/RHY
Mutations in Apoptosis
Genes
• A range of mutations in genes encoding
different elements of various apoptosis
pathways have been recently implicated in
disease pathogenesis.
• It is envisaged that knowledge of the
mutations of apoptosis genes will benefit the
research into the clinical management of a
range of disease states.
CH0576/RHY
•Genes implicated in disease pathogenesis include the
following:– TNF Receptor 1
Familial periodic fever syndrome
– Fas
Autoimmune lymphoproliferative syndrome I (ALPS I);
malignant lymphoma; bladder cancer
– FasL
SLE
– Perforin
Familial haemophagocytic lymphohistiocytosis (FHL)
– Caspase-10
Autoimmune lymphoproliferative syndrome II
– bcl-10
Non-Hodgkin’s lymphoma
– p53
Various malignant neoplasms
– Bax
Colon cancer; haematopoietic malignancies
– bcl-2
Non-Hodgkin’s lymphoma
– c-IAP-2
Low grade MALT lymphoma
– NAIP1
Spinal muscular atrophy
–This list includes mutations in death receptors, death ligands/cytotoxic
effector molecules, intracellular apoptosis inducers, and apoptosis
inhibitory proteins
CH0576/RHY