Apoptosis

• The cells of a multicellular organism are

members of a highly organized community.

• The number of cells within this community is

tightly regulated – not simply by controlling the
rate of cell division, but also by controlling the
rate of cell death.
• If cells are no longer needed, they commit
suicide by activating an intracellular death
programme. This process is therefore termed
programmed cell death, although it is more
commonly called apoptosis derived from a
Greek word meaning “falling off”.
• The amount of apoptosis that occurs in developing and adult
organisms is surprisingly large.

• For example in the developing vertebrate nervous system up

to half or more of the nerve cells normally die soon after
they are formed.

• In a healthy adult human, billions of cells die in the bone

marrow and intestine every hour.

• It appears very wasteful for so many cells to die especially as

most are in perfect heath at the time they commit suicide.
The question needs to be asked what purpose this cell death
serves.
Apoptosis During the Metamorphosis of a
Tadpole into a Frog

As a tadpole changes into a frog, the cells in

the tadpole tail are induced to undergo
apoptosis; as a consequence, the tail is lost.
Sculpting Mouse Digits
• (A) The paw in this mouse
embryo has been stained with
a dye that specifically labels
cells that have undergone
apoptosis. The apoptotic cells
appear as bright green dots
between the developing digits.

• (B) This interdigital cell death

eliminates the tissue between
the developing digits, as seen
one day later, when few, if any,
apoptotic cells can be seen.
• In many other cases cell death helps to regulate
cell numbers.

• In the developing nervous system, cell death

adjusts the number of nerve cells to match the
number of target cells that require innervation.

• In all of these cases the cells die via apoptosis.

• In adult tissues, cell death exactly balances cell division; if
this was not so then the tissue would grow or shrink.

• If part of the liver is removed in an adult rat, liver cell

proliferation increases to make up for the loss.

• Conversely if a rat is treated with Phenobarbital liver cell

division is stimulated and therefore liver enlargement. Once
the drug is stopped the level of apoptosis within the liver
greatly increases until the liver returns to its normal size.

• Thus the liver is kept at a constant size through regulation of

both the cell birth and death rates.
 Cell Death

ELECTRON MICROGRAPHS SHOWING CELLS THAT HAVE DIED

BY (C) NECROSIS OR (A AND B) APOPTOSIS
 Caspases
• The intracellular machinery responsible for apoptosis appears
to be similar in all animal cells. The machinery is based upon a
family of proteases that have a similar cysteine residue at their
active site and cleave at the C side of aspartic acid residues.

• These proteases are termed caspases and are usually

synthesized in the cell as inactive precursors, which are
activated by cleavage at aspartic acids by other caspases,
resulting in an amplifying proteolytic cascade.

• Some of the activated caspases then cleave other key proteins

in the cell.
 Caspases
• In humans and mice approximately 14 caspases have been
identified.

• From a functional perceptive, it is useful to view the caspases

as either upstream (initiator) caspases or downstream
(executioner) caspases.

• Although most caspases are directly involved in cell death, a

few are not, at least in mammals and higher eukaryotes. A
subgroup of caspases including caspase 1,4 and 5 in humans,
is involved in processing of pro-inflammatory cytokines, such
as pro-interleukin 1- beta.
Two cleaved fragments from each procaspase associate
to form an active caspase which is a tetramer of 2 small
and 2 large subunits; the prodomains are usually
discarded
• Initiator caspases activate
executioner caspases
producing a proteolytic
caspase cascade.

• The executioner caspases

then cleave a variety of
key proteins leading to
cell death.
E.g. In healthy cells, the endonuclease CAD
associates with its inhibitor, iCAD.
Activation of executioner caspases in the
cell leads to cleavage of iCAD, which
unleashes the nuclease.
Activated CAD cuts the chromosomal DNA
between nucleosomes, resulting in the
production of DNA fragments
• Initiator procaspases have a long prodomain which
contains a caspase recruitment domain (CARD) that
enables them to assemble into activation
complexes when the cell receives a signal to
undergo apoptosis.

• Once in such complexes the initiator procaspases

are brought into close proximity which is sufficient
to activate them.

• They then cleave each other to start the irreversible

process
Extrinsic Activation of Apoptosis

DISC – Death-inducing signaling complex

FADD – Fas-associated death domain
• Tumor Necrosis factor (TNF) produced by cells of the
immune system, interacts with cells through specific
TNF receptors.

• These receptors have TNF-binding sites on the outer

face of the plasma membrane and a “death domain”
of about 80 amino acid residues that passes the self-
destruct signal through the membrane to cytosolic
proteins such as TRADD (TNF receptor associated
death domain).
 Restraining the Extrinsic Pathway
• Many cells produce inhibitory proteins that act
either extra or intracellularly to restrain the
extrinsic pathway. E.g.

• Cell surface decoy receptors which have a

ligand binding domain but not a death domain;
because they can bind a death ligand but
cannot activate apoptosis, the decoys
competitively inhibit the death receptors.
Restraining the Extrinsic Pathway
• Intracellular blocking proteins
such as FLIP, which resembles
an initiator caspase but lacks
the proteolytic domain;

• it competes with procaspases

8 and 10 for binding sites in
the DISC and inhibits the
activation of these
procaspases
Intrinsic Activation of Apoptosis
 Bcl2 Proteins Regulate the Intrinsic
Pathway by controlling release of
cytochrome c from the IMS

The anti-apoptotic proteins share 4 distinctive Bcl2 homology

domains. The pro-apoptotic bcl2 proteins consist of two subfamilies
– the BH123 proteins and the BH3-only proteins.
Bax and Bak are the two main BH123 proteins in mammals and at least one
must is required for the intrinsic pathway to operate.

Bak is tightly bound to the mitochondrial outer membrane whereas Bax is

mainly located in the cytosol and translocates to the mitochondria after an
apoptotic signal activates it
• Bax and Bak also operate on the surface of the
ER and nuclear membranes

• When activated in response to ER stress they

are thought to release Ca2+ into the cytosol,
which helps activate the intrinsic pathway by a
poorly understood mechanism
• The anti-apoptoptic Bcl2 proteins such as Bcl2 and Bcl-Xl are located on the
cytosolic surface of the outer mitochondrial membrane, the ER and the nuclear
envelope where they help preserve the integrity of the membrane e.g. preventing
inappropriate release of Ca2+

• These proteins bind to and inhibit pro-apoptopic proteins e.g. they bind to Bak and
prevent it form oligomerizing therefore inhibiting Cyt c release.

• A number of these proteins must be inhibited for the intrinsic pathway to induce
apoptosis with the BH3 only proteins mediating this inhibition
• BH3-only proteins are the largest subclass of the Bcl2 family. The cell either
produces them or activates them in response to apoptotic stimulus and they are
thought to promote apoptosis mainly by inhibiting anti-apoptotic Bcl2 proteins.

• Their BH3 domain binds to a long hydrophobic groove on anti-apoptotic proteins

neutralizing their activity. By a poorly understood mechanism this enables the
aggregation of Bax and Bak. Some do bind directly to Bak and Bax and help
trigger aggregation of these proteins
 The extrinsic pathway can recruit the
intrinsic pathway
• This allows an amplification of the caspase cascade. The
BH3-only protein Bid is the link between these two
pathways.

• When the intrinsic pathway is activated caspase 8 cleaves Bid

to form tBid.

• tBid translocates to the mitochondrial outer membrane

where it inhibits anti-apoptotic Bcl2 proteins and triggers the
aggregation of pro apoptotic BH123 proteins to release
cytochrome c and other intermembrane proteins
 IAPs inhibit Caspases
• Inhibitors of apoptosis (IAPs) were first identified
in certain insect viruses which encode IAP proteins
to prevent a host cell that is infected from killing
itself by apoptosis.

• It is now known that most animal cells make IAPs.

• All IAPs have one or more BIR domains which allow

them to bind to and inhibit activated caspases.
 IAPs regulate apoptosis in insect cells
• In Drosophila the IAPs can be overcome by anti-
IAP proteins produced in response to apoptotic
stimuli

• There are 5 anti-IAPs in flies including Reaper,

Grim and Hid.

• Deletion of these three genes prevent insect cells

undergoing apoptosis.
Anti-IAPs are found in mammalian cells
Anti-IAPs are found in mammalian cells
 However their role in apoptosis is still
controversial
• When the genes encoding 2 known mammalian anti-
IAPs called Smac/Diablo and Omi are inactivated in
mouse cells apoptosis appears to be unaffected.

• However it appears that the combined activates of

the Bcl2 proteins, IAPs and anti-IAPs determine the
animal cells sensitivity to apoptotic inducing stimuli
with IAPs and anti-IAPs dominant in insects and Bcl2
protein dominant in mammals
 Survival Factors
e.g. In regulating brain development
Survival Factors
 PCD and Plants

Swiss Cheese Vine (Monstera deliciosa)

 PCD and Plants
• Although the process of apoptosis is mainly found animal tissues, the process of
programmed cell death is also found in plants. Apoptosis is rare in plants but
many other mechanisms exist which result in cell death, many of which are
unique to plants.

• Two examples of PCD are senescence and the cell death associated with the
hypersensitive response (HR) – part of the plants immune response).

• Cell death in plants permits developmental and biochemical plasticity. Almost all
phases of the plant life cycle, from germination through vegetative and
reproductive development, are influenced by PCD. In addition responses to
pathogens and abiotic stresses involve the controlled death of cells.