Download Chem*3560 Lecture 13: Caspases and Programmed Cell Death

Chem*3560
Lecture 13: Caspases and Programmed Cell Death
In a multicellular organism, there are a number of circumstances in which individual cells are sacrificed
for the benefit of the organism as a whole. A process exists for the orderly dissolution of the cellular
components so that they can be absorbed and used by neighbours. This is the process of programmed
cell death or apoptosis (pronounced apo’ptosis, not apop’tosis). The Greek word means “falling off”,
as of autumn leaves.
Catastrophic cell death or Necrosis
Programmed cell death or Apoptosis
Pathological response to injury
Normal physiological response to
internal or external signals
Cytoplasm shrinks without membrane rupture
Cell membranes pinch off into smaller units
Cell contents are packaged to be absorbed by
neighbours; no inflammation
Organelles swell and rupture
Cell membrane lyses
Cell contents spill out
Inflammation
When does apoptosis happen?
In embryonic and fetal development:
sculpting of embryonic form
organization of the nervous system.
In the adult:
on stimulation by the immune system
in response to DNA damage or abnormality, due to
chemical or radiation damage, or viral infection
on withdrawal of supporting hormones
in tumours.
How does apoptosis happen?
Apoptosis occurs when proteolytic enzymes in the cell called Caspases become activated. Caspase
stands for Cys-catalysed Aspartate targeting protease, i.e. Cysteine is the catalytic nucleophile, and
attacks sequences at aspartate. Preferred target sequences follow the polar/non-polar pattern
-Asp-Glu-Val-Asp-, with the cut coming after the second Asp. The sequence -Asp-Glu-Ala-Asp-,
which in single letter codes reads -DEAD- is also a target.
Mammalian cells contain 14 currently known caspases, but many of these are involved in inflammatory
responses rather than apoptosis. Two subgroups of caspases are involved in apoptosis:
Caspases 3 and 7 are designated as effector or executioner caspases because they carry out the
cleavages that shut down the cell’s operations.
Caspases 8 and 9 are designated as initiator caspases, because they activate caspases 3 and 7.
Caspase activation
Caspases are synthesized as
inactive procaspases, from
which the N-terminal region is
cut. A second cut is critical
for activation and divides the
caspase into 20 kDa and 10
kDa segments which remain
attached through H-bonds.
The activating cut severs a
loop (L2 in the figure), and
reorients the catalytic Cys
from an inaccessible position
to one that can interact with
substrate. The sequence at the activating cut site is a caspase target sequence, so caspases
can activate procaspases.
Caspase substrates
The executioner caspases 3 and 7 are highly selective in their targets, but are geared to shut down the
cell’s activities down irrevocably:
Structural nuclear proteins
DNA repair and replication proteins
Cytoskeleton proteins actin, spectrin, keratin
Many protein kinases, particularly those controlling cytoskeleton and cell proliferation
One particular target is the Inhibitor of Caspase Activated DNase, ICAD. When the inhibitor is
destroyed, its companion DNase sets about cutting DNA in the linker regions between nucleosomes,
approximately every 200 base-pairs. The cut DNA appears as a “ladder” pattern of DNA fragments in
gel electrophoresis, which is one of the key experimental indicators that apoptosis is occurring.
The substrates of initiator caspases 8 and 9 are procaspases 3 and 7, as well as their own
procaspases. This means that once the process starts, a positive feedback loop leads to escalating
caspase activation.
How is apoptosis induced?
Apoptosis may be initiated internally, or signalled from outside the cell, frequently induced by the
action of immune system cells such as T-lymphocytes. The internal signal activates procaspase 9,
whereas the external signal activates procaspase 8. Whichever initiator caspase is first activated, the
end result will be the activation of all the apoptosis-related caspases
Internal pathway for inducing apoptosis
Internal control over apoptosis is maintained by a family of proteins called Bcl-2 proteins which are
closely associated with mitochondria. Bcl-2 itself is anti-apoptotic, but a related protein Bax is
pro-apoptotic. So long as the balance is in favour of Bcl-2, the cell functions normally. If the balance is
shifted in favour of Bax, apoptosis may be induced.
When DNA
damage is
detected during
replication, the
damage
response
activates DNA
repair
processes, as
well as the cell
proliferation suppressor called p53. p53 is a transcription factor which controls expression of a number
of proteins that halt cell cycle progression. These include the cyclin kinase inhibitor p21, which
inhibits Cyclin A-Cdk2. This causes the cell cycle to stall while repairs are undertaken. Meanwhile Bax
is being expressed. If DNA repairs take too long, Bax levels build up to the critical point of excess over
Bcl-2, and this results in release of cytochrome c from mitochondria.
When cytochrome c enters the cytoplasm, it causes aggregation of a protein called Apaf-1 (apoptosis
promoting and activating factor). The N-terminus of Apaf-1 has a segment called the Caspase
recruitment domain or CARD, and the CARD domain becomes exposed when Apaf-1 binds
cytochrome C. CARD domains tend to stick to other CARD domains, and Procaspase 9 also has a
CARD domain at its N-terminus (making it a CARD-carrying protein). The result is a localized build
up of Procaspase 9 molecules that pair up around Apaf-1 molecules.
The initiator procaspase has weak activity that is ineffective if the procaspase molecules are dispersed,
but when held close together in pairs, eventually one will activate the other and set the whole
apoptosis process off.
Externally signalled apoptosis
Cell surface membranes carry a variety of proteins that act as receptors for
extracellular signals. A set of receptors called Tumor Necrosis Factor
receptors (TNF-R), and a related receptor called FAS are found on many cell
surfaces. The signals they respond to take the form of protein ligands, secreted
by or carried on the membranes of cells of the immune system.
In the absence of ligand, the receptors are randomly dispersed on the surface
of the cell, and have no effect on the cell’s fate.
When the ligand binds, this induces the receptors to cluster together. The
portion of the receptor inside the membrane carries a protein binding domain
called the death domain, which binds other proteins carrying death
domains. Binding is too weak to act at single receptors, but becomes
effective when receptors group in a cluster.
Death domains are found on small adapter proteins that carry the
death domain at one end and a death effector domain (DED) at the
other end. Death effector domains are also protain binding domains
that associate with like domains. Procaspase 8 carries a death effector
domain at its N-terminus, so procaspase 8 molecules become paired up
when they bind to the receptor-adapter complex. Procaspase 8 has
weak activity that is ineffective if the procaspase molecules are
dispersed, but when paired up, eventually one will activate the
other and set the whole apoptosis process off..
IAPs (inhibitors of apoptosis) protect against
accidents
IAPs are proteins that bind to the catalytic site of caspases, and
trick the caspase by inserting a peptide chain in the reverse direction relative to a normal caspase
substrate. The amount of IAP is small, so it can cope with occasional accidental activation of a
caspase, but if a real signal exists, too much caspase is produced for IAPs to deal with.
The IAP-Caspase complex becomes a target for ubiquitin ligase, and the ubiquitin tag is added to the
unwanted caspase, marking it for destruction.