Page 8 - Academic Press Encyclopedia of Physical Science and Technology 3rd Molecular Biology

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Encyclopedia of Physical Science and Technology EN002G-90 May 17, 2001 20:42

546 Cell Death (Apoptosis)

inner plasma membrane. Several lines of evidences sug- the apoptosome (Fig. 3). Apaf-1 binds to procaspase-9
gest that procaspase-8 may be proteolytically processed via CARD motifs and procaspase-9 is processed using
to the active form by simple oligomerization. However, both Apaf-1 and energy from dATP/ATP hydrolysis to
the precise mechanism is still unclear and may require convert procaspase-9 to the active form. Cytochrome c
unknown additional factor(s) that may be in DISC or on appears to play a role in overcoming the inhibition of the
the inner plasma membrane, may be required to gener- Apaf-1 active site masked by the WD-40 repeat. Once
ate active caspase-8 more efficiently. Once caspase-8 is activated, effector caspases downstream of caspase-9 are
activated in cells, it cleaves effector procaspases located sequentially activated (Fig. 3).
downstream of it such as caspase-3, -6, and -7 in order to It has recently been shown that there are two cell types
amplify the apoptotic signal (Fig. 2). Finally, cells show with different sensitivity to Fas signaling induced by an
a variety of apoptotic features such as described above, agonistic anti-Fas antibody. In type I cells, procaspase-8 is
due to cleavage of more than 100 substrate proteins which rapidly activated following receptor engagement, whereas
contributes to DNA fragmentation, loss of mitochondrial in type II cells the activation of procaspase-8 is delayed,
function, and the maintenance of nuclear structure and although both type I and type II cells show similar kinetics
plasma membrane. of Fas-mediated apoptosis and loss of mitochondrial func-
An alternative apoptotic pathway via mitochondria tion. In addition, Bcl-2 inhibits apoptosis in type II but not
has been found by the in vitro reconstitution assay (de- type I cells. Why do these cells die in a similar manner
scribed in section V). Based on a dATP/ATP-inducible to each other regardless of the amount of active caspase-8
cell-free system, three factors responsible for process- and yet show different blocking activity by Bcl-2? One
ing procaspase-3 have been purified and identified. These explanation is that there are two distinct pathways in Fas-
include Apaf-1, which is found as a mammalian homo- mediated apoptosis, a direct pathway from caspase-8 to
logue of CED-4 known to be another executor of apop- effector caspase in type I cells and a caspase-8-mediated
tosis in C. elegans, cytochrome c (Apaf-2) that is mainly mitochondrial pathway in type II cells. It is likely that
present in the mitochondrial intermembrane space, and Bcl-2 is only able to block the latter pathway. However,
procaspase-9 (Apaf-3). Cytochrome c is released from this hypothesis is still controversial in view of the report
the intermembrane space of mitochondria into the cy- that physiological Fas ligand but not an agonistic antibody
toplasm during apoptosis induced by a variety of apop- kills both types of cells similarly regardless of the Bcl-2
totic stimuli, including DNA-damaging agents, protein ki- expression level. These results might depend on the effi-
nase inhibitors (staurosporine), and death receptors (Fig. ciency of precise trimerization of Fas. The evidence that
3). Moreover, the release of cytochrome c is blocked there are two distinct pathways in the Fas system has come
by anti-apoptotic proteins belonging to the Bcl-2 fam- from the analyses of Bid-deficient mice. If Bid, a Bcl-2
ily such as Bcl-2 and Bcl-xL (Fig. 3). Recent obser- family member, is cleaved by caspase-8, truncated Bid
vations have indicated that cytochrome c-deficient mice (tBid) can translocate from the cytosol to mitochondria,
show an embryonic lethal phenotype with defects in ox- subsequently, cytochrome c is released, executing apop-
idative phosphorylation. In addition, their embryonic fi- tosis. Administration of an agonistic anti-Fas antibody to
broblasts are resistant to stresses such as UV irradia- wild-type mice in vivo causes death with hepatocellular
tion, serum withdrawal and staurosporine. The mech- apoptosis and haemorrhagic necrosis in the liver within 3
anism by which cytochrome c is released from mito- hours, whereas Bid-deficient mice survive treatment with
chondria by apoptotic stimuli remains elusive, although this antibody. In addition, hepatocytes from Bid −/− mice
some hypotheses, including opening of a specific chan- are resistant to this antibody in vitro, suggesting that the
nel for cytochrome c, alteration of the permeability tBid-mediated pathway via mitochondria predominantly
transition pore (PTP) that regulates inner mitochondrial works in hepatocellular apoptosis induced by agonistic
membrane potential, or swelling and subsequent rup- Fas antibody. It will be interesting to determine whether
ture of the outer mitochondrial membrane, have been or not Apaf-1 −/− and caspase-9 −/− conditional knockout
proposed. mice (because of the embryonic lethal phenotype) show
Apaf-1 possesses a region homologous to the a similar response as Bid-deficient mice to the adminis-
procaspase-prodomain, known as the caspase-recruiting tration of the anti-Fas antibody. To investigate the actual
domain (CARD) at the N terminus, a region homolo- signaling pathways activated by the physiological Fas lig-
gous to CED-4 in the middle part and WD-40 repeat and in vivo, it will be necessary to administer soluble Fas
structure that appears to be involved in protein–protein ligand to Bid-deficient mice.
interactions. The released cytochrome c interacts with Some death receptors also activate caspase proteases
two cytosolic proteins, Apaf-1 and procaspase-9, and by ligation with their cognate ligands. Like in TNFRI sig-
dATP/ATP in the cytoplasm to form a complex known as naling, DD regions of activated death receptors including