Genetics of Apoptosis

5.2

Activation of caspase-8

Mammalian caspase-8 and -10, and the fly caspase DREDD contain two DEDs in
their prodomain region (Boldin et al., 1996; Muzio et al., 1996; Chen et al., 1998).
Most of the studies have focused on caspase-8, but given the structural similarities
between DED-containing caspases, it is likely that caspase-10 is activated by a
mechanism similar to that of caspase-8 activation. As DD-containing death receptors
have not been found in the fly, it is unlikely that DREDD activation occurs at the
level of membrane recruitment, although activation may still require dFADD-
mediated oligomerization (Hu and Yang, 2000). Upon binding of the Fas/CD95
ligand, the CD95 receptor recruits a cytosolic adaptor protein, FADD (Fas-associated
death domain), through a homotypic interaction between the CD95 intracellular
DD and the FADD C-terminal DD (Boldin et al., 1995; Chinnaiyan et al, 1995).
FADD contains a DED in its N-terminal region that interacts with the DEDs in the
prodomain of procaspase-8 and recruits procaspase-8 to CD95 (Boldin et al., 1996;
Muzio et al., 1996). Therefore, it seems likely that the aggregation of CD95 by its
ligand brings the DDs in the CD95 intracellular C-termini in close proximity to
create high-affinity binding sites for the FADD DD. The complex formed by CD95,
FADD, procaspase-8, and possibly other proteins is known as DISC (death-inducing
signaling complex) (see Chapter 1). Recruitment of procaspase-8 to the DISC leads
to proximity-induced autocatalytic activation (Muzio et al., 1998).

5.3

The Apaf-1/caspase-9 apoptosome

The activation of caspase-9 requires Apaf-1, dATP, and cytochrome c, which is
released from the mitochondria in cells committed to apoptosis (Li et al., 1997;
Srinivasula et al., 1998). Apaf-1 contains multiple WD40 motifs in the carboxyl
terminus, which appear to act as a negative regulatory domain by preventing Apaf-1
oligomerization and Apaf-1/caspase-9 interaction (Srinivasula et al., 1998; Zou et al.,
1997, 1999). The deletion of the Apaf-1 WD-40 repeats makes Apaf-1 constitutively
active and capable of activating procaspase-9 independently of cytochrome c and
dATP. Thus, binding of cytochrome c and dATP to Apaf-1 causes conformational
changes in the Apaf-1 molecule that expose the amino-terminal CARD, enabling it
to interact with the CARD in procaspase-9. Reconstitution studies using purified
components found that Apaf-1 binds dATP. This is followed by binding of
cytochrome c, an effect which promotes multimerization of Apaf-1 molecules (Zhou
et al., 1997, 1999). The heptameric Apaf-1 complex, often called apoptosome, then
recruits and activates procaspase-9. The Apaf-1/caspase-9 complex acts as a
holoenzyme that cleaves and activates procaspase-3 (Rodriguez and Lazebnik, 1999).
As procaspase-9 processing is not required for its activation (Stennicke et al., 1999),
and since processed caspase-9 is only weakly active in the absence of Apaf-1
(Rodriguez and Lazebnik, 1999), it is likely that the apoptosome primarily functions

38 GENETICS OF APOPTOSIS