Apaf-1, a Human Protein Homologous to C. elegans CED-4, Participates in Cytochrome c–Dependent Activation of Caspase-3

Abstract

We report here the purification and cDNA cloning of Apaf-1, a novel 130 kd protein from HeLa cell cytosol that participates in the cytochrome c-dependent activation of caspase-3. The NH2-terminal 85 amino acids of Apaf-1 show 21% identity and 53% similarity to the NH2-terminal prodomain of the Caenorhabditis elegans caspase, CED-3. This is followed by 320 amino acids that show 22% identity and 48% similarity to CED-4, a protein that is believed to initiate apoptosis in C. elegans. The COOH-terminal region of Apaf-1 comprises multiple WD repeats, which are proposed to mediate protein-protein interactions. Cytochrome c binds to Apaf-1, an event that may trigger the activation of caspase-3, leading to apoptosis.

Hsp27 functions as a negative regulator of cytochrome c-dependent activation of procaspase-3

“…Whereas other studies have demonstrated that release of cyt c from mitochondria induces a cas-3-mediated apoptotic cascade (Li et al, 1997;Liu et al, 1996;Zou et al, 1997), we asked if heat-shock treatment inhibits cyt cmediated activation of cas-3. As demonstrated previously for other cell types, expression of Hsp27 was induced in 293T cells at 6 ± 8 h after mild heat shock (Figure 1a).…”

“…Previous studies have demonstrated that release of cyt c from mitochondria to cytosol is an important step in inducing cas-3-mediated apoptosis (Li et al, 1997;Liu et al, 1996;Zou et al, 1997). To assess whether Hsp27 functions upstream or downstream to cyt c release from mitochondria, L929/pRc and L929/Hsp27 cells were treated with various agents, such as methylmethane sulphonate (MMS), 1-b-D-arabinofuranosyl-cytosine (ara-C) or staurosporine (STSP), that are known to cause the release of mitochondrial cyt c (Kharbanda et al, 1997a), and cytosols were analysed by immunoblotting (IB) with anti-cyt c. The results demonstrate that MMS treatment of both L929/pRc and L929/Hsp27 cells is associated with the release of cyt c ( Figure 3a, upper panel).…”

“…Cyt c forms a complex with Apaf-1 and cas-9, and is necessary at least in part for activation of cas-3 (Li et al, 1997;Liu et al, 1996;Zou et al, 1997). Since Hsp27 functions downstream to cyt c, Apaf-1 and cas-9, we next determined whether Hsp27 associates with cas-3 and thereby inhibits its activity.…”

“…Moreover, Hsp27 functions as an inhibitor of cas-3 activation by associating with and limiting the availability of cas-3 for cleavage by active cas-9. The release of cyt c from mitochondria is induced by exposure to STSP, diverse DNA damaging agents and ischemia (Kharbanda et al, 1997a;Kluck et al, 1997;Li et al, 1997;Liu et al, 1996;Yang et al, 1997;Zou et al, 1997;Narula et al, 1999). Other studies have shown that cyt c release is inhibited by the anti-apoptotic Bcl-2 and Bcl-x L proteins (Kharbanda et al, 1997a;Yang et al, 1997).…”

“…Recent studies have shown that Apaf-1 binds to cas-9 (initiator caspase) in the presence of cyt c and that Apaf-1-mediated oligomerization leads to cas-9 activation (Li et al, 1997). The ®nding that activated cas-9 in turn cleaves and activates cas-3 (executioner caspase) has supported a model in which the Apaf-1/cas-9 complex functions upstream to cas-3 in the apoptotic protease cascade (Li et al, 1997;Xue et al, 1996;Zou et al, 1997). Activation of the Apaf-1/cas-9 complex appears to be a common pathway employed by diverse stimuli that induce apoptosis in mammalian cells (Li et al, 1997).…”