Role of cytochrome c and dATP/ATP hydrolysis in Apaf-1-mediated caspase-9 activation and apoptosis

Abstract

Apaf-1 plays a critical role in apoptosis by binding to and activating procaspase-9. We have identified a novel Apaf-1 cDNA encoding a protein of 1248 amino acids containing an insertion of 11 residues between the CARD and ATPase domains, and another 43 amino acid insertion creating an additional WD-40 repeat. The product of this Apaf-1 cDNA activated procaspase-9 in a cytochrome c and dATP/ATP-dependent manner. We used this Apaf-1 to show that Apaf-1 requires dATP/ATP hydrolysis to interact with cytochrome c, self-associate and bind to procaspase-9. A P-loop mutant (Apaf-1K160R) was unable to associate with Apaf-1 or bind to procaspase-9. Mutation of Met368 to Leu enabled Apaf-1 to self-associate and bind procaspase-9 independent of cytochrome c, though still requiring dATP/ATP for these activities. The Apaf-1M368L mutant exhibited greater ability to induce apoptosis compared with the wild-type Apaf-1. We also show that procaspase-9 can recruit procaspase-3 to the Apaf-1-procaspase-9 complex. Apaf-1(1-570), a mutant lacking the WD-40 repeats, associated with and activated procaspase-9, but failed to recruit procaspase-3 and induce apoptosis. These results suggest that the WD-40 repeats may be involved in procaspase-9-mediated procaspase-3 recruitment. These studies elucidate biochemical steps required for Apaf-1 to activate procaspase-9 and induce apoptosis.

Guarding the Goods. New Insights into the Central Alarm System of Plants

“…The binding of Apaf-1 to its ligand cytochrome C and subsequent oligomerization requires dATP or ATP, and substitution of the nonhydrolyzable analog ATPgS prevents both steps (Hu et al, 1999). This observation indicates that ATP hydrolysis rather than simple ATP binding is required for Apaf-1 to bind ligand and form oligomers.…”

“…Full-length Apaf-1 can be induced to oligomerize in vitro by the addition of cytochrome C (Hu et al, 1999). Apaf-1 binds cytochrome C, and this binding requires both the WD-40 repeat domain and the NB-ARC domain, as well as ATP hydrolysis (Hu et al, 1999).…”

“…Full-length Apaf-1 can be induced to oligomerize in vitro by the addition of cytochrome C (Hu et al, 1999). Apaf-1 binds cytochrome C, and this binding requires both the WD-40 repeat domain and the NB-ARC domain, as well as ATP hydrolysis (Hu et al, 1999). Based on these data, it is hypothesized that the WD-40 repeat domain contains a ligand binding pocket that is unmasked only upon a conformational change induced by ATP hydrolysis.…”

Effects of dietary protein levels on the disease resistance, immune function and physical barrier function in the gill of grass carp ( Ctenopharyngodon idella ) after challenged with Flavobacterium columnare

“…Additionally, IAP inhibits caspases in mammals [90]. Inactivated Apaf-1 inhibits the activation of caspase 9, leading to the suppression of caspase 3 in human embryonic kidney 293T cells [91].…”

Stress in the brain: novel cellular mechanisms of injury linked to Alzheimer's disease

“…Apaf-1 consists of three different domains that include CARD, repeats of tryptophan and aspartate residues (WD-40 repeats), and a nucleotide-binding domain CED-4. Binding of cytochrome c to Apaf-1 results in the removal of the WD-40 domain, masking the CED-4 and CARD domains, and leads to the oligomerization of Apaf-1 under the assistance of dATP/ATP [91]. The oligomerization of Apaf-1 promotes the allosteric activation of caspase 9 by forming the Apaf-1 apoptosome [117].…”