Regulation of the Apaf-1/Caspase-9 Apoptosome by Caspase-3 and XIAP

Abstract

The apoptosome is a multiprotein complex comprising Apaf-1, cytochrome c, and caspase-9 that functions to activate caspase-3 downstream of mitochondria in response to apoptotic signals. Binding of cytochrome c and dATP to Apaf-1 in the cytosol leads to the assembly of a heptameric complex in which each Apaf-1 subunit is bound noncovalently to a procaspase-9 subunit via their respective CARD domains. Assembly of the apoptosome results in the proteolytic cleavage of procaspase-9 at the cleavage site PEPD 315 to yield the large (p35) and small (p12) caspase-9 subunits. In addition to the PEPD site, caspase-9 contains a caspase-3 cleavage site (DQLD 330 ), which when cleaved, produces a smaller p10 subunit in which the NH 2 -terminal 15 amino acids of p12, including the XIAP BIR3 binding motif, are removed. Using purified proteins in a reconstituted reaction in vitro, we have assessed the relative impact of Asp 315 and Asp 330cleavage on caspase-9 activity within the apoptosome. In addition, we characterized the effect of caspase-3 feedback cleavage of caspase-9 on the rate of caspase-3 activation, and the potential ramifications of Asp 330 cleavage on XIAP-mediated inhibition of the apoptosome. We have found that cleavage of procaspase-9 at Asp 330 to generate p35, p10 or p37, p10 forms resulted in a significant increase (up to 8-fold) in apoptosome activity compared with p35/p12. The significance of this increase was demonstrated by the near complete loss of apoptosome-mediated caspase-3 activity when a point mutant (D330A) of procaspase-9 was substituted for wild-type procaspase-9 in the apoptosome. In addition, cleavage at Asp 330 exposed a novel p10 NH 2 -terminal peptide motif (AISS) that retained the ability to mediate XIAP inhibition of caspase-9. Thus, whereas feedback cleavage of caspase-9 by caspase-3 significantly increases the activity of the apoptosome, it does little to attenuate its sensitivity to inhibition by XIAP.

N-(4-Hydroxyphenyl)retinamide induced differentiation with repression of telomerase and cell cycle to increase interferon-γ sensitivity for apoptosis in human glioblastoma cells

“…5). The association of the cytosolic cytochrome c along with Apaf-1 and pro-caspase-9 contributes to the formation of apoptosome leading to activation of caspase-9 [45], which then activates effector caspases such as caspase-3 [46]. We found that apoptosis occurred in all three glioblastoma cell lines with activation of caspase-9 as well as of caspase-3 for apoptosis, which in turn caused ICAD fragmentation (Fig.…”

The inhibitors of apoptosis: there is more to life than Bcl2

“…This proteolytic event further increases the enzymatic activity of caspase-9, and was proposed to remove the peptide sequence that binds XIAP . However, recent work suggests that XIAP BIR3 can still interact with and inhibit the enzyme activity of processed caspase-9 in the apoptosome complex (Zou et al, 2003).…”

A mitochondrial block and expression of XIAP lead to resistance to TRAIL-induced apoptosis during progression to metastasis of a colon carcinoma

“…In this case, removal of XIAP allows for complete caspase-3 maturation through an autocatalytic process. Caspase-3 then cleaves caspase-9 at an alternative site leading to the generation of the p37 fragment seen in the western blot ( Figure 4d; Zou et al, 2003). Autoprocessing of caspase-9 then generates the p35 fragment.…”