Autoactivation of Procaspase-9 by Apaf-1-Mediated Oligomerization

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Abstract

Activation of procaspase-9 by Apaf-1 in the cytochrome c/dATP-dependent pathway requires proteolytic cleavage to generate the mature caspase molecule. To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we designed an in vitro Apaf-1-procaspase-9 activation system using recombinant components. Here, we show that deletion of the Apaf-1 WD-40 repeats makes Apaf-1 constitutively active and capable of processing procaspase-9 independent of cytochrome c an dATP. Apaf-1-mediated processing of procaspase-9 occurs at Asp-315 by an intrinsic autocatalytic activity of procaspase-9 itself. We provide evidence that Apaf-1 can form oligomers and may facilitate procaspase-9 autoactivation by oligomerizing its precursor molecules. Once activated, caspase-9 can initiate a caspase cascade involving the downstream executioners caspase-3, -6, and -7.

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Most cited references 25

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Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3.

Hua Zou, William Henzel, Xuesong Liu … (1997)

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.

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FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis.

Arul M Chinnaiyan, Karen O'Rourke, Muneesh Tewari … (1995)

Using the cytoplasmic domain of Fas in the yeast two-hybrid system, we have identified a novel interacting protein, FADD, which binds Fas and Fas-FD5, a mutant of Fas possessing enhanced killing activity, but not the functionally inactive mutants Fas-LPR and Fas-FD8. FADD contains a death domain homologous to the death domains of Fas and TNFR-1. A point mutation in FADD, analogous to the lpr mutation of Fas, abolishes its ability to bind Fas, suggesting a death domain to death domain interaction. Overexpression of FADD in MCF7 and BJAB cells induces apoptosis, which, like Fas-induced apoptosis, is blocked by CrmA, a specific inhibitor of the interleukin-1 beta-converting enzyme. These findings suggest that FADD may play an important role in the proximal signal transduction of Fas.

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Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death.

Mark Boldin, Tanya M Goncharov, Yury Goltseve … (1996)

Fas/APO-1 and p55 tumor necrosis factor (TNF) receptor (p55-R) activate cellular mechanisms that result in cell death. Upon activation of these receptors, Fas/APO-1 binds a protein called MORT1 (or FADD) and p55-R binds a protein called TRADD. MORT1 and TRADD can also bind to each other. We have cloned a novel protein, MACH, that binds to MORT1. This protein exists in multiple isoforms, some of which contain a region that has proteolytic activity and shows marked sequence homology to proteases of the ICE/CED-3 family. Cellular expression of the proteolytic MACH isoforms results in cell death. Expression of MACH isoforms that contain an incomplete ICE/CED-3 region provides effective protection against the cytotoxicity induced by Fas/APO-1 or p55-R triggering. These findings suggest that MACH is the most upstream enzymatic component in the Fas/APO-1- and p55-R-induced cell death signaling cascades.