A Structure of the Human Apoptosome at 12.8 Å Resolution Provides Insights into This Cell Death Platform

Yu, Xianjun; Acehan, Devrim; Ménétret, Jean‐François; Booth, Christopher R.; Ludtke, Steven J.; Riedl, Stefan J.; Shi, Yigong; Wang, Xiaodong; Akey, Christopher W.

doi:10.1016/j.str.2005.09.006

Cited by 132 publications

(156 citation statements)

References 46 publications

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“…It is commonly accepted that Apaf-1 has to undergo a large conformational change to proceed from the autoinhibited monomeric structure into the apoptosomal assembly. From cryo-EM studies, it is conceivable that these conformational changes include gross structural alterations in the NOD itself, because the crystallographic model of the N-terminal half of Apaf-1 (30) could not be fitted into the cryo-EM map of the apoptosome without major modifications in the subdomain arrangement (21). We propose that these conformational changes are brought about by the ␥-phosphate exerting mechanical pressure on a structural element within the NOD.…”

Section: Discussionmentioning

confidence: 97%

A New Model for the Transition of APAF-1 from Inactive Monomer to Caspase-activating Apoptosome

Reubold

Wohlgemuth

Eschenburg

2009

Journal of Biological Chemistry

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The cytosolic adaptor protein Apaf-1 is a key player in the intrinsic pathway of apoptosis. Binding of mitochondrially released cytochrome c and of dATP or ATP to Apaf-1 induces the formation of the heptameric apoptosome complex, which in turn activates procaspase-9. We have re-investigated the chain of events leading from monomeric autoinhibited Apaf-1 to the functional apoptosome in vitro. We demonstrate that Apaf-1 does not require energy from nucleotide hydrolysis to eventually form the apoptosome. Despite a low intrinsic hydrolytic activity of the autoinhibited Apaf-1 monomer, nucleotide hydrolysis does not occur at any stage of the process. Rather, mere binding of ATP in concert with the binding of cytochrome c primes Apaf-1 for assembly. Contradicting the current view, there is no strict requirement for an adenine base in the nucleotide. On the basis of our results, we present a new model for the mechanism of apoptosome assembly.Cells of most multicellular organisms can terminate themselves conducting the evolutionary conserved program of apoptosis. As the well regulated response to a death signal from outside or inside the cell, a cascade of cysteine proteases, socalled caspases, is activated that eventually leads to the orderly disintegration of cell organelles and chromatin (1-3). In contrast to necrotic cell death, which is accompanied by inflammation and damage of the surrounding tissue, apoptotic cells keep their degraded constituents contained in intact membrane. The cell corpses are then cleanly removed by phagocytes obviating inflammatory and autoimmune responses (4). Apoptosis is vital for development and homeostasis of metazoans (5-7), and malfunction of the human apoptosis machinery is implicated in autoimmune diseases, neurodegenerative disorders, and cancer (8 -11).The intrinsic pathway of apoptosis responds to cytotoxic stress, genomic damage, and developmental cues (12, 13). The pivotal point of this pathway is the release of cytochrome c from the mitochondria through permeabilization of the outer membrane (14, 15). In human cells, transduction of this decisive death signal is achieved via the 142-kDa cytosolic protein Apaf-1, the mammalian ortholog of Caenorhabditis elegans CED-4 (16). Apaf-1 contains an N-terminal caspase recruitment domain (CARD), 2 a central nucleotide binding and oligomerization domain (NOD), and a C-terminal regulatory domain of 13 WD40 repeats. It is thought that the WD40 repeat domain blocks the CARD and NOD until cytochrome c is released into the cytosol (17-20). Binding of cytochrome c to the WD40 repeat domain in the presence of ATP or dATP appears to induce a large conformational change in Apaf-1 that eventually bares the NOD and the effector-binding CARD. Mediated by the NOD, Apaf-1 oligomerizes into a wheel-like heteroheptameric protein complex (21) termed apoptosome. The apoptosome recruits the initiator caspase-9 via homophilic CARD-CARD interaction and the holoenzyme then activates downstream executioner caspases (22-24).So far, there is no consistent view...

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Section: Discussionmentioning

confidence: 97%

A New Model for the Transition of APAF-1 from Inactive Monomer to Caspase-activating Apoptosome

Reubold

Wohlgemuth

Eschenburg

2009

Journal of Biological Chemistry

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“…Because Apaf-1 heptamerizes in the apoptosome, the cooperativity of the inhibition by cIAP1 may result from the interaction of multiple cIAP1 monomers with oligomerized caspase-9 in the apoptosome. Unfortunately, the stoichiometry of caspase-9 in the apoptosome is unknown (38,39).…”

Section: Discussionmentioning

confidence: 99%

cIAP1 Cooperatively Inhibits Procaspase-3 Activation by the Caspase-9 Apoptosome

Burke

Smith

2010

Journal of Biological Chemistry

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Although early studies of inhibitor of apoptosis proteins (IAPs) suggested that cIAP1 directly binds and inhibits caspases similarly to X-linked IAP (XIAP), a recent one found that micromolar concentrations of cIAP1 only weakly inhibit caspase-3, -7, or -9. Here, we show that cIAP1 specifically and cooperatively blocks the cytochrome c-dependent apoptosome in vitro. Hence, cIAP1 prevented the activation of procaspase-3 but had no effect on the processing of procaspase-9 or the activity of prior activated caspase-3. Like cIAP1, XIAP had no effect on procaspase-9 processing and was a more potent inhibitor of procaspase-3 activation than of already activated caspase-3 activity. Inhibition of procaspase-3 activation depended on BIR2 and BIR3 of cIAP1 and was independent of BIR1, RING, CARD, and UBA domains. Smac prevented cIAP1 from inhibiting procaspase-3 activation and reversed the inhibition by prior addition of cIAP1. A procaspase-9 mutant (D315A) that cannot produce the p12 subunit was resistant to inhibition by cIAP1. Therefore, the N-terminal Ala-Thr-Pro-Phe motif of the p12 subunit of the caspase-9 apoptosome facilitates apoptosome blockade. Consequently, cIAP1 cooperatively interacts with oligomerized processed caspase-9 in the apoptosome and blocks procaspase-3 activation. Inhibitor of apoptosis proteins (IAPs)2 are a family of eight human proteins that have one or three baculovirus IAP repeat (BIR) domains. Although IAPs have been thought of as primarily inhibitors of apoptosis, it is now known that they play important roles in mitotic chromosome segregation, cellular morphogenesis, copper homeostasis, and intracellular signal transduction (1, 2). IAPs with three BIR domains (XIAP, cIAP1, cIAP2, and neuronal apoptosis inhibitor protein (NAIP)) can directly bind caspases via BIR2 and BIR3 and down-regulate caspase activity (3-7). Caspases are highly specific cysteinyl proteases that implement the cell death program by processing hundreds of different intracellular proteins after one or a few aspartyl residues (8). XIAP, which is the best characterized member of the family, inhibits the hydrolytic activity of caspase-3, -7, and -9 (9 -11). Similarly to XIAP, NAIP directly binds and inhibits caspase-3, -7, and -9 (6, 7). Recent work on cIAP1 and cIAP2 indicates that they can directly bind processed caspase-3 or -7 (12) but have little effect on the activity of caspase-3, -7, or -9 (13).Importantly, cIAPs are E3 ubiquitin ligases, which catalyze both trans-and autoubiquitination, as are four other IAPs (XIAP, melanoma IAP (also called Livin), ILP2 (also called testis-specific IAP), and Apollon (also called BRUCE)) (14 -21). Interestingly, cIAPs can mediate either nondegradative Lys-63 polyubiquitination or degradative Lys-48 polyubiquitination of a protein target. For example, Lys-63 polyubiquitination of RIP1 mediates TNF␣-evoked NFB activation, whereas Lys-48 auto-or transubiquitination mediates proteasomal degradation of cIAPs or of a cIAP-binding protein such as a caspase or Smac/DIABLO (14,16,(22)(23)(24...

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“…The CARD and the nucleotide-binding domains of Apaf-1 are responsible for the oligomerization in the presence of cytochrome c and dATP, whereas the WD40 domain is believed to interact with cytochrome c. In the absence of apoptotic stimuli, Apaf-1 exists in monomeric form. In the presence of cytochrome c and dATP, Apaf-1 oligomerizes to form a wheel-shaped signaling platform, apoptosome, which contains seven Apaf-1 molecules, each bound to one molecule of cytochrome c and one caspase-9 (Acehan et al, 2002;Yu et al, 2005). Caspase-9, which is activated through an apoptosomeinduced conformational change, further processes the downstream caspases, such as caspase-3 and caspase-7, to carry out the execution of apoptosis (Slee et al, 1999) (Figure 2).…”

Section: Apoptosome and Mitochondria Pathwaymentioning

confidence: 99%

Caspases in apoptosis and beyond

2008

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A Structure of the Human Apoptosome at 12.8 Å Resolution Provides Insights into This Cell Death Platform

Cited by 132 publications

References 46 publications

A New Model for the Transition of APAF-1 from Inactive Monomer to Caspase-activating Apoptosome

A New Model for the Transition of APAF-1 from Inactive Monomer to Caspase-activating Apoptosome

cIAP1 Cooperatively Inhibits Procaspase-3 Activation by the Caspase-9 Apoptosome

Caspases in apoptosis and beyond

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