Pro-apoptotic proteins released from the mitochondria regulate the protein composition and caspase-processing activity of the native Apaf-1/caspase-9 apoptosome complex

The apoptosome is a large caspase-activating ( approximately 700-1400 kDa) complex, which is assembled from Apaf-1 and caspase-9 when cytochrome c is released during mitochondrial-dependent apoptotic cell death. Apaf-1 the core scaffold protein is approximately 135 kDa and contains CARD (caspase recruitment domain), CED-4, and multiple (13) WD40 repeat domains, which can potentially interact with a variety of unknown regulatory proteins. To identify such proteins we activated THP.1 lysates with dATP/cytochrome c and used sucrose density centrifugation and affinity-based methods to purify the apoptosome for analysis by MALDI-TOF mass spectrometry. First, we used a glutathione S-transferase (GST) fusion protein (GST-casp9(1-130)) containing the CARD domain of caspase-9-(1-130), which binds to the CARD domain of Apaf-1 when it is in the apoptosome and blocks recruitment/activation of caspase-9. This affinity-purified apoptosome complex contained only Apaf-1XL and GST-casp9(1-130), demonstrating that the WD40 and CED-4 domains of Apaf-1 do not stably bind other cytosolic proteins. Next we used a monoclonal antibody to caspase-9 to immunopurify the native active apoptosome complex from cell lysates, containing negligible levels of cytochrome c, second mitochondria-derived activator of caspase (Smac), or Omi/HtrA2. This apoptosome complex exhibited low caspase-processing activity and contained four stably associated proteins, namely Apaf-1, pro-p35/34 forms of caspase-9, pro-p20 forms of caspase-3, X-linked inhibitor of apoptosis (XIAP), and cytochrome c, which was only bound transiently to the complex. However, in lysates containing Smac and Omi/HtrA2, the caspase-processing activity of the purified apoptosome complex increased 6-8-fold and contained only Apaf-1 and the p35/p34-processed subunits of caspase-9. During apoptosis, Smac, Omi/HtrA2, and cytochrome c are released simultaneously from mitochondria, and thus it is likely that the functional apoptosome complex in apoptotic cells consists primarily of Apaf-1 and processed caspase-9.     

Dark is a Drosophila homologue of Apaf-1/CED-4 and functions in an evolutionarily conserved death pathway

Here we identify a new gene, dark, which encodes a Drosophila homologue of mammalian Apaf-1 and Caenorhabditis elegans CED-4, cell-death proteins. Like Apaf-1, but in contrast to CED-4, Dark contains a carboxy-terminal WD-repeat domain necessary for interactions with the mitochondrial protein cytochrome c. Dark selectively associates with another protein involved in apoptosis, the fly apical caspase, Dredd. Dark-induced cell killing is suppressed by caspase-inhibitory peptides and by a dominant-negative mutant Dredd protein, and enhanced by removal of the WD domain. Loss-of-function mutations in dark attenuate programmed cell deaths during development, causing hyperplasia of the central nervous system, and other abnormalities including ectopic melanotic tumours and defective wings. Moreover, ectopic cell killing by the Drosophila cell-death activators, Reaper, Grim and Hid, is substantially suppressed in dark mutants. These findings establish dark as an important apoptosis effector in Drosophila and raise profound evolutionary considerations concerning the relationship between mitochondrial components and the apoptosis-promoting machinery.     

The adapter protein apoptotic protease-activating factor-1 (Apaf-1) is proteolytically processed during apoptosis

Apoptotic protease-activating factor-1 (Apaf-1), a key regulator of the mitochondrial apoptosis pathway, consists of three functional regions: (i) an N-terminal caspase recruitment domain (CARD) that can bind to procaspase-9, (ii) a CED-4-like region enabling self-oligomerization, and (iii) a regulatory C terminus with WD-40 repeats masking the CARD and CED-4 region. During apoptosis, cytochrome c and dATP can relieve the inhibitory action of the WD-40 repeats and thus enable the oligomerization of Apaf-1 and the subsequent recruitment and activation of procaspase-9. Here, we report that different apoptotic stimuli induced the caspase-mediated cleavage of Apaf-1 into an 84-kDa fragment. The same Apaf-1 fragment was obtained in vitro by incubation of cell lysates with either cytochrome c/dATP or caspase-3 but not with caspase-6 or caspase-8. Apaf-1 was cleaved at the N terminus, leading to the removal of its CARD H1 helix. An additional cleavage site was located within the WD-40 repeats and enabled the oligomerization of p84 into a approximately 440-kDa Apaf-1 multimer even in the absence of cytochrome c. Due to the partial loss of its CARD, the p84 multimer was devoid of caspase-9 or other caspase activity. Thus, our data indicate that Apaf-1 cleavage causes the release of caspases from the apoptosome in the course of apoptosis.     

Regulation of apoptotic protease activating factor-1 oligomerization and apoptosis by the WD-40 repeat region.

Apoptotic protease activating factor-1 (Apaf-1) has been identified as a proximal activator of caspase-9 in cell death pathways that trigger mitochondrial damage and cytochrome c release. The mechanism of Apaf-1 action is unclear but has been proposed to involve the clustering of caspase-9 molecules, thereby facilitating autoprocessing of adjacent zymogens. Here we show that Apaf-1 can dimerize via the CED-4 homologous and linker domains of the molecule providing a means by which Apaf-1 can promote the clustering of caspase-9 and facilitate its activation. Apaf-1 dimerization was repressed by the C-terminal half of the molecule, which contains multiple WD-40 repeats, but this repression was overcome in the presence of cytochrome c and dATP. Removal of the WD-40 repeat region resulted in a constitutively active Apaf-1 that exhibited greater cytotoxicity in transient transfection assays when compared with full-length Apaf-1. These data suggest a mechanism for Apaf-1 function and reveal an important regulatory role for the WD-40 repeat region.     

Evidence that CED-9/Bcl2 and CED-4/Apaf-1 localization is not consistent with the current model for C. elegans apoptosis induction

In C. elegans, the BH3-only domain protein EGL-1, the Apaf-1 homolog CED-4 and the CED-3 caspase are required for apoptosis induction, whereas the Bcl-2 homolog CED-9 prevents apoptosis. Mammalian B-cell lymphoma 2 (Bcl-2) inhibits apoptosis by preventing the release of the Apaf-1 (apoptotic protease-activating factor 1) activator cytochrome c from mitochondria. In contrast, C. elegans CED-9 is thought to inhibit CED-4 by sequestering it at the outer mitochondrial membrane by direct binding. We show that CED-9 associates with the outer mitochondrial membrane within distinct foci that do not overlap with CED-4, which is predominantly perinuclear and does not localize to mitochondria. CED-4 further accumulates in the perinuclear space in response to proapoptotic stimuli such as ionizing radiation. This increased accumulation depends on EGL-1 and is abrogated in ced-9 gain-of-function mutants. CED-4 accumulation is not sufficient to trigger apoptosis execution, even though it may prime cells for apoptosis. Our results suggest that the cell death protection conferred by CED-9 cannot be solely explained by a direct interaction with CED-4.     

Apaf-1XL is an inactive isoform compared with Apaf-1L

Apaf-1 plays a crucial role in the cytochrome c/dATP-dependent activation of caspase-9 and -3. We found that the human myeloid leukemic K562 cells were more resistant to cytochrome c-induced activation of caspase-9 and -3 in a cell-free system compared with the human T-lymphoblastic subclone CEM/VLB(100) cells. Apaf-1 cDNA sequencing revealed an additional insert of 11 aa between the CARD and CED-4 (ATPase) domains in K562 cells, which was identical to the sequence of Apaf-1XL. Immunoprecipitation of Apaf-1 with caspase-9 after a cell-free reaction demonstrated that Apaf-1XL in the K562 cell line showed a lower binding ability to caspase-9 compared with Apaf-1L protein. The resistance of K562 cells to cytochrome c-dependent apoptosis may be partly due to this Apaf-1XL form. These results suggest that the additional insert between CARD and CED-4 domains might affect Apaf-1 recruitment of caspase-9 during apoptosis.     

ARK, the Apaf-1 related killer in Drosophila, requires diverse domains for its apoptotic activity

In mammals and Drosophila, apoptotic caspases are under positive control of the CED-4-like proteins Apaf-1 and ARK, respectively. In an EMS-mutagenesis screen, we isolated 33 ark mutants as recessive suppressors of hid-induced apoptosis. The ark mutants are loss-of-function alleles characterized by reduced developmental apoptosis. Using the phenotypic series of these alleles, we identified helical domain I in the nucleotide oligomerization domain as critical for ARK's apoptotic activity. Interestingly, the WD40 region may also have an unanticipated positive requirement for the apoptotic activity of ARK. Considering structural information, we discuss the roles of these domains for assembly and activity of the ARK apoptosome, and propose that the WD40 region is anti-apoptotic in the absence of apoptotic signals, and pro-apoptotic in the presence of such signals. Furthermore, a defined null allele reveals that ark is required for most, but not all apoptosis suggesting the existence of an ARK-independent apoptotic pathway.     

The Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not all, programmed cell death

The Apaf-1 protein is essential for cytochrome c-mediated caspase-9 activation in the intrinsic mammalian pathway of apoptosis. Although Apaf-1 is the only known mammalian homologue of the Caenorhabditis elegans CED-4 protein, the deficiency of apaf-1 in cells or in mice results in a limited cell survival phenotype, suggesting that alternative mechanisms of caspase activation and apoptosis exist in mammals. In Drosophila melanogaster, the only Apaf-1/CED-4 homologue, ARK, is required for the activation of the caspase-9/CED-3-like caspase DRONC. Using specific mutants that are deficient for ark function, we demonstrate that ARK is essential for most programmed cell death (PCD) during D. melanogaster development, as well as for radiation-induced apoptosis. ark mutant embryos have extra cells, and tissues such as brain lobes and wing discs are enlarged. These tissues from ark mutant larvae lack detectable PCD. During metamorphosis, larval salivary gland removal was severely delayed in ark mutants. However, PCD occurred normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in D. melanogaster.     

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

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.     

The role of ARK in stress-induced apoptosis in Drosophila cells

The molecular mechanisms of apoptosis are highly conserved throughout evolution. The homologs of genes essential for apoptosis in Caenorhabditis elegans and Drosophila melanogaster have been shown to be important for apoptosis in mammalian systems. Although a homologue for CED-4/apoptotic protease-activating factor (Apaf)-1 has been described in Drosophila, its exact function and the role of the mitochondrial pathway in its activation remain unclear. Here, we used the technique of RNA interference to dissect apoptotic signaling pathways in Drosophila cells. Inhibition of the Drosophila CED-4/Apaf-1-related killer (ARK) homologue resulted in pronounced inhibition of stress-induced apoptosis, whereas loss of ARK did not protect the cells from Reaper- or Grim-induced cell death. Reduction of DIAP1 induced rapid apoptosis in these cells, whereas the inhibition of DIAP2 expression did not but resulted in increased sensitivity to stress-induced apoptosis; apoptosis in both cases was prevented by inhibition of ARK expression. Cells in which cytochrome c expression was decreased underwent apoptosis induced by stress stimuli, Reaper or Grim. These results demonstrate the central role of ARK in stress-induced apoptosis, which appears to act independently of cytochrome c. Apoptosis induced by Reaper or Grim can proceed via a distinct pathway, independent of ARK.     

Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation

The apoptosome is an Apaf-1 cytochrome c complex that activates procaspase-9. The three-dimensional structure of the apoptosome has been determined at 27 A resolution, to reveal a wheel-like particle with 7-fold symmetry. Molecular modeling was used to identify the caspase recruitment and WD40 domains within the apoptosome and to infer likely positions of the CED4 homology motif and cytochrome c. This analysis suggests a plausible role for cytochrome c in apoptosome assembly. In a subsequent structure, a noncleavable mutant of procaspase-9 was localized to the central region of the apoptosome. This complex promotes the efficient activation of procaspase-3. Therefore, the cleavage of procaspase-9 is not required to form an active cell death complex.     

Control of the cell death pathway by Dapaf-1, a Drosophila Apaf-1/CED-4-related caspase activator

We identified a Drosophila Apaf-1/CED-4 homolog gene, dapaf-1. Alternative splicing results in two dapaf-1 mRNA species, which encode distinct forms of caspase activator, Dapaf-1L (Apaf-1 type) and Dapaf-1S (CED-4 type). Distinct caspases were activated by these Dapaf-1 isoforms. Loss of Dapaf-1 function resulted in defective cytochrome c-dependent caspase activities and reduced apoptosis in embryo and in larval brain. Dapaf-1 activities were also involved in cell death induced by ectopic expression of reaper in the compound eye. These data suggest that Dapaf-1/cytochrome c-dependent cell death-inducing machinery is present in Drosophila, and the requirement of Dapaf-1/Apaf-1 in neural cell death is conserved through evolution.     

Failure of Bcl-2 family members to interact with Apaf-1 in normal and apoptotic cells

CED-9 blocks programmed cell death (apoptosis) in the nematode C. elegans by binding to and neutralizing CED-4, an essential activator of the aspartate-directed cysteine protease (caspase) CED-3. In mammals, the CED-9 homologs Bcl-2 and Bcl-xL also block apoptosis by interfering with the activation of CED-3-like caspases. However, it is unknown whether this occurs by binding to the CED-4 homolog Apaf-1. Whilst two groups previously detected an interaction between Bcl-xL and Apaf-1 in immunoprecipitates,1,2 another group found no interaction between Apaf-1 and any of ten individual members of the Bcl-2 family using the same experimental approach.3 In this study, we aimed to resolve this discrepancy by monitoring the binding of Apaf-1 to three Bcl-2 family members within cells. Using immunofluorescence and Western blot analysis, we show that whilst Apaf-1 is a predominantly cytoplasmic protein, Bcl-2, Bcl-xL and Bax mostly reside on nuclear/ER and mitochondrial membranes. This pattern of localization is maintained when the proteins are co-expressed in both normal and apoptotic cells, suggesting that Bcl-2, Bcl-xL or Bax do not significantly sequester cytoplasmic Apaf-1 to intracellular membranes. In addition, we confirm that Apaf-1 does not interact with Bcl-2 and Bcl-xL in immunoprecipitates. Based on these data, we propose that Apaf-1 is not a direct, physiological target of Bcl-2, Bcl-xL or Bax.     

Identification of a Bcl-XL binding region within the ATPase domain of Apaf-1

CED-4, a pro-apoptotic factor in Caenorhabditis elegans, activates the cell death protease CED-3. CED-9 directly binds to CED-4 and represses this. However, it has remained unclear whether a mammalian CED-9 homologue, Bcl-XL, inhibits the function of the mammalian CED-4 homologue, Apaf-1, by direct binding. To analyze the interaction, we adopted a yeast two-hybrid system. Since Bcl-XL and the CED-4-like portion of Apaf-1 failed to exhibit a positive result in the assay, we prepared "fragment libraries" of bcl-XL or apaf-1 cDNA. By screening of the apaf-1 "fragment library," we obtained nine clones interacting with Bcl-XL, all containing the same region within the ATPase domain, designated BBR: the Bcl-XL binding region. Binding of BBR to Bcl-XL was also confirmed by immunoprecipitation assays. Bcl-2, Bcl-w, A1/Bfl-1, and Boo/Diva failed to show the same capacity for binding to BBR as Bcl-XL. These results indicate that Bcl-XL directly binds to a specific region in Apaf-1.     

Bcr-Abl-mediated protection from apoptosis downstream of mitochondrial cytochrome c release

Bcr-Abl, activated in chronic myelogenous leukemias, is a potent cell death inhibitor. Previous reports have shown that Bcr-Abl prevents apoptosis through inhibition of mitochondrial cytochrome c release. We report here that Bcr-Abl also inhibits caspase activation after the release of cytochrome c. Bcr-Abl inhibited caspase activation by cytochrome c added to cell-free lysates and prevented apoptosis when cytochrome c was microinjected into intact cells. Bcr-Abl acted posttranslationally to prevent the cytochrome c-induced binding of Apaf-1 to procaspase 9. Although Bcr-Abl prevented interaction of endogenous Apaf-1 with the recombinant prodomain of caspase 9, it did not affect the association of endogenous caspase 9 with the isolated Apaf-1 caspase recruitment domain (CARD) or Apaf-1 lacking WD-40 repeats. These data suggest that Apaf-1 recruitment of caspase 9 is faulty in the presence of Bcr-Abl and that cytochrome c/dATP-induced exposure of the Apaf-1 CARD is likely defective. These data provide a novel locus of Bcr-Abl antiapoptotic action and suggest a distinct mechanism of apoptosomal inhibition.     

Ordering the Cytochrome c–initiated Caspase Cascade: Hierarchical Activation of Caspases-2, -3, -6, -7, -8, and -10 in a Caspase-9–dependent Manner

Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1–mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c–inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.     

Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation.

To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we produced recombinant full-length Apaf-1 and purified it to complete homogeneity. Here we show using gel filtration that full-length Apaf-1 exists as a monomer that can be transformed to an oligomeric complex made of at least eight subunits after binding to cytochrome c and dATP. Apaf-1 binds to cytochrome c in the absence of dATP but does not form the oligomeric complex. However, when dATP is added to the cytochrome c-bound Apaf-1 complex, complete oligomerization occurs, suggesting that oligomerization is driven by hydrolysis of dATP. This was supported by the observation that ATP, but not the nonhydrolyzable adenosine 5'-O-(thiotriphosphate), can induce oligomerization of the Apaf-1-cytochrome c complex. Like the spontaneously oligomerizing Apaf-530, which lacks its WD-40 domain, the oligomeric full-length Apaf-1-cytochrome c complex can bind and process procaspase-9 in the absence of additional dATP or cytochrome c. However, unlike the truncated Apaf-530 complex, the full-length Apaf-1 complex can release the mature caspase-9 after processing. Once released, mature caspase-9 can process procaspase-3, setting into motion the caspase cascade. These observations indicate that cytochrome c and dATP are required for oligomerization of Apaf-1 and suggest that the WD-40 domain plays an important role in oligomerization of full-length Apaf-1 and the release of mature caspase-9 from the Apaf-1 oligomeric complex.     

Human CARD4 protein is a novel CED-4/Apaf-1 cell death family member that activates NF-kappaB

The nematode CED-4 protein and its human homolog Apaf-1 play a central role in apoptosis by functioning as direct activators of death-inducing caspases. A novel human CED-4/Apaf-1 family member called CARD4 was identified that has a domain structure strikingly similar to the cytoplasmic, receptor-like proteins that mediate disease resistance in plants. CARD4 interacted with the serine-threonine kinase RICK and potently induced NF-kappaB activity through TRAF-6 and NIK signaling molecules. In addition, coexpression of CARD4 augmented caspase-9-induced apoptosis. Thus, CARD4 coordinates downstream NF-kappaB and apoptotic signaling pathways and may be a component of the host innate immune response.     

Requirement of cytochrome c for apoptosis in human cells

During apoptosis, cytochrome c released from mitochondria activates Apaf-1, a cofactor of caspase-9. The evidence that cytochrome c can activate Apaf-1 is abundant, but the proof that cytochrome c is required for apoptosis is limited to two studies that used genetically modified mice. One of these studies concluded that in some tissues apoptosis may require Apaf-1 but not cytochrome c, which indicated the need to analyze the requirement of cytochrome c beyond the mouse models, and in human tumor cells in particular. In this study, we designed tools to silence cytochrome c expression in human cells and tested these tools in an experimental system of oncogenic transformation. We found that cytochrome c was required for apoptosis induced by both DNA damage and, unexpectedly, TNFalpha. Overall, this study established that cytochrome c is required for apoptosis in human cells and provided tools to dissect mechanisms of apoptosis in various experimental models.     

Three-dimensional structure of a double apoptosome formed by the Drosophila Apaf-1 related killer

The Drosophila Apaf-1 related killer (Dark) forms an apoptosome that activates Dronc, an apical procaspase in the intrinsic cell death pathway. To study this process, we assembled a large Dark complex in the presence of dATP. Remarkably, we found that cytochrome c was not required for assembly and when added, cytochrome c did not bind to the Dark complex. We then determined a 3D structure of the Dark complex at 18.8A resolution using electron cryo-microscopy and single particle methods. In the structure, eight Dark subunits form a wheel-like particle and two of these rings associate face-to-face. In contrast, Apaf-1 forms a single ring that is comprised of seven subunits and each Apaf-1 binds a molecule of cytochrome c. We then used relevant crystal structures to model the Dark complex. This analysis shows that a single Dark ring and the Apaf-1 apoptosome share many key features. When taken together, the data suggest that a single ring in the Dark complex may represent the Drosophila apoptosome. Thus, our analysis provides a domain model of this complex and gives insights into its function.