Portrait of a killer: the mitochondrial apoptosome emerges from the shadows

Apoptosis (programmed cell death) is a physiological process used to eliminate superfluous, damaged, infected, or aged cells in multicellular organisms. During apoptosis the cellular architecture is dismantled from within in a highly controlled fashion. Members of the caspase family of cysteine proteases are responsible for the destructive phase of apoptosis. One major pathway to caspase activation involves the formation of a multisubunit protease activation complex called the apoptosome. The apoptosome is assembled in response to signals that provoke mitochondrial outer membrane permeabilization and the release of cytochrome c into the cytosol. Recent studies indicate that the apoptosome is a wheel-like structure consisting of seven molecules of Apaf-1 and a similar number of caspase-9 dimers. Knowledge of the structure of the apoptosome will likely lead to the design of therapeutic modulators of apoptosis.     

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.     

Apo cytochrome c inhibits caspases by preventing apoptosome formation

Caspases are cysteine proteases and potent inducers of apoptosis. Their activation and activity is therefore tightly regulated. There are several mechanisms by which caspases can be activated but one key pathway involves release of holo cytochrome c from mitochondria into the cytoplasm. Cytoplasmic holo cytochrome c binds to apoptotic protease activating factor-1 (Apaf-1), driving the formation of an Apaf-1 oligomer (the apoptosome) which in turn binds and activates caspase-9. Previously we showed that the apo form of cytochrome c (lacking heme) can bind Apaf-1 and block both holo-dependent caspase activation in cell extracts and Bax-induced apoptosis in cells. Here we tested the ability of apo cytochrome c to inhibit caspase-9 activation induced by recombinant Apaf-1. Furthermore, using purified proteins and size exclusion chromatography we show that apo cytochrome c prevents holo cytochrome c-dependent apoptosome formation.     

Physiological concentrations of K+ inhibit cytochrome c-dependent formation of the apoptosome.

In many forms of apoptosis, cytochrome c released from mitochondria induces the oligomerization of Apaf-1 to form a caspase-activating apoptosome complex. Activation of lysates in vitro with dATP and cytochrome c results in the formation of an active caspase-processing approximately 700-kDa apoptosome complex, which predominates in apoptotic cells, and a relatively inactive approximately 1.4-MDa complex. We now demonstrate that assembly of the active complex is suppressed by normal intracellular concentrations of K(+). Using a defined apoptosome reconstitution system with recombinant Apaf-1 and cytochrome c, K(+) also inhibits caspase activation by abrogating Apaf-1 oligomerization and apoptosome assembly. Once assembled, the apoptosome is relatively insensitive to the effects of ionic strength and processes/activates effector caspases. The inhibitory effects of K(+) on apoptosome formation are antagonized in a concentration-dependent manner by cytochrome c. These studies support the hypothesis that the normal intracellular concentrations of K(+) act to safeguard the cell against inappropriate formation of the apoptosome complex, caused by the inadvertent release of small amounts of cytochrome c. Thus, the assembly and activation of the apoptosome complex in the cell requires the rapid and extensive release of cytochrome c to overcome the inhibitory effects of normal intracellular concentrations of K(+).     

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.     

Alpha-fetoprotein positively regulates cytochrome c-mediated caspase activation and apoptosome complex formation.

Previous results have shown that the oncoembryonic marker alpha-fetoprotein (AFP) is able to induce apoptosis in tumor cells through activation of caspase 3, bypassing Fas-dependent and tumor necrosis factor receptor-dependent signaling. In this study we further investigate the molecular interactions involved in the AFP-mediated signaling of apoptosis. We show that AFP treatment of tumor cells is accompanied by cytosolic translocation of mitochondrial cytochrome c. In a cell-free system, AFP mediates processing and activation of caspases 3 and 9 by synergistic enhancement of the low-dose cytochrome c-mediated signals. AFP was unable to regulate activity of caspase 3 in cell extracts depleted of cytochrome c or caspase 9. Using high-resolution chromatography, we show that AFP positively regulates cytochrome c/dATP-mediated apoptosome complex formation, enhances recruitment of caspases and Apaf-1 into the complex, and stimulates release of the active caspases 3 and 9 from the apoptosome. By using a direct protein-protein interaction assay, we show that pure human AFP almost completely disrupts the association between processed caspases 3 and 9 and the cellular inhibitor of apoptosis protein (cIAP-2), demonstrating its release from the complex. Our data suggest that AFP may regulate cell death by displacing cIAP-2 from the apoptosome, resulting in promotion of caspase 3 activation and its release from the complex.     

Cytochrome c promotes caspase-9 activation by inducing nucleotide binding to Apaf-1

We report here the biochemical analysis of the reconstituted de novo procaspase-9 activation using highly purified cytochrome c, recombinant apoptotic protease-activating factor-1 (Apaf-1), and recombinant procaspase-9. Using a nucleotide binding assay, we found that Apaf-1 alone bound dATP poorly and the nucleotide binding to Apaf-1 was significantly stimulated by cytochrome c. The binding of dATP to Apaf-1 induces the formation of a multimeric Apaf-1. cytochrome c complex, apoptosome. Procaspase-9 also synergistically promotes dATP binding to Apaf-1 in a cytochrome c-dependent manner. The dATP bound to apoptosome remained as dATP, not dADP. A nonhydrolyzable ATP analog, ADPCP (beta,gamma-methylene adenosine 5'-triphosphate), was able to support apoptosome formation and caspase activation in place of dATP or ATP. These data indicate that the key event in Apaf-1-mediated caspase-9 activation is cytochrome c-induced dATP binding to Apaf-1.     

PSAP induces a unique Apaf-1 and Smac-dependent mitochondrial apoptotic pathway independent of Bcl-2 family proteins

Presenilin-associated protein (PSAP) has been identified as a mitochondrial proapoptotic protein. However, the mechanism by which PSAP induces apoptosis remains unknown. To this end, we have established an inducible expression system. Using this system, we have examined the roles of B-cell lymphoma 2 (Bcl-2) family proteins, cytochrome c, Smac (Smac/Diablo, second mitochondria-derived activator of caspases/direct IAP binding protein with low PI), and Apaf-1 (apoptotic protease-activating factor) in PSAP-induced apoptosis. Our results demonstrate that knockdown of Apaf-1 abolished PSAP-induced caspase activation and poly(ADP ribose) polymerase (PARP) cleavage, indicating that the apoptosome formation triggered by cytochrome c is crucial for PSAP-induced apoptosis. Our data also demonstrate that knockdown of Smac abolished PSAP-induced caspase activation and PARP cleavage, indicating that, in addition to Apaf-1 or apoptosome formation, Smac is also essential for PSAP-induced apoptosis. However, interestingly, our data demonstrate that overexpression of Bcl-2 and Bcl-xL did not protect cells from PSAP-induced apoptosis, and that knockdown of Bid, Bax, and Bak had no effect on PSAP-induced cytochrome c and Smac release, indicating that PSAP-induced apoptosis is not regulated by Bcl-2 family proteins. These results strongly suggest that PSAP evokes mitochondrial apoptotic cascades via a novel mechanism that is not regulated by Bcl-2 family proteins, but that both the formation of cytochrome c-Apaf-1 apoptosome and the presence of Smac are absolutely required for PSAP-induced apoptosis.     

Initiator caspases in apoptosis signaling pathways

Death receptor- or mitochondrion-dependent apoptosis is initiated by the recruitment and activation of apical caspases in the apoptosis signaling pathways. In death receptor-mediated apoptosis, engagement of death receptors leads to the formation of the death-inducing signaling complex (DISC) containing the death receptors, adaptor proteins, caspase-8 and caspase-10. In mitochondrion-dependent apoptosis, release of cytochrome C into the cytosol results in the formation of apoptosome containing cytochrome C, Apaf-1 and caspase-9. Caspase-8, caspase-10 and caspase-9 are believed to be the initiator caspases at the top of the caspase signaling cascade. Recruitment of caspases to DISC and apoptosome leads to their activation by dimer formation. Recent biochemical and structural analyses of components in the DISC and apoptosome shed new lights on their roles in inducing the onset of apoptosis signaling.     

The role of cytochrome c in caspase activation in Drosophila melanogaster cells

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. We demonstrate here that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals.     

A novel Apaf-1-independent putative caspase-2 activation complex

Caspase activation is a key event in apoptosis execution. In stress-induced apoptosis, the mitochondrial pathway of caspase activation is believed to be of central importance. In this pathway, cytochrome c released from mitochondria facilitates the formation of an Apaf-1 apoptosome that recruits and activates caspase-9. Recent data indicate that in some cells caspase-9 may not be the initiator caspase in stress-mediated apoptosis because caspase-2 is required upstream of mitochondria for the release of cytochrome c and other apoptogenic factors. To determine how caspase-2 is activated, we have studied the formation of a complex that mediates caspase-2 activation. Using gel filtration analysis of cell lysates, we show that caspase-2 is spontaneously recruited to a large protein complex independent of cytochrome c and Apaf-1 and that recruitment of caspase-2 to this complex is sufficient to mediate its activation. Using substrate-binding assays, we also provide the first evidence that caspase-2 activation may occur without processing of the precursor molecule. Our data are consistent with a model where caspase-2 activation occurs by oligomerization, independent of the Apaf-1 apoptosome.     

Apoptosome: the cellular engine for the activation of caspase-9

Characterization of the apoptosome by cryo-EM reveals a wheel-shaped heptameric assembly involving Apaf-1 and cytochrome c. This structure provides a framework for understanding the activation mechanisms of caspase-9, an important initiator caspase in apoptosis.     

Pro-apoptotic apoptosis protease-activating factor 1 (Apaf-1) has a cytoplasmic localization distinct from Bcl-2 or Bcl-x(L)

How Bcl-2 and its pro-survival relatives prevent activation of the caspases that mediate apoptosis is unknown, but they appear to act through the caspase activator apoptosis protease-activating factor 1 (Apaf-1). According to the apoptosome model, the Bcl-2-like proteins preclude Apaf-1 activity by sequestering the protein. To explore Apaf-1 function and to test this model, we generated monoclonal antibodies to Apaf-1 and used them to determine its localization within diverse cells by subcellular fractionation and confocal laser scanning microscopy. Whereas Bcl-2 and Bcl-x(L) were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocalize with them, even when these pro-survival proteins were overexpressed or after apoptosis was induced. Immunogold electron microscopy confirmed that Apaf-1 was dispersed in the cytoplasm and not on mitochondria or other organelles. After the death stimuli, Bcl-2 and Bcl-x(L) precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of larger Apaf-1 complexes, which are steps that presage apoptosis. However, neither Bcl-2 nor Bcl-x(L) could prevent the in vitro activation of Apaf-1 induced by the addition of exogenous cytochrome c. Hence, rather than sequestering Apaf-1 as proposed by the apoptosome model, Bcl-2-like proteins probably regulate Apaf-1 indirectly by controlling upstream events critical for its activation.     

Apaf-1 oligomerizes into biologically active approximately 700-kDa and inactive approximately 1.4-MDa apoptosome complexes

Apaf-1, by binding to and activating caspase-9, plays a critical role in apoptosis. Oligomerization of Apaf-1, in the presence of dATP and cytochrome c, is required for the activation of caspase-9 and produces a caspase activating apoptosome complex. Reconstitution studies with recombinant proteins have indicated that the size of this complex is very large in the order of approximately 1.4 MDa. We now demonstrate that dATP activation of cell lysates results in the formation of two large Apaf-1-containing apoptosome complexes with M(r) values of approximately 1.4 MDa and approximately 700 kDa. Kinetic analysis demonstrates that in vitro the approximately 700-kDa complex is produced more rapidly than the approximately 1.4 MDa complex and exhibits a much greater ability to activate effector caspases. Significantly, in human tumor monocytic cells undergoing apoptosis after treatment with either etoposide or N-tosyl-l-phenylalanyl chloromethyl ketone (TPCK), the approximately 700-kDa Apaf-1 containing apoptosome complex was predominately formed. This complex processed effector caspases. Thus, the approximately 700-kDa complex appears to be the correctly formed and biologically active apoptosome complex, which is assembled during apoptosis.     

Fas-mediated apoptosome formation is dependent on reactive oxygen species derived from mitochondrial permeability transition in Jurkat cells

Generation of reactive oxygen species (ROS) and activation of caspase cascade are both indispensable in Fas-mediated apoptotic signaling. Although ROS was presumed to affect the activity of the caspase cascade on the basis of findings that antioxidants inhibited the activation of caspases and that the stimulation of ROS by itself activated caspases, the mechanism by which these cellular events are integrated in Fas signaling is presently unclear. In this study, using human T cell leukemia Jurkat cells as well as an in vitro reconstitution system, we demonstrate that ROS are required for the formation of apoptosome. We first showed that ROS derived from mitochondrial permeability transition positively regulated the apoptotic events downstream of mitochondrial permeability transition. Then, we revealed that apoptosome formation in Fas-stimulated Jurkat cells was clearly inhibited by N-acetyl-L-cysteine and manganese superoxide dismutase by using both the immunoprecipitation and size-exclusion chromatography methods. To confirm these in vivo findings, we next used an in vitro reconstitution system in which in vitro-translated apoptotic protease-activating factor 1 (Apaf-1), procaspase-9, and cytochrome c purified from human placenta were activated by dATP to form apoptosome; the formation of apoptosome was markedly inhibited by reducing reagents such as DTT or reduced glutathione (GSH), whereas hydrogen peroxide prevented this inhibition. We also found that apoptosome formation was substantially impaired by GSH-pretreated Apaf-1, but not GSH-pretreated procaspase-9 or GSH-pretreated cytochrome c. Collectively, these results suggest that ROS plays an essential role in apoptosome formation by oxidizing Apaf-1 and the subsequent activation of caspase-9 and -3.     

Apoptosomes: engines for caspase activation

Activation of the caspases that initiate apoptosis typically requires cognate scaffold proteins, including CED-4 in Caenorhabditis elegans, Apaf-1 in mammals and Dark in Drosophila. Each scaffold protein oligomerizes procaspases into a complex called the apoptosome, but the regulation and biological roles of the scaffolds differ. Whereas CED-4 is restrained by the Bcl-2 homologue CED-9, Apaf-1 is inhibited by its WD40 repeat region, until it is activated by cytochrome c, derived from damaged mitochondria. Although Dark also has a WD40 region, its activation does not seem to involve cytochrome c. CED-4 is essential for apoptosis in the worm and Dark for many apoptotic responses in the fly, but the Apaf-1/caspase-9 system probably amplifies rather than initiates the mammalian caspase cascade.     

Regulation of apoptosis by the redox state of cytochrome c

Cytochrome c, released from mitochondria into the cytosol, triggers formation of the apoptosome resulting in activation of caspases. This paper reviews the evidence for and against the redox state of cytochrome c regulating apoptosis, and possible mechanisms of this. Three research groups have found that the oxidized form of cytochrome c (Fe(3+)) can induce caspase activation via the apoptosome, while the reduced form (Fe(2+)) cannot. It is unclear whether this is due to the oxidized and reduced forms of cytochrome c having: (i) different affinities for Apaf-1, (ii) different abilities to activate Apaf-1 once bound, or (iii) different affinities for other components of the cell. Experiments replacing the Fe of cytochrome c with redox-inactive metals indicate that cytochrome c does not have to change redox states to activate caspases. In healthy cells, cytosolic cytochrome c is rapidly reduced by various enzymes and/or reductants, which may function to block apoptosis. However, in apoptotic cells, cytosolic cytochrome c is rapidly oxidized by mitochondrial cytochrome oxidase, to which it has access due to permeabilization of the outer membrane. Regulation of the redox state of cytochrome c potentially enables regulation of the intrinsic pathway of apoptosis at a relatively late stage.     

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.     

A structure of the human apoptosome at 12.8 A resolution provides insights into this cell death platform

Apaf-1 and cytochrome c coassemble in the presence of dATP to form the apoptosome. We have determined a structure of the apoptosome at 12.8 A resolution by using electron cryomicroscopy and single-particle methods. We then docked appropriate crystal structures into the map to create an accurate domain model. Thus, we found that seven caspase recruitment domains (CARDs) form a central ring within the apoptosome. At a larger radius, seven copies of the nucleotide binding and oligomerization domain (NOD) associate laterally to form the hub, which encircles the CARD ring. Finally, an arm-like helical domain (HD2) links each NOD to a pair of beta propellers, which bind a single cytochrome c. This model provides insights into the roles of dATP and cytochrome c in assembly. Our structure also reveals how a CARD ring and the central hub combine to create a platform for procaspase-9 activation.     

Positive regulation of apoptosis by HCA66, a new Apaf-1 interacting protein, and its putative role in the physiopathology of NF1 microdeletion syndrome patients

As a component of the apoptosome, a caspase-activating complex, Apaf-1 plays a central role in the mitochondrial caspase activation pathway of apoptosis. We report here the identification of a novel Apaf-1 interacting protein, hepatocellular carcinoma antigen 66 (HCA66) that is able to modulate selectively Apaf-1-dependent apoptosis through its direct association with the CED4 domain of Apaf-1. Expression of HCA66 was able to potentiate Apaf-1, but not receptor-mediated apoptosis, by increasing downstream caspase activity following cytochrome c release from the mitochondria. Conversely, cells depleted of HCA66 were severely impaired for apoptosome-dependent apoptosis. Interestingly, expression of the Apaf-1-interacting domain of HCA66 had the opposite effect of the full-length protein, interfering with the Apaf-1 apoptotic pathway. Using a cell-free system, we showed that reduction of HCA66 expression was associated with a diminished amount of caspase-9 in the apoptosome, resulting in a lower ability of the apoptosome to activate caspase-3. HCA66 maps to chromosome 17q11.2 and is among the genes heterozygously deleted in neurofibromatosis type 1 (NF1) microdeletion syndrome patients. These patients often have a distinct phenotype compared to other NF1 patients, including a more severe tumour burden. Our results suggest that reduced expression of HCA66, owing to haploinsufficiency of HCA66 gene, could render NF1 microdeleted patients-derived cells less susceptible to apoptosis.