Caspase-3 cleaves Apaf-1 into an approximately 30 kDa fragment that associates with an inappropriately oligomerized and biologically inactive approximately 1.4 MDa apoptosome complex

Cytochrome c and dATP/ATP induce oligomerization of Apaf-1 into two distinct apoptosome complexes: an approximately 700 kDa complex, which recruits and activates caspases-9, -3 and -7, and an approximately 1.4 MDa complex, which recruits and processes caspase-9, but does not efficiently activate effector caspases. While searching for potential inhibitors of the approximately 1.4 MDa apoptosome complex, we observed an approximately 30 kDa Apaf-1 immunoreactive fragment that was associated exclusively with the inactive complex. We subsequently determined that caspase-3 cleaved Apaf-1 within its CED-4 domain (SVTD(271) downward arrowS) in both dATP-activated lysates and apoptotic cells to form a prominent approximately 30 kDa (p30) N-terminal fragment. Purified recombinant Apaf-1 p30 fragment weakly inhibited dATP-dependent activation of caspase-3 in vitro. However, more importantly, prevention of endogenous formation of the p30 fragment did not stimulate latent effector caspase processing activity in the large complex. Similarly, the possibility that XIAP, an inhibitor of apoptosis protein (IAP), was responsible for the inactivity of the approximately 1.4 MDa complex was excluded as immunodepletion of this caspase inhibitor failed to relieve the inhibition. However, selective proteolytic digestion of the approximately 1.4 MDa and approximately 700 kDa complexes showed that Apaf-1 was present in conformationally distinct forms in these two complexes. Therefore, the inability of the approximately 1.4 MDa apoptosome complex to process effector caspases most likely results from inappropriately folded or oligomerized Apaf-1.     

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.     

Diarylurea compounds inhibit caspase activation by preventing the formation of the active 700-kilodalton apoptosome complex

The release of mitochondrial proapoptotic proteins into the cytosol is the key event in apoptosis signaling, leading to the activation of caspases. Once in the cytosol, cytochrome c triggers the formation of a caspase-activating protein complex called the apoptosome, whereas Smac/Diablo and Omi/htra2 antagonize the caspase inhibitory effect of inhibitor of apoptosis proteins (IAPs). Here, we identify diarylurea compounds as effective inhibitors of the cytochrome c-induced formation of the active, approximately 700-kDa apoptosome complex and caspase activation. Using diarylureas to inhibit the formation of the apoptosome complex, we demonstrated that cytochrome c, rather than IAP antagonists, is the major mitochondrial caspase activation factor in tumor cells treated with tumor necrosis factor. Thus, we have identified a novel class of compounds that inhibits apoptosis by blocking the activation of the initiator caspase 9 by directly inhibiting the formation of the apoptosome complex. This mechanism of action is different from that employed by the widely used tetrapeptide inhibitors of caspases or known endogenous apoptosis inhibitors, such as Bcl-2 and IAPs. Thus, these compounds provide a novel specific tool to investigate the role of the apoptosome in mitochondrion-dependent death paradigms.     

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(+).     

Caspase-containing complexes in the regulation of cell death and inflammation

Caspases are a family of cysteine proteases that are essential in the initiation and execution of apoptosis and the proteolytic maturation of inflammatory cytokines such as IL-1beta and IL-18. Caspases can be subdivided into those that have a large prodomain and those that have not. In general, apoptotic and inflammatory signalling pathways are initiated when large-prodomain caspases are recruited to large protein complexes via homotypic interactions involving death domain folds. The formation of these specialised multimeric platforms involves three major functions: (1) the sensing of cellular stress, damage, infection or inflammation; (2) multimerisation of the platform; and (3) recruitment and conformational activation of caspases. In this overview we discuss the complexes implicated in the regulation of cell death and inflammatory processes such as the death-inducing signalling complex (DISC), the apoptosome, the inflammasomes and the PIDDosome. We describe their sensing functions, compositions and functional outcomes. Inhibitory protein families such as FLIPs and CARD-only proteins prevent the recruitment of caspases in these sensing complexes, avoiding inappropriate initiation of cell death or inflammation.     

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.     

Recruitment, activation and retention of caspases-9 and -3 by Apaf-1 apoptosome and associated XIAP complexes

During apoptosis, release of cytochrome c initiates dATP-dependent oligomerization of Apaf-1 and formation of the apoptosome. In a cell-free system, we have addressed the order in which apical and effector caspases, caspases-9 and -3, respectively, are recruited to, activated and retained within the apoptosome. We propose a multi-step process, whereby catalytically active processed or unprocessed caspase-9 initially binds the Apaf-1 apoptosome in cytochrome c/dATP-activated lysates and consequently recruits caspase-3 via an interaction between the active site cysteine (C287) in caspase-9 and a critical aspartate (D175) in caspase-3. We demonstrate that XIAP, an inhibitor-of-apoptosis protein, is normally present in high molecular weight complexes in unactivated cell lysates, but directly interacts with the apoptosome in cytochrome c/dATP-activated lysates. XIAP associates with oligomerized Apaf-1 and/or processed caspase-9 and influences the activation of caspase-3, but also binds activated caspase-3 produced within the apoptosome and sequesters it within the complex. Thus, XIAP may regulate cell death by inhibiting the activation of caspase-3 within the apoptosome and by preventing release of active caspase-3 from the complex.     

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.     

凋亡体与细胞凋亡

由细胞色素C（Cytochrome c,Cyt c）、ATP/dATP、凋亡酶激活因子-1（apoptotic protease activating factor-1,Apaf-1）以及procaspase-9（caspase-9的前体）构成的约700 kDa、具有很强的caspase酶激活活性的大分子蛋白复合物——凋亡体（apoptosome）,在哺乳动物线粒体凋亡途径和胚胎发育中至关重要。描述了凋亡体上各因子的结构、功能及其相互关系,线粒体介导的凋亡通路中凋亡体的形成及其调控。     

Caspase activation involves the formation of the aposome, a large (approximately 700 kDa) caspase-activating complex.

In mammals, apoptotic protease-activating factor 1 (Apaf-1), cytochrome c, and dATP activate caspase-9, which initiates the postmitochondrial-mediated caspase cascade by proteolytic cleavage/activation of effector caspases to form active approximately 60-kDa heterotetramers. We now demonstrate that activation of caspases either in apoptotic cells or following dATP activation of cell lysates results in the formation of two large but different sized protein complexes, the "aposome" and the "microaposome". Surprisingly, most of the DEVDase activity in the lysate was present in the aposome and microaposome complexes with only small amounts of active caspase-3 present as its free approximately 60-kDa heterotetramer. The larger aposome complex (M(r) = approximately 700,000) contained Apaf-1 and processed caspase-9, -3, and -7. The smaller microaposome complex (M(r) = approximately 200,000-300,000) contained active caspase-3 and -7 but little if any Apaf-1 or active caspase-9. Lysates isolated from control THP.1 cells, prior to caspase activation, showed striking differences in the distribution of key apoptotic proteins. Apaf-1 and procaspase-7 may be functionally complexed as they eluted as an approximately 200-300-kDa complex, which did not have caspase cleavage (DEVDase) activity. Procaspase-3 and -9 were present as separate and smaller 60-90-kDa (dimer) complexes. During caspase activation, Apaf-1, caspase-9, and the effector caspases redistributed and formed the aposome. This resulted in the processing of the effector caspases, which were then released, possibly bound to other proteins, to form the microaposome complex.     

Mechanical aspects of apoptosome assembly

Killing a cell through apoptosis ultimately rests on the mechanical destruction of the structure and function of cellular machineries. Understanding the mechanics of one particular function is usually the last step in our quest to decipher the underlying molecular mechanism. Execution of apoptosis is initiated by the activation of initiator caspases, which is mediated by specific adaptor protein complexes generally known as apoptosomes. This review discusses the assembly, structure and function of the heptameric Apaf-1 apoptosome, the tetrameric CED-4 complex, the octameric Dark apoptosome, and the death-inducing signaling complex (DISC).     

Redox Regulation of Apoptosis before and after Cytochrome C Release

Programmed cell death, or apoptosis, is one of the most studied areas of modern biology. Apoptosis is a genetically regulated process, which plays an essential role in the development and homeostasis of higher organisms. Mitochondria, known to play a central role in regulating cellular metabolism, was found to be critical for regulating apoptosis induced under both physiological and pathological conditions. Mitochondria are a major source of reactive oxygen species (ROS) but they can also serve as its target during the apoptosis process. Release of apoptogenic factors from mitochondria, the best known of which is cytochrome c, leads to assembly of a large apoptosis-inducing complex called the apoptosome. Cysteine proteases (called caspases) are recruited to this complex and, following their activation by proteolytic cleavage, activate other caspases, which in turn target for specific cleavage a large number of cellular proteins. The redox regulation of apoptosis during and after cytochrome c release is an area of intense investigation. This review summarizes what is known about the biological role of ROS and its targets in apoptosis with an emphasis on its intricate connections to mitochondria and the basic components of cell death.     

Caspase-8 in apoptosis: the beginning of "the end"?

Caspase-8 is a member of the cysteine proteases, which are implicated in apoptosis and cytokine processing. Like all caspases, caspase-8 is synthesized as an inactive single polypeptide chain zymogen procaspase and is activated by proteolytic cleavage, through either autoactivation after recruitment into a multimeric complex or trans-cleavage by other caspases. Thus, ligand binding-induced trimerization of death receptors results in recruitment of the receptor-specific adapter protein Fas-associated death domain (FADD), which then recruits caspase-8. Activated caspase-8 is known to propagate the apoptotic signal either by directly cleaving and activating downstream caspases or by cleaving the BH3 Bcl2-interacting protein, which leads to the release of cytochrome c from mitochondria, triggering activation of caspase-9 in a complex with dATP and Apaf-1. Activated caspase-9 then activates further "downstream caspases," including caspase-8. Knockout data indicate that caspase-8 is required for killing induced by the death receptors Fas, tumor necrosis factor receptor 1, and death receptor 3. Moreover, caspase-8-/- mice die in utero as a result of defective development of heart muscle and display fewer hematopoietic progenitor cells, suggesting that the FADD/caspase-8 pathway is absolutely required for growth and development of specific cell types.     

Caspase-7 is directly activated by the approximately 700-kDa apoptosome complex and is released as a stable XIAP-caspase-7 approximately 200-kDa complex

MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce approximately 1.4-MDa and approximately 700-kDa apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This approximately 700-kDa apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/HtrA2, active caspase-7 is released from the apoptosome and forms a stable approximately 200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the apoptosome and also forms a stable approximately 200-kDa complex with active caspase-7.     

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.     

Construction and analysis of a modular model of caspase activation in apoptosis

Background  

A key physiological mechanism employed by multicellular organisms is apoptosis, or programmed cell death. Apoptosis is triggered by the activation of caspases in response to both extracellular (extrinsic) and intracellular (intrinsic) signals. The extrinsic and intrinsic pathways are characterized by the formation of the death-inducing signaling complex (DISC) and the apoptosome, respectively; both the DISC and the apoptosome are oligomers with complex formation dynamics. Additionally, the extrinsic and intrinsic pathways are coupled through the mitochondrial apoptosis-induced channel via the Bcl-2 family of proteins.     

Small molecule inhibitors of Apaf-1-related caspase- 3/-9 activation that control mitochondrial-dependent apoptosis

Apoptosis is a biological process relevant to human disease states that is strongly regulated through protein-protein complex formation. These complexes represent interesting points of chemical intervention for the development of molecules that could modulate cellular apoptosis. The apoptosome is a holoenzyme multiprotein complex formed by cytochrome c-activated Apaf-1 (apoptotic protease-activating factor), dATP and procaspase-9 that link mitochondria disfunction with activation of the effector caspases and in turn is of interest for the development of apoptotic modulators. In the present study we describe the identification of compounds that inhibit the apoptosome-mediated activation of procaspase-9 from the screening of a diversity-oriented chemical library. The active compounds rescued from the library were chemically optimised to obtain molecules that bind to both recombinant and human endogenous Apaf-1 in a cytochrome c-noncompetitive mechanism that inhibits the recruitment of procaspase-9 by the apoptosome. These newly identified Apaf-1 ligands decrease the apoptotic phenotype in mitochondrial-mediated models of cellular 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.     

Identification of an inhibitor of caspase activation from heart extracts; ATP blocks apoptosome formation

By revealing the biochemistry of apoptosis it is expected we will both improve our understanding of diseases where apoptosis plays an important role and aid the development of therapies for these disorders. Caspases are a family of proteases whose activity is required for apoptosis. In this study, a cell-free system was used to investigate the mechanism of caspase-9 activation in extracts from heart cells. Unlike extracts from other cell types, heart extracts were found to activate caspases poorly. This could be explained by the low levels of Apaf-1 in heart cells. However, subsequent testing showed that heart extracts contained an inhibitor of caspase activation that could block caspase activation in extracts from different cell types. Subsequent purification of the inhibitor of caspase activation from these extracts identified ATP. Caspase-9 is activated by recruitment into a multi-protein complex, the apoptosome, which then activates downstream caspases that kill the cell. Importantly, size exclusion chromatography showed that ATP inhibits apoptosome formation at physiologically relevant concentrations. Together these data support the hypothesis that intracellular ATP concentration is a critical factor in determining whether an apoptotic stimulus can induce apoptosome formation. Thus, the well described fall in intracellular ATP apoptosis is not an epiphenomenon but may be a pro-apoptotic event contributing to cell death.     

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.