Subcellular localization of Apaf-1 in apoptotic rat pituitary cells

A key step in the intrinsic apoptotic pathway is the assembly of the apoptosome complex. The apoptosome components are well known; however, the physiology of the assembly of the apoptosome complex at the cellular level is still poorly defined. The aim of this work was to study the subcellular distribution of the apoptosome scaffold protein apoptotic protease-activating factor 1 (Apaf-1) before and after triggering apoptosis in single somatotrophs. Somatotrophs are the subject of extensive pituitary tissue remodeling in different physiological situations in which the quality and the number of pituitary cells are determined by cell proliferation and apoptosis. We show herein that 2 h after triggering apoptosis with rotenone, Apaf-1 redistributed to the proximity of mitochondria. In addition, the degree of colocalization between Apaf-1 and fluorescently labeled caspase-9 significantly increased during the same period. Furthermore, we show herein for the first time in single cells that the colocalization between Apaf-1 and cytochrome c increases only transiently, indicating a transient interaction between cytochrome c and Apaf-1 during the activation of apoptosis in these cells. cytochrome c; caspase-9; apoptosis; apoptosome complex     

The holo-apoptosome: activation of procaspase-9 and interactions with caspase-3

Activation of procaspase-9 on the apoptosome is a pivotal step in the intrinsic cell death pathway. We now provide further evidence that caspase recruitment domains of pc-9 and Apaf-1 form a CARD-CARD disk that is flexibly tethered to the apoptosome. In addition, a 3D reconstruction of the pc-9 apoptosome was calculated without symmetry restraints. In this structure, p20 and p10 catalytic domains of a single pc-9 interact with nucleotide binding domains of adjacent Apaf-1 subunits. Together, disk assembly and pc-9 binding create an asymmetric proteolysis machine. We also show that CARD-p20 and p20-p10 linkers play important roles in pc-9 activation. Based on the data, we propose a proximity-induced association model for pc-9 activation on the apoptosome. We also show that pc-9 and caspase-3 have overlapping binding sites on the central hub. These binding sites may play a role in pc-3 activation and could allow the formation of hybrid apoptosomes with pc-9 and caspase-3 proteolytic activities.     

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.     

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.     

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.     

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.     

PHAPI, CAS, and Hsp70 promote apoptosome formation by preventing Apaf-1 aggregation and enhancing nucleotide exchange on Apaf-1

During apoptosis, cytochrome c is released from mitochondria to the cytosol, where it binds Apaf-1. The Apaf-1/cytochrome c complex then oligomerizes either into heptameric caspase-9-activating apoptosome, which subsequently activates caspase-3 and caspase-7, or bigger inactive aggregates, depending on the availability of nucleotide dATP/ATP. A tumor suppressor protein, PHAPI, enhances caspase-9 activation by promoting apoptosome formation through an unknown mechanism. We report here the identification of cellular apoptosis susceptibility protein (CAS) and heat shock protein 70 (Hsp70) as mediators of PHAPI activity. PHAPI, CAS, and Hsp70 function together to accelerate nucleotide exchange on Apaf-1 and prevent inactive Apaf-1/cytochrome c aggregation. CAS expression is induced by multiple apoptotic stimuli including UV irradiation. Knockdown of CAS by RNA interference (RNAi) in cells attenuates apoptosis induced by UV light and causes endogenous Apaf-1 to form aggregates. These studies indicated that PHAPI, CAS, and Hsp70 play an important regulatory role during apoptosis.     

Regulation of the Apaf-1/caspase-9 apoptosome by caspase-3 and XIAP

The apoptosome is a multiprotein complex comprising Apaf-1, cytochrome c, and caspase-9 that functions to activate caspase-3 downstream of mitochondria in response to apoptotic signals. Binding of cytochrome c and dATP to Apaf-1 in the cytosol leads to the assembly of a heptameric complex in which each Apaf-1 subunit is bound noncovalently to a procaspase-9 subunit via their respective CARD domains. Assembly of the apoptosome results in the proteolytic cleavage of procaspase-9 at the cleavage site PEPD(315) to yield the large (p35) and small (p12) caspase-9 subunits. In addition to the PEPD site, caspase-9 contains a caspase-3 cleavage site (DQLD(330)), which when cleaved, produces a smaller p10 subunit in which the NH(2)-terminal 15 amino acids of p12, including the XIAP BIR3 binding motif, are removed. Using purified proteins in a reconstituted reaction in vitro, we have assessed the relative impact of Asp(315) and Asp(330) cleavage on caspase-9 activity within the apoptosome. In addition, we characterized the effect of caspase-3 feedback cleavage of caspase-9 on the rate of caspase-3 activation, and the potential ramifications of Asp(330) cleavage on XIAP-mediated inhibition of the apoptosome. We have found that cleavage of procaspase-9 at Asp(330) to generate p35, p10 or p37, p10 forms resulted in a significant increase (up to 8-fold) in apoptosome activity compared with p35/p12. The significance of this increase was demonstrated by the near complete loss of apoptosome-mediated caspase-3 activity when a point mutant (D330A) of procaspase-9 was substituted for wild-type procaspase-9 in the apoptosome. In addition, cleavage at Asp(330) exposed a novel p10 NH(2)-terminal peptide motif (AISS) that retained the ability to mediate XIAP inhibition of caspase-9. Thus, whereas feedback cleavage of caspase-9 by caspase-3 significantly increases the activity of the apoptosome, it does little to attenuate its sensitivity to inhibition by XIAP.     

A chemical inhibitor of Apaf-1 exerts mitochondrioprotective functions and interferes with the intra-S-phase DNA damage checkpoint

QM31 represents a new class of cytoprotective agents that inhibit the formation of the apoptosome, the caspase activation complex composed by Apaf-1, cytochrome c, dATP and caspase-9. Here, we analyzed the cellular effects of QM31, as compared to the prototypic caspase inhibitor Z-VAD-fmk. QM31 was as efficient as Z-VAD-fmk in suppressing caspase-3 activation, and conferred a similar cytoprotective effect. In contrast to Z-VAD-fmk, QM31 inhibited the release of cytochrome c from mitochondria, an unforeseen property that may contribute to its pronounced cytoprotective activity. Moreover, QM31 suppressed the Apaf-1-dependent intra-S-phase DNA damage checkpoint. These results suggest that QM31 can interfere with the two known functions of Apaf-1, namely apoptosome assembly/activation and intra-S-phase cell cycle arrest. Moreover, QM31 can inhibit mitochondrial outer membrane permeabilization, an effect that is independent from its action on Apaf-1.     

Proteolytic Processing of the Caspase-9 Zymogen Is Required for Apoptosome-mediated Activation of Caspase-9

Maturation of the single-chain caspase-9 zymogen through autoproteolytic processing is mediated by the Apaf-1 apoptosome at the onset of apoptosis. Processed caspase-9 and the apoptosome form a holoenzyme with robust proteolytic activity that is 2–3 orders of magnitude higher than that of free processed caspase-9. An unresolved important question is the role of caspase-9 processing, with some experimental data suggesting its dispensability. In this study, we demonstrate that, in contrast to wild-type caspase-9, the unprocessed single-chain caspase-9 triple mutant E306A/D315A/D330A (Casp9-TM) could no longer be adequately activated by the apoptosome. Compared with the protease activity of wild-type caspase-9, that of Casp9-TM in the presence of the apoptosome was drastically reduced. The crippled protease activity of Casp9-TM in the presence of the apoptosome is likely attributable to a markedly reduced ability of Casp9-TM to form homodimers. These data identify an essential role for the autoproteolytic processing of caspase-9 in its activation.     

Taurine inhibits apoptosis by preventing formation of the Apaf-1/caspase-9 apoptosome

Cardiomyocyte apoptosis contributes to cell death during myocardial infarction. One of the factors that regulate the degree of apoptosis during ischemia is the amino acid taurine. To study the mechanism underlying the beneficial effect of taurine, we examined the interaction between taurine and mitochondria-mediated apoptosis using a simulated ischemia model with cultured rat neonatal cardiomyocytes sealed in closed flasks. Exposure to medium containing 20 mM taurine reduced the degree of apoptosis following periods of ischemia varying from 24 to 72 h. In the untreated group, simulated ischemia for 24 h led to mitochondrial depolarization accompanied by cytochrome c release. The apoptotic cascade was also activated, as evidenced by the activation of caspase-9 and -3. Taurine treatment had no effect on mitochondrial membrane potential and cytochrome c release; however, it inhibited ischemia-induced cleavage of caspase-9 and -3. Taurine loading also suppressed the formation of the Apaf-1/caspase-9 apoptosome and the interaction of caspase-9 with Apaf-1. These findings demonstrate that taurine effectively prevents myocardial ischemia-induced apoptosis by inhibiting the assembly of the Apaf-1/caspase-9 apoptosome. ischemia; cultured cardiomyocytes     

A molecular view on signal transduction by the apoptosome

Apoptosomes are signaling platforms that initiate the dismantling of a cell during apoptosis. In mammals, assembly of the apoptosome is the pivotal point in the mitochondrial pathway of apoptosis, and is prompted by binding of cytochrome c to the apoptotic protease-activating factor 1 (Apaf-1) in the presence of ATP. The resulting wheel-like heptamer of seven molecules Apaf-1 and seven molecules cytochrome c binds and activates the initiator caspase-9, which in turn ignites the downstream caspase cascade. In this review we discuss the molecular determinants for the formation of the mammalian apoptosome and caspase activation and describe the related signaling platforms in flies and nematodes.     

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.     

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.     

Caspase-9 plays a marginal role in serum starvation-induced apoptosis

Serum withdrawal represents a potent trigger to induce caspase-dependent apoptosis in a series of cell culture models. In rat 423-cells, caspase-8 and caspase-3 were apparently sufficient to initiate and proceed apoptosis without involving the intrinsic amplification loop via caspase-9. To assess the reasons for this inactivity of an otherwise crucial initiator caspase, we examined the ability for apoptosome assembly in 423-cells. Caspase-9 and Apaf-1 were expressed and cytochrome c released from mitochondria upon serum withdrawal. Although functional apoptosomes were assembled successfully in vitro, caspase-9 processing was found essentially refrained during apoptosis in 423-cells. Cell fractionation experiments revealed that sequestration of caspase-9 to cytoskeletal structures in 423-cells contributed to the observed impairment of apoptosome formation. Altogether, these findings provide evidence that serum starvation-induced apoptosis may occur independently of the intrinsic pathway and that caspase-9 sequestration potentially represents a novel biological antiapoptotic strategy.     

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.     

Apoptosome: a platform for the activation of initiator caspases

Apoptosome refers to the adaptor protein complex that mediates the activation of an initiator caspase at the onset of apoptosis. In mammalian cells, caspase-9, caspase-8, and caspase-2 rely on the apoptotic protease-activating factor 1 (Apaf-1)-apoptosome, death-inducing signaling complex (DISC), and PIDDosome, respectively, for activation. In Drosophila, activation of the caspase-9 homolog Dronc requires assembly of an apoptosome comprised of Dark/Hac-1/Dapaf-1. In Caenorhabditis elegans, activation of the caspase CED-3 is facilitated by the CED-4-apoptosome. Recent biochemical and structural investigation revealed significant insights into the assembly and function of the various apoptosomes. Nonetheless, conclusive mechanisms by which the initiator caspases are activated by the apoptosomes remain elusive. Several models have been proposed to explain the activation process. The induced proximity model summarizes the general process of initiator caspase activation. The proximity-driven dimerization model describes how initiator caspases respond to induced proximity and offers an explanation for their activation. Regardless of how initiator caspases are activated, enhanced activity must be correlated with altered active site conformation. The induced conformation model posits that the activated conformation for the active site of a given initiator caspase is attained through direct interaction with the apoptosome or through homo-oligomerization facilitated by the apoptosome.     

XIAP inhibition of caspase-3 preserves its association with the Apaf-1 apoptosome and prevents CD95- and Bax-induced apoptosis

Ligation of death receptors or formation of the Apaf-1 apoptosome results in the activation of caspases and execution of apoptosis. We recently demonstrated that X-linked inhibitor-of-apoptosis protein (XIAP) associates with the apoptosome in vitro. By utilizing XIAP mutants, we now report that XIAP binds to the 'native' apoptosome complex via a specific interaction with the small p12 subunit of processed caspase-9. Indeed, we provide the first direct evidence that XIAP can simultaneously bind active caspases-9 and -3 within the same complex and that inhibition of caspase-3 by the Linker-BIR2 domain prevents disruption of BIR3-caspase-9 interactions. Recent studies suggest that inhibition of caspase-3 is dispensable for its anti-apoptotic effects. However, we clearly demonstrate that inhibition of caspase-3 is required to inhibit CD95 (Fas/Apo-1)-mediated apoptosis, whereas inhibition of either caspase-9 or caspase-3 prevents Bax-induced cell death. Finally, we illustrate for the first time that XIAP mutants, which are incapable of binding to caspases-9 and -3 are completely devoid of anti-apoptotic activity. Thus, XIAP's capacity to maintain inhibition of caspase-9 within the Apaf-1 apoptosome is influenced by its ability to simultaneously inhibit active caspase-3, and depending upon the apoptotic stimulus, inhibition of caspase-9 or 3 is essential for XIAP's anti-apoptotic activity.     

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.     

Analysis of the composition, assembly kinetics and activity of native Apaf-1 apoptosomes

The Apaf-1 apoptosome is a multi-subunit caspase-activating scaffold that is assembled in response to diverse forms of cellular stress that culminate in apoptosis. To date, most studies on apoptosome composition and function have used apoptosomes reassembled from recombinant or purified proteins. Thus, the precise composition of native apoptosomes remains unresolved. Here, we have used a one-step immunopurification approach to isolate catalytically active Apaf-1/caspase-9 apoptosomes, and have identified the major constituents of these complexes using mass spectrometry methods. Using this approach, we have also assessed the ability of putative apoptosome regulatory proteins, such as Smac/DIABLO and PHAPI, to regulate the activity of native apoptosomes. We show that Apaf-1, caspase-9, caspase-3 and XIAP are the major constituents of native apoptosomes and that cytochrome c is not stably associated with the active complex. We also demonstrate that the IAP-neutralizing protein Smac/DIABLO and the tumor-suppressor protein PHAPI can enhance the catalytic activity of apoptosome complexes in distinct ways. Surprisingly, PHAPI also enhanced the activity of purified caspase-3, suggesting that it may act as a co-factor for this protease.