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.     

Cytochrome c depletion upon expression of Bcl-XS

We have shown previously that Bcl-XS causes acute cell death in 3T3 cells without activating caspases (Fridman, J. S., Benedict, M. A., and Maybaum, J. (1999) Cancer Res. 59, 5999-6004). In this study, we determined that the explanation for lack of caspase activation is the cellular depletion of cytochrome c. Electron microscopy revealed gross structural changes in the mitochondria of Bcl-XS-expressing cells; however, cytochrome c was not detected in cytosolic fractions from these cells. Surprisingly, it was determined that cellular cytochrome c levels decreased as Bcl-XS expression levels increased. Experiments performed to eliminate other possible explanations for the lack of caspase activation showed that these 3T3 cells have a functional cytoplasmic apoptosome, a complex of proteins that form a functional trigger capable of activating the proximal caspase in an apoptotic pathway Chinnaiyan, A. M. (1999) Neoplasia 1, 5-15, as cytosolic extracts from these cells were capable of cleaving pro-caspase-9. These cells were also able to release cytochrome c from their mitochondria after appropriate stimulation, other than Bcl-XS expression (i.e. withdrawal from serum for 24 h), and initiate a cell death that is inhibited by a dominant negative caspase-9. We conclude that lack of caspase activation is due to a Bcl-XS-induced depletion of active cytochrome c, a phenomenon that represents an alternative cell death effector pathway and/or a novel mechanism for regulating caspase activation.     

Defective molecular timer in the absence of nucleotides leads to inefficient caspase activation

In the intrinsic death pathway, cytochrome C (CC) released from mitochondria to the cytosol triggers Apaf-1 apoptosome formation and subsequent caspase activation. This process can be recapitulated using recombinant Apaf-1 and CC in the presence of nucleotides ATP or dATP [(d)ATP] or using fresh cytosol and CC without the need of exogenous nucleotides. Surprisingly, we found that stored cytosols failed to support CC-initiated caspase activation. Storage of cytosols at different temperatures led to the loss of all (deoxy)nucleotides including (d)ATP. Addition of (d)ATP to such stored cytosols partially restored CC-initiated caspase activation. Nevertheless, CC could not induce complete caspase-9/3 activation in stored cytosols, even with the addition of (d)ATP, despite robust Apaf-1 oligomerization. The Apaf-1 apoptosome, which functions as a proteolytic-based molecular timer appeared to be defective as auto-processing of recruited procaspase-9 was inhibited. Far Western analysis revealed that procaspase-9 directly interacted with Apaf-1 and this interaction was reduced in the presence of physiological levels of ATP. Co-incubation of recombinant Apaf-1 and procaspase-9 prior to CC and ATP addition inhibited CC-induced caspase activity. These findings suggest that in the absence of nucleotide such as ATP, direct association of procaspase-9 with Apaf-1 leads to defective molecular timer, and thus, inhibits apoptosome-mediated caspase activation. Altogether, our results provide novel insight on nucleotide regulation of apoptosome.     

Endoplasmic reticulum stress induces apoptosis by an apoptosome-dependent but caspase 12-independent mechanism

The endoplasmic reticulum (ER) is the cellular site of polypeptide folding and modification. When these processes are hampered, an unfolded protein response (UPR) is activated. If the damage is too broad, the mammalian UPR launches the apoptotic program. As a consequence, mobilization of ER calcium stores sensitizes mitochondria to direct proapoptotic stimuli. We make use of a mouse Apaf1-deficient cell system of proneural origin to understand the roles played in this context by the apoptosome, the most studied apoptotic machinery along the mitochondrial pathway of death. We show here that in the absence of the apoptosome ER stress induces cytochrome c release from the mitochondria but that apoptosis cannot occur. Under these circumstances, Grp78/BiP and GADD153/CHOP, both hallmarks of UPR, are canonically up-regulated, and calcium is properly released from ER stores. We also demonstrate that caspase 12, a protease until now believed to play a central role in the initiation of ER stress-induced cell death in the mouse system, is dispensable for the mitochondrial pathway of death to take place.     

Apoptosome inactivation rescues proneural and neural cells from neurodegeneration

Deficiency of the apoptosome component Apaf1 leads to accumulation of supernumerary brain cells in mouse embryos. We observed that neural precursor cells (NPCs) in Apaf1(-/-) embryos escape programmed cell death, proliferate and retain their potential to differentiate. To evaluate the circumstances of Apaf1(-/-) NPC survival and investigate their fate under neurodegenerative conditions, we established cell lines of embryonic origin (ETNA). We found that Apaf1(-/-) NPCs resist common apoptotic stimuli and neurodegenerative inducers such as amyloid-beta peptide (typical of Alzheimer's disease) and mutant G93A superoxide dismutase 1 (typical of familial amyotrophic lateral sclerosis). Similar results were obtained in Apaf1(-/-) primary cells. When death is prevented by Apaf1 deficiency, cytochrome c is released from mitochondria and rapidly degraded by the proteasome, but mitochondria remain intact. Under these conditions, neither activation by cleavage of initiator caspases nor release of alternative apoptotic inducers from mitochondria takes place. In addition, NPCs can still differentiate, as revealed by neurite outgrowth and expression of differentiation markers. Our findings imply that the mitochondrion/apoptosome pathway is the main route of proneural and neural cells to death and that its inhibition prevents them from dismantling in neurodegenerative conditions. Indeed, the ETNA cell model is ideally suited for exploring the potential of novel cell therapies for the treatment of human neurodegenerations.     

The two Drosophila cytochrome C proteins can function in both respiration and caspase activation

Cytochrome C has two apparently separable cellular functions: respiration and caspase activation during apoptosis. While a role of the mitochondria and cytochrome C in the assembly of the apoptosome and caspase activation has been established for mammalian cells, the existence of a comparable function for cytochrome C in invertebrates remains controversial. Drosophila possesses two cytochrome c genes, cyt-c-d and cyt-c-p. We show that only cyt-c-d is required for caspase activation in an apoptosis-like process during spermatid differentiation, whereas cyt-c-p is required for respiration in the soma. However, both cytochrome C proteins can function interchangeably in respiration and caspase activation, and the difference in their genetic requirements can be attributed to differential expression in the soma and testes. Furthermore, orthologues of the apoptosome components, Ark (Apaf-1) and Dronc (caspase-9), are also required for the proper removal of bulk cytoplasm during spermatogenesis. Finally, several mutants that block caspase activation during spermatogenesis were isolated in a genetic screen, including mutants with defects in spermatid mitochondrial organization. These observations establish a role for the mitochondria in caspase activation during spermatogenesis.     

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.     

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。     

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.     

Cytochrome c is dispensable for fas-induced caspase activation and apoptosis.

Cytochrome c is thought to play an important role in the initiation of apoptosis following its release from mitochondria. It is controversial whether such release is also involved in caspase activation and apoptotic cell death after ligation of the cell surface molecule Fas. We addressed this issue by investigating cells from the human cell lines Jurkat and SKW6 which had been treated with the inhibitor of the mitochondrial F0/F1-ATPase, oligomycin. Oligomycin-treatment led, over a wide range of concentrations, to ATP-depletion and, at similar concentrations, abrogated the appearance of caspase-3-like activity caused by stauroporine. Electroporation of cytochrome c protein into intact cells induced caspase activation in both cell lines and significant nuclear apoptosis in Jurkat cells. In ATP-depleted cells, electroporation of cytochrome c induced neither caspase activation nor nuclear fragmentation. Fas-induced caspase activation and nuclear apoptosis, however, were unaffected by the depletion of ATP. Thus, cytochrome c is unlikely to be an important factor in Fas-induced cell death.     

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(+).     

The apoptosome activates caspase-9 by dimerization

The apical protease of the human intrinsic apoptotic pathway, caspase-9, is activated in a polymeric activation platform known as the apoptosome. The mechanism has been debated, and two contrasting hypotheses have been suggested. One of these postulates an allosteric activation of monomeric caspase-9; the other postulates a dimer-driven assembly at the surface of the apoptosome--the "induced proximity" model. We show that both Hofmeister salts and a reconstituted mini-apoptosome activate caspase-9 by a second-order process, compatible with a conserved dimer-driven process. Significantly, replacement of the recruitment domain of the apical caspase of the extrinsic apoptotic pathway, caspase-8, by that of caspase-9 allows activation of this hybrid caspase by the apoptosome. Consequently, apical caspases can be activated simply by directing their zymogens to the apoptosome, ruling out the requirement for allosteric activation and supporting an induced proximity dimerization model for apical caspase activation in vivo.     

Big wheel keeps on turning: apoptosome regulation and its role in chemoresistance

Apoptosis, a form of programmed cell death, enables organisms to maintain tissue homeostasis through deletion of extraneous cells and also serves as a means to eliminate potentially harmful cells. Numerous stress signals have been shown to engage the intrinsic pathway of apoptosis, with the release from mitochondria of proapoptotic factors such as cytochrome c and the subsequent formation of a cytosolic complex between apoptotic protease-activating factor-1 (Apaf-1) and procaspase-9, known as the apoptosome. Recent studies have led to the identification of an array of factors that control the formation and activation of the apoptosome under physiological conditions. Moreover, deregulation of apoptosome function has been documented in various forms of human cancer, and may play a role in both carcinogenesis and chemoresistance. We discuss how the apoptosome is regulated in normal and disease states, and how targeting of apoptosome-dependent, post-mitochondrial stages of apoptosis may serve as a rational approach to cancer treatment.     

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.     

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.     

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.     

Apaf-1-deficient fog mouse cell apoptosis involves hypo-polarization of the mitochondrial inner membrane, ATP depletion and citrate accumulation

To explore how the intrinsic apoptosis pathway is controlled in the spontaneous fog (forebrain overgrowth) mutant mice with an Apaf1 splicing deficiency, we examined spleen and bone marrow cells from Apaf1(+/+) (+/+) and Apaf1(fog/fog) (fog/fog) mice for initiator caspase-9 activation by cellular stresses. When the mitochondrial inner membrane potential (Deltapsim) was disrupted by staurosporine, +/+ cells but not fog/fog cells activated caspase-9 to cause apoptosis, indicating the lack of apoptosome (apoptosis protease activating factor 1 (Apaf-1)/cytochrome c/(d)ATP/procaspase-9) function in fog/fog cells. However, when a marginal ( approximately 20%) decrease in Deltapsim was caused by hydrogen peroxide (0.1 mM), peroxynitritedonor 3-morpholinosydnonimine (0.1 mM) and UV-C irradiation (20 J/m(2)), both +/+ and fog/fog cells triggered procaspase-9 auto-processing and its downstream cascade activation. Supporting our previous results, procaspase-9 pre-existing in the mitochondria induced its auto-processing before the cytosolic caspase activation regardless of the genotypes. Cellular ATP concentration significantly decreased under the hypoactive Deltapsim condition. Furthermore, we detected accumulation of citrate, a kosmotrope known to facilitate procaspase-9 dimerization, probably due to a feedback control of the Krebs cycle by the electron transfer system. Thus, mitochondrial in situ caspase-9 activation may be caused by the major metabolic reactions in response to physiological stresses, which may represent a mode of Apaf-1-independent apoptosis hypothesized from recent genetic studies.     

Delay in Apoptosome Formation Attenuates Apoptosis in Mouse Embryonic Stem Cell Differentiation

Differentiation is an inseparable process of development in multicellular organisms. Mouse embryonic stem cells (mESCs) represent a valuable research tool to conduct in vitro studies of cell differentiation. Apoptosis as a well known cell death mechanism shows some common features with cell differentiation, which has caused a number of ambiguities in the field. The research question here is how cells could differentiate these two processes from each other. We have investigated the role of the mitochondrial apoptotic pathway and cell energy level during differentiation of mESCs into the cardiomyocytes and their apoptosis. p53 expression, cytochrome c release, apoptosome formation, and caspase-3/7 activation are observed upon induction of both apoptosis and differentiation. However, remarkable differences are detected in time of cytochrome c appearance, apoptosome formation, and caspase activity upon induction of both processes. In apoptosis, apoptosome formation and caspase activity were observed rapidly following the cytochrome c release. Unlike apoptosis, the release of cytochrome c upon differentiation took more time, and the maximum caspase activity was also postponed for 24 h. This delay suggests that there is a regulatory mechanism during differentiation of mESCs into cardiomyocytes. The highest ATP content of cells was observed immediately after cytochrome c release 6 h after apoptosis induction and then decreased, but it was gradually increased up to 48 h after differentiation. These observations suggest that a delay in the release of cytochrome c or delay in ATP increase attenuate apoptosome formation, and caspase activation thereby discriminates apoptosis from differentiation in mESCs.