A novel enhancer of the Apaf1 apoptosome involved in cytochrome c-dependent caspase activation and apoptosis

Apaf1/CED4 family members play central roles in apoptosis regulation as activators of caspase family cell death proteases. These proteins contain a nucleotide-binding (NB) self-oligomerization domain and a caspase recruitment domain (CARD). A novel human protein was identified, NAC, that contains an NB domain and CARD. The CARD of NAC interacts selectively with the CARD domain of Apaf1, a caspase-activating protein that couples mitochondria-released cytochrome c (cyt-c) to activation of cytosolic caspases. Cyt-c-mediated activation of caspases in cytosolic extracts and in cells is enhanced by overexpressing NAC and inhibited by reducing NAC using antisense/DNAzymes. Furthermore, association of NAC with Apaf1 is cyt c-inducible, resulting in a mega-complex (>1 MDa) containing both NAC and Apaf1 and correlating with enhanced recruitment and proteolytic processing of pro-caspase-9. NAC also collaborates with Apaf1 in inducing caspase activation and apoptosis in intact cells, whereas fragments of NAC representing only the CARD or NB domain suppress Apaf1-dependent apoptosis induction. NAC expression in vivo is associated with terminal differentiation of short lived cells in epithelia and some other tissues. The ability of NAC to enhance Apaf1-apoptosome function reveals a novel paradigm for apoptosis regulation.     

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.     

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.     

Intracellular K(+) inhibits apoptosis by suppressing the Apaf-1 apoptosome formation and subsequent downstream pathways but not cytochrome c release

Cellular ionic homeostasis, fundamentally K(+) homeostasis, has been implicated as a critical regulator of apoptosis. The intracellular K(+) efflux on apoptotic insult and suppression of apoptosis by high concentration of extracellular K(+) or after inhibition of this efflux by K(+) channel blockers have established the crucial role of K(+) in turning on the apoptotic machinery. Several contrasting observations have reported the antiapoptotic effect of intracellular K(+) concentration to be the result of inhibition of cytochrome c release from mitochondria, but the exact inhibitory mechanism remains obscure. However, here we show the blockage of K(+) efflux during apoptosis did not affect cytochrome c release from the mitochondria, still completely inhibited the formation of the apoptosome comprising Apaf-1, cytochrome c, caspase-9 and other accessories. As a consequence of this event, procaspase-9, -3, -8 and other death-related proteins were not processed. Furthermore, physiological concentrations of K(+) also inhibited the processing of procaspase-3 by purified caspase-8 or -9, the nucleosomal DNA fragmentation by purified DFF40/CAD and the nuclear fragmentation to varying extents. Altogether, these findings suggest that the efflux of K(+) is prerequisite not only for the formation of the apoptosome but also for the downstream apoptotic signal-transduction pathways.     

HSP27 inhibits cytochrome c-dependent activation of procaspase-9.

We have previously shown that the small heat shock protein HSP27 inhibited apoptotic pathways triggered by a variety of stimuli in mammalian cells. The present study demonstrates that HSP27 overexpression decreases U937 human leukemic cell sensitivity to etoposide-induced cytotoxicity by preventing apoptosis. As observed for Bcl-2, HSP27 overexpression delays poly(ADP-ribose)polymerase cleavage and procaspase-3 activation. In contrast with Bcl-2, HSP27 overexpression does not prevent etoposide-induced cytochrome c release from the mitochondria. In a cell-free system, addition of cytochrome c and dATP to cytosolic extracts from untreated cells induces the proteolytic activation of procaspase-3 in both control and bcl-2-transfected U937 cells but fails to activate procaspase-3 in HSP27-overexpressing cells. Immunodepletion of HSP27 from cytosolic extracts increases cytochrome c/dATP-mediated activation of procaspase-3. Overexpression of HSP27 also prevents procaspase-9 activation. In the cell-free system, immunodepletion of HSP27 increases LEDH-AFC peptide cleavage activity triggered by cytochrome c/dATP treatment. We conclude that HSP27 inhibits etoposide-induced apoptosis by preventing cytochrome c and dATP-triggered activity of caspase-9, downstream of cytochrome c release.     

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.     

The mitochondrial apoptosome: a killer unleashed by the cytochrome seas.

The caspase family of cysteine proteases have emerged as central regulators of apoptosis. Diverse cellular stresses trigger caspase activation by promoting release of mitochondrial components, including cytochrome c, into the cytoplasm. In turn, cytochrome c promotes the assembly of a caspase-activating complex termed the apoptosome. In this article, the apoptosome and its role in life and death decisions of cells are discussed.     

Physiological concentrations of bile salts inhibit recovery of ischemic-injured porcine ileum

We have previously shown rapid in vitro recovery of barrier function in porcine ischemic-injured ileal mucosa, attributable principally to reductions in paracellular permeability. However, these experiments did not take into account the effects of luminal contents, such as bile salts. Therefore, the objective of this study was to evaluate the role of physiological concentrations of deoxycholic acid in recovery of mucosal barrier function. Porcine ileum was subjected to 45 min of ischemia, after which mucosa was mounted in Ussing chambers and exposed to varying concentrations of deoxycholic acid. The ischemic episode resulted in significant reductions in transepithelial electrical resistance (TER), which recovered to control levels of TER within 120 min, associated with significant reductions in mucosal-to-serosal (3)H-labeled mannitol flux. However, treatment of ischemic-injured tissues with 10(-5) M deoxycholic acid significantly inhibited recovery of TER with significant increases in mucosal-to-serosal (3)H-labeled mannitol flux, whereas 10(-6) M deoxycholic acid had no effect. Histological evaluation at 120 min revealed complete restitution regardless of treatment, indicating that the breakdown in barrier function was due to changes in paracellular permeability. Similar effects were noted with the application of 10(-5) M taurodeoxycholic acid, and the effects of deoxycholic acid were reversed with application of the Ca(2+)-mobilizing agent thapsigargin. Deoxycholic acid at physiological concentrations significantly impairs recovery of epithelial barrier function by an effect on paracellular pathways, and these effects appear to be Ca(2+) dependent.     

Physiological concentrations of melatonin inhibit nitric oxide synthase in rat cerebellum

In the present paper we show the inhibitory effect of melatonin on rat cerebellar nitric oxide synthase (NOS) activity. NO production was monitored by the stoichiometric conversion of L-arginine to L-citrulline. The inhibitory effect of melatonin was dose-dependent, with an IC₅₀ value of about 0.1 mM. However, a significant inhibition of enzyme activity (> 22%) was observed at 1 nM melatonin which is in the range of the physiological serum concentration of the hormone at night. The inhibitory effect of melatonin was observed exclusively in the presence of Ca⁺⁺. Results suggest a new and important role of the pineal hormone melatonin on central nervous system processes, i.e., by modulating NO production.     

Physiological and pathological roles of Apaf1 and the apoptosome

Different cellular pathways can lead to apoptosis. Apaf1 is the molecular core of the apoptosome, a multiproteic complex mediating the so-called mitochondrial pathway of cell death. The importance of this pathway during development has been clearly demonstrated by knocking out key genes. Also, the relevance of Apaf1 dosage during development has been recently underlined. Moreover, a growing body of evidences seems to point out a possible role of the mitochondria-dependent apoptosis in different pathologies. In particular, we discuss here some recent evidences regarding the putative role of the apoptosome in neurodegeneration and cancer.     

Relationship between intracellular K+ concentrations and K+ fluxes in growing and contact-inhibited cells.

The K+ content and the K+ flux were measured in the cell lines ME2 and MF2 isolated from plasmocytoma MOPC 173. Both cell lines were shown to have the seem K+ content and the same K+ steady state flux per unit of surface area. In ME2 cells, no modification of the exchange movement was observed during contact inhibition. However, contact-inhibited cells exhibited an increased resistance to depletion, characterized by a lower K+ net movement. The (Na+ plus K+)-ATPase measured in homogenates is poorly correlated to in vivo cation fluxes both because of the enhancement due, presumably, to the drop of K+ concentration on the cytoplasmic face of the membrane and because of losses during preparation which can be conspicuous, especially in contact-inhibited cells. The K+ net flux is considerable increased when the intracellular K+ level is reduced after preincubation of the cells in a K+ -free medium. Thus, internal K+ seems to regulate the K+ influx.     

The Physiological Relevance of Na+-Coupled K+-Transport

Plant roots utilize at least two distinct pathways with high and low affinities to accumulate K+. The system for high-affinity K+ uptake, which takes place against the electrochemical K+ gradient, requires direct energization. Energization of K+ uptake via Na+ coupling has been observed in algae and was recently proposed as a mechanism for K+ uptake in wheat (Triticum aestivum L.). To investigate whether Na+ coupling has general physiological relevance in energizing K+ transport, we screened a number of species, including Arabidopsis thaliana L. Heynh. ecotype Columbia, wheat, and barley (Hordeum vulgare L.), for the presence of Na+-coupled K+ uptake. Rb+-flux analysis and electrophysiological K+-transport assays were performed in the presence and absence of Na+ and provided evidence for a coupling between K+ and Na+ transport in several aquatic species. However, all investigated terrestrial species were able to sustain growth and K+ uptake in the absence of Na+. Furthermore, the addition of Na+ was either without effect or inhibited K+ absorption. The latter characteristic was independent of growth conditions with respect to Na+ status and pH. Our results suggest that in terrestrial species Na+-coupled K+ transport has no or limited physiological relevance, whereas in certain aquatic angiosperms and algae this type of secondary transport energization plays a significant role.     

Superoxide dismutase does not inhibit the oxidation of cytochrome c and cytochrome oxidase.

An electrometric system was used to measure Ca⁺⁺ uptake by sarcoplasmic reticulum vesicles (SR). The method permits continuous recording of Ca⁺⁺ uptake and thus the valuation of kinetic parameters. Furthermore, the ultrasensitivity of the method permits to follow changes in Ca⁺⁺ concentration below 10^?6 M.     

Aging augments interstitial K+ concentrations in active muscle of rats.

Skeletal muscle performance declines with advancing age, and the underlying mechanism is not completely understood. A large body of convincing evidence has demonstrated a crucial role for interstitial K+ concentration ([K+]o) in modulating contractile function of skeletal muscle. The present study tested the hypothesis that during muscle contraction there is a greater accumulation of [K+]o in aged compared with adult skeletal muscle. Twitch muscle contraction was induced by electrical stimulation of the sciatic nerves of 8- and 32-mo-old Fischer 344 x Brown Norway rats. Levels of [K+]o were measured continuously by a microdialysis technique with the probes inserted into the gastrocnemius muscle. Stimulation at 1, 3, and 5 Hz elevated muscle [K+]o by 52, 64, and 88% in adult rats, and by 78, 98, and 104% in aged rats, respectively, and the increase was significantly higher in aged than in adult rats. Recovery for [K+]o, as measured by the time for [K+]o to recover by 20 and 50% from peak response after stimulation, was slower in aged rats. Ouabain (5 mM), a specific inhibitor of the Na+-K+ pump, was added in the perfusate to inhibit the reuptake of K+ into the cells to assess the role of the pump in the overall K+ balance. Ouabain elevated muscle [K+]o at rest, and the effect was significantly attenuated in aged animals. The present data demonstrated an augmented [K+]o in aged skeletal muscle compared with adult skeletal muscle, and the data suggested that an alteration in the function of the Na+-K+ pump may contribute, in part, to the deficiency in K+ balance in skeletal muscle of aged rats.     

Physiological and pathophysiological roles of ATP-sensitive K+ channels

ATP-sensitive potassium (K(ATP)) channels are present in many tissues, including pancreatic islet cells, heart, skeletal muscle, vascular smooth muscle, and brain, in which they couple the cell metabolic state to its membrane potential, playing a crucial role in various cellular functions. The K(ATP) channel is a hetero-octamer comprising two subunits: the pore-forming subunit Kir6.x (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea receptor SUR (SUR1 or SUR2). Kir6.x belongs to the inward rectifier K(+) channel family; SUR belongs to the ATP-binding cassette protein superfamily. Heterologous expression of differing combinations of Kir6.1 or Kir6.2 and SUR1 or SUR2 variant (SUR2A or SUR2B) reconstitute different types of K(ATP) channels with distinct electrophysiological properties and nucleotide and pharmacological sensitivities corresponding to the various K(ATP) channels in native tissues. The physiological and pathophysiological roles of K(ATP) channels have been studied primarily using K(ATP) channel blockers and K(+) channel openers, but there is no direct evidence on the role of the K(ATP) channels in many important cellular responses. In addition to the analyses of naturally occurring mutations of the genes in humans, determination of the phenotypes of mice generated by genetic manipulation has been successful in clarifying the function of various gene products. Recently, various genetically engineered mice, including mice lacking K(ATP) channels (knockout mice) and mice expressing various mutant K(ATP) channels (transgenic mice), have been generated. In this review, we focus on the physiological and pathophysiological roles of K(ATP) channels learned from genetic manipulation of mice and naturally occurring mutations in humans.     

The regulatory properties of yeast pyruvate kinase. Effects of NH4+ and K+ concentrations.

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied at 25 degrees C and pH 6.2 as a function of the concentrations of ADP, phosphoenolpyruvate, Mg2+ and either NH4+ or K+. The data were analysed by the exponential model for four substrates, obtained by extension of the model described by Ainsworth, Kinderlerer & Gregory [(1983) Biochem. J. 209, 401-411]. On that basis, it was concluded that NH4+ binding is almost non-interactive but leads to the appearance of positive interaction in the velocity response to increase in its concentration because of positive interactions with phosphoenolpyruvate and Mg2+. The data obtained with K+ lead to the same conclusions and differ only in suggesting that NH4+ is bound more strongly to the enzyme than is K+. Both data sets are used as the basis for a discussion of the substrate interactions of pyruvate kinase and it appears therefrom that the heterotropic interactions accord with what is known of the events that take place at the active site during catalysis. The paper also reports a determination of the dissociation constants for the NH4+ complexes with ADP and phosphoenolpyruvate and an examination of the simultaneous activation of pyruvate kinase by K+ and NH4+ ions.     

Sodium and buffer cations inhibit dephosphorylation of (Na+ + K+)-ATPase

Effects of various cations on the dephosphorylation of (Na+ + K+)-ATPase, phosphorylated by ATP in 50 mM imidazole buffer (pH 7.0) at 22 degrees C without added Na+, have been studied. The dephosphorylation in imidazole buffer without added K+ is extremely sensitive to K+-activation (Km K+ = 1 microM), less sensitive to Mg2+-activation (Km Mg2+ = 0.1 mM) and Na+-activation (Km Na+ = 63 mM). Imidazole and Na+ effectively inhibit K+-activated dephosphorylation in linear competitive fashion (Ki imidazole 7.5 mM, Ki Na+ 4.6 mM). The Ki for Na+ is independent of the imidazole concentration, indicating different and non-interacting inhibitory sites for Na+ and imidazole. Imidazole inhibits Mg2+-activated dephosphorylation just as effective as K+-activated dephosphorylation, as judged from the Ki values for imidazole in the two processes. Tris buffer and choline chloride, like imidazole, inhibit dephosphorylation in the presence of residual K+ (less than 1 microM), but less effectively in terms of I50 values and extent of inhibition. Tris inhibits to the same extent as choline. This indicates different inhibitory sites for Tris or choline and for imidazole. These findings indicate that high steady-state phosphorylation levels in Na+-free imidazole buffer are due to the induction of a phosphorylating enzyme conformation and to the inhibition of (K+ + Mg2+)-stimulated dephosphorylation.     

Mathematical modeling of the formation of apoptosome in intrinsic pathway of apoptosis

Caspase-9 is the protease that mediates the intrinsic pathway of apoptosis, a type of cell death. Activation of caspase-9 is a multi-step process that requires dATP or ATP and involves at least two proteins, cytochrome c and Apaf-1. In this study, we mathematically model caspase-9 activation by using a system of ordinary differential equations (an ODE model) generated by a systems biology tool Simpathica—a simulation and reasoning system, developed to study biological pathways. A rudimentary version of “model checking” based on comparing simulation data with that obtained from a recombinant system of caspase-9 activation, provided several new insights into regulation of this protease. The model predicts that the activation begins with binding of dATP to Apaf-1, which initiates the interaction between Apaf-1 and cytochrome c, thus forming a complex that oligomerizes into an active caspase-9 holoenzyme via a linear binding model with cooperative interaction rather than through network formation.