No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Simultaneous release of adenylate kinase and cytochrome c in cell death
... As control, ATP was repleted in the presence of the inhibitors by supplying the cells with glucose. Under these conditions, a variety of substances known to induce apoptosis in cells with normal ATP levels caused swelling and necrotic lysis in ATP-depleted cells within a time frame in which energy failure per se did not have lethal effects [1,3,19,29]. In this model, we examined the effects of Bcl-2 on cell death, loss of mitochondrial membrane potential, and cytochrome c release. ...
... To determine cytochrome c release from mitochondria, cytosolic fractions were isolated after selective lysis of the plasma membrane with digitonin as described previously [3,29]. Briefly, 4 ϫ 10 6 cells were resuspended in 250 l phosphate-buffered saline and lysed by adding 250 l of a digitonin (100 g/ml)/sucrose (500 mM) solution under vigorous vortexing, and further incubation for 30 s. Cytosolic fractions were quickly isolated and removed from organelles and cell debris by centrifugation at 14,000g for 60 s at 4°C. ...
... Once the mitochondria break, not only the proper mitochondrial function is lost, but also death-promoting proteins, e.g., cytochrome c, apoptosis-inducing factor, or caspases, are released [9,37,38]. Since the release of mitochondrial proteins is observed in both apoptosis and necrosis [3,29,39,40], the shape of death may be decided by downstream events such as caspase activation, while the extent of cell death may be controlled by steps upstream or within mitochondria [36 and references therein]. Bcl-2 block of death pathways upstream of the point where apoptosis and necrosis signalling pathways diverge would therefore be most effective. ...
The intracellular ATP concentration decides on the onset of either apoptosis or necrosis in Jurkat cells exposed to death stimuli. Bcl-2 can block apoptotic demise, which occurs preferably under conditions of high cellular ATP levels. Here, we investigated the effects of Bcl-2 on the necrotic type of cell demise that prevails under conditions of energy loss. ATP levels were modulated by using mitochondrial inhibitors, such as rotenone or S-nitrosoglutathione, in medium either lacking glucose or supplemented with glucose to stimulate glycolytic ATP generation. Under conditions of ATP depletion, staurosporine (STS) induced >90% necrosis in vector control-transfected cells, whereas bcl-2-transfected cells were protected. Thus, the antiapoptotic protein Bcl-2 can reduce the overall amount of cell death in ATP-depleted cells regardless whether it occurs by apoptosis or necrosis. Cytochrome c release, normally preceding STS-induced necrosis, was also inhibited by Bcl-2. However, Bcl-2 did not prevent an initial STS-induced drop of the mitochondrial membrane potential (ΔΨm). Therefore, the mechanisms whereby Bcl-2 prevents cell death and favors retention of cytochrome c in the mitochondria require neither the maintenance of mitochondrial ΔΨ nor the maintenance of normal ATP levels.
... Release of Mitochondrial Proteins. Cytosolic fractions from Jurkat cells were isolated by a quick cell lysis method with digitonin (33). While the mixture was being vortexed, lysis buffer [9.4 µg of digitonin/10 6 cells and 500 mM sucrose in PBS (2 mM NaH2PO4, 16 mM Na2HPO4, and 150 mM NaCl)] was quickly added to the cell suspension (4 × 10 6 cells in PBS). ...
... Cytochrome c Staining. The cytochrome c distribution was examined by immunocytochemistry (33). Briefly, Jurkat cells were prelabeled for 60 min with the mitochondria-specific aldehyde-fixable dye Mitotracker Red (20 nM, Molecular Probes) before treatment with TBT. ...
... An early step in the apoptotic program is the release of proapoptotic proteins from the mitochondrial intermembrane space, such as AIF (38) or cytochrome c (39). This is accompanied by the release of other proteins primarily located within the same subcellular compartment, such as adenylate kinase (ADK) (33). We followed the loss of ADK as an easily quantifiable indicator for the loss of the barrier function of the outer mitochondrial membrane. ...
The toxicity of tributyltin chloride (TBT) involves Ca(2+) overload, cytoskeletal damage, and mitochondrial failure leading to cell death by apoptosis or necrosis. Here, we examined whether the intracellular ATP level modulates the mode of cell death after exposure to TBT. When Jurkat cells were energized by the mitochondrial substrate, pyruvate, low concentrations of TBT (1-2 microM) triggered an immediate depletion of intracellular ATP followed by necrotic death. When ATP levels were maintained by the addition of glucose, the mode of cell death was typically apoptotic. Glycolytic ATP production was required for apoptosis at two distinct steps. First, maintenance of adequate ATP levels accelerated the decrease of mitochondrial membrane potential, and the release of the intermembrane proteins adenylate kinase and cytochrome c from mitochondria. A possible role of the adenine nucleotide exchanger in this first ATP-dependent step is suggested by experiments performed with the specific inhibitor, bongkrekic acid. This substance delayed cytochrome c release in a manner similar to that caused by ATP depletion. Second, caspase activation following cytochrome c release was only observed in ATP-containing cells. Bcl-2 had only a minor effect on TBT-triggered caspase activation or cell death. We conclude that intracellular ATP concentrations control the mode of cell death in TBT-treated Jurkat cells at both the mitochondrial and caspase activation levels.
... AK2 catalyzes the reversible adenine nucleotide phosphoryl transfer in the reaction 2 ADP ↔ ATP + AMP in the IMS. Loss of AK2 leads to impaired mitochondrial function (Burkart et al, 2011;Six et al, 2015), hampers induction of the endoplasmic reticulum unfolded protein response (Burkart et al, 2011), and sensitizes cells to induction of apoptosis (Single et al, 1998;Kohler et al, 1999;Lee et al, 2007). Human patients with defects in AK2 suffer from an autosomal recessive form of severe combined immunodeficiency (SCID) named reticular dysgenesis (RD) (Lagresle-Peyrou et al, 2009;Pannicke et al, 2009). ...
... We demonstrated that absence of AK2 processing results in increased cellular levels of AK2 and in the accumulation of folded, active, cytosolic AK2 (Figs 5 and 6E). AK2 activity is linked to metabolic signaling (Dzeja et al, 2004(Dzeja et al, , 2011Dzeja & Terzic, 2009), to maintenance of OXPHOS efficiency (Burkart et al, 2011;Six et al, 2015), and to induction of the ER unfolded protein response (Burkart et al, 2011) as well as apoptosis (Single et al, 1998;Kohler et al, 1999;Lee et al, 2007). Control of DPP8/9 activity might thus serve in modulating AK2 function, e.g., during differentiation or mitochondrial biogenesis. ...
Plasticity of the proteome is critical to adapt to varying conditions. Control of mitochondrial protein import contributes to this plasticity. Here, we identified a pathway that regulates mitochondrial protein import by regulated N-terminal processing. We demonstrate that dipeptidyl peptidases 8/9 (DPP8/9) mediate the N-terminal processing of adenylate kinase 2 (AK2) en route to mitochondria. We show that AK2 is a substrate of the mitochondrial disulfide relay, thus lacking an N-terminal mitochondrial targeting sequence and undergoing comparatively slow import. DPP9-mediated processing of AK2 induces its rapid proteasomal degradation and prevents cytosolic accumulation of enzymatically active AK2. Besides AK2, we identify more than 100 mitochondrial proteins with putative DPP8/9 recognition sites and demonstrate that DPP8/9 influence the cellular levels of a number of these proteins. Collectively, we provide in this study a conceptual framework on how regulated cytosolic processing controls levels of mitochondrial proteins as well as their dual localization to mitochondria and other compartments.
... Outer membrane rupture will release any inter-membrane protein in addition to cytochrome c. Single et al. (531) exposed Jurkat cells to a variety of apoptotic stimuli and monitored the mitochondrial release of cytochrome c and adenylate kinase by selectively permeabilizing the plasma membrane with digitonin. The authors found that cycloheximide, campothecin, or actinomycin D, which each induce apoptosis in these cells, caused a parallel release of cytochrome c and adenylate kinase, as did staurosporine, which induces necrosis in these cells (531), consistent with a nonselective permeabilization or rupture of the outer mitochondrial membrane. ...
... Single et al. (531) exposed Jurkat cells to a variety of apoptotic stimuli and monitored the mitochondrial release of cytochrome c and adenylate kinase by selectively permeabilizing the plasma membrane with digitonin. The authors found that cycloheximide, campothecin, or actinomycin D, which each induce apoptosis in these cells, caused a parallel release of cytochrome c and adenylate kinase, as did staurosporine, which induces necrosis in these cells (531), consistent with a nonselective permeabilization or rupture of the outer mitochondrial membrane. ...
... Determination of adenylate kinase and cytochrome c release. Cytosolic fractions were obtained as described previously [32]: Jurkat cells (4 ϫ 10 6 ) in 250 l phosphate-buffered saline [2 mM NaH 2 PO 4 , 16 mM Na 2 HPO 4 , 150 mM NaCl (PBS)] were lysed by quickly adding 250 l of digitonin (9.4 g digitonin/10 6 cells) in 500 mM sucrose under vigorous vortexing. After 30 s at room temperature, organelles and cell debris were separated from the lysate by centrifugation at 14,000g for 60 s at 4°C. ...
... Cytochrome c content was determined in cytosolic fractions (derived from 3 ϫ 10 5 cells/lane) by SDS-PAGE followed by Western blot using a primary antibody against cytochrome c (1.5 g/ml, clone 7H8.2C12, Pharmingen, Hamburg, Germany) as described in detail previously [31,32]. Blots were developed with a polyclonal IgGhorseradish peroxidase (1:1000, Pharmingen) followed by enhanced chemiluminescence detection (ECL, Amersham). ...
Under pathological conditions, the mode of cell death, apoptosis or necrosis, is relevant for the subsequent fate of the tissue. Cell demise may be shaped by endogenous mediators such as nitric oxide (NO) which interfere with subroutines of the death program. Here we show that apoptosis of Jurkat cells elicited by either staurosporine (STS) or anti-CD95 antibodies in glucose-free medium is converted to necrosis by NO donors. In the presence of NO, release of mitochondrial cytochrome c was delayed and activation of execution caspases was prevented. Stimulated cells died nonetheless. The switch in the mode of cell death was due to NO-dependent failure of mitochondrial energy production. Restoration of intracellular ATP by glucose supplementation recovered the cells' ability to activate caspases and undergo apoptosis. In this system, the apoptosis/necrosis conversion promoted by NO was not mediated by cyclic guanosine monophosphate-dependent mechanisms, poly-(ADP-ribose)-polymerase (PARP) activation, or inhibition of caspases due to S-nitrosylation and glutathione depletion. In contrast, depleting intracellular ATP with rotenone, an inhibitor of mitochondrial complex I mimicked the effect of NO. The findings presented here suggest that NO can decide the shape of cell death by lowering intracellular ATP below the level required to allow the coordinated execution of apoptosis.
... Outer membrane rupture will release any inter-membrane protein in addition to cytochrome c. Single et al. (531) exposed Jurkat cells to a variety of apoptotic stimuli and monitored the mitochondrial release of cytochrome c and adenylate kinase by selectively permeabilizing the plasma membrane with digitonin. The authors found that cycloheximide, campothecin, or actinomycin D, which each induce apoptosis in these cells, caused a parallel release of cytochrome c and adenylate kinase, as did staurosporine, which induces necrosis in these cells (531), consistent with a nonselective permeabilization or rupture of the outer mitochondrial membrane. ...
... Single et al. (531) exposed Jurkat cells to a variety of apoptotic stimuli and monitored the mitochondrial release of cytochrome c and adenylate kinase by selectively permeabilizing the plasma membrane with digitonin. The authors found that cycloheximide, campothecin, or actinomycin D, which each induce apoptosis in these cells, caused a parallel release of cytochrome c and adenylate kinase, as did staurosporine, which induces necrosis in these cells (531), consistent with a nonselective permeabilization or rupture of the outer mitochondrial membrane. ...
Mitochondria play a central role in the survival and death of neurons. The detailed bioenergetic mechanisms by which isolated mitochondria generate ATP, sequester Ca(2+), generate reactive oxygen species, and undergo Ca(2+)-dependent permeabilization of their inner membrane are currently being applied to the function of mitochondria in situ within neurons under physiological and pathophysiological conditions. Here we review the functional bioenergetics of isolated mitochondria, with emphasis on the chemiosmotic proton circuit and the application (and occasional misapplication) of these principles to intact neurons. Mitochondria play an integral role in both necrotic and apoptotic neuronal cell death, and the bioenergetic principles underlying current studies are reviewed.
... Antigen-antibody complexes were detected with Western Lightning Chemoluminescent Reagent (PerkinElmer, Boston, MA). To detect translocation of cytochrome c from the mitochondria to cytosol, cells were fractionated by a rapid digitonin lysis procedure (Single et al., 1998). Cells were incubated on ice for 5 min in digitonin lysis buffer (DLB; 75 mM NaCl, 1 mM NaH 2 PO 4 , 8 mM Na 2 HPO 4 , 250 mM sucrose) containing 20 ug digitonin per 1×10 6 cells. ...
... Hepa-V3 and Hepa-CR17 cells were treated with 20 uM As3+ for the time periods indicated. (A) Cytosolic and mitochondrial fractions were isolated by a digitonin lysis procedure (Single et al., 1998) and cytochrome c expression in the fractions was assessed by immunoblotting. (B) Caspase-9-like activity was measured utilizing the fluorogenic substrate Ac-LEHD-AMC. ...
Trivalent arsenite (As(3+)) is a known human carcinogen capable of inducing both cellular transformation and apoptotic cell death by mechanisms involving the production of reactive oxygen species. The tripeptide antioxidant glutathione (GSH) constitutes a vital cellular defense mechanism against oxidative stress. While intracellular levels of GSH are an important determinant of cellular susceptibility to undergo apoptotic cell death, it is not known whether cellular GSH biosynthetic capacity per se regulates As(3+)-induced apoptosis. The rate-limiting enzyme in GSH biosynthesis is glutamate cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modifier (GCLM) subunit. To determine whether increased GSH biosynthetic capacity enhanced cellular resistance to As(3+)-induced apoptotic cell death, we utilized a mouse liver hepatoma (Hepa-1c1c7) cell line stably overexpressing both GCLC and GCLM. Overexpression of the GCL subunits increased GCL holoenzyme formation and activity and inhibited As(3+)-induced apoptosis. This cytoprotective effect was associated with a decrease in As(3+)-induced caspase activation, cleavage of caspase substrates and translocation of cytochrome c to the cytoplasm. In aggregate, these findings demonstrate that enhanced GSH biosynthetic capacity promotes resistance to As(3+)-induced apoptosis by preventing mitochondrial dysfunction and cytochrome c release and highlight the role of the GSH antioxidant defense system in dictating hepatocyte sensitivity to As(3+)-induced apoptotic cell death.
... The mode of release of the two proteins is still controversial. Previous work showed that cyt c release is paralleled by the release of other intermembrane proteins such as adenylate kinase (ADK) (Single et al., 1998). These experiments are supporting the theory that unselective release of mitochondrial proteins occurrs during apoptosis. ...
... An early step in the apoptotic program is the release of proapoptotic proteins from the mitochondrial inter-membrane space, such as AIF (Zamzami et al., 1996) or cytochrome c (Liu et al., 1996). This is accompanied by the release of other proteins primarily located within the same subcellular compartment, such as adenylate kinase (ADK) (Single et al., 1998). We followed the loss of ADK, as an easily quantifiable indicator for the loss of the barrier function of the outer mitochondrial membrane. ...
Necrosis and apoptosis can occur simultaneously in heart infarction, stroke, and ischemia or in chemical poisoning. By regulating the intracellular ATP level, mitochondria might influence the mode of cell death execution. In cerebellar neurons the endogenous mediator nitric oxide and the exogenous mitochondrial toxin MPP+ triggered indirect excitotoxicity. Apoptosis under this condition was characterised by: i) initial intracellular ATP depletion, ii) [Ca2+]i increase, iii)?DYm dissipation and iv) cyt c release. The putative food excitotoxin aspartame (APM) triggered NMDA-R-dependent excitotoxicity in CGC. Caspases were not activated in APM-induced apoptosis. Apoptosis inducing factor (AIF) was translocated from mitochondria to the nucleus in APM and in glutamate treated cells. Release of AIF preceded cytochrome c release. Jurkat cells exposed to staurosporine (STS) undergo apoptosis in a caspase-dependent manner. ATP depletion converted STS-induced cell death from apoptosis to necrosis. TBT decreased intracellular ATP and triggered apoptosis or necrosis in Jurkat cells depending on the concentration used. ATP levels in the cells modulate the switch between apoptosis and necrosis. Two different steps are involved in this phenomenon: i) release of mitochondrial proteins and ii) caspase activation. Restoration of ATP led to DYm loss, mitochondrial swelling, cyt c and adenylate kinase release and caspase activation. Under ATP depleted conditions, release of mitochondrial protein and loss of DYm were delayed and caspases were not activated. Mitochondria can play a role at all stages of cell death. Mitochondrial are involved in the initiation of cell death in excitotoxicity. Mitochondrial damage (calcium, nitric oxide, toxins) can induce enhancing loops within the death program. Release of mitochondrial proapoptotic proteins and ATP levels can decide if caspase-dependent apoptosis, caspase-independent apoptosis or necrosis is the mode of cell death. Necrosis and apoptosis can occur simultaneously in heart infarction, stroke, and ischemia or in chemical poisoning. By regulating the intracellular ATP level, mitochondria might influence the mode of cell death execution. In cerebellar neurons the endogenous mediator nitric oxide and the exogenous mitochondrial toxin MPP+ triggered indirect excitotoxicity. Apoptosis under this condition was characterised by: i) initial intracellular ATP depletion, ii) [Ca2+]i increase, iii)?DYm dissipation and iv) cyt c release. The putative food excitotoxin aspartame (APM) triggered NMDA-R-dependent excitotoxicity in CGC. Caspases were not activated in APM-induced apoptosis. Apoptosis inducing factor (AIF) was translocated from mitochondria to the nucleus in APM and in glutamate treated cells. Release of AIF preceded cytochrome c release. Jurkat cells exposed to staurosporine (STS) undergo apoptosis in a caspase-dependent manner. ATP depletion converted STS-induced cell death from apoptosis to necrosis. TBT decreased intracellular ATP and triggered apoptosis or necrosis in Jurkat cells depending on the concentration used. ATP levels in the cells modulate the switch between apoptosis and necrosis. Two different steps are involved in this phenomenon: i) release of mitochondrial proteins and ii) caspase activation. Restoration of ATP led to DYm loss, mitochondrial swelling, cyt c and adenylate kinase release and caspase activation. Under ATP depleted conditions, release of mitochondrial protein and loss of DYm were delayed and caspases were not activated. Mitochondria can play a role at all stages of cell death. Mitochondrial are involved in the initiation of cell death in excitotoxicity. Mitochondrial damage (calcium, nitric oxide, toxins) can induce enhancing loops within the death program. Release of mitochondrial proapoptotic proteins and ATP levels can decide if caspase-dependent apoptosis, caspase-independent apoptosis or necrosis is the mode of cell death.
... Opening of the PTP increases the permeability of certain ions across the mitochondrial membrane, resulting in mitochondrial swelling and rupture of the mitochondrial outer membrane [157]. Thereby, opening of the PTP may cause irreversible ultrastructure alterations of mitochondria [158] and allows the passive release of mitochondrial proteins [159,160]. Proapoptotic proteins that are released from the mitochondrial intermembrane space into the cytoplasm include cytochrome c [82], caspases [98] and apoptosis inducing factor (AIF) [159]. ...
... The release of cytochrome c from mitochondria was analyzed by selective digitonin permeabilization, as described previously [160], with the following modifications: At the indicated time points the culture medium was exchanged for 300 µl permeabilization buffer ...
Die Depolymerisierung von Microtubuli führte in Kleinhirnkörnerzellen zur Zerstörung des neuronalen Netzwerks und folgendem apoptotischen Zelltod. Die Apoptose war durch mitochondriale Cytochrome c Freisetzung, Aktivierung von Caspasen, Chromatinkondensation, oligonukleosomale DNA-Fragmentierung und Umverteilung von Phosphatidylserin in die äußere Schicht der Plasmamembran gekennzeichnet. Die meisten dieser apoptotischen Veränderungen wurden durch die Gabe von Caspaseinhibitoren verhindert, obwohl Cytochrome c aus den Mitochondrien freigesetzt und das axodendritische Netzwerk zerstört wurde. Wenn der intrazelluläre ATP-Spiegel abgesenkt wurde, wurden sowohl Caspasenaktivierung als auch apoptotische Kernveränderungen und die Phosphatidylserinumverteilung verhindert, während der primäre Effekt, die Zerstörung von Microtubuli und der Neuritenverlust weiterhin stattfand. Umgekehrt führte die Wiederherstellung des ATP-Gehalts zur erneuten Ausprägung aller apoptotischen Merkmale. Daher wird angenommen, daß für die Durchführung der Apoptose genügend ATP vorhanden sein muss und könnte erklären, wieso unter energiedefizienten Zuständen degenerierende Neuronen persistieren können. Die Zerstörung der Microtubuli und Neuritenverlust wurden weder durch Caspaseninhibition noch durch das anti-apoptotische Bcl-2 verhindert, dennoch waren die neuronalen Zellkörper vor Apoptose geschützt. Nach einer gewissen Zeit starben allerdings auch diese Neuronen. Dies impliziert, daß unterschiedliche Todesprogramme verantwortlich für Neuritenverlust und für Apoptose existieren. Der verzögerte und unter Caspaseinhibition auftretende Zelltod war durch eine partielle Chromatinkondensierung, hochmolekulare DNA-Fragmentierung und Phosphatidylserinumverteilung gekennzeichnet. Proteasominhibitoren reduzierten diese Caspase-unabhängige Apoptose, was daraufschliessen läßt, daß die Proteasen des Proteasoms die Durchführung des apoptotischen Zelltods übernehmen können. In cerebellar granule cells, microtubule disassembling by colchicine induced degeneration of the axodendritic network followed by apoptotic demise of neuronal somata. Colchicine-induced apoptosis was accompanied by mitochondrial cytochrome c release, activation of execution caspase-3, advanced chromatin condensation, oligonucleosomal DNA fragmentation, and phosphatidylserine translocation to the outer surface of the plasma membrane. In the presence of peptide caspase inhibitors, apoptotic changes were prevented, although cytochrome c was released from mitochondria and the axodendritic network was still destroyed. Depletion of intracellular ATP blocked caspase activation, nuclear apoptotic features, and phosphatidylserine exposure, while the primary effect of microtubule disruption and neurite loss still occurred. Conversely, repletion of intracellular ATP restored all apoptotic features. Thus, sufficient ATP is required for the execution of neuronal apoptosis and energy deficiency may lead to the persistence of degenerating and dysfunctional neurons. Neither caspase inhibition by ATP depletion or pharmacological agents nor antiapoptotic Bcl-2 prevented microtubule breakdown or neurite loss. But neuronal somata were protected from apoptosis under these conditions. However, after a lag period caspase-inhibited as well as Bcl-2 overexpressing neurons underwent delayed cell death. This implies that death programs for neurite degeneration and apoptosis can occur independently from each other and that block of caspases may not rescue neurons from demise under degenerative conditions. The delayed caspase-independent death was characterized by partial chromatin condensation, large scale DNA fragmentation and phosphatidylserine exposure. Inhibitors of the proteasome reduced caspase-independent apoptosis, implying that noncaspase proteases can take over execution of apoptosis in neurons.
... Taken together, these results suggest that Hsp60, Hsp10, cytochrome c and AK are released simultaneously from mitochondria. These findings are in agreement with a recent report showing the simultaneous release of AK and cytochrome c during apoptotic death of Jurkat cells (Single et al., 1998; KöKö hler et al., 1999). Taken together, these observations strongly suggest that the release of intermembrane proteins from mitochondria is likely to be the result of permeabilization of the outer mitochondrial membrane, which initially leads to release of intermembrane space proteins only. ...
... The dissociation of Hsps from active caspase-3 co-incided with the release from mitochondria of Hsps and other intermembrane space proteins including cytochrome c and AK. The release of cytochrome c has been well documented in several cell systems (Bossy-Wetzel et al., 1997; Kim et al., 1997; Kluck et al., 1997a; Yang et al., 1997) and the release of AK was recently reported to occur simultaneously with that of cytochrome c (Single et al., 1998; KöKö hler et al., 1999). However, the significance of AK release or its contribution to the apoptotic process, if any, is not clear. ...
Activation of pro-caspase-3 is a central event in the execution phase of apoptosis and appears to serve as the convergence point of different apoptotic signaling pathways. Recently, mitochondria were found to play a central role in apoptosis through release of cytochrome c and activation of caspases. Moreover, a sub-population of pro-caspase-3 has been found to be localized to this organelle. In the present study, we demonstrate that pro-caspase-3 is present in the mitochondrial fraction of Jurkat T cells in a complex with the chaperone proteins Hsp60 and Hsp10. Induction of apoptosis with staurosporine led to the activation of mitochondrial pro-caspase-3 and its dissociation from the Hsps which were released from mitochondria. The release of Hsps occurred simultaneously with the release of other mitochondrial intermembrane space proteins including cytochrome c and adenylate kinase, prior to a loss of mitochondrial transmembrane potential. In in vitro systems, recombinant Hsp60 and Hsp10 accelerated the activation of pro-caspase-3 by cytochrome c and dATP in an ATP-dependent manner, consistent with their function as chaperones. This finding suggests that the release of mitochondrial Hsps may also accelerate caspase activation in the cytoplasm of intact cells.
... Previous studies showed that after StADK expression was inhibited in potato, the content of adenylate and starch would be significantly increased [9]. During cell death, cytochrome C in ADK and mitochondria is released into the cytoplasm [10]. Under salt stress, both the expression level and activity of adenosine kinase change, and the DNA methylation level will be affected [11]. ...
Adenylate kinase (ADK) is a key enzyme that is widely distributed in animals and plants. It plays an important role in growth and stress response. However, ADK genes in potato (StADK) have been little reported. It is of great significance to identify ADK members and understand the molecular mechanism of stress response and tolerance. Based on the potato genome data, 23 StADK genes were identified at a genome-wide level. We then performed a comprehensive study using a bioinformatics method. The results of the evolutionary tree showed that StADK proteins were divided into four groups, and they were highly homologous to the Arabidopsis thaliana ADK members. Meanwhile, our study found that they existed on eight chromosomes, and we obtained three pairs of fragment duplications. Furthermore, we detected the six selected StADK genes using qRT-PCR, and the results confirmed that the genes are involved in the regulation of cold, ABA, salt, H2O2 and drought stresses. Our study provides a theoretical basis for studying the function of the potato ADK genes and lays a solid foundation for further understanding the molecular mechanism of the potato ADK genes under various environmental stresses.
... JNK activation is also associated with hepatic apoptosis (52). The death signaling pathway leading to apoptosis involves the activation of caspase-3 and release of cytochrome C (53). ...
Objectives:
Signal transduction of mitogen-activated protein kinases (MAPKs) is activated during ischemia. In this study, c-Jun N-terminal Kinase (JNK) and p38 MAPK (p38) gene and protein expression were evaluated as two members of the MAPK family during liver ischemia-reperfusion in rats.
Materials and methods:
Thirty-two male Wistar rats were divided into four groups of eight: Vehicle, ischemia-reperfusion (IR), ischemia-reperfusion+silibinin (IR+SILI), and SILI. The IR and IR+SILI groups differed from the other two groups in that they underwent one hour of ischemia followed by three hr of reperfusion. The Vehicle and IR groups received normal saline while the SILI and IR+SILI groups were treated with silibinin (50 mg/kg). At the end of the reperfusion time, blood and ischemic liver tissue were collected for further experiments.
Results:
The expression of JNK and p38 gene, the amount of serum hepatic injury indices, and malondialdehyde (MDA) in the IR group increased significantly compared with the vehicle group. The JNK and p38 gene expression decreased significantly in the IR + SILI group compared with the IR group. Glutathione peroxidase (GPx) and total antioxidant capacity (TAC) levels decreased in the IR group while increasing in the IR+SILI group. Histological examination showed that silibinin significantly reduced the severity of hepatocyte degradation. Western blot results were completely consistent with real-time PCR results.
Conclusion:
The possible pathways of the protective effect of silibinin against hepatic ischemia damages is to reduce the expression of the p38 and JNK gene and protein.
... Subsequently, we explored the potential link between purine nucleotide metabolism and cell apoptosis. Nucleotides are helpful for promoting the apoptosis of certain cells (38,39) . In the present study, the results showed that splenic expression of ADA was down-regulated at day 42 and the gene expression of APRT was up-regulated by supplementing with vitamin C in vitro. ...
The nutrition condition of earlier life has a long-term influence on health of its later period. In order to the explore effects of in ovo feeding of vitamin C on splenic development, splenic metabolism and apoptosis were detected in embryo, adult chickens and in vitro . A total of 360 fertile eggs were selected and randomly assigned to 2 treatments: the control group (CON) and vitamin C group (VC) were injected with saline and vitamin C on embryonic day 11, respectively. Functional enrichment of differentially expressed genes by transcriptome on embryonic day 19 suggested that purine nucleotide metabolism might be a potential pathway for the in ovo feeding of vitamin C to regulate spleen development. Additionally, the in ovo feeding of vitamin C significantly increased splenic vitamin C content on post-hatch day 21. Meanwhile, the splenic expression of adenosine deaminase, serine/threonine kinase 1 and proliferating cell nuclear antigen were down-regulated, whereas the expression of cysteinyl aspartate specific proteinase 9 was up-regulated in VC group. On post-hatch day 42, the in ovo feeding of vitamin C significantly down-regulated the splenic expression of B-cell lymphoma 2, and increased the mRNA level of cysteinyl aspartate specific proteinase 9. The in ovo feeding of vitamin C could regulate the expression of genes related to adenylate metabolism and increased the apoptosis rate in vitro , which consistent with the result in vivo . Together, these results illustrated that the in ovo feeding of vitamin C regulated splenic development and maturation by affecting purine nucleotide metabolism pathway and promoting apoptosis.
... Separation of membrane/organelle fraction from cytosol was performed based on previously published methods [48]. Cells were incubated with digitonin buffer (8.75 µg digitonin/10 6 cells, 75 mM NaCl, 1 mM NaH 2 PO 4 , 8 mM Na 2 HPO 4 , 250 mM sucrose and protease/phosphatase inhibitor mixture) for 2 min on ice. ...
Anti-apoptotic BCL2 proteins are important targets for cancer therapy as cancers depend on their activity for survival. Direct inhibitors of MCL1 have entered clinical trials, although their efficacy may be limited by toxicity. An alternative approach may be to induce the pro-apoptotic protein NOXA which selectively inhibits MCL1 in cells. Many compounds originally proposed as inhibitors of the BCL2 family were subsequently found to induce the pro-apoptotic protein NOXA through the unfolded protein response. In the present study, we compared various putative BH3 mimetics across a panel of carcinoma cell lines and measured expression of NOXA protein and mRNA, as well as the kinetics of NOXA induction. We found that AT101 [(-)-gossypol] induces high levels of NOXA in carcinoma cell lines yet cells survive. When combined with an appropriate BCL2 or BCL-XL inhibitor, NOXA-dependent sensitization occurs. NOXA protein continues to accumulate for many hours after AT101 is removed, providing a window for administering these combinations. As MCL1 promotes drug resistance and overall survival, we propose that NOXA induction is an alternative therapeutic strategy to target MCL1 and either kill cancer cells that are dependent on MCL1 or sensitize cancer cells to other BCL2 inhibitors.
... Data are contradictory as to whether FTP and mitochondrial depolarisation is an essential component o f the apoptotic machinery, or simply an accompanying phenomenon resulting from caspase activation. Support for the third model comes from the observation that 1) other intermembrane proteins such as apoptosis inducing factor (AIF) and adenylate kinase-2 can be detected in apoptotic cytosols (Single et al., 1998;Susin et al., 1999); 2) inhibitors o f FT pore opening impede the collapse o f Av|/m and inhibit apoptosis (Zamzami et al., 1996); 3) BAX and BAK have been reported to bind directly to ANT and VDAC (Marzo et al., 1998a;Marzo et al., 1998b;Shimizu et al., 1999); and 4) BCL-2 expression can stabilise the mitochondrial membrane potential (Vander Heiden et al., 1997). However, opposing evidence shows that mitochondrial depolarisation was not required for cytochrome c release, as 1) cytochrome c redistribution can occur before loss of Ai|/m (Bossy-Wetzel et al., 1998); 2) loss o f Av|/m but not cytochrome c release can be prevented by caspase inhibitors in UV treated cells (Goldstein et al., 2000); and 3) translocation o f cytochrome c into the cytosol can occur ...
The basic region leucine zipper (bZIP) transcription factor CES-2 (cell death specification) controls the apoptotic fate of a subset of neurones in the pharynx of the nematode C.elegans. Conserved components of the cell death pathway have previously been shown to function across species. The aim of the work described in this thesis was to use ces-2 to identify critical genes involved in apoptotic control in mammalian cells. Preliminary experiments were performed to ascertain whether mammalian cells contained endogenous CES-2 like proteins, and whether the C.elegans protein, when expressed in mammalian cells, is correctly localised. The former was confirmed in HeLa nuclear extracts utilising the specific DNA binding sequence of CES-2 in an electrophoretic mobility shift assay; and the latter by immunofluorescence studies established that expression of CES-2 in several mammalian cell types was confined to the nucleus, consistent with its role as a nuclear transcription factor. The bZIP family of proteins function as specific homo- and hetero-dimers. The strategy used, therefore, to identify CES-2-like or CES-2-interacting proteins was by two-hybrid screening of a HeLa cDNA library. Two mammalian bZIP transcription factors were identified which interacted specifically with CES-2, CHOP (C/EBP homologous protein) and ATF4/CREB-2 (activating transcription factor 4 / cAMP response element binding protein-2). The bZIP domain of CES-2 was sufficient for these interactions and this was confirmed by co-immunoprecipitation studies. Since CHOP is upregulated in response to ER-stress and apoptosis induced by the N-linked glycosylation inhibitor tunicamycin, the effect of expressing CES-2 on the response of HeLa cells to tunicamycin was investigated. In contrast to its pro-apoptotic role in the C.elegans neurones, overexpression of CES-2 in HeLa cells, in both transient and stable transfectants, increased the resistance of the cells to apoptosis induced by tunicamycin. Experiments using thapsigargin, calcium ionophore and taxol suggested that CES-2 protection depended on the upregulation of CHOP. Results obtained from expressing the CES-2 bZIP domain alone point to protection being by a dominant negative mechanism to down-regulate CHOP activity. Analysis of the second CES-2 interacting protein showed that ATF4 is also upregulated by agents that induce ER-stress, with a time course similar to CHOP. In the light of recent published data that ATF4 activates CHOP by binding its promoter element, CES-2 binding to ATF4 may also affect the regulation of ATF4-dependent chop transcription. Thus CES-2 is able to bind proteins positioned at different levels of the ER-stress pathway to influence apoptosis. The results presented in this thesis demonstrate that CES-2 can function in mammalian cells and suggest that CES-2-like proteins exist as conserved elements of the apoptotic program.
... The interplay between intracellular ATP levels and the release of the intermembrane proteins AK and cytochrome c (Cyt c), can be used to determine mitochondrial dysfunction on the molecular level and ultimately as indicators of cell viability and toxicity. AK release during stressful cellular states is responsible for the up and/or down regulation of intracellular ATP levels, and additionally the release of AK and Cyt c activates the execution caspase cascade resulting in apoptosis/necrosis (Single et al., 1998;Leist et al., 1999;Rasda and Bernardi, 2007;Hardie, 2003;Singh et al., 2011). ...
Abstract
Ethnopharmacology relevance
Traditional herbal medicines are utilized by 27 million South Africans. Xysmalobium undulatum (Uzara) is one of the most widely used traditional medicinal plants in Southern Africa. A false belief in the safety of herbal medicine may result in liver injury. Herb-induced liver injury (HILI) range from asymptomatic elevation of liver enzymes, to cirrhosis and in certain instances even acute liver failure. Various in vitro and in vivo models are available for the pre-clinical assessment of drug and herbal hepatotoxicity. However, more reliable and readily available in vitro models are needed, which are capable of bridging the gap between existing models and real human exposure. Three-dimensional (3D) spheroid cultures offer higher physiological relevance, overcoming many of the shortcomings of traditional two-dimensional cell cultures.
Aims of this study
This study investigated the hepatotoxic and anti-prolific effects of the crude X. undulatum aqueous extract during a sub-chronic study (21 days), in both a 3D HepG2/C3A spheroid model and the Sprague Dawley rat model.
Methods
HepG2/C3A spheroids were treated with a known hepatotoxin, valproic acid, and crude X. undulatum aqueous extract for 21 days with continuous evaluation of cell viability and proliferation. This was done by evaluating cell spheroid growth, intracellular adenosyl triphosphate (ATP) levels and extracellular adenylate kinase (AK). Sprague Dawley rats were treated with the same compounds over 21 days, with evaluation of in vivo toxicity effects on serum chemistry.
Results
The results from the in vitro study clearly indicated hepatotoxic effects and possible liver damage following treatment with valproic acid, with associated growth inhibition, loss of cell viability and increased cytotoxicity as indicated by reduced intracellular ATP levels and increased AK levels. These results were supported by the increased in vivo levels of AST, ALT and LDH following treatment of the Sprague Dawley rats with valproic acid, indicative of hepatic cellular damage that may result in hepatotoxicity. The in vitro 3D spheroid model was also able to predict the potential concentration dependant hepatotoxicity of the crude X undulatum aqueous extract. Similarly, the results obtained from the in vivo Sprague Dawley model indicated moderate hepatotoxic potential.
Conclusion
The data from both the 3D spheroid model and the Sprague Dawley model were able to indicate the potential concentration dependant hepatotoxicity of the crude X undulatum aqueous extract. The results obtained from this study also confirmed the ability of the 3D spheroid model to effectively and reliably predict the long-term outcomes of possible hepatotoxicity.
... Separation of membrane/organelle fraction from cytosol was performed based on previously published methods (Single et al. 1998;Ganju and Eastman 2003). Cells were incubated with digitonin buffer (8.75 μg digitonin/ 10 6 cells, 75 mM NaCl, 1 mM NaH 2 PO 4 , 8 mM Na 2 HPO 4 , 250 mM sucrose and protease/phosphatase Raji) were incubated with 0-10 μM 6-HTBN. ...
Nuphar alkaloids, originally isolated from water lilies, induce apoptosis in mammalian cells in less than 1 h, making them possibly the fastest known inducers. However, the mechanism by which this rapid apoptosis occurs remains unknown. We have investigated canonical aspects of apoptosis to determine how the nuphar alkaloid, (+)-6-hydroxythiobinupharidine (6HTBN), induces apoptosis. 6HTBN induced rapid apoptosis in various leukemia, lymphoma, and carcinoma cell lines, suggesting that its mechanism is cell-type independent. It also circumvented resistance of patient-derived chronic lymphocytic leukemia cells generated by co-culture on survival-promoting stroma. Intriguingly, 6HTBN failed to induce apoptosis in platelets. The mechanism of apoptosis involves activation of caspase 9 and caspase 3, but not caspase 8 as previously reported. The release of cytochrome c from mitochondria occurred even in the absence of BAX/BAK and in cells that retained mitochondrial membrane potential. These results suggest a novel mechanism of apoptosis that has previously not been reported. The molecular target of the nuphar alkaloids remains to be determined.
... Separation of membrane/organelle fraction from cytosol was performed based on previously published methods 19 . Cells were incubated with digitonin buffer (8.75 µg/10 6 cells digitonin, 75 mM NaCl, 1 mM NaH 2 PO 4 , 8 mM Na 2 HPO 4 , 250 mM sucrose and protease/phosphatase inhibitor mixture) for 2 min on ice. ...
Targeting anti-apoptotic BCL2 family proteins has become an attractive therapeutic strategy for many cancers, and the BCL2-selective inhibitor ABT-199 (venetoclax) has obtained clinical success. However, MCL1 can promote drug resistance and overall cancer cell survival. Thus, there is a critical need to develop an effective drug that antagonizes MCL1. However, most putative MCL1 inhibitors have been misclassified as they fail to directly inhibit MCL1 in cells, but rather induce the pro-apoptotic protein NOXA. We have investigated three putative MCL1 inhibitors: MIM1, UMI-77, and A-1210477. All three compounds were developed in cell-free assays and then found to be cytotoxic, and hence assumed to directly target MCL1 in cells. Here, we investigated whether these compounds directly inhibit MCL1 or inhibit MCL1 indirectly through the induction of NOXA. Both MIM1- and UMI-77-induced NOXA through the unfolded protein response pathway, and sensitized leukemia cells to ABT-199; this cytotoxicity was dependent on NOXA suggesting that these compounds do not directly target MCL1. A-1210477 was the only compound that did not induce NOXA, but it still sensitized cells to ABT-199. A-1210477 induced accumulation of MCL1 protein consistent with it binding and preventing MCL1 degradation. However, at concentrations used in several prior studies, A-1210477 also induced cytochrome c release, caspase activation, and apoptosis in a BAX/BAK-independent manner. Furthermore, the release of cytochrome c occurred without loss of mitochondrial membrane potential. This apoptosis was extremely rapid, sometimes occurring within 0.5–1 h. Hence, we have identified a novel mechanism of apoptosis that circumvents the known mechanisms of cytochrome c release. It remains to be determined whether these unexpected mechanisms of action of putative BH3 mimetics will have therapeutic potential.
... Next, we monitored the adenylate kinase (AK) activity that is released in the culture supernatant upon plasma membrane damage 22 . As expected, for both MR766 and PRVABC59 strains, we found a progressive increase of AK activity released in the supernatant as compared to uninfected controls (Fig. 6b). ...
Zika virus (ZIKV) is a neurotropic agent that targets the developing fetal brain in women infected during pregnancy. In addition to the developing central nervous system, ZIKV has been recently shown to infect cells of the peripheral nervous system (PNS), highlighting its potential to cause acute peripheral neuropathies in adults, such as Guillain-Barré Syndrome (GBS). Here we show that myelinating dorsal root ganglia (DRG) explants obtained from interferon-alpha/beta receptor knock-out mice are productively infected by ZIKV. Virus replication is cytopathic in both peripheral neurons and myelinating Schwann cells leading to myelin disruption. These results confirm and extend previous observations suggesting that the PNS is indeed a potential site of ZIKV infection, replication and cytopathicity.
... During stressful states, AK up-or down-regulates ATP production, influencing cell viability and toxicity. Furthermore, during the active release of AK and Cyt c the execution caspase cascade is activated, resulting in cell death (Single et al. 1998;Leist et al 1999;Hardie 2003;Anderson and Borlak 2007;Rasola and Bernardi 2007). ...
Xysmalobium undulatum (Uzara) is one of the most widely used indigenous traditional herbal remedies in Southern Africa. Commercially available Uzara plant material was used to prepare a crude aqueous extract, of which the toxicity potential was investigated in the hepatic HepG2/C3A cell line in both traditional two-dimensional (2D) and rotating three-dimensional (3D) spheroid cell cultures. These cultures were treated over a period of 4 days at concentrations of 200, 350, 500, and 750 mg/kg plant extract to protein content. Basic physiological parameters of the cell cultures were measured during exposure, including cell proliferation, glucose uptake, intracellular adenosine triphosphate levels and adenylate kinase release. The results indicated that all physiological parameters monitored were affected in a dose dependent manner, with the highest concentration of Uzara crude water extract (750 mg/kg) resulting in toxicity. Anti-proliferating effects of Uzara crude water extract were observed in both the 2D and 3D cell cultures, with the most pronounced effects at concentrations of 350, 500 and 750 mg/kg. Discrepancies between results obtained from the 2D and 3D cell culture models may be attributed to the type of repair system that is initiated upon exposure, depending on where cells are within the cell cycle. DNA repair systems differ in cells within the G1 phase and non-diving cells, (i.e. cells found predominantly in in vitro 3D and the in vivo situation).
... Whole cell lysates were prepared by using radioimmunoprecipitation assay buffer (1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS in PBS) supplemented with 100X protease inhibitor cocktail (Roche Applied Science) and phosphatase inhibitor cocktail (Cell Signaling Technology, Inc., Danvers, MA, USA). The mitochondrial and cytosolic fractions were separated using the digitonin method (27). Briefly, 5x10 6 Nude mice and tumor inoculations. ...
Choroidal melanoma is the most common primary intraocular tumor in adults. Cepharanthine (CEP), a natural alkaloid extracted from the roots of Stephania cepharantha Hayata, has been demonstrated to inhibit the proliferation of various cancer cells. However, its potential anticancer effect in choroidal melanoma has not been clarified yet. In the present study, it was identified that CEP may potently inhibit the proliferation of human choroidal melanoma cells, induce cell death and cell cycle arrest, and activate cellular apoptotic proteins, including Bcl-2-associated X protein, caspase and poly(ADP-ribose) polymerase. Results also revealed that CEP induced the cellular production of reactive oxygen species (ROS) and led to cytochrome c release, whereas concurrent treatment with N-acetyl-L-cysteine (a ROS scavenger) attenuated the situation. In addition, CEP was also revealed to activate c-Jun N-terminal kinase (JNK) 1 and 2, whereas inhibition of JNK1/2 partially abrogated the proliferation inhibitory effect of CEP, indicating that JNK1 and JNK2 were involved in CEP-triggered cellular apoptosis. In addition, the anticancer effects of CEP were also observed in a choroidal melanoma xenograft model. In summary, the results of the present study demonstrated that CEP is effective in suppressing human choroidal melanoma cell and tumor cell proliferation, and that CEP may therefore represent a potentially novel therapeutic agent for the treatment of choroidal melanoma.
... 3a). ZIKV infection of both untreated T-HESC and dT-HESC induced cytopathicity and cell death as determined by the levels of adenylate kinase (AK) released in the culture supernatants28 with kinetics similar to those of virus replication(Fig. 3b).ZIKV induces the expression of IFN-β and ISGs. ...
Zika virus (ZIKV) is a recently re-emerged flavivirus transmitted to humans by mosquito bites but also from mother to fetus and by sexual intercourse. We here show that primary human endometrial stromal cells (HESC) are highly permissive to ZIKV infection and support its in vitro replication. ZIKV envelope expression was detected in the endoplasmic reticulum whereas double-stranded viral RNA colocalized with vimentin filaments to the perinuclear region. ZIKV productive infection also occurred in the human T-HESC cell line together with the induction of interferon-β (IFN-β) and of IFN-stimulated genes. Notably, in vitro decidualization of T-HESC with cyclic AMP and progesterone upregulated the cell surface expression of the ZIKV entry co-receptor AXL and boosted ZIKV replication by ca. 100-fold. Thus, endometrial stromal cells, particularly if decidualized, likely represent a crucial cell target of ZIKV reaching them, either via the uterine vasculature in the viremic phase of the infection or by sexual viral transmission, and a potential source of virus spreading to placental trophoblasts during pregnancy.
... Fresh cells were washed with PBS and pelleted by centrifugation at 150 x g for 7 min. Cytosolic fractions were isolated by selective lysis of the plasma membrane with 35 μg of digitonin per 4 × 10 6 N2a cells (Single et al. 1998) or with 25 μg/ml of digitonin for rat cardiomyocytes (Dedkova and Blatter 2012) followed by centrifugation at 1000 x g for 10 min. Supernatants containing cytosolic fraction of proteins were centrifuged at 10,000 g for 15 min to pellet any remaining cellular debris. ...
The adenylate kinase (AK) isoforms network plays an important role in the intracellular energy transfer processes, the maintenance of energy homeostasis, and it is a major player in AMP metabolic signaling circuits in some highly-differentiated cells. For this purpose, a rapid and sensitive method was developed that enables to estimate directly and semi-quantitatively the distribution between cytosolic AK1 and mitochondrial AK2 localized in the intermembrane space, both in isolated cells and tissue samples (biopsy material). Experiments were performed on isolated rat mitochondria or permeabilized material, including undifferentiated and differentiated neuroblastoma Neuro-2a cells, HL-1 cells, isolated rat heart cardiomyocytes as well as on human breast cancer postoperative samples. In these samples, the presence of AK1 and AK2 could be detected by high-resolution respirometry due to the functional coupling of these enzymes with ATP synthesis. By eliminating extra-mitochondrial ADP with an excess of pyruvate kinase and its substrate phosphoenolpyruvate, the coupling of the AK reaction with mitochondrial ATP synthesis could be quantified for total AK and mitochondrial AK2 as a specific AK index. In contrast to the creatine kinase pathway, the AK phosphotransfer pathway is up-regulated in murine neuroblastoma and HL-1 sarcoma cells and in these malignant cells expression of AK2 is higher than AK1. Differentiated Neuro-2a neuroblastoma cells exhibited considerably higher OXPHOS capacity than undifferentiated cells, and this was associated with a remarkable decrease in their AK activity. The respirometric method also revealed a considerable difference in mitochondrial affinity for AMP between non-transformed cells and tumor cells.
... For Immunocytochemistry, CGC were grown on glass-bottomed culture dishes, exposed to A2E or solvent, fixed with 4% paraformadehyde and permeabilized with 0.1% Triton X-100 (for cytochrome c) or 0.1% NaDodSO 4 (for AIF). Staining of cytochrome c (anti-cytochrome c antibody; clone 6H2.B4, Pharmingen, Hamburg, Germany) was performed as described 38,39 . Rabbit anti-AIF serum 29 was used at a dilution of 1:500. ...
Age-related macular degeneration (AMD) is the leading cause of severe visual impairment in humans living in industrialized countries. A precondition for AMD appears to be the accumulation of the age pigment lipofuscin in lysosomes of retinal pigment epithelial (RPE) cells. Here we show that A2E (N-retinyl-N-retinylidene ethanolamine) the major fluorophore of lipofuscin, induces apoptosis in RPE and other cells at concentrations found in human retina. Apoptosis is accompanied by appearance of the pro-apoptotic proteins cytochrome c and apoptosis inducing factor (AIF) in the cytoplasm and the nucleus but does not involve activation of caspase-3. Biochemical examinations show that A2E inhibits mitochondrial function by specifically targeting cytochrome oxidase (COX). With both, isolated mitochondria and purified COX, A2E inhibits oxygen consumption synergistically with light. Inhibition is reversed by addition of cytochrome c or cardiolipin. Loss of RPE cell viability through inhibition of mitochondrial function might constitute a pivotal step towards the progressive degeneration of the central retina. We present a working hypothesis that suggests that A2E, once released from lysosomes (by lysosomal rupture or by ‘overflow’ of the lysosomal capacity) can target mitochondria and inhibit mitochondrial function. This causes the release of the pro-apoptotic proteins and the induction of cell death.
... We hypothesized that a reduction in cell proliferation rather than cell death was responsible for the decreased cell density occurring in stimulated wells. Stimulation of 50 Hz decreased cell number through a direct effect on cell cycle, and not cell death, as evidenced by the assessment of adenylate kinase (AK) release (Single et al., 1998), confirmed by caspase-3 immunoassay, as shown in Figure 2A-C. Incorporation of BrdU in stimulated cells is also greatly diminished in comparison to the controls, further demonstrating a direct effect of electrical stimulation on cell cycle (Fig. 3A). ...
Very low intensity alternating current decreases cell proliferation. Electric fields impact cellular functions by activation of ion channels or by interfering with cell membrane integrity. Ion channels can regulate cell cycle and play a role in tumorigenesis. In the absence of thermal influences, low-frequency, low-intensity, alternating current (AC) directly affects cell proliferation without a significant deleterious contribution to cell survival. However, to be effective, exposure to AC stimulation also requires a permissive role for GIRK2 (or KIR3.2) potassium channels.
... The supernatant containing the nuclear fraction was collected by centrifugation at 12,000 g for 2 min and stored at -80 0 C. The supernatant obtained at previous step was centrifuged at 6000×g for 10 min at 4 0 C andthe pellet containing mitochondrial fraction was then washed with mitochondrial storage buffer and centrifuged at 6000×g for 20 min.The pellet then was resuspendedin mitochondrial storage buffer. Digitonin soluble and insoluble fractions were prepared as previously described (29). Briefly, 2×10 6 cells were suspended in PBS and mixed properly with an equal volume of digitonin (150µg/ml) in sucrose (0.5 M). ...
Apoptosis is one of the mechanisms used by host cells to get rid of unwanted intracellular organisms, and often found to be subverted by pathogens through use of host anti-apoptotic proteins. In the present study, with the help of in vitro and in vivo approaches, we documented that the macrophage anti-apoptotic protein myeloid cell leukemia 1 (MCL-1) is exploited by the intra-macrophage parasite Leishmania donovani to protect their "home" from actinomycin D-induced mitochondria-dependent apoptosis. Amongst all the anti apoptotic BCL-2 family members, infection preferentially up-regulated expression of MCL-1 at both the mRNA and protein levels, and compared to infected control, MCL-1 silenced infected macrophages documented enhanced caspase activity and increased apoptosis when subjected to actinomycin D treatment. Phosphorylation kinetics and ChIP assay demonstrated that infection induced MCL-1 expression was regulated by the transcription factor CREB and silencing of CREB resulted in reduced expression of MCL-1 and increased apoptosis. During infection, MCL-1 was found to be localized in mitochondria and this was significantly reduced in Tom70 silenced macrophages, suggesting the active role of TOM70 in MCL-1 transport. In the mitochondria, MCL-1 interacts with the major pro-apoptotic protein BAK and prevents BAK-BAK homo-oligomer formation thereby preventing cytochrome c release mediated mitochondrial dysfunction. Silencing of MCL-1 in the spleen of infected mice showed decreased parasite burden and increased induction of splenocyte apoptosis. Collectively our results showed that L. donovani exploited macrophage anti-apoptotic protein MCL-1 to prevent BAK mediated mitochondria dependent apoptosis thereby protecting its niche, which is essential for disease progression.
... In particular, AK2 deficiency in Drosophila melanogaster is embryonically lethal (Fujisawa et al., 2009;Chen et al., 2012). Moreover, it has been suggested that AK2 deficiency may induce the unfolded protein response (Burkart et al., 2011) and apoptosis (Single et al., 1998;Köhler et al., 1999;Lee et al., 2007). The intracellular localization of AK2 is strictly limited to the mitochondrial intermembrane space (Dzeja et al., 2007) where it is required to generate ADP. ...
Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.
... Fresh cells were washed with PBS and pelleted by centrifugation at 150 x g for 7 min. Cytosolic fractions were isolated by selective lysis of the plasma membrane with 35 μg of digitonin per 4 × 10 6 N2a cells (Single et al. 1998) or with 25 μg/ml of digitonin for rat cardiomyocytes (Dedkova and Blatter 2012) followed by centrifugation at 1000 x g for 10 min. Supernatants containing cytosolic fraction of proteins were centrifuged at 10,000 g for 15 min to pellet any remaining cellular debris. ...
Tubulin, a well-known component of the microtubule in the cytoskeleton, has an important role in the transport and positioning of mitochondria in a cell type dependent manner. This review describes different functional interactions of tubulin with cellular protein complexes and its functional interaction with the mitochondrial outer membrane. Tubulin is present in oxidative as well as glycolytic type muscle cells, but the kinetics of the in vivo regulation of mitochondrial respiration in these muscle types is drastically different. The interaction between VDAC and tubulin is probably influenced by such factors as isoformic patterns of VDAC and tubulin, post-translational modifications of tubulin and phosphorylation of VDAC. Important factor of the selective permeability of VDAC is the mitochondrial creatine kinase pathway which is present in oxidative cells, but is inactive or missing in glycolytic muscle and cancer cells. As the tubulin-VDAC interaction reduces the permeability of the channel by adenine nucleotides, energy transfer can then take place effectively only through the mitochondrial creatine kinase/phosphocreatine pathway. Therefore, closure of VDAC by tubulin may be one of the reasons of apoptosis in cells without the creatine kinase pathway. An important question in tubulin regulated interactions is whether other proteins are interacting with tubulin. The functional interaction may be direct, through other proteins like plectins, or influenced by simultaneous interaction of other complexes with VDAC.
... Apoptosis-inducing factor (AIF) is released simultaneously with cytochrome c, and translocates to the nucleus where it causes chromatin condensation and high molecular weight DNA fragmentation Susin et al., 1999b). In addition adenylate kinase, caspase-2, -3, and -9 as well as heat shock proteins hsp-10 and -60 are released from mitochondria upon apoptosis-induction (Kohler et al., 1999;Krajewski et al., 1999;Samali et al., 1999;Single et al., 1998;Susin et al., 1999a). The heat shock proteins have been shown to accelerate the maturation of caspase-3 (Xanthoudakis et al., 1999). ...
... For example, second mitochondria-derived activator of caspases (Smac)/direct inhibitor-of-apoptosis protein-binding protein with low pI (DIABLO) promotes apoptosis by inhibiting the inhibitor of apoptosis proteins (25)(26). Adenylate kinase-2 (AK-2) is another intermembrane space protein that is released into the cytosol, although its role in apoptosis has not yet been defined (27)(28). ...
... All steps were performed on ice or at 4°C. In some cases, an alternative protocol was used in which cytosolic fractions were obtained by selective plasma membrane permeabilization with digitonin (Single et al., 1998). Briefly, 2 ϫ 10 6 cells were lysed by including for 1 to 2 min in lysis buffer (75 mM NaCl, 8 mM Na 2 HPO 4 , 1 mM NaH 2 PO 4 , 1 mM EDTA, and 350 g/ml digitonin). ...
The ability of the protein kinase C down-regulator bryostatin 1 to potentiate 1-β-d-arabinofuranosylcytosine (ara-C)-induced apoptosis was examined in human leukemia cells (U937) over-expressing the antiapoptotic protein Bcl-xL. Coadministration of bryostatin 1 with ara-C resulted in enhanced cytosolic release of cytochrome c and Smac/DIABLO, procaspase-3 and -9 activation, loss of mitochondrial membrane potential (Δψm), poly(ADP-ribosyl)phosphorylase degradation, apoptosis, and loss of clonogenic survival in U937/Bcl-xL cells, although effects were not as marked as in empty-vector control cells. Whereas the broad caspase inhibitor ZVAD-fluoromethyl ketone blocked ara-C/bryostatin 1-mediated caspase activation, loss of Δψm , and apoptosis in U937 cells, it failed to diminish cytochrome c release. In contrast, ectopic expression of Bcl-xL blocked cytochrome c redistribution as well as all other events involved in ara-C/bryostatin 1-mediated apoptosis. The ability of ectopic expression of cytokine response modifier A to attenuate, albeit partially, bryostatin 1-mediated potentiation of ara-C-related apoptosis suggested a contributory role for activation of the extrinsic pathway in this phenomenon. Finally, the FF1ATPase inhibitor oligomycin effectively blocked cytochrome c release as well as loss of Δψm and apoptosis in U937/Bcl-xL cells. Together, these findings support the concept that bryostatin 1 potentiates ara-C lethality in human leukemia cells ectopically expressing Bcl-xL by diminishing the capacity of this antiapoptotic protein to antagonize cytochrome c release. In addition, they raise the possibility that activation of caspase cascades operating independently of Bcl-xL-associated mitochondrial actions may also contribute to enhanced lethality.
... Cytochrome c and AK also appeared simultaneously in the cytosol of cells undergoing necrosis, a different type of cell death. Diffusion of cytochrome c from mitochondria to cytosol is induced by the activation of apoptotic pathway genes, such as caspases [19]. Protein disulfide-isomerase (PDI) is a 55-kDa molecular chaperone that has two functions; namely, the maintenance of proper protein folding and the regulation of the redox state of proteins by catalyzing the thiol-disulfide exchange reaction. ...
... Cytochrome c and AK also appeared simultaneously in the cytosol of cells undergoing necrosis, a different type of cell death. Diffusion of cytochrome c from mitochondria to cytosol is induced by the activation of apoptotic pathway genes, such as caspases [19]. Protein disulfide-isomerase (PDI) is a 55-kDa molecular chaperone that has two functions; namely, the maintenance of proper protein folding and the regulation of the redox state of proteins by catalyzing the thiol-disulfide exchange reaction. ...
We have previously demonstrated that differentially expressed proteins affect abnormal development and function of cloned term placenta. This is associated with cloned fetus morbidity and mortality. We also frequently observed loss of the cloned fetus and failed development during early pregnancy periods. To confirm the pattern of important gene expression in cloned placenta during pre- and post-implantation, we investigated expression pattern of proteins in early stage (21 days) domestic cat placentas of cloned embryo transfer (CEP; n=2) and artificial insemination (CP; n=4) derived pregnancy. The differentially expressed proteins were investigated by 2-DE and MALDI-TOF/MS. Twenty-three proteins were up- and down-regulated at least 1.5-fold in the CEP (P<0.05) compared with the CP. Differentially expressed proteins were analysed using PDQest program and statistically analysed by 1-way ANOVA using the SPSS software. In CEP, 13 proteins were up-regulated, such as 78-kDa glucose-regulated protein (GRP78), annexin A2 (ANXA2), protein DJ-1 (DJ1), adenylate kinase isoenzyme 1 (AK1), protein disulfide-isomerase A3 (PDIA3), heat shock protein β-1 (HSPB1), actin, cytoplasmic 1 (ACTB), serum albumin (ALB), protein disulfide-isomerase A6 (PDIA6), G protein-regulated inducer of neurite outgrowth 1 (GRIN1) and triosephosphate isomerase (TIM). In contrast, 10 proteins were down-regulated, such as vinculin (VCL), triosephosphate isomerase (TIM), heterogeneous nuclear ribonucleoprotein H (hnRNPH), tropomyosin α-4 (TPM4), 60-kDa heat shock protein, mitochondrial (Hsp60), serum albumin (ALB), calumenin (CALU), keratin type 1 (CK1) and prohibitin (PHB). To validate the identified proteins in the CEP compared with the CP, we investigate a peptide sequences using MALDI-TOF/TOF tandem mass spectrometry. The sequence information obtained a high ions score from NCBI and Swiss-Prot databases. In conclusion, we did identify abnormal expression of proteins that might be associated with impaired development of CEP, which may endanger the cloned fetus during early pregnancy.
... Cytochrome c and AK also appeared simultaneously in the cytosol of cells undergoing necrosis, a different type of cell death. Diffusion of cytochrome c from mitochondria to cytosol is induced by the activation of apoptotic pathway genes, such as caspases [19]. Protein disulfide-isomerase (PDI) is a 55-kDa molecular chaperone that has two functions; namely, the maintenance of proper protein folding and the regulation of the redox state of proteins by catalyzing the thiol-disulfide exchange reaction. ...
The normal placenta formation and development affect subsequent survival and development of fetus. Somatic cell nuclear transfer (SCNT) is still associated with functional and structural abnormalities of placentation in cloned pregnancy. Moreover, the molecular basis of placenta derived from cloned embryos is poorly understood. To determine the abnormal expression of proteins of cloned term placenta (CTP; n=3) compared with control placenta (n=3), a proteomic analysis was performed using 2-DE, MALDI-TOF MS, and MALDI-TOF/TOF MS/MS. The cloned kittens were killed 1 month after Caesarean section. The results showed that 42 proteins were significantly up/down expressed in the CTP (P<0.05). In CTP, thirty proteins were up-regulated, such as apoptosis-related cathepsin D (CD), annexin A1, and heat shock protein 27, and 12 proteins were down-regulated, such as prohibitin (PHB). Abnormal expression of CD and PHB may be related to induced reactive oxygen species (ROS) resulting in promoted cellular senescence and apoptosis. The expression patterns of CD and PHB were validated by Western blotting and immunofluorescence assay. Additionally, we also confirmed the abnormal expression of SOD (up-regulated) and CAT (down-regulated) enzymes in the CTP. Increased generation of ROS in the CTP was related to decreased mitochondrial membrane potential as detected in placenta tissues by MitoTracker green FM (MTG; 1μM, Invitrogen, Carlsbad, CA). ROS-induced mitochondrial damage accelerated telomere loss [as detected by IQ-fluorescent in situ hybridization (FISH)] compared with control. ROS is an important modulator of telomere loss and that telomere-driven cellular senescence is primarily a stress responsive. Thus, the data suggest that combined abnormal proteins expression were associated with hindered development of cloned placenta and viability of fetus. Therefore, if we can solve this problem, the efficiency of SCNT and transgenic cat production will be increased, which in turn, may be applied as a potential means of restoration of endangered animals.
... Separation of mitochondrial and cytosolic fractions was obtained with the digitonin method [14] The supernatant was removed as the cytosolic fraction and the pellet was resuspended in the same volume of buffer not containing digitonin. 20 μL Laemmli sample buffer supplemented with 10% dithiothreitol and 10% SDS was added to all samples. ...
Visible spectroscopy was used to measure real-time changes in the oxidation state of cytochrome c (cyt c) and the a-cytochromes (cyt aa(3)) of cytochrome oxidase during mitochondrial outer membrane permeabilization (MOMP) initiated by anisomycin in HL-60 cells. The oxidation state of mitochondrial cyt c was found to be approximately 62% oxidized before MOMP and became approximately 70% oxidized after MOMP. In contrast, the cytosolic pool of cyt c was found to be almost fully reduced. This oxidation change allows cyt c release to be continuously and quantitatively monitored in real time. Anoxia and antimycin were used to fully reduce and fully oxidize, respectively, the mitochondrial pool of cyt c and it was found that the release of cyt c was independent of it oxidation state consistent with a simple model of cyt c passively diffusing down a concentration gradient through a pore or tear in the outer membrane. After MOMP was complete, the flux of cyt c diffusing back into the mitochondria was measured from the residual mitochondrial oxygen consumption after complete inhibition of the bc(1) with antimycin and myxothiazol. The outer membrane was found to be highly permeable after MOMP implying that the reduction of cyt c in the cytosol must be very rapid. The permeability of the outer membrane measured in this study would result in the release of cyt c with a time constant of less than 1 s.
... Beim Lyse und mehrere Zentrifugationsschritte eine Subfraktionierung durchführen (Single et al., 1998). Eine Möglichkeit hierfür ist die Digitonin-Lyse. ...
In Keratinozyten wird sowohl durch UVB als auch durch PUVA-Bestrahlung Apoptose induziert. Wir untersuchten die in Keratinozyten durch UVB, PUVA, UVA und Todesliganden wie TRAIL ausgelösten Apoptosewege näher. UVB und PUVA, nicht aber UVA-Bestrahlung lösen in vitro Keratinozytenapoptose aus. 2-4 h nach UVB beobachteten wir die Aktivierung von Caspasen. Nach PUVA setzt die Aktivierung von Caspasen wesentlich später ein, nämlich erst 12 h nach Bestrahlung. Passend dazu, ist ein Verlust des mitochondrialen Transmembranpotentials 6-8 h nach UVB und 12-14 h nach PUVA detektierbar. Die Überexpression des Proteins Bcl-2 verhindert den Verlust des mitochondrialen Transmembranpotentials und die Caspase-Aktivierung nach UVB, und vermittelt auch einen klonogen Schutz, unabhängig von der Bildung reaktiver Sauerstoffradikale. Im Gegensatz dazu verzögert es den Verlust des Transmembranpotentials und die Caspase-Aktivierung nach PUVA nur und verhindert sie nicht. PUVA-bestrahlte Zellen können sich nicht weiter teilen, sind also durch Bcl-2 nicht klonogen geschützt.
... The report about an initial, caspase-independent cytochrome c release followed by a caspase-mediated drastic loss of mitochondrial cytochrome c may proof the existence of such amplifying feedback loops at least in some models (Chen et al., 2000). However, in other models of apoptosis, caspase inhibition did not influence cytochrome c release at all, therefore excluding such proteolytic feedback loops (Single et al., 1998). ...
Konstanz, Univ., Diss., 2000. Computerdatei im Fernzugriff.
... However, as an early event in apoptosis, protein release from mitochondria followed by translocation to different cellular compartments was demonstrated to result from mitochondrial dysfunction. Irreversible alterations of mitochondrial ultrastructures lead to the passive release of mitochondrial proteins (Single et al., 1998; Susin et al., 1996). Proapoptotic proteins that are released from the mitochondrial intermembrane space into the cytoplasm include cytochrome c (Liu et al., 1996), caspases (Susin et al., 1999a), AIF (Susin et al., 1996), the endonuclease G (endoG) (Li et al., 2001), Smac/Diablo (Adrain et al., 2001) and the serine protease Omi/HtrA2 (van Loo et al., 2002b). ...
Apoptosis is a controlled process of cell demise which plays an essential role in development, organ homeostasis and disease. Shrinkage and fragmentation of the nucleus are among the most striking morphological features of cell death by apoptosis, but little is known about the underlying mechanisms. The objective of the present study was to perform a comprehensive analysis of the nuclear proteome during apoptosis using a mass spectrometry based analysis platform. Subsequently, the candidate proteins should be characterised biochemically. The work was divided into three major parts. In the first part, a cell-free apoptosis reaction consisting of isolated nuclei and cytosolic extracts had to be established. First, CD95-L induced apoptosis in Jurkat T-cells was characterised. Then, cytosolic extracts from control cells as well as from cells undergoing CD95-L induced apoptosis were isolated using the detergent digitonin. These extracts were free from nuclear, mitochondrial and ER-proteins. Nuclei in high-purity were prepared from mouse liver. The successful induction of cell-free apoptosis was monitored by the caspase-dependent cleavage of the nuclear proteins PARP-1 and lamin B as well as by oligonucleosomal DNA fragmentation. In the second part, a method had to be established which was suitable for the relative quantification of the highly positive charged nuclear proteins. The method applied here is named Isotope Coded Protein Label (ICPL) and is based upon the differential isotopic labelling of all free amino groups, at lysines and at the N-terminus, followed by the identification and quantification of peptides using mass spectrometry. The proteins were labelled with two different isoforms of the reagent nicotinoyloxy-succinimide, a light (4 hydrogen, 4H) and a deuterated, heavy form (4 deuteriums, 4D). The chemical reaction of free amino groups with nicotinoyloxy-succinimide leads to their modification with nicotinic acid resulting in a neutralisation of the positively charged lysines and thus shifting the isoelectric point towards the acidic area. This shift improved the separation of nuclear alkaline proteins (> pI 10) in the following 2D-gel-electrophoresis that followed. In a high-throughput approach, nuclei from cell-free control reactions were labelled with the light ICPL-reagent (H4) and nuclei from apoptotic reactions were reacted with the heavy reagent (D4) before both samples were combined. Then, the complexity of this protein mixture was reduced by 2D-gel-electrophoresis. The most prominent 384 spots were excised from the stained gels, digested with trypsin and analysed by mass spectrometry. Three independent sets of experiments, consisting of freshly isolated components were performed. This led to the identification of 13 nuclear proteins the level of which were reproducibly altered between the control and apoptosis reaction. These identified proteins can be classified mainly into two groups of protein: proteins involved in chromatin organisation and architecture (HMG B1/B2, DEK, HCC-1, Histone H1.0/H1.2/H4) and proteins involved in RNA-transport and -metabolism (hnRNP A2/B1, hnRNP C1/C2, U2 snRNPA). Three further proteins were identified which did not belong to either group: hsp 70, lamin B2 and PP1. In the third part of this work, some of the identified proteins were characterised using biochemical and immunochemical methods, leading to the following conclusions: 1) Neither HMGB1 nor HMGB2 are proteolytically processed in apoptosis. In addition, no translocation between the nucleus and the cytoplasm takes place. Most probably, both proteins are post-translationally modified in the course of apoptosis. 2) Proteolysis of hnRNP A2/B1 was not observed in apoptosis. A translocation of hnRNP A2/B1 out of the nucleus explains most likely constant reduction of its protein level observed by the proteome approach. 3) No proteolytic cleavage of hnRNP C1/C2 was detected in apoptosis, whereas a translocation from the nucleus to the cytoplasm was observed. Additionally, post-translational modification may influence this translocation process. 4) Neither translocation from the nucleus nor proteolytic cleavage was observed for the proto-oncogene DEK. Nevertheless, the binding affinity of DEK toward DNA changed during apoptosis. This effect is dependent on caspases and on the protein kinase CK2 and is most likely due to changes of the phosphorylation status of DEK. Apoptose, die kontrollierte Form des Zelltodes, spielt eine zentrale Rolle in der Embryonalentwicklung, der Organ-Homöostase und zahlreichen Erkrankungen. Obwohl das Schrumpfen und die Fragmentierung des Zellkerns zu den auffälligsten morphologischen Veränderungen in der apoptotischen Zelle gehören, ist wenig über die Mechanismen, die hierzu führen, bekannt. Ziel dieser Arbeit war es, mit Hilfe eines neuartigen, auf Massenspektrometrie beruhenden Proteomansatzes eine umfassende Analyse der apoptose-bedingten Veränderungen der Proteine des Zellkerns durchzuführen und diese anschließend biochemisch zu charakterisieren. Die Untersuchungen ließen sich in drei experimentelle Teile gliedern. Im ersten Teil der Arbeit konnte eine zellfreie Apoptose-Reaktion, bestehend aus isolierten Zellkernen und zytosolischen Extrakten, etabliert werden. Es wurde zunächst die CD95-L stimulierte Apoptose in Jurkat T-Zellen zeitlich charakterisiert. Anschließend wurden zytosolische Extrakte sowohl aus Kontrollzellen als auch aus CD95-L-behandelten Zellen mittels des Detergens Digitonin gewonnen. Diese konnten frei von Kern-, Mitochondrien- und ER-Proteinen hergestellt werden. Hochreine Zellkerne wurden aus Mausleber isoliert. Die erfolgreiche Induktion der Apoptose unter zellfreien Bedingungen wurde anhand der Spaltung der nukleären Caspasesubstrate PARP-1 und Lamin B sowie der oligonukleosomalen DNA Fragmentierung verifiziert. Im zweiten Teil der Arbeit wurde eine Methode zum Vergleich zweier Proteome etabliert, mit der bevorzugt die stark positiv geladenen Kernproteine untersucht werden konnten. Die gewählte Methode, das Isotope Coded Protein Label (ICPL), beruht auf einer differentiellen isotopischen Markierung aller freien Aminogruppen, an Lysinen und dem N-Terminus, und der anschließenden Identifizierung und Quantifizierung der Proteine mittels Massen-spekrometrie. Hierzu werden die Proteine mit zwei unterschiedlichen Formen des Reagens Nicotinoyloxy-succinimid, einer leichten (4 Wasserstoffe - H4) bzw. einer schweren Form (4 Deuteriums - D4), derivatisiert. Die Reaktion von freien Aminogruppen mit Nicotinoyloxy-succinimid führt zu einer kovalenten Modifizierung mit Nikotinsäure, diese neutralisiert zusätzlich die positive Ladung der Lysine und führt so zu einer Verschiebung des isoelektrischen Punktes in den sauren Bereich. Dies ermöglichte eine verbesserte Trennung basischer Proteine (< pI 10) während der anschließenden 2D-Gel-Elektrophorese. In einem experimentellen Großansatz wurden zunächst Kernproteine aus zellfreien Kontrollreaktionen mit dem leichten ICPL-Reagenz (H4) und Proteine aus apoptotischen Reaktionen (30 und 60 min CD95-L) mit dem schweren ICPL-Reagenz (D4) markiert und anschließend vereinigt. Um die Proteinkomplexität zu reduzieren, wurde dieses Gemisch mittels 2D-Gel-Elektrophorese aufgetrennt. Die 384 deutlichsten Proteinspots aus dem gefärbten 2D-Gel wurden ausgestochen, tryptisch verdaut und massenspektrometrisch analysiert. Nach insgesamt dreimaliger Durchführung dieses Ansatzes mit jeweils neu und unabhängig voneinander isolierten Komponenten konnten insgesamt 13 Kernproteine identifiziert werden, deren Niveau zwischen kontroll- und apoptotischem Ansatz reproduzierbar verändert war. Die identifizierten Kandidaten ließen sich vorrangig zwei Proteingruppen zuordnen: Proteine, die an der Organisation und Architektur von Chromatin beteiligt sind (HMG B1/B2, DEK, HCC-1, Histone H1.0/H1.2/H4) und Proteine, die bei RNA-Transport und -Metabolismus eine Rolle spielen (hnRNP A2/B1, hnRNP C1/C2, U2 snRNPA). Zusätzlich konnten noch drei weitere Proteine identifiziert werden: Hsp 70, Lamin B2, PP1. Im dritten Teil der Arbeit wurden einige Kandidaten biochemisch charakterisiert, woraus folgende Teilergebnisse abgeleitet werden konnten: 1. Weder HMGB1 noch HMGB2 werden während der Apoptose proteolytisch gespalten, noch kommt es zu einer Translokation zwischen Kern und Zytoplasma. Wahrscheinlich ist, daß beide Proteine im Laufe der Apoptose posttranslational modifiziert werden. 2. Proteolyse von hnRNP A2/B1 in der Apoptose konnte nicht beobachtet weren. Eine Translokation von hnRNP A2/B1 vom Zellkern ins Zytoplasma ist vermutlich der Grund für die stetige Abnahme des Proteinlevels im Proteomansatz. 3. Eine proteolytische Spaltung von hnRNP C1/C2 in der Apoptose wurde nicht detektiert. Hingegen konnte eine Translokation von hnRNP C1/C2 während der Apoptose in das Zytoplasma beobachtet werden. Ein Einfluß von posttranslationalen Modifikationen auf diese Translokation ist nicht auszuschließen. 4. Weder eine Translokation aus dem Kern noch eine proteolytische Spaltung konnte für das Proto-Onkogen DEK in der Apoptose beobachtet werden. Vielmehr nimmt im Laufe der Apoptose die Bindungsaffinität von DEK für DNA ab, was wahrscheinlich auf eine Veränderung des Phosphorylierungsstatus des Proteins zurückzuführen ist. Außerdem ist diese apoptose-spezifische Veränderung der Bindungsaffinität abhängig von Caspasen und der Protein Kinase CK2.
... Permeability transition is a process observed during apoptosis, when the mitochondrial inner membrane, which is characterised by highly selective permeability to solutes, undergoes important changes causing characteristic features: swelling of mitochondria due to imbalanced ion distribution (Vander Heiden et al., 1997); dissipation of mitochondrial membrane potential (∆Ψ m ) ; induction of the release of proteins, that are localised in the matrix or intermembrane space of mitochondria ("proapoptotic" proteins, cytochrome c, apoptosis inducing factor (AIF) (Daugas et al., 2000;Lorenzo et al., 1999), adenylate kinase (Single et al., 1998), procaspases-2 and 9 (Susin et al., 1999a;Susin et al., 1996)), to the cytosol. ...
Oligodendrocytes are target of an autoimmune attack in demyelinating diseases. The crucial mediator causing oligodendrocyte loss and myelin degradation has not yet been defined. In order to investigate degeneration of murine oligodendrocytes in vitro, a culture enriched in oligodendrocytes was established. 30 of all cells developed to mature oligodendrocytes, expressing myelin oligodendrocyte glycoprotein. In this culture, no sensitivity of oligodendrocytes to inflammatory cytokines or to stimulation of CD95 was observed. Inhibition of transcription or translation did not sensitise oligodendrocytes for cytokines or CD95-ligation. This result finds correlates and contrary examples in the literature and indicates that these stimuli are not the only ones involved in oligodendrocyte damage in vivo. Althoug ganglioside levels are alterated in demyelination, gangliosides have not been studied before with regard to demyelination. Disialoganglioside GD3, but no structurally similar ganglioside, damaged oligodendrocytes with high cell type specificity. Parallel evaluation of chemically synthesised and bovine brain-derived GD3 ensured the purity of the effect excluding toxcitiy caused by contamination. GD3 induced apoptotic cell death in oligodendrocytes, causing chromatin condensation, phosphatidylserine exposure, cytochrome c release, activation of caspase-3 and maintenance of cellular integrity. The involvement of caspases seemed to be of minor importance in GD3 induced oligodendrocyte death, as their inhibition did not prevent cell death. GD3-caused oligodendrocyte death was inhibited in cells overexpressing human bcl-2. We conclude that mitochondria play an essential role in GD3 triggered apoptosis. That microglia synthesises GD3 in vivo was observed before. This study shows that microglia is able to release GD3 into the culture medium upon activation by inflammatory stimuli. This provides evidence for GD3 as a microglia-derived mediator during inflammation. Oligodendrocytes are target of an autoimmune attack in demyelinating diseases. The crucial mediator causing oligodendrocyte loss and myelin degradation has not yet been defined. In order to investigate degeneration of murine oligodendrocytes in vitro, a culture enriched in oligodendrocytes was established. 30 of all cells developed to mature oligodendrocytes, expressing myelin oligodendrocyte glycoprotein. In this culture, no sensitivity of oligodendrocytes to inflammatory cytokines or to stimulation of CD95 was observed. Inhibition of transcription or translation did not sensitise oligodendrocytes for cytokines or CD95-ligation. This result finds correlates and contrary examples in the literature and indicates that these stimuli are not the only ones involved in oligodendrocyte damage in vivo. Althoug ganglioside levels are alterated in demyelination, gangliosides have not been studied before with regard to demyelination. Disialoganglioside GD3, but no structurally similar ganglioside, damaged oligodendrocytes with high cell type specificity. Parallel evaluation of chemically synthesised and bovine brain-derived GD3 ensured the purity of the effect excluding toxcitiy caused by contamination. GD3 induced apoptotic cell death in oligodendrocytes, causing chromatin condensation, phosphatidylserine exposure, cytochrome c release, activation of caspase-3 and maintenance of cellular integrity. The involvement of caspases seemed to be of minor importance in GD3 induced oligodendrocyte death, as their inhibition did not prevent cell death. GD3-caused oligodendrocyte death was inhibited in cells overexpressing human bcl-2. We conclude that mitochondria play an essential role in GD3 triggered apoptosis. That microglia synthesises GD3 in vivo was observed before. This study shows that microglia is able to release GD3 into the culture medium upon activation by inflammatory stimuli. This provides evidence for GD3 as a microglia-derived mediator during inflammation.
... Furthermore, SOx (104 kD) was released from rat liver mitochondria incubated in cytosol (Fig. 3). Several intermembrane space proteins, including AK (Kohler et al., 1999;Samali et al., 1999;Single et al., 1998), a cytochrome c-GFP fusion protein (Heiskanen et al., 1999), AIF (Susin et al., 1997(Susin et al., , 1999b, and certain caspases (Mancini et al., 1998;Susin et al., 1999a) have been reported to be coreleased with cytochrome c in other systems, often in the absence of a permeability transition. ...
During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.
This chapter discusses the study of mitochondria in apoptosis. Accumulating evidence has shown that mitochondria also play an integral role in the death of a cell by apoptosis. Because of the mitochondrial cytochrome c release and its regulation by Bcl-2 family members appears to be a common mitochondrial change during apoptosis, the chapter focuses on the methods used to study cytochrome c release. The chapter also discusses methods to examine other mitochondrial parameters that may relate to the apoptosis, including the identification of proteins released from the mitochondria concomitantly with cytochrome c. Many inducers of apoptosis have shown to induce the release of cytochrome c from the mitochondria of cultured cells. Radiation, topoisomerase inhibitors, protein synthesis inhibitors, and protein kinase inhibitors are all commonly used apoptotic stimuli that trigger cytochrome c release. None of these toxins appears directly to induce the release of cytochrome c; rather they use signaling pathways that may involve Bax, Bid, or other proapoptotic Bcl-2 family members. Various toxins act directly on mitochondria to induce cytochrome c release. Some of these toxins induce a phenomenon known as permeability transition (PT) in isolated mitochondria.
Three-dimensional (3D) protein structures are of great importance for the rational design and structure-based discovery of specific inhibitors. Homology models of proteins play a significant role when no experimental three dimensional structures are available. LeishBase is a database of 347 Leishmania major proteins whose structures have been modeled by homology modeling. Detailed structural and biological details of Leishmania major proteins can provide a better understanding of their function and a way to identify potential targets involved in disease pathology. Also, an effort has been made to identify and prioritize targets among the 347 proteins present in this database. A list of top 12 targets is provided that may be probable Leishmania major drug targets. A collection of organized data under a common relational platform will give significant leverage in drug discovery effort against Leishmaniasis.
Apoptosis and necrosis may share initiation, signaling and execution mechanisms. Therefore, it is difficult to find unambiguous markers for the mode of cell death. The most clearly distinguishing features of apoptosis were the activation of caspases, and the selective uptake by phagocytes. The present data now show that phagocytosis is principally possible also in caspase-independent cell death. Jurkat cells induced to undergo necrosis by combining toxins with ATP-depleting agents were phagocytosed in a PS-dependent fashion barely after plasma membrane lysis. Lysed Jurkat cells showed a caspase- and Ca2+-independent PS exposure on their surface. In addition, such cells were preferred by macrophages and were removed more efficiently than apoptotic cells. Jurkat cell death triggered with staurosporine in the presence of the pan-caspase inhibitor zVAD-fmk was characterized by delayed necrosis and the absence of PS-translocation. Nevertheless, such cells where removed by macrophages before plasma membrane lysis occurred by mechanisms that might involve CD14. Caspase-independent PS exposure could be provoked by Ca2+ influx in Jurkat cells using an ionophore or in neurons by glutamate receptor stimulation, respectively. Ca2+-stressed cells were efficiently phagocytosed before membrane lysis. Protein kinase C inhibitors prevented both Ca2+-mediated PS exposure and phagocytosis. MCF7 and LoVo36 cells induced to undergo a caspase-independent apoptosis-like cell death by HSP70-depletion were efficiently removed by macrophages. Overexpression of caspase-3 in MCF7 cells did not influence this phagocytosis. The TNFa-response of macrophages to pro-inflammatory stimuli could be abolished by Jurkat and LoVo36 cells dying by classical apoptosis and ATP-depleted necrotic Jurkat cells, but not by heat-killed Jurkat cells or HSP70-depleted LoVo36-cells, suggesting that some necrotic cells may provoke pro-inflammatory responses.
The activation of the death receptors TNF-receptor-1 (TNF-R1) or CD95, respectively, is a hallmark of inflammatory or viral liver disease. The present thesis investigated the regulation of death receptor-triggered apoptosis by glutathione alterations using different murine in vivo models and an in vitro model of CD95-mediated apoptosis. The findings provide evidence that (i) a depletion of hepatic GSH levels precludes the execution of death receptor-mediated cell death; and that (ii) the molecular target affected by GSH depletion is the activation of caspase-8 at the CD95 DISC (death-inducing signaling complex) and possibly the activation of caspases via the apoptosome. The data suggest that a dysregulation of cell demise at chronically decreased hepatic GSH affects the pathological processes and thus possibly the outcome of liver disease.
Although much emphasis has been laid on the role of caspase in cell death, recent data indicate that, in many instances, mammalian cell death is caspase-independent. Thus, in many examples of mammalian cell death the 'decision' between death and life is upstream or independent of caspase activation. Similarly, it is unclear whether PCD of plants and fungi involves the activation of caspase-like enzymes, and no caspase-like gene has thus far been cloned in these phyla. Apoptosis inducing factor (AIF) is a new mammalian, caspase-independent death effector which, upon apoptosis induction, translocates from its normal localization, the mitochondrial intermembrane space, to the nucleus. Once in the nucleus, AIF causes chromatin condensation and large scale DNA fragmentation to fragments of approximately 50 kbp. The AIF cDNA from mouse and man codes for a protein which possesses three domains (i) an amino-terminal presequence which is removed upon import into the intermembrane space of mitochondria; (ii) a spacer sequence of approximately 27 amino acids; and (iii) a carboxyterminal 484 amino acid oxidoreductase domain with strong homology to oxidoreductases from other vertebrates (X. laevis), non-vertebrate animals (C. elegans, D. melanogaster), plants, fungi, eubacteria, and archaebacteria. Functionally important amino acids involved in the interaction with the prosthetic groups flavin adenine nucleotide and nicotinamide adenine nucleotide are strongly conserved between AIF and bacterial oxidoreductase. Several eukaryotes possess a similar domain organisation in their AIF homologs, making them candidates to be mitochondrial oxidoreductases as well as caspase-independent death effectors. The phylogenetic implications of these findings are discussed.
Programmed cell death (apoptosis) is used by multicellular organisms during development and to maintain homeostasis within mature tissues. One of the first genes shown to regulate apoptosis was bcl-2. Subsequently, a number of Bcl-2-related proteins have been identified. Despite overwhelming evidence that Bcl-2 proteins are evolutionarily conserved regulators of apoptosis, their precise biochemical function remains controversial. Three biochemical properties of Bcl-2 proteins have been identified: their ability to localize constitutively and/or inducibly to the outer mitochondrial, outer nuclear and endoplasmic reticular membranes, their ability to form heterodimers with proteins bearing an amphipathic helical BH3 domain, and their ability to form ion-conducting channels in synthetic membranes. The discovery that mitochondria can play a key part in the induction of apoptosis has focused attention on the role that Bcl-2 proteins may have in regulating either mitochondrial physiology or mitochondria-dependent caspase activation. Here we attempt to synthesize our current understanding of the part played by mitochondria in apoptosis with a consideration of how Bcl-2 proteins might control cell death through an ability to regulate mitochondrial physiology.
Mitochondrial membrane permeabilization is a rate-limiting step of cell death. This process is, at least in part, mediated by opening of the permeability transition pore complex (PTPC) Several soluble proteins from the mitochondrial intermembrane space and matrix are involved in the activation of catabolic hydrolases including caspases and nucleases. We therefore investigated the composition of a mixture of proteins released from purified mitochondria upon PTPC opening. This mixture was subjected to a novel proteomics/mass spectrometric approach designed to identify a maximum of peptides. Peptides from a total of 79 known proteins or genes were identified. In addition, 21 matches with expressed sequence tags (EST) were obtained. Among the known proteins, several may have indirect or direct pro-apoptotic properties. Thus endozepine, a ligand of the peripheral benzodiazepin receptor (whose occupation may facilitate mitochondrial membrane permeabilization), was found among the released proteins. Several proteins involved in protein import were also released, namely the so-called X-linked deafness dystonia protein (DDP) and the glucose regulated protein 75 (grb75), meaning that protein import may become irreversibly disrupted in mitochondria of apoptotic cells. In addition, a number of catabolic enzymes are detected: arginase 1 (which degrades arginine), sulfite oxidase (which degrades sulfur amino acids), and epoxide hydrolase. Although the functional impact of each of these proteins on apoptosis remains elusive, the present data bank of mitochondrial proteins released upon PTPC opening should help further elucidation of the death process.
Hepatocyte apoptosis is crucial in several forms of liver disease. Here, we examined in different models of murine liver injury whether and how metabolically induced alterations of hepatocyte ATP levels control receptor-mediated apoptosis. ATP was depleted either in primary hepatocytes or in vivo by various phosphate-trapping carbohydrates such as fructose. After the activation of the tumor necrosis factor (TNF) receptor or CD95, the extent of hepatocyte apoptosis and liver damage was quantified. TNF-induced cell death was completely blocked in ATP-depleted hepatocyte cultures, whereas apoptosis mediated by CD95 was enhanced. Similarly, acute TNF-induced liver injury in mice was entirely inhibited by ATP depletion with ketohexoses, whereas CD95-mediated hepatotoxicity was enhanced. ATP depletion prevented mitochondrial cytochrome c release, loss of mitochondrial membrane potential, activation of type II caspases, DNA fragmentation, and cell lysis after exposure to TNF. The extent of apoptosis inhibition correlated with the severity of ATP depletion, and TNF-induced apoptosis was restored when ATP was repleted by increasing the extracellular phosphate concentration. Our study demonstrates that TNF-induced hepatic apoptosis can be selectively and reversibly blocked upstream of mitochondrial dysfunction by ketohexose-mediated ATP depletion.
Caspase activation, exposure of phosphatidylserine (PS) on the outer surface of the plasma membrane, and rapid phagocytic removal of dying cells are key features of apoptosis. Nonapoptotic/necrotic modes of death occur independent of caspase activation, but the role of phagocytosis is largely unknown. To address this issue, we studied phagocytosis by human monocyte-derived macrophages (HMDM) and rat microglial cells. Target cells (Jurkat) were stimulated by several different methods that all caused caspase-independent death. First, we induced necrosis by combining toxins with ATP-depleting agents. Under these conditions, neither PS was exposed nor were such cells phagocytosed before their death. However, once the plasma membrane integrity was lost, the dead cells were rapidly and efficiently engulfed by HMDM. Next, we triggered Jurkat cell death with staurosporine in the presence of the pan-caspase inhibitor zVAD-fmk. Under these conditions, death occurred by delayed necrosis and without exposure of PS. Nevertheless, such lethally challenged cells were phagocytosed before the loss of membrane integrity. Finally, we triggered Ca2+ influx in Jurkat cells with an ionophore, or in neurons by glutamate receptor stimulation, respectively. In both models, PS was exposed on the cell surface. Ca2+-stressed cells were phagocytosed starting at 30 min after stimulation. Protein kinase C inhibitors prevented Ca2+-mediated PS exposure and phagocytosis. Essentially, similar phagocytosis data were obtained for all models with HMDM and microglia. We conclude that also cells dying nonapoptotically and independent of caspase activation may be recognized and removed before, or very quickly after, membrane lysis.
Apoptosis and necrosis are considered conceptually and morphologically distinct forms of cell death. Here, we report that demise of human T cells caused by two classic apoptotic triggers (staurosporin and CD95 stimulation) changed from apoptosis to necrosis, when cells were preemptied of adenosine triphosphate (ATP). Nuclear condensation and DNA fragmentation did not occur in cells predepleted of ATP and treated with either of the two inducers, although the kinetics of cell death were unchanged. Selective and graded repletion of the extramitochondrial ATP/pool with glucose prevented necrosis and restored the ability of the cells to undergo apoptosis. Pulsed ATP/depletion/repletion experiments also showed that ATP generation either by glycolysis or by mitochondria was required for the active execution of the final phase of apoptosis, which involves nuclear condensation and DNA degradation.
Bcl-xL, an antiapoptotic member of the Bcl-2 family, inhibits programmed cell death in a broad variety of cell types. Recent reports
have demonstrated that cytochromec is released from mitochondria during apoptosis and have suggested that this release may be a critical step in the activation
of proapoptotic caspases and subsequent cell death. Furthermore, it has been demonstrated that Bcl-2 can prevent the release
of cytochromec from mitochondria in cells triggered to undergo apoptosis. This has led to the hypothesis that the antiapoptotic effects
of Bcl-2 family members are due specifically to their ability to prevent cytochrome c release thus preventing subsequent cytochrome c-dependent caspase activation. In the present report, we use microinjection techniques to investigate the relationship between
cytochromec release, induction of apoptosis, and Bcl-xLactivity in intact cells. We demonstrate that microinjection of cytochrome c into the cytosol of human kidney 293 cells results in a dose-dependent induction of apoptosis. In contrast, MCF7 breast carcinoma
cells (stably transfected to express the Fas antigen CD95, and denoted MCF7F) that lack detectable levels of caspase 3 (CPP32),
are totally resistant to microinjection of cytochrome c. However, transfection of MCF7F cells with an expression plasmid coding for pro-caspase 3, but not other pro-caspases, restores
cytochrome c sensitivity. Although MCF7F cells are insensitive to cytochrome c microinjection, they rapidly undergo apoptosis in a caspase-dependent manner in response to either tumor necrosis factor
or anti-Fas plus cycloheximide, and these deaths are strongly inhibited by Bcl-xL expression. Furthermore, microinjection of cytochrome c does not overcome these antiapoptotic effects of Bcl-xL. Our results support the concept that the release of cytochrome c into the cytoplasm can promote the apoptotic process in cells expressing pro-caspase 3 but that cytochromec release is not sufficient to induce death in all cells. Importantly, the ability of Bcl-xL to inhibit cell death in the cytochrome c-insensitive MCF7F cells cannot be due solely to inhibition of cytochrome c release from mitochondria.
The major heat shock protein, Hsp70, is an effective inhibitor of apoptosis. To study its mechanism of action, we created tumor cell lines with altered Hsp70 levels. The expression levels of Hsp70 in the cells obtained correlated well with their survival following treatments with tumor necrosis factor, staurosporine and doxorubicin. Surprisingly, the surviving Hsp70-expressing cells responded to the apoptotic stimuli by activation of stress-activated protein kinases, generation of free radicals, early disruption of mitochondrial transmembrane potential, release of cytochrome c from mitochondria and activation of caspase-3-like proteases in a manner essentially similar to that of the dying cells with low Hsp70 levels. However, Hsp70 inhibited late caspase-dependent events such as activation of cytosolic phospholipase A2 and changes in nuclear morphology. Furthermore, Hsp70 conferred significant protection against cell death induced by enforced expression of caspase-3. Thus, Hsp70 rescues cells from apoptosis later in the death signaling pathway than any known anti-apoptotic protein, making it a tempting target for therapeutic interventions.
A cell-free system based on cytosols of normally growing cells is established that reproduces aspects of the apoptotic program in vitro. The apoptotic program is initiated by addition of dATP. Fractionation of cytosol yielded a 15 kDa protein that is required for in vitro apoptosis. The absorption spectrum and protein sequence revealed that this protein is cytochrome c. Elimination of cytochrome c from cytosol by immunodepletion, or inclusion of sucrose to stabilize mitochondria during cytosol preparation, diminished the apoptotic activity. Adding back cytochrome c to the cytochrome c-depleted extracts restored their apoptotic activity. Cells undergoing apoptosis in vivo showed increased release of cytochrome c to their cytosol, suggesting that mitochondria may function in apoptosis by releasing cytochrome c.
Cell death, a scheduled event during development and tissue turnover, or the ultimate consequence of toxic or pathologic insults seems to involve a relatively limited number of execution pathways. This reflects the evolution of an organized sequence of events perhaps converging onto final common pathways that are used to dispose of unwanted or injured cells. In many cases, the ordered execution of this internal death program leads to typical morphological and biochemical changes that have been termed apoptosis. Apoptosis, often equated with developmental or programmed cell death, has been opposed to unscheduled or accidental cell lysis/necrosis. However, increasing evidence suggests that the two forms of cell demise share similar characteristics, at least in the signaling and early progression phase. Recent studies have shown that, when the intensity of the insult is very high and/or when ATP generation is deficient, cells fail to execute the ordered changes ensuing in apoptosis. Then cell lysis/necrosis supervenes before the processes leading to nuclear condensation and exposure of surface molecules can be completed. Thus, apoptosis and necrosis seem to represent only different shapes of cell demise, resulting from a more or less complete execution of the internal death program.
We report here the purification of the third protein factor, Apaf-3, that participates in caspase-3 activation in vitro. Apaf-3 was identified as a member of the caspase family, caspase-9. Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. Activated caspase-9 in turn cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminished caspase-3 activation. Mutation of the active site of caspase-9 attenuated the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade that is triggered by cytochrome c and dATP.
Apoptosis is a type of cell death whose morphological appearance relies on the activation of caspase-family cysteine proteases. Recently, it has become clear that inhibition of caspases does not always prevent irreversible loss of cellular function, although it does prevent the acquisition of apoptotic morphology. Alterations in mitochondrial membrane structure and function can occur in a caspase-independent fashion and have a higher predictive value for cell death than caspase activation. Here, Douglas Green and Guido Kroemer argue that caspases might have a dual function in the apoptotic process: first, as signal-transduction molecules that act as facultative inducers of mitochondrial membrane changes, and, second, as processing enzymes that orchestrate the apoptotic phenotype. They propose a model for initiation of apoptosis in which mitochondria and caspases engage in a self-amplifying pathway of mutual activation.
The central executioners of apoptosis: caspases or mitochondria?
- D Green
- G Kroemer
Green D and Kroemer G (1998) The central executioners of apoptosis: caspases or
mitochondria? Trends Cell Biol. 8: 267 ± 271
Cellspecific induction of apoptosis by microinjection of cytochrome c -Bcl-x L has activity independent of cytochrome c release
- F Li
- A Srinivasan
- Y Wang
- Rc Armstrong
- Kj Tomaselli
- Lc Fritz
Li F, Srinivasan A, Wang Y, Armstrong RC, Tomaselli KJ and Fritz LC (1997) Cellspecific induction of apoptosis by microinjection of cytochrome c -Bcl-x L has
activity independent of cytochrome c release. J. Biol. Chem. 272: 30299 ± 30305