Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Apaf-1 plays a crucial role in the cytochrome c/dATP-dependent activation of caspase-9 and -3. We found that the human myeloid leukemic K562 cells were more resistant to cytochrome c-induced activation of caspase-9 and -3 in a cell-free system compared with the human T-lymphoblastic subclone CEM/VLB(100) cells. Apaf-1 cDNA sequencing revealed an additional insert of 11 aa between the CARD and CED-4 (ATPase) domains in K562 cells, which was identical to the sequence of Apaf-1XL. Immunoprecipitation of Apaf-1 with caspase-9 after a cell-free reaction demonstrated that Apaf-1XL in the K562 cell line showed a lower binding ability to caspase-9 compared with Apaf-1L protein. The resistance of K562 cells to cytochrome c-dependent apoptosis may be partly due to this Apaf-1XL form. These results suggest that the additional insert between CARD and CED-4 domains might affect Apaf-1 recruitment of caspase-9 during apoptosis.
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PMID:Apaf-1XL is an inactive isoform compared with Apaf-1L. 1126 2

We have found a novel alternative splicing product of the apoptotic protease activating factor 1 (APAF-1), termed APAF-1-ALT, in the LNCaP human prostate cancer cell line. APAF-1-ALT harbors the caspase recruitment domain and an incomplete CED-4 like/ATPase domain, but lacks the WD-40 repeat units. The LNCaP cell expressed the full-length APAF-1 weakly and APAF-1-ALT rather abundantly, especially after mycoplasma infection. LNCaP underwent a retarded DNA damage-induced apoptosis, which was independent of caspase 9 activity. APAF-1-ALT functioned less effectively in inducing apoptosis than did APAF-1-XL, the full-length APAF-1, in transient transfection. Moreover, APAF-1-ALT interfered with APAF-1-XL's ability to induce apoptosis in transient double transfection experiment. These results indicate that APAF-1-ALT is a molecule that is deficient and impeded for mediating apoptosis and that it may contribute to the resistance to DNA damage-induced treatment observed in LNCaP.
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PMID:APAF-1-ALT, a novel alternative splicing form of APAF-1, potentially causes impeded ability of undergoing DNA damage-induced apoptosis in the LNCaP human prostate cancer cell line. 1280 98

The key role of mitochondria in the apoptotic process is well understood, but not many data are available regarding the specific role of mitochondrial DNA mutations in determining cell fate. We investigated whether two mitochondrial DNA mutations (L217R and L156R) associated with maternally-inherited Leigh syndrome may play a specific role in triggering the apoptotic cascade. Considering that different nuclear genetic factors may influence the expression of mtDNA mutations, we used a 143BTK(-) osteosarcoma cell line deprived from its own mtDNA in order to insert mutated mtDNAs. Analysis of mitochondrial features in these cybrids indicated that both mitochondrial DNA mutations produced evidence of biochemical, functional and ultrastructural modifications of mitochondria, and that these modifications were associated with an increased apoptotic proneness. Cybrids were highly susceptible to two different apoptotic stimuli, tumour necrosis factor-alpha and Staurosporin. The mechanism involved was the mitochondrial 'intrinsic' pathway, i.e. the caspase 9-driven cascade. More importantly, our results also indicated that the polarization state of the mitochondrial membrane, i.e. a constitutive hyperpolarization detected in cybrid clones, played a specific role. Interestingly, the different effects of the two mutations in terms of susceptibility to apoptosis probably reflect the deeper bioenergetic defect associated with the L217R mutation. This work provides the first evidence that hyperpolarization of mitochondria may be a 'risk factor' for cells with a deep ATPase dysfunction, such as cells from patients with maternally-inherited Leigh syndrome.
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PMID:Maternally-inherited Leigh syndrome-related mutations bolster mitochondrial-mediated apoptosis. 1522 5

Normal human ectocervical epithelial (hECE) cells undergo apoptosis in culture. Baseline apoptosis could be increased by shifting cells to serum-free medium and blocked by lowering extracellular calcium. Treatment with the ATPase apyrase attenuated baseline apoptosis, suggesting that extracellular ATP and purinergic mechanisms control the apoptosis. Treatment with ATP and the P2X7 receptor analog 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) increased apoptosis significantly, in a time- and dose-related manner. The threshold of ATP effect was 0.5 microM in hECE cells and approximately 1 microM in CaSki cancer cells. The apoptotic effect of BzATP was additive in part to that of tumor necrosis factor (TNF)-alpha, and it could be attenuated by lowering extracellular calcium and by treatment with the caspase-9 inhibitor Leu-Glu-His-Asp-O-methyl-fluoromethylketone (LEHD-FMK). Treatment with BzATP activated caspase-9, and, in contrast to TNF-alpha, it had only a mild effect on caspase-8. Both BzATP and TNF-alpha activated caspase-3, suggesting that BzATP activates predominantly the mitochondrial apoptotic pathway. Both hECE and CaSki cells secrete ATP into the extracellular fluid, and mean ATP activity in conditioned medium was approximately 0.5 microM, which is in the range of values that suffice to activate the P2X7 receptor. On the basis of these findings we propose a novel autocrine-paracrine mechanism of cervical cell apoptosis that operates by P2X7 receptor control of cytosolic calcium and utilizes the mitochondrial apoptotic pathway.
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PMID:P2X7 receptor-mediated apoptosis of human cervical epithelial cells. 1526 6

In the HeLa tumor cell line, we studied the characteristics of the dual effect of digitalis compounds on cell growth (proliferation and death). In addition, we explored whether both effects occur by means of the same mechanism. HeLa cell cultures were exposed to increasing concentrations (0.01 nM-10 microM) of ouabain, strophantidin, digoxin, and digoxigenin at 24-96 h intervals. Cell growth in treated cultures was compared with cell growth under nontreated conditions. Additionally, we studied changes in nuclear morphology, as well as in genomic DNA degradation, cytochrome c release, and caspase-9 and -3 presence and processing induced by toxic concentrations of digitalis. Digitalis compounds increased HeLa cell number when exposed to concentrations <10 nM during a 48 h period. Ethacrynic acid (a nonsteroid inhibitor for Na+/K+-ATPase) did not induce cell growth at these concentrations. Digitalis concentrations >10 nM induced cell death in a concentration- and exposure period-dependent fashion. Changes in nuclear morphology, DNA fragmentation, mitochondrial cytochrome c release, and proteolytic processing of caspases-9 and -3, suggest apoptotic cell death. The IC50 for the inducing effect of apoptosis by ouabain at 96 h was 18 nM and corresponds with the IC50 for the Na+/K+-ATPase inhibition in HeLa cells. In conclusion, the dual effect of digitalis compounds on HeLa cells growth is concentration and time-dependent. The apoptosis-inducing effect correlates with inhibition of Na+/K+-ATPase. Proliferation does not appear to be mediated through this pathway. The apoptosis-induction pathway is possibly cytochrome c-dependent.
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PMID:Proliferation and apoptosis of HeLa cells induced by in vitro stimulation with digitalis. 1650 6

Oxidative stress plays an important role in mediating ventricular remodeling and dysfunction in heart failure (HF), but its mechanism of action has not been fully elucidated. In this study we determined whether a combination of antioxidant vitamins reduced myocyte apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular (SR) Ca2+ ATPase downregulation in HF after myocardial infarction (MI) and whether these effects were associated with amelioration of left ventricular (LV) remodeling and dysfunction. Vitamins (vitamin C 300 mg and vitamin E 300 mg) were administered to rabbits 1 week after MI or sham operation for 11 weeks. The results showed that MI rabbits exhibited cardiac dilation and LV dysfunction measured by fractional shortening and the maximal rate of pressure rise (dP/dt), an index of contractility. These changes were associated with elevation of oxidative stress, decreases of mitochondrial Bcl-2 and cytochrome c proteins, increases of cytosolic Bax and cytochrome c proteins, caspase 9 and caspase 3 activities and myocyte apoptosis, and downregulation of beta-adrenergic receptor sensitivity and SR Ca2+ ATPase. Combined treatment with vitamins C and E diminished oxidative stress, increased mitochondrial Bcl-2 protein, decreased cytosolic Bax, prevented cytochrome c release from mitochondria to cytosol, reduced caspase 9 and caspase 3 activities and myocyte apoptosis, blocked beta-adrenergic receptor desensitization and SR Ca2+ ATPase downregulation, and attenuated LV dilation and dysfunction in HF after MI. The results suggest that antioxidant therapy may be beneficial in HF.
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PMID:Vitamins C and E attenuate apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular Ca2+ ATPase downregulation after myocardial infarction. 1667 21

In previous studies, we have shown that cerebral hypoxia results in increased activity of caspase-9, the initiator caspase, and caspase-3, the executioner of programmed cell death. We have also shown that cerebral hypoxia results in high affinity Ca2+-ATPase-dependent increase in nuclear Ca2+ -influx in the cerebral cortex of newborn piglets. The present study tests the hypothesis that inhibiting nuclear Ca2+ -influx by pretreatment with clonidine, an inhibitor of high affinity Ca2+ -ATPase, will prevent the hypoxia-induced increase in caspase-9 and caspase-3 activity in the cerebral cortex of newborn piglets. Thirteen newborn piglets were divided into three groups, normoxic (Nx, n=4), hypoxic (Hx, n=4), and hypoxic treated with clonidine (100 mg/kg) (Hx-Cl, n=5). Anesthetized, ventilated animals were exposed to an FiO2 of 0.21 (Nx) or 0.07 (Hx) for 60 min. Cerebral tissue hypoxia was documented biochemically by determining levels of ATP and phosphocreatine (PCr). Caspase-9 and -3 activity were determined spectrofluoro-metrically using specific fluorogenic synthetic substrates. ATP (micromoles/g brain) was 4.6 +/- 0.3 in Nx, 1.7 +/- 0.4 in Hx (P < 0.05 vs. Nx), and 1.5 +/- 0.2 in Hx-Cl (P < 0.05 vs. Nx). PCr (micromoles/g brain) was 3.6 +/- 0.4 in Nx, 1.1 +/- 0.3 in Hx (P < 0.05 vs. Nx), and 1.0 +/- 0.2 in Hx-Cl (P < 0.05 vs. Nx). Caspase-9 activity (nmoles/mg protein/h) was 0.548 +/- 0.0642 in Nx and increased to 0.808 +/- 0.080 (P < 0.05 vs. Nx and Hx-Cl) in the Hx and 0.562 +/- 0.050 in the Hx-Cl group (p = NS vs. Nx). Caspase-3 activity (nmoles/mg protein/h) was 22.0 +/- 1.3 in Nx and 32 +/- 6.3 in Hx (P < 0.05 vs. Nx) and 18.8 +/- 3.2 in the Hx-Cl group (P < 0.05 vs. Hx). The data demonstrate that clonidine administration prior to hypoxia prevents the hypoxia-induced increase in the activity of caspase-9 and caspase-3. We conclude that the high afinity Ca2+ -ATPase-dependent increased nuclear Ca2+ during hypoxia results in increased caspase-9 and caspase-3 activity.
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PMID:Mechanism of activation of caspase-9 and caspase-3 during hypoxia in the cerebral cortex of newborn piglets: the role of nuclear Ca2+ -influx. 1726 55

Apoptotic protease activating factor-1 (Apaf-1) is a critical regulator of apoptosis and a crucial part of the apoptosome that is assembled in response to several cellular stresses like hypoxia. We have previously shown that hypoxia results in increased influx of nuclear Ca(2+) and increased expression of nuclear apoptotic proteins. The present study investigates that Apaf-1 is expressed during hypoxia in the cerebral cortex of newborn piglets and that administration of clonidine prevents the hypoxia induced increase expression of Apaf-1. Studies were conducted in 19 newborn piglets, 6 normoxic (Nx), 7 hypoxic (Hx FiO(2) of 0.05-0.07 for 1h) and 6 clonidine-treated hypoxic (Hx-Clo) piglets. Tissue hypoxia was confirmed biochemically by determining the levels of high energy phosphates ATP and phosphocreatine (PCr). Neuronal nuclei, mitochondrial membranes and cytosolic fractions were isolated and separated by 12% SDS-PAGE and probed with specific antibodies to Apaf-1. The expression of Apaf-1 in neuronal nuclei was 48.86+/-5.27 in Nx, 108.43+/-6.37 in Hx and 78.53+/-7.00 in Hx-Clo. The Apaf-1 expression of in mitochondrial fraction was 72.73+/-11.76 in Nx, 132.27+/-16.15 in Hx and 85.17+/-5.64 in Hx-Clo. Similarly, the expression of Apaf-1 in cytosolic fraction was 86.79+/-6.97 in Nx, 193.95+/-15.41 in Hx and 111.07+/-7.91 in Hx-Clo. In summary, the results show that hypoxia results in increased expression of Apaf-1 proteins in neuronal nuclear, mitochondrial and cytosolic fractions. Administration of a high affinity Ca(2+)-ATPase, prevented the hypoxia induced increased expression of Apaf-1 protein, suggesting that the hypoxia-induced increased expression of Apaf-1 proteins is nuclear Ca(2+)-influx mediated. We conclude that cerebral hypoxia-induced increase in Apaf-1 protein will lead to increased activation of procaspase-9 to caspase-9 in the cytosolic compartment leading to a cascade of hypoxic neuronal death.
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PMID:Mechanisms of expression of apoptotic protease activating factor-1 (Apaf-1) in nuclear, mitochondrial and cytosolic fractions of the cerebral cortex of newborn piglets. 1727 90

Calcium ion is essential for cellular functions including signal transduction. Uncontrolled calcium stress has been linked causally to a variety of neurodegenerative diseases. Thapsigargin, which inhibits Ca(2+)-ATPase in the endoplasmic reticulum (ER) and blocks the sequestration of calcium by the ER, induced apoptotic cell death (chromatin condensation and nuclear fragmentation) accompanied by GRP78 protein expression and caspase-3 activation in rat fetal cortical neurons (days in vitro 9-10). Blockade of N-methyl-D-aspartate (NMDA) receptors with NMDA antagonists induced apoptosis without GRP78 protein expression. Apoptosis accompanied both caspase-9 and caspase-3 activation. We then examined whether GSK-3 is involved in thapsigargin-induced cell death by using GSK-3 inhibitors. We assayed the effects of selective GSK-3 inhibitors, SB216763, alsterpaullone and 1-azakenpaullone, on thapsigargin-induced apoptosis. These inhibitors completely protected cells from thapsigargin-induced apoptosis. In addition, GSK-3 inhibitors inhibited caspase-9 and caspase-3 activation accompanied by thapsigargin-induced apoptosis. These results suggest that thapsigargin induces caspase-dependent apoptosis mediated through GSK-3beta activation in rat cortical neurons.
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PMID:Caspase-dependent apoptosis induced by thapsigargin was prevented by glycogen synthase kinase-3 inhibitors in cultured rat cortical neurons. 1740 51

We have shown that hypoxia results in increased influx of nuclear Ca++ and increased expression of nuclear apoptotic proteins. The present study tests the hypothesis that hypoxia alters the distribution of pro-apoptotic proteins Bad and Bax, and the anti-apoptotic proteins Bcl-xl, and Bcl-2 in the nuclear, mitochondrial and cytosolic compartments of the cerebral cortex of newborn piglets and the administration of Clonidine, an inhibitor of high affinity nuclear Ca++ -ATPase, will prevent the hypoxia-induced increase in apoptotic proteins' expression. Studies were conducted in 19 newborn piglets, 6 normoxic (Nx), 7 hypoxic and 6 Clonidine-treated hypoxic (Hx-Clo). Tissue hypoxia was documented biochemically by measuring cerebral tissue ATP and phosphocreatine (PCr) levels. Bax and Bad protein expression increased in all the three compartments during hypoxia, while there was no significant change in the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl. In Clonidine pretreated hypoxic group, the hypoxia-induced increased expression of pro-apoptotic proteins Bad and Bax was prevented in all the three fractions. We conclude that hypoxia results in increased expression of pro-apoptotic proteins in nuclear, mitochondrial and cytosolic compartments and that the increased expression of pro-apoptotic proteins during hypoxia is nuclear Ca++ -influx-dependent. We propose that during hypoxia the increased ratio of (pro-apoptotic Bad and Bax/anti-apoptotic Bcl-xl and Bcl-2) in all the three compartments, will lead to altered mitochondrial and nuclear membrane permeability as well as caspase-9 activation in the cytosolic compartment.
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PMID:Effect of hypoxia on expression of apoptotic proteins in nuclear, mitochondrial and cytosolic fractions of the cerebral cortex of newborn piglets: the role of nuclear Ca++ -influx. 1829 86


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