Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P42574 (caspase-3)
 45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutamate toxicity in traumatic brain injury, ischemia, and Huntington's disease causes cortical neuron death and dysfunction. We tested the efficacy of calpain and caspase-3 inhibitors alone and in combination to prevent neuronal death and preserve electrophysiological functions in rat primary cortical neurons following glutamate exposure. Cortical neurons exposed to 0.5 microM glutamate for 24 h committed mostly apoptotic death as determined by Wright staining and ApopTag assay. Levels of expression, formation of active forms, and activities of calpain and caspase-3 were increased following glutamate exposure. Also, in situ double labeling identified conformationally active caspase-3-p20 fragment and chromatin condensation in apoptotic neurons. Pretreatment of cortical neurons with 0.2 microM N-benzyloxylcarbonyl-Leu-Nle-aldehyde (calpain-specific inhibitor) and 100 microM N-benzyloxylcarbonyl-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-fluoromethyl ketone (caspase-3-specific inhibitor) provided strong neuroprotection. Standard patch-clamp techniques were used to measure the whole-cell currents associated with Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors. The lack of a change in capacitance indicated that neurons treated with inhibitor(s) plus glutamate did not undergo apoptotic shrinkage and maintained the same size as the control neurons. Whole-cell currents associated with Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors were similar in amplitude and activation/inactivation kinetics for cells untreated and treated with inhibitor(s) and glutamate. Spontaneous synaptic activity as observed by miniature end-plate currents was also similar. Prevention of glutamate-induced apoptosis by calpain and caspase-3 inhibitors preserved normal activities of crucial ion channels such as Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors in neurons. Our studies strongly imply that calpain and caspase-3 inhibitors may also provide functional neuroprotection in the animal models of traumatic brain injury and neurodegenerative diseases.
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PMID:Inhibition of calpain and caspase-3 prevented apoptosis and preserved electrophysiological properties of voltage-gated and ligand-gated ion channels in rat primary cortical neurons exposed to glutamate. 1650 8

Previous studies have shown that cerebral hypoxia results in increased activity of caspase-9, a key initiator of programmed cell death. We have also shown increased nitric oxide (NO) free radical generation during hypoxia in the cerebral cortex of newborn piglets. The present study tests the hypothesis that hypoxia-induced increase in caspase-9 activity in the cerebral cortex of newborn piglets is mediated by NO derived from neuronal nitric oxide synthase (nNOS). To test this hypothesis, cytosolic caspase-9 activity was determined in 15 newborn piglets divided into three groups: normoxic (Nx, n=5), hypoxic (Hx, n=5), and Hx pretreated with 7-nitroindazole sodium salt (7-NINA), a selective nNOS inhibitor, 1mg/kg, i.p., 1h prior to hypoxia (Hx+7NI, n=5). The hypoxic piglets were exposed to an FiO(2) of 0.06 for 1h. Tissue hypoxia was documented by ATP and phosphocreatinine (PCr) levels. The cytosolic fraction was obtained from the cerebral cortical tissue following centrifugation at 100,000 x g for 1h and caspase-9 activity was assayed using Ac-Leu-Glu-His-Asp-amino-4-methyl coumarin, a specific fluorogenic substrate for caspase-9. Caspase-9 activity was determined spectroflourometrically at 460 nm using 380 nm as excitation wavelength. ATP levels (micromol/g brain) were 4.35+/-0.21 in the Nx 1.43+/-0.28 in the Hx (p<0.05 versus Nx), and 1.73+/-0.33 in the Hx+7-NINA group (p<0.05 versus Nx, p=NS versus Hx). PCr levels (micromol/g brain) were 3.80+/-0.26 in the Nx, 0.96+/-0.20 in the Hx (p<0.05 versus Nx), and 1.09+/-0.39 in the Hx+7 NINA group (p<0.05 versus Nx, p=NS versus Hx). Cytosolic caspase-9 activity (nmol/mg protein/h), increased from 1.27+/-0.15 in the Nx to 2.13+/-0.14 in the Hx (p<0.05 versus Nx) compared to 1.10+/-0.21 in the Hx+7-NINA group (p<0.05 versus Hx, p=NS versus Nx). Caspase-3 activity (nmol/mg protein/h) also increased from 9.39+/-0.73 in Nx to 18.94+/-3.64 in Hx (p<0.05 versus Nx) compared to 8.04+/-1.05 in the Hx+7-NINA group (p<0.05 versus Hx, p=NS versus Nx). The data show that administration of 7-NINA, an nNOS inhibitor, prevented the hypoxia-induced increase in caspase-9 activity that leads to increase in caspase-3 activity. Since nNOS inhibition blocked the increase in caspase-9 activity during hypoxia, we conclude that hypoxia-induced increase in caspase-9 activity is mediated by nNOS derived NO. We propose that the NO generated during hypoxia leads to activation of caspase-9 and results in initiation of caspase-cascade-dependent hypoxic neuronal death.
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PMID:Effect of neuronal nitric oxide synthase inhibition on caspase-9 activity during hypoxia in the cerebral cortex of newborn piglets. 1654 6

The pathway of interferon-gamma (IFN-gamma)-induced suppression in tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis of fibroblast-like synovial cells (FLS) was investigated. rTRAIL triggered FLS apoptosis in a type II cell death manner, whereas IFN-gamma pretreatment significantly inhibited TRAIL-mediated apoptosis. As disruption of mitochondrial transmembrane potential (DeltaPsim), Leu-Glu-His-Asp ase (IETD ase) activity, and the appearance of hypodiploid DNA + cells were markedly suppressed in IFN-gamma-treated FLS in response to TRAIL, IFN-gamma-induced suppression was supposed to achieve at upstream of caspase-8. IFN-gamma rapidly phosphorylated signal transducers and activators of transcription 1 (STAT1), STAT3, and STAT6 as well as ERK, whereas enhanced neither phosphorylation of Akt nor nuclear translocation of nuclear factor kappaB (NF-kappaB) p65. Janus kinase (JAK)-induced phosphorylation of STAT1/3/6, which acts at translational regulation, seemed to be crucial because chemical inhibition of JAK as well as cycloheximide (CHX) abolished both the phosphorylation of STAT1/3/6 and the IFN-gamma-induced inhibitory effect. Although ERK was phosphorylated through IFN-gamma, chemical inhibition of ERK by PD98059 did not abolish the IFN-gamma-induced inhibitory effect. The authors tried to determine the responsible molecules; however, expression of TRAIL receptors; pro-caspase-3/-8/-9; Fas-associated death domain protein (FADD); tumor necrosis factor receptor 1-associated death domain protein (TRADD); silencer of death domain (SODD); FLICE inhibitory protein (FLIP); and Bcl-2, Bcl-xL, and Bax in FLS was not modulated by IFN-gamma. Although the authors have not yet clarified the precise mechanism, these data suggest that IFN-gamma/JAK/STAT pathway, which is supposed to be activated in inflammatory rheumatoid arthritis (RA) synovial tissues, contributes to form apoptosis resistance phenotype of the cells in situ, leading to a marked increase in cellularity of synovial cells.
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PMID:Significant inhibition of TRAIL-mediated fibroblast-like synovial cell apoptosis by IFN-gamma through JAK/STAT pathway by translational regulation. 1658 46

Apoptosis signaling pathways are implicated in the pathogenesis of temporal lobe epilepsy (TLE), but the role of endoplasmic reticulum (ER) stress and ER-localized apoptosis signaling components remains largely unexplored. Presently, we investigated ER stress and ER localization of proapoptotic Bcl-2 family members and initiator and effector caspases in resected hippocampus from patients with intractable TLE and compared findings with autopsy controls. Hippocampal immunoreactivity for KDEL (Lys-Asp-Glu-Leu), a motif in ER stress chaperones glucose-regulated proteins 78 and 94, and calnexin, was significantly higher in TLE hippocampus compared with controls. The ER-containing microsomal fraction in control brain contained Bid, Bim, and caspase 3, whereas Bad and caspases 6, 7, and 9 were very low or absent. In contrast, caspases 6, 7, and 9 were present within the microsomal fraction of TLE brain. Furthermore, cleaved caspases 7 and 9 were detected in TLE samples but not controls, and KDEL-expressing neurons coexpressed cleaved caspase 9. Potentially adaptive changes were also detected, including lowered Bim levels in this fraction, and binding of caspase 7 to the X-linked inhibitor of apoptosis protein. These data suggest seizures may induce ER stress and trigger proapoptotic signaling pathways in the ER that are counteracted by antiapoptotic signals in chronic human TLE.
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PMID:Endoplasmic reticulum stress and apoptosis signaling in human temporal lobe epilepsy. 1665 83

The molecular basis for the substrate specificity of human caspase-3 has been investigated using peptide analog inhibitors and substrates that vary at the P2, P3, and P5 positions. Crystal structures were determined of caspase-3 complexes with the substrate analogs at resolutions of 1.7 A to 2.3 A. Differences in the interactions of caspase-3 with the analogs are consistent with the Ki values of 1.3 nM, 6.5 nM, and 12.4 nM for Ac-DEVD-Cho, Ac-VDVAD-Cho and Ac-DMQD-Cho, respectively, and relative kcat/Km values of 100%, 37% and 17% for the corresponding peptide substrates. The bound peptide analogs show very similar interactions for the main-chain atoms and the conserved P1 Asp and P4 Asp, while interactions vary for P2 and P3. P2 lies in a hydrophobic S2 groove, consistent with the weaker inhibition of Ac-DMQD-Cho with polar P2 Gln. S3 is a surface hydrophilic site with favorable polar interactions with P3 Glu in Ac-DEVD-Cho. Ac-DMQD-Cho and Ac-VDVAD-Cho have hydrophobic P3 residues that are not optimal in the polar S3 site, consistent with their weaker inhibition. A hydrophobic S5 site was identified for caspase-3, where the side-chains of Phe250 and Phe252 interact with P5 Val of Ac-VDVAD-Cho, and enclose the substrate-binding site by conformational change. The kinetic importance of hydrophobic P5 residues was confirmed by more efficient hydrolysis of caspase-3 substrates Ac-VDVAD-pNA and Ac-LDVAD-pNA compared with Ac-DVAD-pNA. In contrast, caspase-7 showed less efficient hydrolysis of the substrates with P5 Val or Leu compared with Ac-DVAD-pNA. Caspase-3 and caspase-2 share similar hydrophobic S5 sites, while caspases 1, 7, 8 and 9 do not have structurally equivalent hydrophobic residues; these caspases are likely to differ in their selectivity for the P5 position of substrates. The distinct selectivity for P5 will help define the particular substrates and signaling pathways associated with each caspase.
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PMID:Structural and kinetic analysis of caspase-3 reveals role for s5 binding site in substrate recognition. 1678 34

Direct IAP binding protein with low pI/second mitochondrial activator of caspases, HtrA2/Omi and GstPT/eRF3 are mammalian proteins that bind via N-terminal inhibitor of apoptosis protein (IAP) binding motifs (IBMs) to the baculoviral IAP repeat (BIR) domains of IAPs. These interactions can prevent IAPs from inhibiting caspases, or displace active caspases, thereby promoting cell death. We have identified several additional potential IAP antagonists, including glutamate dehydrogenase (GdH), Nipsnap 3 and 4, CLPX, leucine-rich pentatricopeptide repeat motif-containing protein and 3-hydroxyisobutyrate dehydrogenase. All are mitochondrial proteins from which N-terminal import sequences are removed generating N-terminal IBMs. Whereas most of these proteins have alanine at the N-terminal position, as observed for previously described antagonists, GdH has an N-terminal serine residue that is essential for X-linked IAP (XIAP) interaction. These newly described IAP binding proteins interact with XIAP mainly via BIR2, with binding eliminated or significantly reduced by a single point mutation (D214S) within this domain. Through this interaction, many are able to antagonise XIAP inhibition of caspase 3 in vitro.
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PMID:Identification of mammalian mitochondrial proteins that interact with IAPs via N-terminal IAP binding motifs. 1679 1

Orexin-A and orexin-B are regulatory peptides involved in the control of feeding, sleep-wakefulness, and exerting various endocrine and metabolic actions. Recently we demonstrated that orexins, acting at OX(1) receptor (OX(1)R), are proapoptotic peptides. The aim of this study was to investigate the role of the receptor subtype OX(2)R in the control of apoptosis. Orexins caused a caspase-dependent cell death by apoptosis and a drastic cell growth inhibition in Chinese hamster ovary cells transfected with OX(2)R cDNA. On addition of either orexin (10(-6) m) for 48 h, apoptosis was demonstrated by DNA fragmentation, chromatin condensation, annexin-V binding, and activation of caspase-3 and caspase-9. Orexins were active on apoptosis and cell growth inhibition in the range of concentrations between 10(-10) and 10(-5) m with an EC(50) of 5 x 10(-8) m peptides. No effect of orexins could be detected in parental Chinese hamster ovary cells. A rat pancreatic acinar cell line, AR42J, which expresses OX(2)R but not OX(1)R, also underwent growth suppression and apoptosis on treatment with orexins. Suppression of AR42J cell growth by 10(-6) m orexin was more than 75% after 24 h. Induction of annexin-V-labeled AR42J cell number was dose dependent, with EC(50) of 5.1 x 10(-8) m orexin-A and 9.8 x 10(-8) m orexin-B. The OX(2)R agonist [Ala (11), d-Leu (15)]orexin-B promoted effects on cell growth and apoptosis, which were similar to those elicited by orexins. The OX(1)R antagonist SB33487 did not alter orexin-induced inhibition of growth or orexin-induced stimulation of apoptosis in AR42J cells. For the first time, we provide functional and pharmacological evidence for a role of the OX(2)R in orexin-induced apoptosis.
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PMID:Orexin-induced apoptosis: the key role of the seven-transmembrane domain orexin type 2 receptor. 1685 48

The mechanisms by which infections induce diaphragm dysfunction remain poorly understood. The purpose of this study was to determine which caspase pathways (i.e., the extrinsic, death receptor-linked caspase-8 pathway, and/or the intrinsic, mitochondrial-related caspase-9 pathway) are responsible for endotoxin-induced diaphragm contractile dysfunction. We determined 1) whether endotoxin administration (12 mg/kg IP) to mice induces caspase-8 or -9 activation in the diaphragm; 2) whether administration of a caspase-8 inhibitor (N-acetyl-Ile-Glu-Thr-Asp-CHO, 3 mg/kg iv) or a caspase-9 inhibitor (N-acetyl-Leu-Glu-His-Asp-CHO, 3 mg/kg iv) blocks endotoxin-induced diaphragmatic weakness and caspase-3 activation; 3) whether TNF receptor 1-deficient mice have reduced caspase activation and diaphragm dysfunction following endotoxin; and 4) whether cytokines (TNF-alpha or cytomix, a mixture of TNF-alpha, interleukin-1beta, interferon-gamma, and endotoxin) evoke caspase activation in C(2)C(12) myotubes. Endotoxin markedly reduced diaphragm force generation (P < 0.001) and induced increases in caspase-3 and caspase-8 activity (P < 0.03), but failed to increase caspase-9. Inhibitors of caspase-8, but not of caspase-9, prevented endotoxin-induced reductions in diaphragm force and caspase-3 activation (P < 0.01). Mice deficient in TNF receptor 1 also had reduced caspase-8 activation (P < 0.001) and less contractile dysfunction (P < 0.01) after endotoxin. Furthermore, incubation of C(2)C(12) cells with either TNF-alpha or cytomix elicited significant caspase-8 activation. The caspase-8 pathway is strongly activated in the diaphragm following endotoxin and is responsible for caspase-3 activation and diaphragm weakness.
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PMID:The extrinsic caspase pathway modulates endotoxin-induced diaphragm contractile dysfunction. 1721 30

We have developed a two-component system involving reconstituted caspase (recCaspase) for selective and/or conditional ablation of targeted cells. Caspases, the executioners of programmed cell death, are normally synthesized as inactive zymogens and are activated by proteolytic processing of their subunits. We show here, using two different caspases, Caenorhabditis elegans CED-3 and human Caspase-3, that coexpression of the subunits generates constitutively active caspase activity that leads to cell death. This recCaspase activity, however, occurred only when the subunits associated through binding of linked antiparallel leucine-zipper domains. We exploited the dual-component nature of recCaspases by expressing the individual subunits from combinations of promoters either to target selectively the subset of cells for apoptosis or induce cell death in specific cells at specific times during development. The high degree of target specificity and tight regulation of induction of recCaspase would be advantageous in creating animal models that are ablated for specific cells and in other targeted cell killings.
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PMID:Targeted cell killing by reconstituted caspases. 1728 33

We have previously shown that the activity and the expression of caspase-9 and caspase-3 were increased during hypoxia in the cerebral cortex of newborn piglets. The present study was conducted to test the hypothesis that the hypoxia-induced activation of caspase-3 in the cerebral cortex of newborn piglets is mediated by caspase-9. Twenty-two newborn piglets were randomly assigned to four groups: normoxic (Nx), normoxic pretreated with a selective caspase-9 inhibitor, Z-Leu-Glu(OMe)-His-Asp(OMe)-Fluoromethyl ketone (Z-LEHD-FMK) (Nx+LEHD), hypoxic (Hx), and hypoxic pretreated with Z-LEHD-FMK (Hx+LEHD). Cerebral tissue hypoxia was confirmed biochemically by measuring ATP and phosphocreatine. Caspase-9 and -3 activities were determined spectrofluorometrically. The expression of caspase-9 and -3 proteins was measured by Western blot analysis using active enzyme specific antibodies. Cytosolic caspase-9 activity (nmol/mg protein/h) was 3.70+/-0.40 in Nx, 3.56+/-0.31 in Nx+LEHD (p=NS versus Nx), 4.99+/-0.64 in Hx (p<0.05 versus Nx), and 3.73+/-0.80 in Hx+LEHD (p<0.05 versus Hx, p=NS versus Nx). Cytosolic caspase-3 activity (nmol/mg protein/h) was 7.80+/-1.17 in Nx, 8.15+/-0.87 in Nx+LEHD (p=NS versus Nx), 13.07+/-0.78 in Hx (p<0.05 versus Nx), and 10.05+/-2.09 in Hx+LEHD (p<0.05 versus Hx) The density (ODxmm(2)) of active caspase-9 protein was 18.52+/-1.89 in Nx, 20.53+/-1.12 in Nx+LEHD (p=NS versus Nx), 32.36+/-5.03 in Hx (p<0.05 versus Nx), and 19.94+/-3.59 in Hx+LEHD (p<0.05 versus Hx, p=NS versus Nx). The density (ODxmm(2)) of active caspase-3 protein was 55.87+/-8.73 in Nx, 55.69+/-8.18 in Nx+LEHD (p=NS versus Nx), 94.10+/-12.05 in Hx (p<0.05 versus Nx), and 56.12+/-14.56 in Hx+LEHD (p<0.05 versus Hx, p=NS versus Nx). These data show that administration of a selective caspase-9 inhibitor, Z-LEHD-FMK, prior to hypoxia prevents the hypoxia-induced increase in caspase-3 activity and the expression of active caspase-3 protein. We conclude that the hypoxia-induced activation of caspase-3 during hypoxia in the cerebral cortex of newborn piglets is mediated by caspase-9.
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PMID:Mechanism of caspase-3 activation during hypoxia in the cerebral cortex of newborn piglets. 1755 17


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