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:3.4.22.56 (caspase-3)
35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Poly(ADP-ribose)polymerase (PARP, EC 2.4.2.30), an abundant nuclear protein activated by DNA nicks, mediates cell death in vitro by nicotinamide adenine dinucleotide (NAD) depletion after exposure to nitric oxide. The authors examined whether genetic deletion of PARP (PARP null mice) or its pharmacologic inhibition by 3-aminobenzamide (3-AB) attenuates tissue injury after transient cerebral ischemia. Twenty-two hours after reperfusion following 2 hours of filamentous middle cerebral artery occlusion, ischemic injury was decreased in PARP-/- and PARP+/- mice compared with PARP+/+ litter mates, and also was attenuated in 129/SV wild-type mice after 3-AB treatment compared with controls. Infarct sparing was accompanied by functional recovery in PARP-/- and 3-AB-treated mice. Increased poly(ADP-ribose) immunostaining observed in ischemic cell nuclei 5 minutes after reperfusion was reduced by 3-AB treatment. Levels of NAD--the substrate of PARP--were reduced 2 hours after reperfusion and were 35% of contralateral levels at 24 hours. The decreases were attenuated in PARP-/- mice and in 3-AB-treated animals. Poly(ADP-ribose)polymerase cleavage by caspase-3 (CPP-32) has been proposed as an important step in apoptotic cell death. Markers of apoptosis, such as oligonucleosomal DNA damage, total DNA fragmentation, and the density of terminal deoxynucleotidyl transferase dUTP nick-end-labelled (TUNEL +) cells, however, did not differ in ischemic brain tissue of PARP-/- mice or in 3-AB-treated animals versus controls, although there were differences in the number of TUNEL-stained cells reflecting the decrease in infarct size. Thus, ischemic brain injury activates PARP and contributes to cell death most likely by NAD depletion and energy failure, although the authors have not excluded a role for PARP in apoptotic cell death at earlier or later stages in ischemic cell death. Inhibitors of PARP activation could provide a potential therapy in acute stroke.
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PMID:Ischemic brain injury is mediated by the activation of poly(ADP-ribose)polymerase. 939 Jun 45

The reaction of superoxide and nitric oxide results in the formation of peroxynitrite, a long lived and highly reactive oxidant species. It has been suggested that the formation of peroxynitrite in vivo may contribute to cell death in some neurological conditions. We have examined the effect of peroxynitrite on cell death in the NSC34 spinal cord cell line. A brief (30 min) exposure to either peroxynitrite or hydrogen peroxide caused delayed cell death with an EC50 for both of approximately 1 mM. Cell death was prevented by the RNA synthesis inhibitor actinomycin D and included DNA damage as an early event. We sought to clarify the potential role of the DNA binding enzyme poly(ADP-ribose) polymerase (PARP) in cell death in these cells. Several PARP inhibitors [benzamide, 3-aminobenzamide, nicotinamide, and 6(5H)-phenanthridinone] prevented cell death, but the inactive analogue benzoic acid did not. However, there was no evidence of cleavage of PARP, which occurs in apoptosis via the activation of the caspase CPP32. Therefore, we suggest that PARP contributes to neuronal injury as an early event, probably by lethal NAD depletion, without any requirement for proteolytic cleavage.
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PMID:Peroxynitrite and hydrogen peroxide induced cell death in the NSC34 neuroblastoma x spinal cord cell line: role of poly (ADP-ribose) polymerase. 945 43

We have presently determined the effect of inhibition of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) on the occurrence of apoptosis in insulin-producing cells. The ADP-ribosylation activities of intact cells were decreased by incubation of RINm5F cells for 16 h with the PARP inhibitors nicotinamide (NA) (20-50 mM) or 3-aminobenzamide (3-ABA) (10 mM). Exposure to 20-50 mM NA or 10 mM 3-ABA both resulted in massive apoptosis in RINm5F cells. A 24 h exposure to 50 mM nicotinamide induced apoptosis in fetal but not adult rat islet cells. In addition, exposure of RINm5F cells to 50 mM NA for 12-24 h induced the appearance of the 85 kDa proteolytic PARP fragment, indicating activation of the ICE-like protease caspase-3. Incubation with 20-50 mM NA did not induce any consistent effects upon transcription factor NF-kappaB activity, demonstrating that this pathway is not involved in induction of apoptosis by NA. It is concluded that in insulin-producing cells with a high mitotic rate, inhibition of ADP-ribosylation--and consequently of auto-modification and release of PARP bound to DNA strand breaks--leads to activation of programmed cell death.
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PMID:Nicotinamide-induced apoptosis in insulin producing cells is associated with cleavage of poly(ADP-ribose) polymerase. 970 78

Neutrophils undergo constitutive apoptosis when aged ex vivo. Recent studies have indicated roles for Fas/CD95 and the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase system in this process. We have investigated the role of protein kinase C (PKC) in neutrophil death. We show that there is proteolysis and activation of the novel isoform PKCdelta in aged neutrophils and that this process is accelerated by the addition of an agonistic Fas antibody. PKCdelta proteolysis occurs before the onset of any detectable features of apoptosis and pharmacologic inhibition of this enzyme inhibits neutrophil apoptosis. PKCdelta cleavage and activation is dependent on caspase-8/FADD-like interleukin-1beta converting enzyme (FLICE)-mediated processing of caspase-3/CPP32. Neutrophil survival is prolonged by the addition of broad spectrum (BD.fmk) or caspase-8 targeted (zIETD.fmk) peptide caspase inhibitors. Inhibition of PKCdelta does not prevent apoptosis triggered by factor withdrawal in immature hematopoietic cells, including normal human CD34(+) progenitors indicating that within a given lineage, the mechanisms of apoptosis may be differentiation-stage-specific. Ex vivo aging of neutrophils leads to the increasing production of reactive oxygen species and this is attenuated in cells treated with either caspase or PKCdelta inhibitors. Proteolytically activated PKCdelta acts as a molecular link between the Fas/CD95 receptor and the NADPH-oxidase system and plays a central role in regulating the process of neutrophil apoptosis.
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PMID:Caspase-mediated proteolysis and activation of protein kinase Cdelta plays a central role in neutrophil apoptosis. 1038 25

Production of alpha-1-antitrypsin by human monocytes is an important factor in controlling tissue damage by proteases in the microenvironment of inflammation. Increases of four- to eightfold in levels of native and fragmented forms of alpha-1-antitrypsin have been detected in inflammatory loci in vivo. In this study we have extended our previous observation that the carboxyl-terminal peptide (C-36) of alpha-1-antitrypsin produced by specific proteinase cleavage, when added in its fibrillar form at concentrations of 5 microM or more to monocytes in culture, induces cytotoxic effects. Experiments with synthetic amyloid-forming peptides suggest fibril cytotoxicity to be mediated via a common oxidative stress mechanism. We undertook to determine whether C-36 fibril cytotoxicity also involves this common pathway. Monocytes stimulated with C-36 fibrils for 1 h showed significant elevation in monocyte chemoattractant protein-1 expression, induced reduced nicotinamide-adenine dinucleotide phosphate oxidase activity, increased intracellular lipid peroxidation, altered mitochondrial membrane potential, and increased cytosolic cytochrome c and caspase-3 activity. Treatment of monocytes with C-36 fibrils after 24 h also resulted in increased cytosolic cathepsin D activity, suggesting that lysosomes may also be destabilized over longer periods of time. In contrast, native alpha-1-antitrypsin only showed concentration and time-dependent effects on chemoattractant protein-1 expression, and these appear to be independent of oxidative stress. These results indicate that the cytotoxicity of the fibrillar fragment is mediated via oxidative mechanisms and support important multiple roles for native and also for cleaved forms of alpha-1-antitrypsin in monocyte recruitment and activation during inflammatory processes such as atherosclerosis.
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PMID:Fibrillogenic C-terminal fragment of alpha-1-antitrypsin activates human monocytes via oxidative mechanisms. 1151 75

Microvascular endothelial cell (EC) apoptosis or programmed cell death (PCD) during free radical injury may be involved in the development of cerebral ischemic and degenerative diseases. Yet, the cellular mechanisms that mediate cerebral EC injury require further definition. We therefore used the agent nicotinamide as an investigative tool in EC cultures to examine the role of free radical nitric oxide (NO)-induced PCD. EC injury was evaluated by the trypan blue dye exclusion method, DNA fragmentation, membrane phosphatidylserine (PS) exposure, cysteine protease activity, mitochondrial membrane potential, and mitogen-activated protein kinase phosphorylation. We demonstrate that cerebrovascular PCD consists of two distinct pathways that involve the degradation of genomic DNA and the exposure of membrane PS residues. Each of these pathways is reversible in nature and is controlled independently by caspase 8, caspase 1, and caspase 3. As a cytoprotectant, nicotinamide is novel in the vascular system and functions at two levels. Nicotinamide not only maintains the mitochondrial membrane potential and the prevention of cytochrome c release, but also prevents the induction of caspase-8-, caspase-1- and caspase-3-like activities linked to the DNA repair enzyme poly(ADP-ribose) polymerase through mechanisms that are independent from the MAP kinase systems of p38 and JNK. The work begins to identify therapeutic strategies for the protection of the cerebral vasculature during both acute and chronic degenerative disorders.
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PMID:Nicotinamide modulates mitochondrial membrane potential and cysteine protease activity during cerebral vascular endothelial cell injury. 1201 85

Excessive nitric oxide (NO) production has been implicated in the pathophysiology of cardiomyocyte (CMC) apoptosis and necrosis induced by ischemia/reperfusion, inflammation and NO-donating chemicals. Although caspases are known to be involved in apoptosis, the present study examined whether caspases also play a role in NO-induced CMC necrosis. Neonatal rat CMCs were labeled with Annexin-V and propidium iodide, and apoptosis and necrosis were analyzed by confocal images and fluorescence activated cell sorter analysis. CMC apoptosis and necrosis were also evaluated by determining DNA fragmentation in the cell and the supernatant fractions. Treatment of CMCs with the NO donor, diethylenetriamine NO (DETA/NO) or S-nitroso-N-acetyl-penicillamine (SNAP) at concentrations of 10 and 100 microM for 24h induced predominantly apoptosis over necrosis, but a higher concentration (1mM) of DETA/NO or SNAP provoked both apoptosis and necrosis. The lower doses of DETA/NO-induced apoptosis was associated with a gradual increase in caspase-3 activity over 24h without appreciable activation of poly ADP-ribose polymerase (PARP), while the higher dose of DETA/NO induced a marked increase in caspase-3 activity and CMC apoptosis until 2h after the treatment, and increased necrotic CMCs thereafter associated with robust activation of PARP. The caspase inhibitor Z-DEVD-FMK but not the poly ADP-ribose polymerase (PARP) inhibitor 3-aminobenzamide (3-AB) abolished caspase-3 activation and CMC apoptosis induced by 100 microM DETA/NO. However, both Z-DEVD-FMK and 3-AB abolished PARP activation and CMC necrosis induced by 1mM DETA/NO. The amount of nicotinamide adenine dinucleotide (NAD) and adenine nucleotides in CMCs was not significantly affected by treatment with 10 and 100 microM DETA/NO, but was significantly reduced by treatment with 1mM DETA/NO without a decline of adenylate energy charge. The depletion of NAD and adenine nucleotides was abrogated by Z-DEVD-FMK and 3-AB. These results suggest that caspase activation play a crucial role in CMC apoptosis induced by lower concentrations of NO as well as in CMC necrosis induced by a higher concentration of and a longer exposure to NO. NO-induced CMC necrosis is likely mediated by PARP activation which occurs as a consequence of caspase activation.
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PMID:Nitric oxide induces caspase-dependent apoptosis and necrosis in neonatal rat cardiomyocytes. 1223 74

Tumor necrosis factor alpha (TNF-alpha) is a cytokine with multiple roles in the immune system, including the induction and potentiation of cellular functions in neutrophils (PMNs). TNF-alpha also induces apoptotic signals leading to the activation of several caspases, which are involved in different steps of the process of cell death. Inhibition of caspases usually increases cell survival. Here, we found that inhibition of caspases by the general caspase inhibitor zVAD-fmk did not prevent TNF-alpha-induced PMN death. After 6 hours of incubation, TNF-alpha alone caused PMN death with characteristic apoptotic features (typical morphologic changes, DNA laddering, external phosphatidyl serine [PS] exposure in the plasma membrane, Bax clustering and translocation to the mitochondria, and degradation of mitochondria), which coincided with activation of caspase-8 and caspase-3. However, in the presence of TNF-alpha, PMNs died even when caspases were completely inhibited. This type of cell death lacked nuclear features of apoptosis (ie, no DNA laddering but aberrant hyperlobulated nuclei without typical chromatin condensation) and demonstrated no Bax redistribution, but it did show mitochondria clustering and plasma membrane PS exposure. In contrast, Fas-triggered PMN apoptosis was completely blocked by zVAD-fmk. Experiments with scavengers of reactive oxygen species (ROS) and with inhibitors of mitochondrial respiration, with PMN-derived cytoplasts (which lack mitochondria) and with PMNs from patients with chronic granulomatous disease (which have impaired nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) indicated that TNF-alpha/zVAD-fmk-induced cell death depends on mitochondria-derived ROS. Thus, TNF-alpha can induce a "classical," caspase-dependent and a "nonclassical" caspase-independent cell death.
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PMID:Tumor necrosis factor alpha induces a caspase-independent death pathway in human neutrophils. 1239 8

The current procedure for isolation of islet cells from the pancreas for transplantation by enzymatic digestion is accompanied by significant islet cell loss. Therapeutic strategies aimed at the inhibition of islet cell damage could be expected to increase islet yield and improve cell viability, thereby making more efficient use of available donor tissue. The aim of the present work was to examine the effects of caspase and PARP-1 inhibition on islet survival. We demonstrate that following isolation, islets become increasingly necrotic and display a PARP-1 cleavage pattern typical of necrotic cells, characterized by the appearance of a 50 kDa cleavage product. Caspase inhibition using Z-VAD-fmk resulted in increased necrosis in both human and canine islets by a nicotinamide-sensitive mechanism. Necrosis was also induced by DEVD-fmk, but not by YVAD-cmk, indicating that only inhibitors of caspase-3 were able to cause necrosis. Moreover, increased mitochondrial depolarization was observed in islets following 72 h in culture, which correlated with increased expression of Bax. Mitochondrial depolarization was also visible in islets treated with both Z-VAD-fmk and nicotinamide, indicating that mitochondrial dysfunction may account for the necrotic-like death observed in the absence of PARP-1 and caspase activity. Our results demonstrate that inhibition of PARP-1 cleavage results in increased levels of PARP-1-mediated necrotic cell death, highlighting the importance of PARP-1 cleavage in assuring the execution of the apoptotic program. Taken together, these findings reveal the interdependence of necrosis and apoptosis in isolated islets, suggesting therapeutic strategies which target early events in cell death signaling in order to prevent multiple forms of islet cell death.
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PMID:Inhibition of caspase-mediated PARP-1 cleavage results in increased necrosis in isolated islets of Langerhans. 1510 93

Nicotinamide (vitamin B(3)) reduces the infarct volume following focal cerebral ischemia in rats; however, its mechanism of action has not been reported. After cerebral ischemia and/or reperfusion, reactive oxygen species (ROS) and reactive nitrogen species may be generated by inflammatory cells through several cellular pathways, which can lead to intracellular calcium influx and cell damage. Therefore, we investigated the mechanisms of action of nicotinamide in neuroprotection under conditions of hypoxia/reoxygenation. Results showed that nicotinamide significantly protected rat primary cortical cells from hypoxia by reducing lactate dehydrogenase release with 1 h of oxygen-glucose deprivation (OGD) stress. ROS production and calcium influx in neuronal cells during OGD were dose-dependently diminished by up to 10 mM nicotinamide (p < 0.01). This effect was further examined with OGD/reoxygenation (H/R). Cells were stained with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI) or antibodies against anti-microtubule-associated protein-2 and cleaved caspase-3. Apoptotic cells were studied using Western blotting of cytochrome c and cleaved caspase-3. Results showed that vitamin B(3) reduced cell injury, caspase-3 cleavage and nuclear condensation (DAPI staining) in neuronal cells under H/R. In addition, nicotinamide diminished c-fos and zif268 immediate-early gene expressions following OGD. Taken together, these results indicate that the neuroprotective effect of nicotinamide might occur through these mechanisms in this in vitro ischemia/reperfusion model.
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PMID:Protective effect of nicotinamide on neuronal cells under oxygen and glucose deprivation and hypoxia/reoxygenation. 1515 82


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