Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.56 (caspase-3)
 35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Accumulating evidence strongly suggests that apoptosis contributes to neuronal cell death in a variety of neurodegenerative contexts. Activation of the cysteine protease caspase-3 appears to be a key event in the execution of apoptosis in the central nervous system (CNS). As a result, mice null for caspase-3 display considerable neuronal expansion usually resulting in death by the second week of life. At present, 14 caspase family members have been identified and subdivided into three subgroups on the basis of preference for specific tetrapeptide motifs using a positional scanning combinatorial substrate library. Caspase-3 is a group II member (2, 3, 7) categorized by an absolute substrate requirement for aspartic acid in the P4 position of the scissile bond. The preferred cleavage motif (DExD) for group II caspases is found in many structural, metabolic and repair proteins essential for cellular homeostasis. Consistent with the proposal that apoptosis plays a central in role human neurodegenerative disease, caspase-3 activation has recently been observed in stroke, spinal cord trauma, head injury and Alzheimer's disease. Indeed, peptide-based caspase inhibitors prevent neuronal loss in animal models of head injury and stroke suggesting that these compounds may be the forerunners of non-peptide small molecules that halt apoptosis processes implicated in these neurodegenerative disorders. A clear link between an hereditary neurodegenerative disorder and failed caspase inhibition has recently been proposed for spinal muscular atrophy (SMA). In severe SMA, the neuronal specific inhibitor of apoptosis (IAP) family member known as NAIP is often dysfunctional due to missense and truncation mutations. IAPs such as NAIP potently block the enzymatic activity of group II caspases (3 and 7) suggesting that NAIP mutations may permit unopposed developmental apoptosis to occur in sensory and motor systems resulting in lethal muscular atrophy. Conversely, adenovirally-mediated overexpression of NAIP or the X-linked IAP called XIAP reduces the loss of CA1 hippocampal neurons following transient forebrain ischemia. Taken together, these findings suggest that anti-apoptotic strategies may some day have utility in the treatment of neurodegenerative disease. The present review will summarize some of the recent evidence suggesting that apoptosis inhibitors may become a practical therapeutic approach for both acute and chronic neurodegenerative conditions.
 ...
PMID:Neuroprotection by the inhibition of apoptosis. 1076 48

The killing of L929 mouse fibroblasts by tumor necrosis factor-alpha (TNF-alpha) in the presence of 0.5 microg/ml actinomycin D (Act D) is prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A (CyA) in combination with the phospholipase A(2) inhibitor aristolochic acid (ArA). The MPT is accompanied by the release of cytochrome c from the mitochondria, caspase-8 and caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), and DNA fragmentation, all of which were inhibited by CyA plus ArA. The caspase-3 inhibitor z-Asp-Glu-Val-aspartic acid fluoromethyl-ketone (Z-DEVD-FMK) did not prevent the loss of viability or the redistribution of cytochrome c, but it did prevent caspase-3 activation, PARP cleavage, and DNA fragmentation. Inhibition of the MPT reduced the activation of caspase-8 to the level occurring with TNF-alpha alone (no ActD). The caspase-8 inhibitor z-Ile-Glu(OMe)-Thr-Asp(OMe) fluoromethylketone (Z-IETD-FMK) did not prevent the cell killing and decreased only slightly the translocation of Bid to the mitochondria. These data indicate that induction of the MTP by TNF-alpha causes a release of cytochrome c, caspase-3 activation with PARP cleavage and DNA fragmentation. The loss of viability is dependent on the MPT but independent of the activation of caspase-3. The activation of caspase-8 is not dependent on the MPT. There is no evidence linking this enzyme to the loss of viability. Thus, the killing of L929 fibroblasts by TNF-alpha can occur in the absence of either caspase-3 or caspase-8 activity. Alternatively, cell death can be prevented despite an activation of caspase-8.
 ...
PMID:Cytochrome c-dependent activation of caspase-3 by tumor necrosis factor requires induction of the mitochondrial permeability transition. 1085 32

A functional assay for proteolytic processing of the amyloid precursor protein (APP) was set up in yeast. This consisted of a membrane-bound chimeric protein containing the beta-secretase cleaved C-terminal fragment of APP fused to the Ga14 transcription factor. Using this chimera in a GAL-reporter yeast strain, an expression library of human cDNAs was screened for clones that could activate the GAL-reporter genes by proteolytic processing of the membrane-bound APP-Gal4. Two human proteases, caspase-3 and caspase-8, were identified and confirmed to act by a mechanism that involved proteolysis at the site in the APP-Gal4 chimera that corresponded to the natural caspase cleavage site in APP, thus linking a readily scorable phenotype to proteolytic processing of APP. The activation of caspase-3 involved a mechanism that was independent of aspartic acid residue 175 at the cleavage site normally required for processing of caspase-3.
 ...
PMID:A yeast genetic assay for caspase cleavage of the amyloid-beta precursor protein. 1091 20

Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P(4), P(1) and P(1)' subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S(4) subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S(1) and S(1)' subsites that flank the scissile peptide bond. The S(1) subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P(1)' position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P(1)' subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.
 ...
PMID:Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8. 1094 72

Caspase-8 is an initiator enzyme in the Fas-mediated pathway of which the downstream executioner caspase-3 is a physiological target. Caspases are cysteine proteases that are specific for substrates with an aspartic acid residue at the P(1) position and have an optimal recognition motif that incorporates four amino acid residues N-terminal to the cleavage site. Caspase-8 has been classified as a group III caspase member because it shows a preference for a small hydrophobic residue at the P(4) substrate position. We report the X-ray crystallographic structure of caspase-8 in complex with benzyloxycarbonyl-Asp-Glu-Val-Asp-aldehyde (Z-DEVD), a specific group II caspase inhibitor. The structure shows that the inhibitor interacts favourably with the enzyme in subsite S(4). Kinetic data reveal that Z-DEVD (K(i) 2 nM) is an almost equally potent inhibitor of caspase-8 as the specific group III inhibitor Boc-IETD-aldehyde (K(i) 1 nM). In view of this finding, the original classification of caspases into three specificity groups needs to be modified, at least for caspase-8, which tolerates small hydrophobic residues as well as the acidic residue Asp in subsite S(4). We propose that the subsite S(3) must be considered as an important specificity-determining factor.
 ...
PMID:Caspase-8 specificity probed at subsite S(4): crystal structure of the caspase-8-Z-DEVD-cho complex. 1096 57

Fas-mediated apoptosis results in the activation of caspases, which subsequently cleave cellular substrates that are essential for normal cell viability. In the present study, we show that the Ras-related GTP-binding protein Cdc42 is susceptible to caspase-catalyzed proteolysis in a number of cell lines, including NIH3T3 fibroblasts, human breast cancer cells (e.g. T47D), and COS-7 cells. Both caspase-3 and caspase-7 were able to catalyze the cleavage of Cdc42, whereas caspase-6 and caspase-8 were without effect. The susceptibility to the caspase-stimulated degradation is specific; although Rac can also serve as a caspase substrate, neither Rho nor Ras is degraded. Caspase sensitivity is conferred by a consensus sequence (DXXD) that lies immediately upstream of the Rho insert regions (residues 122-134) of Cdc42 and Rac. The removal of a stretch of residues (120) that includes the insert region or site-directed mutagenesis of either aspartic acid 118 or 121 within a constitutively active background (i.e. Cdc42(F28L)) as well as a wild-type Cdc42 background yields Cdc42 molecules that provide a marked protection against Fas ligand-induced apoptosis. Overall, these results are consistent with a model in which Cdc42 acts downstream of Fas, perhaps to influence the rate of apoptosis, with the ultimate caspase-mediated degradation of Cdc42 then allowing for a maximal apoptotic response.
 ...
PMID:Cdc42 is a substrate for caspases and influences Fas-induced apoptosis. 1127 72

This study was designed to identify the role of a recently identified Ca(2+)/calmodulin-dependent protein kinase (CaMK)-like kinase (CaMKLK) in neuronal apoptosis. For this purpose, we studied proteolytic cleavage of CaMKLK by caspases in vitro and in neuronal NG108 cells. In addition, we have investigated the effect of overexpression of wild type and mutant CaMKLK proteins on staurosporine- and serum deprivation-induced apoptosis of NG108 cells. We found that CaMKLK is a substrate for caspase-3 and -8, both in vitro and in NG108 cells during staurosporine- and serum withdrawal-induced apoptosis. Substitution of an aspartic acid residue at position 62 in an asparagine residue within a putative caspase cleavage site completely blocked cleavage of CaMKLK, strongly indicating that (59)DEND(62) is the caspase recognition site. Overexpression of an Asp(62) --> Asn CaMKLK mutant protected NG108 cells from staurosporine-induced apoptosis to a similar extent as Bcl-x(L). In contrast, overexpression of wild type CaMKLK did not lead to protection. Moreover, microinjection of Asp(62) --> Asn CaMKLK protected NG108 cells from serum deprivation-induced apoptosis, while overexpression of a caspase-generated noncatalytic N-terminal CaMKLK fragment exacerbated apoptosis. Together, our data suggest that cleavage of CaMKLK and generation of the noncatalytic N-terminal domain of CaMKLK facilitate neuronal apoptosis.
 ...
PMID:Caspase-mediated cleavage of the Ca2+/calmodulin-dependent protein kinase-like kinase facilitates neuronal apoptosis. 1147 89

Apoptosis induced in the IL3-dependent murine pro-B lymphocytic (FL5.12) cell line by the 5-lipoxygenase activating protein inhibitor MK886 is accompanied by the rapid loss of the anti-apoptotic bcl-x(L) and bcl-2, but not the proapoptotic bax proteins (Datta et al., J. Biol. Chem. 273, 28163-28169, 1998). Since several reports indicate important roles for noncaspase proteases in apoptosis, the participation of lysosomes, as well as serine, cysteine, or aspartic acid proteases, in the effects of MK886 were investigated. Consistent with the involvement of various proteases, lysosomal degranulation was evident, as observed by a decrease in acridine orange fluorescence at 2 h and an increase in cytosolic beta-hexosaminidase activity at 4 h after treating FL5.12 cells with 10 microM MK886. The disappearance of bcl-x(L) from FL5.12 cells upon MK886 treatment was prevented in a dose-dependent manner by pretreatment with leupeptin, pepstatin, phenylmethylsulfonyl fluoride, or the broad-spectrum caspase inhibitor Boc-D-FMK. Each of the noncaspase protease inhibitors partially inhibited MK886-induced apoptosis as measured by phosphatidylserine externalization and DNA fragmentation. The noncaspase inhibitors also blocked about half of the increase in caspase-3-like activity. Boc-D-FMK completely inhibited this enzyme and prevented apoptosis. None of the inhibitors were able to directly inhibit activated caspase-3 in cell lysates, suggesting their effects were upstream of caspase activation. These observations suggest the involvement of various proteases, possibly originating from lysosomes, upstream of active caspase-3, in the loss of bcl-x(L) protein and in the signaling pathway of MK886-induced apoptosis in FL5.12 cells. This pathway may be unique to MK886 since these same protease inhibitors had only minimal effects on etoposide-induced apoptosis and the accompanying moderate loss of bcl-x(L) in FL5.12 cells.
 ...
PMID:Proteolytic loss of bcl-x(L) in FL5.12 Cells undergoing apoptosis induced by MK886. 1148 88

Apoptosis is dependent on the activation of a group of proteolytic enzymes called caspases. Caspase activation can be detected by immunoblotting using caspase-specific antibodies or by caspase activity measurement employing pro-fluorescent substrates that become fluorescent upon cleavage by the caspase. Most of these methods require the preparation of cell extracts and, therefore, are not suitable for the detection of active caspases within the living cell. Using FAM-VAD-FMK, we have developed a simple and sensitive assay for the detection of caspase activity in living cells. FAM-VAD-FMK is a carboxyfluorescein (FAM) derivative of benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethyl ketone (zVAD-FMK), which is a potent broad-spectrum inhibitor of caspases. FAM-VAD-FMK enters the cell and irreversibly binds to activated caspases. Cells containing bound FAM-VAD-FMK can be analyzed by flow cytometry, fluorescence microscopy, or a fluorescence plate reader. Using FAM-VAD-FMK, we have measured caspase activation in live non-adherent and adherent cells. We show that FAM-VAD-FMK labeled Jurkat and HeLa cells that had undergone apoptosis following treatment with camptothecin or staurosporine. Non-stimulated negative control cells were not stained. Pretreatment with the general caspase inhibitor zVAD-FMK blocked caspase-specific staining in induced Jurkat and HeLa cells. Pretreatment of staurosporine-induced Jurkat cells with FAM-VAD-FMK inhibited affinity labeling of caspase-3, -6, and -7, blocked caspase-specific cell staining, and led to the inhibition of apoptosis. In contrast, the fluorescent control inhibitor FAM-FA-FMK had no effect. Measurement of caspase activation in 96-well plates showed a 3- to 5-fold increase in FAM-fluorescence in staurosporine-treated cells compared to control cells. In summary, we show that FAM-VAD-FMK is a versatile and specific tool for detecting activated caspases in living cells.
 ...
PMID:Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors. 1157 May 4

Activation of ionotropic glutamate receptors can induce neuronal apoptosis in vitro and in vivo. We showed previously that activation of the N-methyl-D-aspartic acid (NMDA) subtype of glutamate receptors in a low Ca(2+) and low Na(+) condition induced apoptotic neuronal death, and that the K(+) efflux via NMDA receptor channels was likely a key event in NMDA-induced apoptosis. Since non-NMDA receptors, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and kainate receptors, are also permeable to K(+), we tested the hypothesis that stimulating K(+) efflux via non-NMDA receptor channels could induce apoptosis in cultured cortical neurons. Using a Ca(2+)-free and Na(+)-free external solution, application of kainate revealed outward membrane currents carried by K(+) efflux. In a low Ca(2+)/low Na(+) medium, a 5-h exposure to 50-500 microM AMPA in the presence of the NMDA receptor antagonist MK801 induced dose-dependent neuronal death 24 h after the onset of the insult, accompanied by intracellular K(+) reduction and caspase-3 activation. The AMPA-induced cell death was attenuated by the caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) and by the protein synthesis inhibitor cycloheximide. Reducing K(+) efflux by raising extracellular K(+) concentration from 5 to 25 mM attenuated AMPA-triggered cell death, the Ca(2+) channel antagonist nifedipine showed no effect on the AMPA toxicity. Kainate induced similar neuronal death sensitive to attenuation by Z-VAD-FMK or elevated extracellular K(+).We suggest that the non-NMDA receptor-mediated K(+) efflux may participate in apoptotic process and that blocking excessive K(+) efflux mediated by NMDA and non-NMDA receptors may selectively prevent neuronal apoptosis under certain pathological conditions.
 ...
PMID:Role of K(+) efflux in apoptosis induced by AMPA and kainate in mouse cortical neurons. 1173 31


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>