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)

Employing the degenerate primer-dependent polymerase chain reaction approach used recently to clone human Mch2, we have identified and cloned the insect Spodoptera frugiperda target of the baculovirus antiapoptotic protein p35. This protein named Sf caspase-1 belongs to the family of caspases and is highly related to human Mch3 and CPP32 in sequence and specific activity. The proenzyme of Sf caspase-1 is 299 amino acids in length and can undergo autocatalytic processing in Escherichia coli to an active enzyme heterocomplex. Autoprocessing occurs at Asp-28, Asp-184, and Asp-195 to generate the large p19/p18 and small p12 subunits. Sf caspase-1 is able to induce apoptosis in Sf9 cells and is capable of cleaving p35 to similar sized fragments as observed with extracts from p35 null mutant baculovirus-infected Sf9 cells. Sf caspase-1 activity is potently inhibited by p35, suggesting that it is an important target of this antiapoptotic protein. Finally, the Sf9 nuclear immunophilin FKBP46 was identified as a death-associated substrate for Sf caspase-1.
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PMID:Spodoptera frugiperda caspase-1, a novel insect death protease that cleaves the nuclear immunophilin FKBP46, is the target of the baculovirus antiapoptotic protein p35. 899 5

Apoptosis induced in rat hepatocytes by transforming growth factor beta1 (TGF-beta1) was accompanied by the activation of interleukin-1beta converting enzyme (ICE)-like proteases. Cell lysates were isolated at various times after TGF-beta1 treatment and analyzed for ICE and CPP32-like activity, using N-acetyl-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin (Ac-YVAD.AMC) and benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (Z-DEVD.AFC), respectively. CPP32-like but not ICE protease activity increased in a time dependent manner and preceded the onset of apoptosis. Kinetic studies in cell lysates indicated that more than one CPP32-like protease was being activated. This was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting of TGF-beta1-treated cells, which showed limited processing of CPP32 as shown by the appearance of the catalytically active p17 subunit. Loss of pro-Mch3alpha was also observed but the catalytically active p19 subunit was not detected. Staurosporine, which induced a much greater level of hepatocyte apoptosis, produced a concomitant increase in CPP32/Mch3alpha processing as shown by the appearance of the p17/p19 subunits and the corresponding increase in CPP32-like protease activity. Apoptosis, CPP32/Mch3alpha processing and the increase in CPP32-like protease activity induced by TGF-beta1 and staurosporine were abolished in hepatocytes pretreated with Z-Asp-Glu-Val-Asp (OMe) fluoromethylketone (Z-DEVD.FMK) or Z-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK). These peptide analogues were potent inhibitors of CPP32-like protease activity in lysates. Pretreatment of hepatocytes with cycloheximide also blocked TGF-beta1-induced apoptosis and the increase in CPP32-like activity. Unlike Z-VAD.FMK and Z-DEVD.FMK, cycloheximide did not inhibit CPP32-like protease activity in cell lysates. Thus, cycloheximide may block apoptosis by inhibiting the synthesis of a protein, which is involved in the upstream events responsible for the activation of the CPP32-like protease activity. Our studies have identified two of the CPP32-like proteases, namely CPP32 and Mch3alpha, which are activated during the execution phase of hepatocyte apoptosis.
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PMID:Processing/activation of CPP32-like proteases is involved in transforming growth factor beta1-induced apoptosis in rat hepatocytes. 918 77

We have investigated the ability of Sf-caspase-1 and two mammalian caspases, caspase-1 and caspase-3, to induce apoptosis in Spodoptera frugiperda Sf-21 insect cells. While the transient expression of the pro-Sf-caspase-1 did not induce apoptosis, expression of the pro-domain deleted form, p31, or coexpression of the two subunits of mature Sf-caspase-1, p19 and p12, induced apoptosis in Sf-21 cells. The behavior of Sf-caspase-1 resembled that of the closely related mammalian caspase, caspase-3, and contrasted with that of the mammalian caspase-1, the pro-form of which was active in inducing apoptosis in Sf-21 cells. The baculovirus caspase inhibitor P35 blocked apoptosis induced by active forms of all three caspases. In contrast, members of the baculovirus inhibitor of apoptosis (IAP) family failed to block active caspase-induced apoptosis. However, during viral infection, expression of OpIAP or CpIAP blocked the activation of pro-Sf-caspase-1 and the associated induction of apoptosis. Thus, the mechanism by which baculovirus IAPs inhibit apoptosis is distinct from the mechanism by which P35 blocks apoptosis and involves inhibition of the activation of pro-caspases like Sf-caspase-1.
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PMID:Baculovirus inhibitors of apoptosis (IAPs) block activation of Sf-caspase-1. 939 Oct 73

Divalent cations, including Zinc and Manganese ions, are important modulators of cell activation. We investigated the ability of these two divalent cations to modulate apoptosis in human Burkitt lymphoma B cells line (Ramos). We found that Zinc (from 10 to 50 microM) inhibited Manganese-induced caspase-3 activation and apoptosis of Ramos cells. Higher concentration of Zinc (50 to 100 microM) did not prevent Manganese-mediated apoptosis but rather increased cell death among Ramos cells. This Zinc-mediated cell death was associated with apoptotic features such as cell shrinkage, the presence of phosphatidylserine residues on the outer leaflet of the cells, chromatin condensation, DNA fragmentation and decrease of mitochondrial transmembrane potential. Zinc-mediated apoptosis was associated with caspase-9 and caspase-3 activation as revealed by the appearance of active p35 fragment of caspase-9 and p19 and p17 of caspase-3 as well as in vivo cleavage of PARP and of a cell-permeable fluorogenic caspase-3 substrate (Phiphilux-G(1)D(2)). Both Zinc-mediated apoptosis and caspase-3 activation were prevented by the cell-permeable, broad-spectrum inhibitor of caspases (zVAD-fmk) or overexpression of bcl-2. In addition, we show that Zinc-induced loss of transmembrane mitochondrial potential is a caspase-independent event, since it is not modified by the presence of zVAD-fmk, which is inhibited by overexpression of bcl-2. These results indicate that depending on its concentration, Zinc can exert opposite effects on caspase-3 activation and apoptosis in human B lymphoma cells: concentrations below 50 microM inhibit caspase-3 activation and apoptosis whereas higher concentrations of Zinc activate a death pathway associated with apoptotic-like features and caspase-3 activation.
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PMID:Zinc-mediated regulation of caspases activity: dose-dependent inhibition or activation of caspase-3 in the human Burkitt lymphoma B cells (Ramos). 1131 17

The mitochondrial pathway is critical for the efficient execution of death receptor-initiated apoptosis in certain cell types. Questions remain as to why the mitochondria are required in that scenario. We investigated the molecular events that determined the need for the mitochondria by using an in vivo model of anti-Fas-induced hepatocyte apoptosis. In wild-type mice, Fas stimulation resulted in normal activation of caspase-3, with the generation of the active p19-p12 complex. In bid-deficient mice, caspase-3 activation was arrested after the initial cleavage at Asp(175). This allowed the generation of the p12 small subunit, but the p20 large subunit could not be further processed to the p19 subunit. The p20-p12 complex generated by Fas stimulation in bid-deficient hepatocytes was inactive, arresting the death program. Failure of p20/p12 caspase-3 to mature and to exhibit activity was because of the inhibition by the inhibitor-of-apoptosis proteins (IAPs), such as XIAP, and also to a low caspase-8 activity. This block could be overcome in wild-type mice by two mechanisms. Smac was released from mitochondria early following Fas activation and was competitively bound to the IAPs to reverse their effects. XIAP could also be cleaved, and this occurred later and was likely mediated by enhanced caspase activities. Both mechanisms were dependent on Bid and thus were not operative in bid-deficient hepatocytes. In conclusion, mitochondrial activation by Bid is required for reversing the IAP inhibition through Smac release. It is also required for the alternative activation of caspases through cytochrome c release, as demonstrated previously. Together, these events ensure a successful progression of the death program initiated by the death receptor activation in the hepatocyte.
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PMID:Relief of extrinsic pathway inhibition by the Bid-dependent mitochondrial release of Smac in Fas-mediated hepatocyte apoptosis. 1268 80

Until recently, the ability of ARF (human p14(ARF), murine p19(ARF)) tumour-suppressor protein, encoded by the INK4A/ARF locus, to inhibit cell growth in response to various stimuli was related to its ability to stabilize p53 through the so-called ARF/MDM2/p53 pathway. However, recent data have demonstrated that ARF is not implicated in this unique p53-dependent pathway. By use of transient and stable expression, we show here that human p14(ARF) inhibits the growth of human tumoral cells lacking functional p53 by inducing a transient G(2) arrest and subsequently apoptosis. This p14(ARF)-induced G(2) arrest was correlated with inhibition of CDC2 activity, inactivation of CDC25C phosphatase and induction of the CDK inhibitor p21(WAFI). Apoptosis was demonstrated using Hoechst 33352 staining, proteolytic activation of caspase-3 and PARP cleavage. Similar results were obtained in experiments with cells synchronized by hydroxyurea block. Importantly, we were able to reproduce these effects 'in vivo' by showing that p14(ARF) inhibits the growth of p53 nullizygous human tumours in nude mice and induces the regression of p53 -/- established tumours. In these experiments, tumoral regression was associated with inhibition of cell proliferation as well as induction of apoptosis confirming the data obtained in cell lines.
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PMID:p14ARF induces G2 arrest and apoptosis independently of p53 leading to regression of tumours established in nude mice. 1266 Aug 18

The human INK4a locus encodes two structurally unrelated tumor suppressor proteins, p16 INK4a and p14 ARF (p19 ARF in the mouse), which are frequently inactivated in human cancer. Both the proapoptotic and cell cycle-regulatory functions of p14 ARF were initially proposed to be strictly dependent on a functional p53/mdm-2 tumor suppressor pathway. However, a number of recent reports have implicated p53-independent mechanisms in the regulation of cell cycle arrest and apoptosis induction by p14 ARF. Here, we show that the G1 cell cycle arrest induced by p14 ARF entirely depends on both p53 and p21 in human HCT116 and DU145 carcinoma cells. In contrast, neither loss of p53 nor p21 impaired apoptosis induction by p14 ARF as evidenced by nuclear DNA fragmentation, phosphatidyl serine exposure, and caspase activation, which included caspase-3/7- and caspase-9-like activities. However, lack of functional p21 resulted in the accumulation of cells in G2/M phase of the cell cycle and markedly enhanced p14 ARF-induced apoptosis that was, nevertheless, efficiently inhibited by the cell permeable broad-spectrum caspase inhibitor zVAD-fmk (valyl-alanyl-aspartyl-(O)-methyl)-fluoromethylketone). Thus, loss of cell cycle restriction point control in the absence of p21 may interfere with p14 ARF-induced apoptosis. Finally, these data indicate that the signaling events required for G1 cell cycle arrest and apoptosis induction by p14 ARF dissociate upstream of p53.
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PMID:Loss of p21 disrupts p14 ARF-induced G1 cell cycle arrest but augments p14 ARF-induced apoptosis in human carcinoma cells. 1575 Jun 19

In order to improve medical treatment of ischemic injury such as myocardial infarction, it is important to elucidate hypoxia-induced changes to endothelial cells. An in vitro blood vessel model, in which HUVECs are stimulated to form a network of capillary-like tubes, was used to analyze hypoxia-induced morphological and biochemical changes. When exposed to hypoxia, the network of capillary tubes broke down into small clusters. This tube breakdown was accompanied by chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, and activation of two apoptotic signals, caspase-3 and p38. We investigated what roles caspase cascade and p38 play in hypoxia-induced apoptosis and tube breakdown by using zVAD-fmk and SB203580, specific inhibitors of these two apoptotic signals, respectively. Chromatin condensation and cell nuclear fragmentation and tube breakdown were effectively inhibited by SB203580, but not by zVAD-fmk. SB203580 caused dephosphorylation of p38, which indicates that p38 was autophosphorylated. Inhibition by zVAD-fmk caused slight MW increase in p17 and emergence of p19, which indicates that the inhibitor caused partial processing of caspase-3. Inhibition of p38 suppressed activation of caspase-3 but not vice versa. In addition, these two inhibitors were shown to differentially inhibit cleavage of so-called caspase substrates. SB203580 inhibited cleavage of PARP and lamin A/C, while zVAD-fmk inhibited cleavage of lamin A/C but not that of PARP. Taken together, these results show that p38 is located upstream of caspase cascade and that, although caspase-3 is activated, a p38-regulated caspase-independent pathway is crucial for the execution of hypoxia-induced apoptosis and tube breakdown.
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PMID:Hypoxia-induced apoptosis and tube breakdown are regulated by p38 MAPK but not by caspase cascade in an in vitro capillary model composed of human endothelial cells. 1737 51

Human T-cell leukemia virus type-1 (HTLV-1) induces adult T-cell leukemia/lymphoma (ATL/L), a fatal lymphoproliferative disorder, and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic progressive disease of the central nervous system after a long period of latent infection. Although the mechanism of transformation and leukemogenesis is not fully elucidated, there is evidence to suggest that the viral oncoprotein Tax plays a crucial role in these processes through the regulation of several pathways including NF-kappaB and the cell cycle pathways. The observation that NF-kappaB, which is strongly induced by Tax, is indispensable for the maintenance of the malignant phenotype of HTLV-1 by regulating the expression of various genes involved in cell cycle regulation and inhibition of apoptosis provides a possible molecular target for these infected cells. To develop potential new therapeutic strategies for HTLV-1 infected cells, in this present study, we initially screened a battery of NF-kappaB and CDK inhibitors (total of 35 compounds) to examine their effects on the growth and survival of infected T-cell lines. Two drugs namely BMS-345541 and Purvalanol A exhibited higher levels of growth inhibition and apoptosis in infected cell as compared to uninfected cells. BMS-345541 inhibited IKKbeta kinase activity from HTLV-1 infected cells with an IC50 (the 50% of inhibitory concentration) value of 50 nM compared to 500 nM from control cells as measured by in vitro kinase assays. The effects of Purvalanol A were associated with suppression of CDK2/cyclin E complex activity as previously shown by us. Combination of both BMS-345541 and Purvalanol A showed a reduced level of HTLV-1 p19 Gag production in cell culture. The apparent apoptosis in these infected cells were associated with increased caspase-3 activity and PARP cleavage. The potent and selective apoptotic effects of these drugs suggest that both BMS-345541 and Purvalanol A, which target both NF-kappaB and CDK complex and the G1/S border, might be promising new agents in the treatment of these infected patients.
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PMID:Two specific drugs, BMS-345541 and purvalanol A induce apoptosis of HTLV-1 infected cells through inhibition of the NF-kappaB and cell cycle pathways. 1854 67

P14(ARF) (p19(ARF) in the mouse) plays a central role in the regulation of cellular proliferation. Although the capacity of p14(ARF) to induce a cell cycle arrest in G1 phase depends on a functional p53/p21-signaling axis, the G2 arrest triggered by p14(ARF) is p53/p21-independent. Using isogeneic HCT116 cells either wild-type or homozygously deleted for p21, 14-3-3sigma or both, we further investigated the cooperative effect of p21 and 14-3-3sigma on cell cycle regulation and apoptosis induction by p14(ARF). In contrast to DNA damage, which induces mitotic catastrophe in 14-3-3sigma-deficient cells, we show here that the expression of p14(ARF) triggers apoptotic cell death, as evidenced by nuclear DNA fragmentation and induction of pan-caspase activities, irrespective of the presence or absence of 14-3-3sigma. The activation of the intrinsic mitochondrial apoptosis pathway by p14(ARF) was confirmed by cytochrome c release from mitochondria and induction of caspase-9- (LEHDase) and caspase-3/7-like (DEVDase) activities. Moreover, 14-3-3sigma/p21 double-deficient cells were exceedingly sensitive to apoptosis induction by p14(ARF) as compared to wild-type cells or cells lacking either gene alone. Notably, p14(ARF)-induced apoptosis was preceded by an arrest in the G2 phase of cell cycle, which coincided with downregulation of cdc2 (cdk1) protein expression and lack of its nuclear localization. This indicates that p14(ARF) impairs mitotic entry by targeting the distal DNA damage-signaling pathway and induces apoptotic cell death, rather than mitotic catastrophe, out of a transient G2 arrest. Furthermore, our data delineate that the disruption of G2/M cell cycle checkpoint control critically determines the sensitivity of the cell toward p14(ARF)-induced mitochondrial apoptosis.
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PMID:Cooperative effect of p21Cip1/WAF-1 and 14-3-3sigma on cell cycle arrest and apoptosis induction by p14ARF. 1880 27


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