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)

The cysteine protease CPP32 has been expressed in a soluble form in Escherichia coli and purified to >95% purity. The three-dimensional structure of human CPP32 in complex with the irreversible tetrapeptide inhibitor acetyl-Asp-Val-Ala-Asp fluoromethyl ketone was determined by x-ray crystallography at a resolution of 2.3 A. The asymmetric unit contains a (p17/p12)2 tetramer, in agreement with the tetrameric structure of the protein in solution as determined by dynamic light scattering and size exclusion chromatography. The overall topology of CPP32 is very similar to that of interleukin-1beta-converting enzyme (ICE); however, differences exist at the N terminus of the p17 subunit, where the first helix found in ICE is missing in CPP32. A deletion/insertion pattern is responsible for the striking differences observed in the loops around the active site. In addition, the P1 carbonyl of the ketone inhibitor is pointing into the oxyanion hole and forms a hydrogen bond with the peptidic nitrogen of Gly-122, resulting in a different state compared with the tetrahedral intermediate observed in the structure of ICE and CPP32 in complex with an aldehyde inhibitor. The topology of the interface formed by the two p17/p12 heterodimers of CPP32 is different from that of ICE. This results in different orientations of CPP32 heterodimers compared with ICE heterodimers, which could affect substrate recognition. This structural information will be invaluable for the design of small synthetic inhibitors of CPP32 as well as for the design of CPP32 mutants.
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PMID:Structure of recombinant human CPP32 in complex with the tetrapeptide acetyl-Asp-Val-Ala-Asp fluoromethyl ketone. 904 80

A growing body of evidence has suggested that oxidative stress causes cardiac injuries during ischemia/reperfusion. Extracellular signal-regulated kinases (ERKs) have been reported to play pivotal roles in many aspects of cell functions and to be activated by oxidative stress in some types of cells. In this study, we examined oxidative stress-evoked signal transduction pathways leading to activation of ERKs in cultured cardiomyocytes of neonatal rats, and determined their role in oxidative stress-induced cardiomyocyte injuries. ERKs were transiently and concentration-dependently activated by hydrogen peroxide (H2O2) in cardiac myocytes. A specific tyrosine kinase inhibitor, genistein, suppressed H2O2-induced ERK activation, while inhibitors of protein kinase A and C or Ca2+ chelators had no effects on the activation. When CSK, a negative regulator of Src family tyrosine kinases, or dominant-negative mutant of Ras or of Raf-1 kinase was overexpressed, activation of transfected ERK2 by H2O2 was abolished. The treatment with H2O2 increased the number of cells stained positive by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and induced formation of DNA ladder and activation of CPP32, suggesting that H2O2 induced apoptosis of cardiac myocytes. When H2O2-induced activation of ERKs was selectively inhibited by PD98059, the number of cardiac myocytes which showed apoptotic death was increased. These results suggest that Src family tyrosine kinases, Ras and Raf-1 are critical for ERK activation by hydroxyl radicals and that activation of ERKs may play an important role in protecting cardiac myocytes from apoptotic death following oxidative stress.
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PMID:Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats. 931 82

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

Caspase-3(-like) proteases play important roles in controlling mammalian apoptosis. However, the downstream events from the caspase-3(-like) protease activation to death of cells are still unclear. Previously, we reported that hydrogen peroxide (H2O2) was generated by the activation of caspase-3(-like) proteases in the process of tyrosine kinase inhibitor-induced apoptosis in human small cell lung carcinoma Ms-1 cells. In the present study, we examined whether generation of H2O2 is a critical event for the apoptotic pathway downstream of caspase-3(-like) protease activation by various anticancer drugs. Anticancer drugs such as camptothecin, vinblastine, inostamycin, and adriamycin induced activation of caspase-3(-like) proteases and apoptosis. Generation of H2O2 was commonly detected after treatment with each of the four anticancer drugs, and scavenging of H2O2 caused cells to fail to undergo apoptosis. Moreover, anticancer drug-induced H2O2 production was inhibited not only by an inhibitor of caspase-3(-like) proteases but also by diphenyleneiodonium chloride, an inhibitor of flavonoid-containing enzymes such as NADPH oxidase. However, activation of caspase-3(-like) proteases was not inhibited by diphenyleneiodonium chloride. These findings suggest that activation of caspase-3(-like) proteases by various anticancer drugs causes generation of H2O2 presumably through the activation of NADPH oxidase, thereby inducing apoptosis. Therefore, H2O2 may function as a common mediator for apoptosis induced by various anticancer drugs.
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PMID:Requirement of caspase-3(-like) protease-mediated hydrogen peroxide production for apoptosis induced by various anticancer drugs. 975 37

Recent studies indicate that arsenic may generate reactive oxygen species to exert its toxicity. However, the mechanism is still unclear. In this study, we demonstrate that arsenite is able to induce apoptosis in a concentration- and time-dependent manner; however, arsenate is unable to do so. An increase of intracellular peroxide levels was accompanied with arsenite-induced apoptosis, as demonstrated by flow cytometry using DCFH-DA. N-Acetyl-L-cysteine (a thiol-containing antioxidant), diphenylene iodonium (an inhibitor of NADPH oxidase), 4,5-dihydro-1,3-benzene disulfonic acid (a selective scavenger of O2-), and catalase significantly inhibit arsenite-induced apoptosis and intracellular fluorescence intensity. In contrast, allopurinol (an inhibitor of xanthine oxidase), indomethacin (an inhibitor of cyclooxygenase), superoxide dismutase, or PDTC had no effect on arsenite-induced cell death. Activation of CPP32 activity, PARP (a DNA repair enzyme) degradation, and release of cytochrome c from mitochondria to the cytosol are involved in arsenite-induced apoptosis, and Bcl-2 antagonize arsenite-induced apoptosis by a mechanism that interferes in the activity of CPP32. These results lead to a working hypothesis that arsenite-induced apoptosis is triggered by the generation of hydrogen peroxide through activation of flavoprotein-dependent superoxide-producing enzymes (such as NADPH oxidase), and hydrogen peroxide might play a role as a mediator to induce apoptosis through release of cytochrome c to cytosol, activation of CPP32 protease, and PARP degradation.
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PMID:Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis. 976 29

A novel anticancer drug, cytotrienin A, isolated from Streptomyces sp., induces apoptosis (or programmed cell death) in human promyelocytic leukemia HL-60 cells within 4 h. To elucidate the mechanism of this process, we performed an in-gel kinase assay using myelin basic protein (MBP) as a substrate and found the activation of kinase with an apparent molecular mass of 36 kDa (p36 MBP kinase). The dose of cytotrienin A required to activate p36 MBP kinase was consistent with that required to induce apoptotic DNA fragmentation in HL-60 cells. This p36 MBP kinase was activated with kinetics distinct from the activation of JNK (c-Jun N-terminal kinase)/stress-activated protein kinase and p38 MAPK (mitogen-activated protein kinase). Importantly, the p36 MBP kinase was immunologically different from MAPK superfamily molecules such as ERK1, JNK isoforms, and p38 MAPK. In addition, the p36 MBP kinase activation and apoptotic DNA fragmentation were inhibited by antioxidants such as N-acetylcysteine and reduced-form glutathione. The p36 MBP kinase activation was also observed during hydrogen peroxide (H2O2) and okadaic acid-induced apoptosis. Although a specific inhibitor of caspase-3-like proteases (Ac-DEVD-CHO) or a specific inhibitor of caspase-1-like proteases (Ac-YVAD-CHO) did not block the cytotrienin A-, H2O2-, or okadaic acid-induced apoptosis, a broad specificity inhibitor of caspases (Z-Asp-CH2-DCB) strongly inhibited the apoptosis of HL-60 cells. Surprisingly, Z-Asp-CH2-DCB inhibited the activation of p36 MBP kinase induced by cytotrienin A or H2O2, but did not inhibit the activation of JNK/stress-activated protein kinase and p38 MAPK. Taken together, these results indicate that p36 MBP kinase activation is downstream of the activation of Z-Asp-CH2-DCB-sensitive caspases, and reactive oxygen species could be included in the apoptotic events. Moreover, according to the Western blotting using the antibodies against MST1/Krs2 or MST2/Krs1, it is suggested that the p36 MBP kinase is an active proteolytic product of MST1/Krs2 and MST2/Krs1, which are originally cloned by virtue of its homology to the budding yeast Ste20 kinase. Thus, the p36 MBP kinase might be a common component of the diverse signaling pathways leading to apoptosis, and controlling this p36 MBP kinase pathway might be a novel strategy for cancer chemotherapy.
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PMID:Caspase-mediated activation of a 36-kDa myelin basic protein kinase during anticancer drug-induced apoptosis. 980 95

Recent studies have suggested that hydrogen peroxide (H2O2), a reactive compound formed endogenously in the breakdown of superoxide, may mediate the induction of apoptosis in various cell types in response to external stimuli. However, the role of H2O2 in the apoptotic pathway has not been clearly established. The purpose of this study was to determine if H2O2 treatment could induce apoptosis through the activation of caspases. Doses of H2O2 ranging from 10 microM to 100 microM, when added to HL-60 cells, resulted in the cleavage of poly(ADP-ribose) polymerase (PARP) from its native 113 Kd form to a processed 89 Kd fragment, indicative of cells undergoing apoptosis. PARP was predominantly in the fragmented form when doses of 20 microM and greater were used. A time course study of changes in PARP processing in H2O2-treated cells revealed that 10 and 50 microM H2O2 required 6 and 3 h, respectively, to specifically degrade PARP, suggesting that the H2O2-induced PARP cleavage is both time and concentration dependent. Since PARP is cleaved by CPP32 (caspase-3), we next determined if H2O2 was capable of effecting changes in CPP32 activity. The caspase activity was assayed using a colorimetric substrate, DEVD-pNa. Results of these experiments showed that H2O2 increased caspase activity at 3 h, corresponding to the time of appearance of fragmented PARP. Also, CPP32 activity and PARP processing were both significantly suppressed by caspase-3 inhibitors. Taken together, these results suggest that H2O2 mediates specific cleavage of PARP and possibly apoptosis by activating caspase 3.
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PMID:Activation of caspase 3 in HL-60 cells exposed to hydrogen peroxide. 1004 34

Protein phosphorylation in a human glioblastoma cell line, T98G, was examined after exposure to oxidative stress in vitro. Hydrogen peroxide (1 mM) markedly induced tyrosine phosphorylation of focal adhesion kinase (FAK) and serine phosphorylation of Akt at 1 h after stimulation. Concommitantly, the association of FAK with phosphatidylinositide 3'-OH-kinase (PI 3-kinase) was also observed by the hydrogen peroxide stimulation. When T98G cells were incubated with wortmannin, a PI 3-kinase inhibitor, both PI 3-kinase activity and phosphorylation of Akt were inhibited, whereas apoptosis by oxidative stress was accelerated. Concomitant with apoptosis, elevated level of CPP32 protease activity (caspase-3) was observed, with decreases in Bcl-2 protein and increases in Bax protein. These results suggested that in the signal transduction pathway from FAK to PI 3-kinase, Akt promotes survival. Thus, it became apparent that FAK is the upstream signal protein of the PI 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis in T98G cells.
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PMID:FAK is the upstream signal protein of the phosphatidylinositol 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis of a human glioblastoma cell line. 1018 51

Apoptosis has been associated with oxidative stress in biological systems. Caspases have been considered to play a pivotal role in the execution phase of apoptosis. However, which caspases function as executioners in reactive oxygen species (ROS)-induced apoptosis is not known. The present study was performed to identify the major caspases acting in ROS-induced apoptosis. Treatment of HL-60 cells with 50 microM hydrogen peroxide (H2O2) for 4 h induced the morphological changes such as condensed and/or fragmented nuclei, increase in caspase-3 subfamily protease activities, reduction of the procaspase-3 and a DNA fragmentation. To determine the role of caspases in H2O2-induced apoptosis, caspase inhibitors, acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone (Ac-YVAD-cmk), acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and acetyl-Val-Glu-Ile-Asp-aldehyde (Ac-VEID-CHO), selective for caspase-1 subfamily, caspase-3 subfamily and caspase-6, respectively, were loaded into the cells using an osmotic lysis of pinosomes method. Of these caspase inhibitors, only Ac-DEVD-CHO completely blocked morphological changes, caspase-3 subfamily protease activation and DNA ladder formation in H2O2-treated HL-60 cells. This inhibitory effect was dose-dependent. These results suggest that caspase-3, but not caspase-1 is required for commitment to ROS-triggered apoptosis.
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PMID:Hydrogen peroxide-induced apoptosis in HL-60 cells requires caspase-3 activation. 1019 75

Our objective is to clarify the role of reactive oxygen species (ROS) in the atrophying tail of anuran tadpoles (tail apoptosis). Changes in catalase, superoxide dismutase (SOD) and caspase activity, genomic DNA, and nitric oxide (NO) generation were investigated biochemically using Rana japonica tadpole tails undergoing regression during thyroid hormone enhancement. DNA fragmentation and ladder formation with concomitant shortening of tadpole tail were induced by DL-thyroxine (T4) in culture medium. Catalase activity was also decreased by T4 treatment. T4 was also found to increase NO synthase (NOS) activity in cultured tadpole tail with concomitant increase in the concentration of NO2- plus NO3- (NOx) in the culture medium. Additional treatment with N-monomethyl-L-arginine (NMMA), a potent inhibitor of NOS, suppressed the enhancing effects of T4 on tail shortening and catalase activity reduction. It was also found that treatment with isosorbide dinitrate (ISDN), a NO generating drug, alone also had an enhancing effect on tail shortening and catalase activity reduction similar to that seen with T4. Both NO and an NO donor (ISDN) strongly suppressed catalase activity. Kinetic analysis revealed that catalase activity decreased and caspase-3-like activity increased during normal tadpole tail atrophy (apoptosis). These results suggested that T4 enhances NO generation, thereby strongly inhibiting catalase activity, resulting in an increase in hydrogen peroxide, and that the oxidative stress elicited by excess hydrogen peroxide might activate cysteine-dependent aspartate-directed protease-3 (caspase-3-like protease), which is thought to cause DNA fragmentation, leading to apoptosis.
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PMID:Thyroxine enhancement and the role of reactive oxygen species in tadpole tail apoptosis. 1023 45


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