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)

Using an in vitro translation assay to screen a human brain cDNA library, we isolated the microtubule-associated protein Tau and determined it to be a caspase-3 substrate whose C-terminal cleavage occurred during neuronal apoptosis. DeltaTau, the 50-kDa cleavage product, was detected by Western blot in apoptotic cortical cells probed with anti-PHF-1 and anti-Tau-5 antibodies, but not anti-T-46 antibody which recognizes the C-terminus. Overexpression of DeltaTau in SK-N-BE2(C) cells significantly increased the incidence of cell death. Staurosporine-induced Tau cleavage was blocked by 20 microM z-Asp-Glu-Val-Asp-chloromethylketone, a caspase-3 inhibitor, and in vitro, Tau was selectively cleaved by caspase-3 or calpain, a calcium-activated protease, but not by caspases-1, -8, or -9. (D421E)-Tau, a mutant in which Asp421 was replaced with a Glu, was resistant to cleavage by caspase-3 and tended to suppress staurosporine-induced cell death more efficiently than did wild-type Tau in both transient and stable expression systems. Finally, the incidence of DeltaTau-induced cell death was augmented by expression of Abeta precursor protein (APP) or Swedish APP mutant. Taken together, these results suggest that the caspase-3 cleavage product of Tau may contribute to the progression of neuronal cell death in Alzheimer's disease.
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PMID:Proapoptotic effects of tau cleavage product generated by caspase-3. 1116 50

We describe a method that has allowed us to measure the synthesis, turnover and assembly of alpha- and beta-erythroid and nonerythroid spectrins in cultured rat hippocampal neurons. For these studies, rat hippocampal cultures containing 74.5-83.0% neurons were established. B-27 (Gibco) supplement has been used to obtain an excellent long-term viability (up to 5 weeks) of hippocampal neurons in culture. For the synthesis, turnover, and assembly experiments the neurons were labeled with [35S]methionine, and chased with 10-fold excess of cold methionine for the turnover experiments. The cells were then lysed and immunoprecipitated with alpha, beta-erythroid, alpha, and beta-nonerythroid spectrin antibodies. Immunoprecipitated [35S]methionine-labeled spectrins of hippocampal neurons grown in vitro produced bands in 5% polyacrylamide minigels strong enough to be detected by the high sensitivity screens of a phosphorimager to generate graphs from which the synthesis or half-lives of alpha, beta-erythroid, alpha, and beta-nonerythroid spectrins were calculated. This method can be used to study the role of calpain, caspase-3, and the ubiquitin-proteasome system on the synthesis and turnover of erythroid and nonerythroid spectrins in resting and depolarized rat hippocampal neurons in culture.
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PMID:Measurement of the synthesis, turnover, and assembly of alpha- and beta-erythroid and nonerythroid spectrins in cultured rat hippocampal neurons. 1122 13

Primary septo-hippocampal cell cultures were incubated in varying concentrations of tumor necrosis factor (TNF-alpha; 0.3-500 ng/ml) to examine proteolysis of the cytoskeletal protein alpha-spectrin (240 kDa) to a signature 145 kDa fragment by calpain and to the apoptotic-linked 120-kDa fragment by caspase-3. The effects of TNF-alpha incubation on morphology and cell viability were assayed by fluorescein diacetate-propidium iodide (FDA-PI) staining, assays of lactate dehydrogenase (LDH) release, nuclear chromatin alterations (Hoechst 33258), and internucleosomal DNA fragmentation. Incubation with varying concentrations of TNF-alpha produced rapid increases in LDH release and nuclear PI uptake that were sustained over 48 hr. Incubation with 30 ng/ml TNF-alpha yielded maximal, 3-fold, increase in LDH release and was associated with caspase-specific 120-kDa fragment but not calpain-specific 145-kDa fragment as early as 3.5 hr after injury. Incubation with the pan-caspase inhibitor, carbobenzosy- Asp-CH(2)-OC (O)-2-6-dichlorobenzene (Z-D-DCB, 50-140 microM) significantly reduced LDH release produced by TNF-alpha. Apoptotic-associated oligonucleosomal-sized DNA fragmentation on agarose gels was detected from 6 to 72 hr after exposure to TNF-alpha. Histochemical changes included chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. Results of this study suggest TNF-alpha may induce caspase-3 activation but not calpain activation in septo-hippocampal cultures and that this activation of caspase-3 at least partially contributes to TNF-alpha-induced apoptosis.
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PMID:TNF-alpha stimulates caspase-3 activation and apoptotic cell death in primary septo-hippocampal cultures. 1128 41

Gas2 is a caspase-3 substrate that plays a role in regulating microfilament and cell shape changes during apoptosis. Here we provide evidence that overexpression of Gas2 efficiently increases cell susceptibility to apoptosis following UV irradiation, etoposide and methyl methanesulfonate treatments, and that these effects are dependent on increased p53 stability and transcription activity. To investigate possible pathways linking Gas2 to p53, a yeast two-hybrid screen swas performed, indicating m-calpain as a strong Gas2- interacting protein. Moreover, we demonstrate that Gas2 physically interacts with m-calpain in vivo and that recombinant Gas2 inhibits calpain-dependent processing of p53. Importantly, the Gas2 dominant-negative form (Gas2171-314) that binds calpain but is unable to inhibit its activity abrogates Gas2's ability to stabilize p53, to enhance p53 transcriptional activity and to induce p53-dependent apoptosis. Finally, we show that Gas2 is able to regulate the levels of p53 independently of Mdm2 status, suggesting that, like calpastatin, it may enhance p53 stability by inhibiting calpain activity.
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PMID:The death substrate Gas2 binds m-calpain and increases susceptibility to p53-dependent apoptosis. 1138 5

The basic treatment of leishmaniasis consists in the administration of pentavalent antimonials. The mechanisms that contribute to pentavalent antimonial toxicity against the intracellular stage of the parasite (i.e., amastigote) are still unknown. In this study, the combined use of several techniques including DNA fragmentation assay and in situ and cytofluorometry terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling methods and YOPRO-1 staining allowed us to demonstrate that potassium antimonyl tartrate, an Sb(III)-containing drug, was able to induce cell death associated with DNA fragmentation in axenic amastigotes of Leishmania infantum at low concentrations (10 microg/ml). This observation was in close correlation with the toxicity of Sb(III) species against axenic amastigotes (50% inhibitory concentration of 4.75 microg/ml). Despite some similarities to apoptosis, nuclease activation was not a consequence of caspase-1, caspase-3, calpain, cysteine protease, or proteasome activation. Altogether, our results demonstrate that the antileishmanial toxicity of Sb(III) antimonials is associated with parasite oligonucleosomal DNA fragmentation, indicative of the occurrence of late events in the overall process of apoptosis. The elucidation of the biochemical pathways leading to cell death could allow the isolation of new therapeutic targets.
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PMID:Antimonial-mediated DNA fragmentation in Leishmania infantum amastigotes. 1140 24

Early preneoplastic cells (sup+) exhibit increased susceptibility to apoptosis, which is lost in late stage preneoplastic cells (sup-). Sup+ cells, which undergo apoptosis when cultured in low serum, show little or no DNA binding activity to nuclear factor (NF)-kappa B either in 10% or 0.2% serum. In contrast sup- cells, which are resistant to apoptosis in low serum, show a sustained constitutive activation of NF-kappa B. The constitutive activation of NF-kappa B observed in sup- cells is not due to loss of I kappa B alpha. We considered that the activation of NF-kappa B in sup- cells might be secondary to an increase in cytosolic Ca(2+), since sup- cells have a cytosolic Ca(2+) level that is double that in sup+ cells. In support of a role for Ca(2+), lowering cytosolic Ca(2+) in sup- cells by addition of the cell-permeable Ca(2+) chelator 1,2 bis(O-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) reduced cytosolic Ca(2+) by approximately 31% relative to untreated sup- cells, concomitant with a 65% reduction in NF-kappa B DNA binding activity and a reduction in I kappa B kinase (IKK) activity. In sup- cells in low serum, addition of BAPTA-AM also resulted in a significant ( approximately 50%) increase in caspase-3 activity. Raising extracellular Ca(2+) in sup+ cells resulted in a slight activation of I kappa B kinase and in enhanced NF-kappa B DNA binding activity. Using proteasome and calpain inhibitors, we determined that the basal activity of NF-kappa B in sup- cells is largely proteasome-independent, but sensitive to calpain inhibitors. Taken together these data suggest that the elevated Ca(2+) in sup- cells causes a modest activation of IKK, which likely contributes to the enhanced basal activation of NF-kappa B in sup- cells; however, the predominant effect of Ca(2+) appears to be mediated by Ca(2+)-enhanced degradation by calpain.
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PMID:Elevated calcium in preneoplastic cells activates NF-kappa B and confers resistance to apoptosis. 1147 82

Immunofluorescence microscopy revealed the rearrangement and gradual dissociation of paxillin from focal adhesion sites during apoptosis. In vitro, cleavage of paxillin by caspase-3 generated a 42-kDa fragment, among other products, while cleavage by calpain generated a different set of fragments. In Rat-1 cells, cleavage of paxillin by caspase-3 was suppressed by zVAD-fmk or zDEVD-cmk, making caspase-3 a likely executioner during etoposide-induced apoptosis. In contrast, the cleavage of paxillin and p130cas in apoptotic L929 cells was blocked by calpain-specific inhibitors, which also reduced the death rate by 23 to 44%. Therefore, The disassembly and degradation of p130cas and paxillin during apoptosis may controlled by both caspases and calpains, depending upon their cellular contexts. Our findings also suggest that focal adhesion proteins paxillin and p130cas take part in integrin-mediated signaling for cell survival, and that their cleavage by caspase and/or calpain may not only disrupt focal adhesion complexes, but may also impede cell survival signaling.
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PMID:Degradation of focal adhesion proteins paxillin and p130cas by caspases or calpains in apoptotic rat-1 and L929 cells. 1151 Nov 2

Endoplasmic reticulum (ER) stress elicits protective responses of chaperone induction and translational suppression and, when unimpeded, leads to caspase-mediated apoptosis. Alzheimer's disease-linked mutations in presenilin-1 (PS-1) reportedly impair ER stress-mediated protective responses and enhance vulnerability to degeneration. We used cleavage site-specific antibodies to characterize the cysteine protease activation responses of primary mouse cortical neurons to ER stress and evaluate the influence of a PS-1 knock-in mutation on these and other stress responses. Two different ER stressors lead to processing of the ER-resident protease procaspase-12, activation of calpain, caspase-3, and caspase-6, and degradation of ER and non-ER protein substrates. Immunocytochemical localization of activated caspase-3 and a cleaved substrate of caspase-6 confirms that caspase activation extends into the cytosol and nucleus. ER stress-induced proteolysis is unchanged in cortical neurons derived from the PS-1 P264L knock-in mouse. Furthermore, the PS-1 genotype does not influence stress-induced increases in chaperones Grp78/BiP and Grp94 or apoptotic neurodegeneration. A similar lack of effect of the PS-1 P264L mutation on the activation of caspases and induction of chaperones is observed in fibroblasts. Finally, the PS-1 knock-in mutation does not alter activation of the protein kinase PKR-like ER kinase (PERK), a trigger for stress-induced translational suppression. These data demonstrate that ER stress in cortical neurons leads to activation of several cysteine proteases within diverse neuronal compartments and indicate that Alzheimer's disease-linked PS-1 mutations do not invariably alter the proteolytic, chaperone induction, translational suppression, and apoptotic responses to ER stress.
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PMID:Endoplasmic reticulum stress-induced cysteine protease activation in cortical neurons: effect of an Alzheimer's disease-linked presenilin-1 knock-in mutation. 1157 34

During apoptotic and excitotoxic neuron death, challenged mitochondria release the pro-apoptotic factor cytochrome c. In the cytosol, cytochrome c is capable of binding to the apoptotic protease-activating factor-1 (APAF-1). This complex activates procaspase-9 in the presence of dATP, resulting in caspase-mediated execution of apoptotic neuron death. Many forms of Ca(2+)-mediated neuron death, however, do not lead to prominent activation of the caspase cascade despite significant release of cytochrome c from mitochondria. We demonstrate that elevation of cytosolic Ca(2+) induced prominent degradation of APAF-1 in human SH-SY5Y neuroblastoma cells and in a neuronal cell-free apoptosis system. Loss of APAF-1 correlated with a reduced ability of cytochrome c to activate caspase-3-like proteases. Ca(2+) induced the activation of calpains, monitored by the cleavage of full-length alpha-spectrin into a calpain-specific 150-kDa breakdown product. However, pharmacological inhibition of calpain activity indicated that APAF-1 degradation also occurred via calpain-independent pathways. Our data suggest that Ca(2+) inhibits caspase activation during Ca(2+)-mediated neuron death by triggering the degradation of the cytochrome c-binding protein APAF-1.
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PMID:Ca(2+)-induced inhibition of apoptosis in human SH-SY5Y neuroblastoma cells: degradation of apoptotic protease activating factor-1 (APAF-1). 1157 34

Although a number of increased CSF proteins have been correlated with brain damage and outcome after traumatic brain injury (TBI), a major limitation of currently tested biomarkers is a lack of specificity for defining neuropathological cascades. Identification of surrogate biomarkers that are elevated in CSF in response to brain injury and that offer insight into one or more pathological neurochemical events will provide critical information for appropriate administration of therapeutic compounds for treatment of TBI patients. Non-erythroid alpha II-spectrin is a cytoskeletal protein that is a substrate of both calpain and caspase-3 cysteine proteases. As we have previously demonstrated, cleavage of alpha II-spectrin by calpain and caspase-3 results in accumulation of protease-specific spectrin breakdown products (SBDPs) that can be used to monitor the magnitude and temporal duration of protease activation. However, accumulation of alpha II-spectrin and alpha II-SBDPs in CSF after TBI has never been examined. Following a moderate level (2.0 mm) of controlled cortical impact TBI in rodents, native alpha II-spectrin protein was decreased in brain tissue and increased in CSF from 24 h to 72 h after injury. In addition, calpain-specific SBDPs were observed to increase in both brain and CSF after injury. Increases in the calpain-specific 145 kDa SBDP in CSF were 244%, 530% and 665% of sham-injured control animals at 24 h, 48 h and 72 h after TBI, respectively. The caspase-3-specific SBDP was observed to increase in CSF in some animals but to a lesser degree. Importantly, levels of these proteins were undetectable in CSF of uninjured control rats. These results indicate that detection of alpha II-spectrin and alpha II-SBDPs is a powerful discriminator of outcome and protease activation after TBI. In accord with our previous studies, results also indicate that calpain may be a more important effector of cell death after moderate TBI than caspase-3.
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PMID:Accumulation of non-erythroid alpha II-spectrin and calpain-cleaved alpha II-spectrin breakdown products in cerebrospinal fluid after traumatic brain injury in rats. 1157 38


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