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Query: UNIPROT:P42574 (caspase-3)
 45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A previous paper from this laboratory reported the activation of a caspase-3-like protease by a digitonin-treated lysosomal fraction [FEBS Lett. 435, 233-236, 1998]. In this study, we examined the effects of specific inhibitors of lysosomal cysteine proteases, such as cathepsins B, S, and L, on the activation of caspase-3 to find out which cathepsin is responsible for the activation. Pro-caspase-3 in the cytosol was cleaved by a lysosomal protease(s) contained in the supernatant of a digitonin-treated crude mitochondrial fraction containing lysosomes (ML) and the cleaved product was detected by Western blotting using anti-caspase-3 antibody. The activation of caspase-3 by the lysosomal protease(s) was pH dependent and the optimum pH for activation was pH 6.6-6.8. This activation was not inhibited by CA-074, a specific inhibitor of cathepsin B, but was strongly inhibited by CLIK-066 and CLIK-181, specific inhibitors of cathepsin L. The inhibitory effect of CLIK-060, a specific inhibitor of cathepsin S, was very weak. Furthermore, the activation of caspase-3 was enhanced by addition of purified cathepsin L only in the presence of the supernatant of the digitonin-treated ML. These results suggested that a cathepsin L-type protease activity might participate in the activation mechanism of caspase-3 in the presence of the supernatnat from the ML.
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PMID:Participation of a cathepsin L-type protease in the activation of caspase-3. 1069 61

In most cases, apoptosis is considered to involve mitochondrial dysfunction with sequential release of cytochrome c from mitochondria, resulting in activation of caspase-3. However, we found that etoposide induced apoptosis in P39 cells, a myelodysplastic syndrome-derived cell line, without the release of cytochrome c. Furthermore, in etoposide-treated P39 cells, no changes in mitochondrial membrane potential (delta psi m) were detected by flow cytometry. Flow cytometry using a pH-sensitive probe demonstrated that lysosomal pH increased during early apoptosis in P39 cells treated with etoposide. A reduction in the ATP level preceded the elevation of lysosomal pH. In addition, specific inhibitors of vacuolar H+-ATPase induced apoptosis in P39 cells but not in HL60 cells. Although etoposide-induced activation of caspase-3 was followed by DNA ladder formation in P39 cells, E-64d, an inhibitor of lysosomal thiol proteases, specifically suppressed etoposide-induced activation of caspase-3. Western blotting analysis provided direct evidence for the involvement of a lysosomal enzyme, cathepsin L. These findings indicate that lysosomal dysfunction induced by a reduction in ATP results in leakage of lysosomal enzymes into the cytosolic compartment and that lysosomal enzyme(s) may be involved in activation of caspase-3 during apoptosis in P39 cells treated with etoposide.
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PMID:Caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line P39. 1130 62

L-2,5-Dihydrophenylalanine (DHPA), a phenylalanine analogue, induced apoptosis in human promyelocytic leukemia cells (HL-60). This apoptosis was demonstrated by morphological changes of the cells, such as fragmentation of nuclei and chromatin condensation, and by some evidence found in biochemical analysis, such as DNA ladder and activation of caspase 3. The DHPA-induced apoptosis was prevented by a pan-caspase inhibitor, Z-VAD-fmk, and a cysteine protease inhibitor, E-64d, which inhibits calpains and cathepsin B and L. A calpain inhibitor, Z-LL-H, did not affect this apoptosis. A cathepsin B specific inhibitor, CA074-Me, prevented only chromatin condensation. However, E-64d and a cathepsin L specific inhibitor, Z-FY(t-Bu)-dmk, protected the cells from both chromatin condensation and oligonucleosomal DNA fragmentation. As proceeding to the apoptotic process, the activities of both cathepsin B and L increased gradually. These results indicated that DHPA was an inducer of cathepsin-dependent apoptosis in HL-60 cells.
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PMID:L-2,5-dihydrophenylalanine, an inducer of cathepsin-dependent apoptosis in human promyelocytic leukemia cells (HL-60). 1177 36

Invasion and metastasis of certain tumors are accompanied by increased mRNA protein levels and enzymatic activity of cathepsin L. Cathepsin L has also been suggested to play a role in the proteolytic cascades associated with apoptosis. To investigate the role of cathepsin L in brain tumor invasion and apoptosis, the human glioma cell line, IPTP, was stably transfected with full-length antisense and sense cDNA of cathepsin L. Down-regulation of cathepsin L by antisense cDNA significantly impaired (up to 70%) glioma cell invasion in vitro and markedly increased glioma cell apoptosis induced by staurosporine. Compared to control and parental cell lines, antisense down-regulation of cathepsin L was associated with an earlier induction of caspase-3 activity. Up-regulation of cathepsin L activity by sense cDNA was associated with reduced apoptosis and later induction of caspase-3 activity. Moreover, down-regulation of cathepsin L lowered the expression of antiapoptotic protein Bcl-2, whereas up-regulation increased the expression of Bcl-2, indicating that cathepsin L acts upstream of caspase-3. These data show that cathepsin L is an important protein mediating the malignancy of gliomas and its inhibition may diminish their invasion and lead to increased tumor cell apoptosis by reducing apoptotic threshold.
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PMID:Selective suppression of cathepsin L by antisense cDNA impairs human brain tumor cell invasion in vitro and promotes apoptosis. 1253 3

Our recent studies show little evidence for increased granulosa cell apoptosis during atresia in teleost follicles, in direct contrast to the mammalian model. Histological evidence suggests that atresia in many oviparous vertebrates involves proteolytic degradation of the energy-rich yolk storage proteins within the oocyte. This study tests the hypothesis that physiological conditions that promote atresia (hormone withdrawal) lead to increased lysosomal protease activity in rainbow trout oocytes. We subjected rainbow trout ovarian follicles to conditions that promote atresia (serum-free culture) for up to 72 hr, and measured the activity of lysosomal proteases using routine enzymatic assays. Furthermore, we used high performance liquid chromatography to quantify the increase in free amino acids resulting from proteolysis of yolk proteins. Concomitantly, we evaluated the extent of follicular apoptosis during prolonged serum-free culture, using caspase-3-like activity and DNA fragmentation as indicators of apoptosis. Our results show a significant, time-dependent increase in cathepsin L-like, but not cathepsin D-like, activity levels during culture in serum-free medium; increased cathepsin L-like activity is confirmed by a significant increase in oocyte free amino acid content after 72 hr culture. In contrast, we detected only a transient increase in apoptosis during prolonged serum-free culture, as revealed through both radioactive 3'end-labeling of oligonucleosomal DNA fragments, and caspase-3-like activity. The results of this study provide the first evidence for a novel mechanism of follicular atresia in teleosts involving cathepsin-mediated yolk proteolysis.
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PMID:Yolk proteolysis in rainbow trout oocytes after serum-free culture: evidence for a novel biochemical mechanism of atresia in oviparous vertebrates. 1270 34

Identification of relevant substrates is essential for elucidation of in vivo functions of peptidases. The recent availability of the complete genome sequences of many eukaryotic organisms holds the promise of identifying specific peptidase substrates by systematic proteome analyses in combination with computer-based screening of genome databases. Currently available proteomics and bioinformatics tools are not sufficient for reliable endopeptidase substrate predictions. To address these shortcomings the bioinformatics tool 'PEPS' (Prediction of Endopeptidase Substrates) has been developed and is presented here. PEPS uses individual rule-based endopeptidase cleavage site scoring matrices (CSSM). The efficiency of PEPS in predicting putative caspase 3, cathepsin B and cathepsin L cleavage sites is demonstrated in comparison to established algorithms. Mortalin, a member of the heat shock protein family HSP70, was identified by PEPS as a putative cathepsin L substrate. Comparative proteome analyses of cathepsin L-deficient and wild-type mouse fibroblasts showed that mortalin is enriched in the absence of cathepsin L. These results indicate that CSSM/PEPS can correctly predict relevant peptidase substrates.
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PMID:Toward computer-based cleavage site prediction of cysteine endopeptidases. 1288 57

We investigated the mechanism of apoptosis induced by bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPase. Bafilomycin A(1) significantly inhibited the growth of MKN-1 human gastric cancer cells. Bafilomycin A(1) induced apoptosis as demonstrated by DNA ladder formation and the TUNEL method. We designed a flow cytometric assay to detect the alteration in lysosomal pH using a fluorescent probe, fluorescein isothiocyanate-conjugated dextran. This assay revealed that bafilomycin A(1) dramatically increased lysosomal pH. However, bafilomycin A(1) induced neither significant decrease in mitochondrial transmembrane potential nor the release of mitochondrial cytochrome c into the cytoplasm. Western blotting showed that cathepsin D, but not cathepsin L, was released into the cytoplasm. The activity of caspase-3 was significantly increased by bafilomycin A(1). However, cathepsin D did not directly cleave procaspase-3. These findings suggest that bafilomycin A(1)-induced apoptosis in MKN-1 cells is mediated by other proteases released after lysosomal dysfunction followed by activation of caspase-3 in a cytochrome c-independent manner. The present study showed that flow cytometric analysis of lysosomal pH can be useful to evaluate lysosomal protease-mediated apoptosis.
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PMID:Vacuolar H+-ATPase inhibitor induces apoptosis via lysosomal dysfunction in the human gastric cancer cell line MKN-1. 1456 21

High doses of Ag can paradoxically suppress immune responses in vivo. This Ag-specific unresponsiveness (termed high dose tolerance) involves extrathymic mechanisms in mature T lymphocytes. To investigate these mechanisms, we used the in vitro model of PBL activated with anti-CD3 or PHA. In these conditions, increasing mitogen concentrations resulted in a reduction of the proliferative response, associated with an increased percentage of apoptotic cells. Apoptosis did not require prior exposure to IL-2, it was not the consequence of CD178/CD95 or TNF/TNFR interactions, and was therefore clearly distinct from activation-induced cell death. Although the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) decreased DNA fragmentation, cytochrome c release and caspase-9 and caspase-3 activation were not implicated, suggesting that this apoptosis did not primarily involve the intrinsic mitochondrial pathway. E64d, a cysteine protease inhibitor, as well as specific inhibitors of cathepsin B and cathepsin L conferred protection. We further demonstrated that cathepsin B and cathepsin L were released from the lysosomes and catalytically active in the cytosol. Release of cathepsin B and cathepsin L was the consequence of lysosomal membrane permeabilization without complete disruption of the cytosol-lysosome pH gradient. These results demonstrate a role for cathepsins in supraoptimal activation-induced apoptosis in vitro and suggest their possible participation in high dose tolerance in vivo.
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PMID:Cathepsin-dependent apoptosis triggered by supraoptimal activation of T lymphocytes: a possible mechanism of high dose tolerance. 1510 Feb 81

Tumorigenesis is associated with several changes that alter the cellular susceptibility to programmed cell death. Here, we show that immortalization and transformation sensitize cells in particular to the cysteine cathepsin-mediated lysosomal death pathway. Spontaneous immortalization increased the susceptibility of wild-type murine embryonic fibroblasts (MEFs) to tumor necrosis factor (TNF)-mediated cytotoxicity >1000-fold, whereas immortalized MEFs deficient for lysosomal cysteine protease cathepsin B (CathB) retained the resistant phenotype of primary cells. This effect was specific for cysteine cathepsins, because also lack of cathepsin L (a lysosomal cysteine protease), but not that of cathepsin D (a lysosomal aspartyl protease) or caspase-3 (the major executioner protease in classic apoptosis) inhibited the immortalization-associated sensitization of MEFs to TNF. Oncogene-driven transformation of immortalized MEFs was associated with a dramatic increase in cathepsin expression and additional sensitization to the cysteine cathepsin-mediated death pathway. Importantly, exogenous expression of CathB partially reversed the resistant phenotype of immortalized CathB-deficient MEFs, and the inhibition of CathB activity by pharmacological inhibitors or RNA interference attenuated TNF-induced cytotoxicity in immortalized and transformed wild-type cells. Thus, tumorigenesis-associated changes in lysosomes may counteract cancer progression and enhance therapeutic responses by sensitizing cells to programmed cell death.
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PMID:Sensitization to the lysosomal cell death pathway upon immortalization and transformation. 1528 36

Serum and potassium deprivation-induced neuronal death on the primary culture of rat cerebellar granule neurons is being widely used as an in vitro model of neurodegeneration and neuronal apoptosis. In our experiments, serum and potassium deprivation for 12 h induced neuronal death in approximately 20% of cerebellar granule neurons as demonstrated by Trypan Blue assay. Neuronal death was accompanied by a transient increase in the intralysosomal cathepsin L activity, which preceded neuronal death. During this time, the lysosomal membrane integrity remained preserved and no leakage of cathepsin L into the cytosol was seen. Ultrastructural analysis revealed the appearance of multiple vacuoles and autophagosomes in the cytoplasmatic compartment of serum- and potassium-deprived granule neurons. Addition of selective cathepsin L inhibitors or of the autophagy inhibitor 3-methyladenine provided partial protection against serum and potassium deprivation-induced death. Our data also show that combining cathepsin L inhibitors and caspase-3 inhibitors leads to a synergistic neuroprotective effect against serum and potassium deprivation. The results of the current study suggest that activation of the autophagosomal--lysosomal compartment plays an important role in neuronal death induced by serum and potassium deprivation in cultured cerebellar granule cells.
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PMID:Up-regulation of lysosomal cathepsin L and autophagy during neuronal death induced by reduced serum and potassium. 1617 44


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