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)

Cytotoxic T lymphocytes induce apoptosis in target cells through the CD95(APO-1/Fas) and the perforin/granzyme B (GrB) pathway. The exact substrate of GrB in vivo is still unknown, but to induce apoptosis GrB requires the activity of caspases in target cells. We show here that in HeLa target cells induction of apoptosis through the perforin/GrB pathway resulted in minor direct cleavage of CPP32 (caspase-3) by GrB. Most caspase-3 cleavage resulted from activation of an upstream caspase. Moreover, target cells derived from caspase-3(-/-) mice displayed GrB-induced poly(ADP-ribose) polymerase (PARP) cleavage with only partially reduced efficiency compared to wild-type target cells. This indicates that other PARP-cleaving caspases can be activated during perforin/GrB-induced cell death. In contrast to caspase-3, FLICE (caspase-8) was directly cleaved by GrB in HeLa cells. We therefore conclude that FLICE not only plays a central role in CD95(APO-1/Fas)-induced apoptosis but can also be directly activated during perforin/GrB-induced apoptosis.
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PMID:Cleavage of FLICE (caspase-8) by granzyme B during cytotoxic T lymphocyte-induced apoptosis. 946 39

Granzyme B (GranB), a serine protease stored in the granules of cytotoxic T lymphocytes and natural killer cells, can initiate target cell apoptosis. To produce large amounts of purified active enzyme, recombinant murine granzyme B (rGranB) was expressed from baculovirus in insect cells. The expressed rGranB is secreted into the culture medium and can be readily purified to homogeneity by one-step affinity chromatography to yield 1.5 mg enzyme per liter insect cell medium. RGranB is recognized by a GranB-specific anti-peptide antibody and is active against synthetic substrate Boc-Ala-Ala-Asp-SBzl with kinetic constant (kcat/Km 45,000 M-1s-1) comparable to purified human GranB, RGranB processes the caspase pro-CPP32 into its enzymatically active form and induces DNA fragmentation in isolated nuclei in the presence of cytosolic factors. The ability to express enzymatically active rGranB using the baculovirus system will help elucidate the role of this granzyme in the immune response.
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PMID:Expression and purification of enzymatically active recombinant granzyme B in a baculovirus system. 948 Aug 18

Recently it has been reported that caspase-3 activation occurs in stimulated T-lymphocytes without associated apoptosis (Miossec, C., Dutilleul, V., Fassy, F., and Diu-Hercend, A. (1997) J. Biol. Chem. 272, 13459-13462). To explore this phenomenon, human peripheral blood lymphocytes (PBLs) were stimulated with mitogenic lectins or anti-CD3 antibody, and the proteolytic processing of different caspases and caspase substrates was analyzed by immunoblotting. Proteolytic processing of caspases-3 and -7 and the caspase substrates poly(ADP-ribose) polymerase, GDP dissociation inhibitor, and PKCdelta was observed when PBLs were activated in vitro, and lysates were prepared using RIPA buffer which contains 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS. In contrast, when a lysis buffer containing 2% SDS was used, the caspases remained in their zymogen pro-forms, and no proteolytic processing of caspase substrates was detected. Moreover, in experiments using intact cells and a cell-permeable fluorigenic caspase substrate, no caspase activity was observed in activated T-cells, whereas it was clearly detected when PBLs were treated with the apoptosis-inducing anticancer drug etoposide. Since the granzyme B is a direct activator of caspase-3 and its expression is induced following T-cell activation, we tested the effects of anti-GraB, an engineered serpin that specifically inhibits GraB. When the activated T-lymphocytes were lysed in RIPA buffer containing anti-GraB, no proteolytic processing or activation of caspase-3 was observed, strongly suggesting that release of GraB or similar proteases from their storage sites in cytotoxic granules during the lysis procedure is responsible for caspase activation. These findings demonstrate that T-cells do not process caspases upon activation and caution about the method of cell lysis used when studying granzyme-expressing cells.
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PMID:Granzyme release and caspase activation in activated human T-lymphocytes. 950 96

Caspases are specific proteases involved in apoptosis, and their inhibition by specific peptide inhibitors can inhibit apoptosis. With these inhibitors we examined the relationship of caspases and sphingolipids involved in the induction of apoptosis of human leukemic HL60 cells. We have previously shown that sphingosine (Sph) and its methylated derivative dimethylsphingosine (DMS) effectively induce apoptosis in HL60 cells. Using these lipids as well as ceramide analogues we found both similarities and differences in the caspase involvement in apoptosis induced by the two distinct lipid types. The wide-spectrum caspase inhibitor Z-VAD-FMK and Z-DEVD-FMK, an inhibitor of the downstream caspases 3 (CPP32, Yama) and 7, both inhibited apoptosis induced by all the lipids tested. Z-AAD-FMK which inhibits the serine protease Granzyme B, inhibited Sph/DMS induced apoptosis, but little or no effect on ceramide induced apoptosis. Granzyme B shares a substrate sequence preference with upstream caspases capable of activating themselves and other caspases downstream. Z-IETD-FMK, which inhibits caspase 8/FLICE also inhibited Sph/DMS induced apoptosis with no inhibition of apoptosis induced by either ceramide. Together, these data indicate that Sph/DMS act independently from ceramide in the apoptosis pathway and further suggest that Sph/DMS act earlier in the pathway than ceramide and are involved upstream of even the early proteases, whereas the point of action for ceramide is downstream of the early proteases but upstream from the late caspases.
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PMID:Inhibition of sphingolipid induced apoptosis by caspase inhibitors indicates that sphingosine acts in an earlier part of the apoptotic pathway than ceramide. 954 Oct 7

Focal adhesion kinase (Fak) is a non-receptor protein-tyrosine kinase that stimulates cell spreading and motility by promoting the formation of contact sites between the cell and the extracellular matrix (focal adhesions). It suppresses apoptosis by transducing survival signals that emanate from focal adhesions via the clustering of transmembrane integrins by components of the extracellular matrix. We demonstrate that Fak is cleaved by caspases at two distinct sites during apoptosis. The sites were mapped to DQTD772, which was preferentially cleaved by caspase-3, and VSWD704, which was preferentially cleaved by caspase-6 and cytotoxic T lymphocyte-derived granzyme B. The cleavage of Fak during apoptosis separates the tyrosine kinase domain from the focal adhesion targeting (FAT) domain. The carboxyl-terminal fragments that are generated suppress phosphorylation of endogenous Fak and thus resemble a natural variant of Fak, FRNK, that inhibits Fak activity by preventing the localization of Fak to focal adhesions. The cleavage of Fak by caspases may thus play an important role in the execution of the suicide program by disabling the anti-apoptotic function of Fak. Interestingly, rodent Fak lacks an optimal caspase-3 consensus cleavage site although it is cleaved in murine cells undergoing apoptosis at an upstream site. This appears to be the first example of a caspase substrate where the cleavage sites are not conserved between species.
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PMID:Caspases cleave focal adhesion kinase during apoptosis to generate a FRNK-like polypeptide. 964 76

The caspase family of proteases has previously been implicated in the biochemical cascade leading to apoptotic cell death. Recently caspase-3 was reported to be cleaved into its catalytically active subunits (17 and 13 kDa) following phytohemagglutinin (PHA) activation of peripheral blood mononuclear cells (C. Miossec et al., J. Biol. Chem. 272, 13459-13462). More recently, J. M. Zapata and colleagues (J. Biol. Chem. 273, 6916-6920, 1998), however, proposed that caspase-3 activity detected during T-cell activation was due to a methodological artifact related to the composition of the cell lysis buffer. Here we show that in PHA-activated Jurkat T-cells using the recommended lysis buffer detailed by Zapata et al., a caspase-3-like protease is activated and is accompanied by cleavage of PARP and alpha-spectrin into cleavage products suggestive of caspase-3 proteolytic activation. LDH release did not increase following PHA stimulation in this paradigm. Two caspase inhibitors, carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-D-DCB) and acetyl-Asp-Glu-Val-Asp-CHO, blocked IL-2 release in a dose-dependent manner. Caspase-3-like protease-generated PARP and alpha-spectrin breakdown product formation was also reduced by Z-D-DCB. In addition, Jurkat T-cells costimulated with anti-CD3 plus anti-CD28 produced significant levels of IL-2 that were also blocked by these caspase inhibitors. Importantly, IL-2 was determined in cell culture supernatants, thus avoiding a cell lysis step that might have enabled activation of caspase-3 by granzyme B. Collectively, these data support the role of caspase-3-like protease activity in Jurkat T-cell activation and demonstrate that caspase-3 like activity is necessary for IL-2 release in PHA-activated and anti-CD3/anti-CD28 costimulated Jurkat T-cells.
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PMID:Caspase-3-like activity is necessary for IL-2 release in activated Jurkat T-cells. 977 Mar 73

Granzyme B (GrB) is predicted to trigger apoptosis by activating preferred caspases, but the zymogens that are directly processed by the granzyme and the requirements for these interactions remain unclarified. We examined this dilemma by comparing the kinetics and pattern of GrB-mediated activation of the executioner caspase-7 in vitro and in vivo. GrB rapidly activates procaspase-7 in vitro by cleaving between the large and small subunits leaving the propeptide intact. During GrB-mediated apoptosis, the caspase-7 propeptide is removed and cleavage occurs between the subunits. Strikingly, caspase-7 is unprocessed in caspase-3-deficient MCF-7 cells exposed to GrB but is rapidly activated when the cells are solubilized. Transfection with caspase-3 restores the removal of the caspase-7 propeptide and the capacity of GrB to subsequently activate the caspase. The data suggest that GrB activates caspase-3, which then removes the propeptide of caspase-7 allowing activation by GrB. Thus GrB initiates the death pathway by processing the accessible caspase-3, and the caspase-7 propeptide regulates trans-activation of the zymogen by granzyme. As a consequence, two proteases, caspase-3 and GrB, are required to activate procaspase-7.
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PMID:Granzyme B mimics apical caspases. Description of a unified pathway for trans-activation of executioner caspase-3 and -7. 985 92

Caspases are cysteinyl aspartate-specific proteinases, many of which play a central role in apoptosis. Here, we report the identification of a new murine caspase homologue, viz. caspase-14. It is most related to human/murine caspase-2 and human caspase-9, possesses all the typical amino acid residues of the caspases involved in catalysis, including the QACRG box, and contains no or only a very short prodomain. Murine caspase-14 shows 83% similarity to human caspase-14. Human caspase-14 is assigned to chromosome 19p13.1. Northern blot analysis revealed that mRNA expression of caspase-14 is undetectable in all mouse adult tissues examined except for skin, while it is abundantly expressed in mouse embryos. In contrast to many other caspase family members, murine caspase-14 is not cleaved by granzyme B, caspase-1, caspase-2, caspase-3, caspase-6, caspase-7 or caspase-11, but is weakly processed into p18 and p11 subunits by murine caspase-8. No aspartase activity of murine caspase-14 could be generated by bacterial or yeast expression. Transient overexpression of murine caspase-14 in mammalian cells did not elicit cell death and did not interfere with caspase-8-induced apoptosis. In conclusion, caspase-14 is a member of the caspase family but no proteolytic or biological activities have been identified so far. The high constitutive expression levels in embryos and specific expression in adult skin suggest a role in ontogenesis and skin physiology.
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PMID:Identification of a new caspase homologue: caspase-14. 1020 98

Lymphocyte granule-mediated apoptosis occurs by perforin-mediated intracellular delivery of granule-associated serine proteases (granzymes). A granule-associated proteoglycan, namely serglycin, that contains chondroitin 4-sulfate (CS) glycosaminoglycans is present in the granules of cytotoxic cells. Serglycin acts as scaffold for packaging the positively charged granzymes and probably chaperones the proteases secreted extracellularly. To learn how the interaction of granzyme B (GrB) with serglycin might influence the apoptotic potential of this proteases, we have evaluated a model system where desalted CS is combined with isolated human granzyme. CS-GrB complexes were very stable, remaining undissociated in salt concentrations upwards to 500 mM (pH 7.4). On the basis of a capture enzyme immunoassay that accurately detects GrB, equivalent amounts of active free and CS-GrB, delivered by perforin or adenovirus, efficiently induced apoptosis in Jurkat cells and produced a similar time-dependent increase in caspase-3-like activity. CS-GrB processed isolated caspases-3 and -7 less efficiently than free granzyme. However, when added to cytosolic extracts, rates of processing were nearly equivalent for the two forms, suggesting cationic GrB may nonspecifically bind cytosolic proteins, leading to reduce proteolytic activity. Finally, GrB was found to be exocytosed from lymphocyte-activated killer cells as a neutral, high macromolecular weight complex, which possessed apoptotic activity. Collectively, the results indicate that neutral, high m.w. GrB has the capacity to induce cell death and will be useful to study the mechanism of cytotoxic cell-mediated apoptosis in vitro.
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PMID:Apoptosis induced by granzyme B-glycosaminoglycan complexes: implications for granule-mediated apoptosis in vivo. 1022 10

Ligation of the Fas receptor induces death-inducing signaling complex (DISC) formation, caspase activation, and subsequent apoptotic death of several cell types. Epstein-Barr virus (EBV)-positive group III Burkitt's lymphoma (BL) cell lines have a marked resistance to Fas-mediated apoptosis, although expressing each of the DISC components, Fas/ APO-1-associated death domain protein (FADD), and caspase-8 (FLICE/MACH/Mch5). The apoptotic pathway distal to the DISC is intact because ceramide analogs, staurosporine, and granzyme B activate caspase-3 and induce apoptosis. Fas resistance was not explained by the putative death-attenuating caspase-8 isoforms. However, while Fas-activated cytosolic extracts from sensitive cells were capable of processing both procaspase-8 and procaspase-3 into active subunit forms, resistant cell extracts did not possess either of these activities. Accordingly, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed higher transcript levels for the FLICE-inhibitory protein (FLIP(L)) in resistant cells and the ratio of caspase-8 to FLIP(L) measured by competition RT-PCR analysis directly correlated with susceptibility to Fas-mediated apoptosis of all cell lines. In addition, modification of the caspase-8/FLIP(L) ratio by caspase-8 or FLIP(L) overexpression was able to alter the susceptibility status of the cell lines tested. Our results imply that the relative levels of caspase-8 and FLIP(L) are an important determinant of susceptibility to Fas-mediated apoptosis.
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PMID:Modulation of caspase-8 and FLICE-inhibitory protein expression as a potential mechanism of Epstein-Barr virus tumorigenesis in Burkitt's lymphoma. 1047 98


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