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PubMed1998

Identification and characterization of murine caspase-14, a new member of the caspase family.

We report here the identification and characterization of a new member of the mouse caspase family, named caspase-14. Northern blot analysis of mRNA from various tissues with caspase-14-specific probe showed a major transcript size of approximately 2.4 kb and variant transcripts of 2.0 kb and 1.5 kb. The major transcript is detected mainly in the liver and to a lesser extent in the brain and kidney. Caspase-14 cDNA encodes a 257-amino acid-long protein that has significant homology to other members of the caspase family. Like other caspases, caspase-14 has a conserved active site, pentapeptide QACRG. However, it lacks an NH2-terminal prodomain or a caspase recruitment domain, suggesting that it could be a downstream caspase that depends on other initiator caspases for activation. Consistent with this, procaspase-14 can be processed in vitro by the death receptor-associated caspase-8 and caspase-10 but not other caspases, and in vivo after stimulation of cells with anti-Fas agonist antibody or Tumor Necrosis Factor-Related Apoptosis Inducing Ligand. Furthermore, procaspase-14 can be cleaved by granzyme B. These observations suggest that caspase-14 may play a role in death receptor and granzyme B-induced apoptosis.

Amino Acid Sequence↗
PubMed2000

Caspases and mitochondria in c-Myc-induced apoptosis: identification of ATM as a new target of caspases.

The mechanism(s) of c-Myc transcription factor-induced apoptosis is still obscure. The activation of c-Myc has been found to lead into the processing/activation of caspases (caspase-3), but the significance of this for the cell demise is debatable. Here we report that several targets of caspases (PKCdelta, MDM2, PARP, replication factor C, 70 kDa U1snRNP, fodrin and lamins) are cleaved during c-Myc-induced apoptosis in Rat-1 MycER cells, indicating an important role for caspases in the apoptotic process. We further found that the ATM (ataxia telangiectasia mutated)--protein is a novel key substrate of caspases. In in vitro assays, purified recombinant ATM protein was found to be cleaved by the effector caspases 3 and 7. The functional significance of the ATM cleavage is supported by the finding that ectopic expression of ATM protected in part against apoptosis. We also show that c-Myc-induced apoptosis involves loss of mitochondrial transmembrane potential, release of cytochrome c from mitochondria into the cytosol and subsequent processing of caspase-9. The cleavage of caspase-9 is, however, minimal and a much later event than the processing/activation of caspase-3, suggesting that it is not the apical caspase. Evidence is provided that there is, nevertheless, an upstream caspase(s) regulating the functions of caspase-3 and mitochondria. Additionally, it was found that p53 becomes upregulated, together with its transcriptional targets MDM2 and p21, upon c-Myc induction, but this occurs also at a later time than the activation of caspase-3.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Granzyme B mimics apical caspases. Description of a unified pathway for trans-activation of executioner caspase-3 and -7.

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.

Apoptosis↗
PubMed2003

Caspase-1 and caspase-8 cleave and inactivate cellular parkin.

Lesions in the parkin gene cause early onset Parkinson's disease by a loss of dopaminergic neurons, thus demonstrating a vital role for parkin in the survival of these neurons. Parkin is inactivated by caspase cleavage, and the major cleavage site is after Asp126. Caspases responsible for parkin cleavage were identified by several experimental paradigms. Transient coexpression of caspases and wild type parkin in HEK-293 cells identified caspase-1, -3, and -8 as efficient inducers of parkin cleavage whereas caspase-2, -7, -9, and -11 did not induce cleavage. A D126A parkin mutation abrogates cleavage induced by caspase-1 and -8, but not by caspase-3. In anti-Fas-treated Jurkat T cells, parkin cleavage was inhibited by caspase inhibitors hFlip and CrmA (but not by X-linked inhibitor of apoptosis (XIAP)), indicating that caspase-8 (but not caspase-3) is responsible for the parkin cleavage in this model. Moreover, induction of apoptosis in caspase-3-deficient MCF7 cells, either by caspase-1 or -8 overexpression or by tumor necrosis factor-alpha treatment, led to parkin cleavage. These results demonstrate that caspase-1 and -8 can directly cleave parkin and suggest that death receptor activation and inflammatory stress can cause loss of the ubiquitin ligase activity of parkin, thus causing accumulation of toxic parkin substrates and triggering dopaminergic cell death.

Apoptosis↗
PubMed2002

Caspase-10 is recruited to and activated at the native TRAIL and CD95 death-inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8.

The involvement of the death adaptor protein FADD and the apoptosis-initiating caspase-8 in CD95 and TRAIL death signalling has recently been demonstrated by the analysis of the native death-inducing signalling complex (DISC) that forms upon ligand-induced receptor cross-linking. However, the role of caspase-10, the other death-effector-domain-containing caspase besides caspase-8, in death receptor signalling has been controversial. Here we show that caspase-10 is recruited not only to the native TRAIL DISC but also to the native CD95 DISC, and that FADD is necessary for its recruitment to and activation at these two protein complexes. With respect to the function of caspase-10, we show that it is not required for apoptosis induction. In addition, caspase-10 can not substitute for caspase-8, as the defect in apoptosis induction observed in caspase-8-deficient cells could not be rescued by overexpression of caspase-10. Finally, we demonstrate that caspase-10 is cleaved during CD95-induced apoptosis of activated T cells. These results show that caspase-10 activation occurs in primary cells, but that its function differs from that of caspase-8.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Protection against Fas receptor-mediated apoptosis in hepatocytes and nonparenchymal cells by a caspase-8 inhibitor in vivo: evidence for a postmitochondrial processing of caspase-8.

Lymphocytes can kill target cells including hepatocytes during various inflammatory diseases by Fas receptor-mediated apoptosis. Caspase-8 is activated at the receptor level, thereby initiating the processing of downstream effector caspases. The aim of this study was to investigate the time course of caspase-8 activation and to evaluate the efficacy of the caspase-8 inhibitor IETD-CHO in a model of Fas-induced apoptosis in vivo. C3Heb/FeJ mice were treated with the anti-Fas antibody Jo-2 (0.6 mg/kg). Western blot analysis demonstrated increased cytochrome c in the cytosol (20 min), which was followed by the progressive activation of caspase-3, -9 (40-120 min), and caspase-8 (120 min). At 90 and 120 min, extensive hemorrhage was observed, indicating damage to sinusoidal lining cells. In addition, high plasma ALT levels (997 +/- 316 U/L) and histological evaluation indicated severe parenchymal cell injury. Parenchymal and nonparenchymal cells showed a similar increase in caspase-3 activity and DNA fragmentation. Treatment with IETD-CHO (10 mg/kg) attenuated the increase in caspase-3 activity and DNA fragmentation by 80-90% and completely prevented hemorrhage and parenchymal cell damage. IETD-CHO also prevented the early release of mitochondrial cytochrome c and the processing of caspase-3, -8, and -9. Thus, our data support the hypothesis that Fas-mediated apoptosis is dependent on caspase-8 activation in hepatocytes and nonparenchymal cells. However, the bulk of procaspase-8 is processed late, suggesting that only a small amount of procaspase-8 may actually be activated at the Fas receptor. This initial signal may be amplified by further activation of caspase-8 by effector caspases, i.e., after mitochondrial activation. Caspase-8 is a promising therapeutic target for inhibition of Fas-mediated apoptosis.

Animals↗
PubMed2004

Caspase-7 expanded function and intrinsic expression level underlies strain-specific brain phenotype of caspase-3-null mice.

Caspase-3-deficient mice of the 129S1/SvImJ (129) strain show severe brain development defects resulting in brain overgrowth and perinatal lethality, whereas on the C57BL/6J (B6) background, these mice develop normally. We therefore sought to identify the strain-dependent ameliorating gene. We biochemically isolated caspase-7 from B6-caspase-3-null (Casp3-/-) tissues as being the enzyme with caspase-3-like properties and capability of performing a caspase-3 surrogate function, apoptotic DNA fragmentation. Moreover, we show that, in contrast to the human enzymes, mouse caspase-7 is as efficient as caspase-3 at cleaving and thus inactivating ICAD (inhibitor of caspase-activated DNase), the inhibitor of apoptotic DNA fragmentation. Low levels of caspase-7 expression and activation correlate with lack of DNA fragmentation in 129-Casp3-/- apoptotic precursor neurons, whereas B6-Casp3-/- cells, which can fragment their DNA, show higher levels of caspase-7 expression and activation. The amount of caspase-7 activation in apoptotic precursor neurons is independent of the presence of caspase-3. Together, our findings demonstrate for the first time a strong correlation between caspase-7 activity, normal brain development, and apoptotic DNA fragmentation in Casp3-/- mice.

Animals↗
PubMed1999

Spontaneous human monocyte apoptosis utilizes a caspase-3-dependent pathway that is blocked by endotoxin and is independent of caspase-1.

Apoptosis is an important mechanism for regulating the numbers of monocytes and macrophages. Caspases (cysteine-aspartate-specific proteases) are key molecules in apoptosis and require proteolytic removal of prodomains for activity. Caspase-1 and caspase-3 have both been connected to apoptosis in other model systems. The present study attempted to delineate what role these caspases play in spontaneous monocyte apoptosis. In serum-free conditions, monocytes showed a commitment to apoptosis as early as 4 h in culture, as evidenced by caspase-3-like activity. Apoptosis, as defined by oligonucleosomal DNA fragmentation, was prevented by a generalized caspase inhibitor, z-VAD-FMK, and the more specific caspase inhibitor, z-DEVD-FMK. The caspase activity was specifically attributable to caspase-3 by the identification of cleavage of procaspase-3 to active forms by immunoblots and by cleavage of the fluorogenic substrate DEVD-AFC. In contrast, a caspase-1 family inhibitor, YVAD-CMK, did not protect monocytes from apoptosis, and the fluorogenic substrate YVAD-AFC failed to show an increase in activity in apoptotic monocytes. When cultured with LPS (1 microgram/ml), monocyte apoptosis was prevented, as was the activation of caspase-3. Unexpectedly, LPS did not change baseline caspase-1 activity. These findings link spontaneous monocyte apoptosis to the proteolytic activation of caspase-3.

Apoptosis↗
PubMed1998

Processing/activation of caspases, -3 and -7 and -8 but not caspase-2, in the induction of apoptosis in B-chronic lymphocytic leukemia cells.

Chlorambucil and prednisolone, two commonly used drugs in the treatment of chronic lymphocytic leukemia (CLL), induce apoptosis in CLL cells. We have investigated the involvement in this apoptotic cell death of caspases, which cleave critical cellular substrates thereby acting as the executioners of the apoptotic process. Induction of spontaneous or chlorambucil/prednisolone-induced apoptosis of freshly isolated B-CLL cells in culture resulted in the activation of the 'effector' caspases, -3 and -7, but generally not of caspase-2. Activation of caspases-3 and -7 was accompanied by the proteolysis of the DNA repair enzyme, poly (ADP-ribose) polymerase. Induction of apoptosis was also accompanied by the processing of caspase-8, the extent of which varied between patients. Induction of apoptosis and processing of all the caspases was inhibited by the cell permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.fmk). Our results demonstrate a key role for the activation and processing of caspases in the execution phase of apoptosis in CLL cells. Apoptosis of CLL cells resulted in the selective activation of some but not all caspases. Our results suggest that the dysregulation of apoptosis observed in CLL may be due to the signalling leading to the activation of caspases rather than a deletion of pro-caspases. High levels of caspase-8 in CLL cells in conjunction with low levels of CD95 receptor may offer new therapeutic opportunities for the treatment of CLL.

Aged↗
PubMed2004

Bcl-2-regulated apoptosis and cytochrome c release can occur independently of both caspase-2 and caspase-9.

Apoptosis in response to developmental cues and stress stimuli is mediated by caspases that are regulated by the Bcl-2 protein family. Although caspases 2 and 9 have each been proposed as the apical caspase in that pathway, neither is indispensable for the apoptosis of leukocytes or fibroblasts. To investigate whether these caspases share a redundant role in apoptosis initiation, we generated caspase-2(-/-)9(-/-) mice. Their overt phenotype, embryonic brain malformation and perinatal lethality mirrored that of caspase-9(-/-) mice but were not exacerbated. Analysis of adult mice reconstituted with caspase-2(-/-)9(-/-) hematopoietic cells revealed that the absence of both caspases did not influence hematopoietic development. Furthermore, lymphocytes and fibroblasts lacking both remained sensitive to diverse apoptotic stimuli. Dying caspase-2(-/-)9(-/-) lymphocytes displayed multiple hallmarks of caspase-dependent apoptosis, including the release of cytochrome c from mitochondria, and their demise was antagonized by several caspase inhibitors. These findings suggest that caspases other than caspases 2 and 9 can promote cytochrome c release and initiate Bcl-2-regulated apoptosis.

Animals↗
PubMed2006

Activation of caspase-8 and caspase-12 pathways by 7-ketocholesterol in human retinal pigment epithelial cells.

PURPOSE: To determine whether caspase or cathepsin pathways are activated in human retinal pigment epithelial cells (ARPE-19) after exposure to 7-ketocholesterol (7kCh). METHODS: ARPE-19 cells were exposed to 7kCh with or without z-VAD-fmk, a pan-caspase inhibitor. Caspase-3, -8, and -9 activities were measured by a fluorochrome inhibitor of caspase (FLICA) assay. Caspase-12 activity was detected by Western blotting. RT-PCR was performed for 18s, mortalin-2, cathepsins B, D, and L/V2. RESULTS: At 24 hours, 7kCh-treated cultures had increased caspase-8 (P < 0.001) and caspase-3 (P < 0.001) activities compared with vehicle-treated cultures. 7kCh-induced caspase-3 activation was blocked by z-VAD-fmk (P < 0.001). Caspase-9 was not activated by 7kCh treatment (P > 0.05). Procaspase-12 was cleaved into its active form after treatment with 7kCh for 24 hours. At 6 hours, the RNA level for mortalin-2, a pro-survival gene, was upregulated. ARPE-19 cells did not express RNA for cathepsins B, D, or L/V2 under any conditions. CONCLUSIONS: In ARPE-19 cells, 7kCh-induced apoptosis uses the receptor-mediated caspase-8 pathway and the endoplasmic reticulum stress-induced caspase-12 pathway but not the mitochondrial caspase-9 pathway. The cathepsin pathways are not involved in 7kCh-induced cell death. These data demonstrate that 7kCh causes a loss of cell viability through caspase-dependent apoptosis and can act as an oxidative stressor leading to retinal pigment epithelial cell atrophy. Elucidating the specific apoptotic pathways involved may have therapeutic potential for AMD and other retinal diseases.

Amino Acid Chloromethyl Ketones↗
PubMedDate not supplied

Caspase 1 and caspase 8 in HIV infected patients with and without tuberculosis.

BACKGROUND: Caspase 8 is involved in apoptosis mediated by Fas and p55 tumor necrosis factor receptor ligation in HIV infection. Apoptosis is partially mediated by interleukin-1beta-converting enzyme (caspase-1). AIMS: We determined apoptosis, using caspase-1 and caspase-8, among patients with HIV infection, with and without tuberculosis (TB), those with TB alone and healthy individuals. SETTING AND DESIGN: Cross-sectional analysis of caspase-1 and caspase-8 among patients with HIV infection, with and without TB, those with TB alone and healthy individuals. MATERIALS AND METHODS: Nineteen HIV infected patients with TB (HIV+/TB+) and 20 with HIV infection without TB (HIV+/TB-) were studied. Fifteen individuals with TB alone were disease controls (HIV-/TB+) and 20 were healthy controls (HIV-/TB-). Caspases were measured by single-step ELISA using commercially available monoclonal antibodies. STATISTICAL ANALYSIS: Two-way ANOVA and Pearson's correlation coefficient. RESULTS: Mean CD4 counts of HIV+/TB+ were lower than HIV+/TB- (p<0.05). OD value of caspase 1 in HIV+/TB+ was 0.295+0.05, while that in HIV+/TB- it was 0.302+0.18. It was 0.293+0.07 in HIV-/TB+ and in HIV-/TB- the values were 0.287+0.06. OD value of caspase 8 in HIV+/TB+ was 0.307+ 0.07, lower than HIV+/TB- (0.927+0.25). It was 0.008+0.03 in HIV-/TB+ and in HIV-/TB-, 0.074+0.004. Values of caspase 8 in patients with HIV infection (with/without TB) were higher than those with TB alone or healthy individuals (p<0.01). Levels of caspase 8 in HIV+/TB- were higher than patients with HIV+/TB+ (p<0.01). CONCLUSION: Levels of caspase-1 are not different irrespective of presence or otherwise of TB and HIV infection. Fas-related apoptosis is higher in HIV infection. With concomitant TB, levels of caspase 8 were lower as compared with those without TB.

Adult↗
PubMed1999

Caspase-8 mediates caspase-3 activation and cytochrome c release during singlet oxygen-induced apoptosis of HL-60 cells.

We reported previously that singlet oxygen, generated by irradiation of rose bengal with visible light, induced apoptosis in human promyelocytic leukemia HL-60 cells. However, the mechanism of apoptosis caused by this reactive oxygen species is unclear. In this study, we demonstrate that singlet oxygen induced caspase-3 activation and Z-DEVD-FMK, a caspase-3 inhibitor, blocked apoptosis induction, while caspase-1 activity was not detectable and the caspase-1 inhibitor Z-YVAD-FMK had a very limited effect on apoptosis. This suggests that the activation of caspase-3 by singlet oxygen is essential for the commitment of cells to undergo apoptosis. Further studies showed that singlet oxygen induced an increase in caspase-8 activity and a reduction in mitochondrial cytochrome c. Time course analysis indicated that the cleavage of caspase-8 precedes that of caspase-3. In addition, blockade of caspase-8 by Z-IETD-FMK inhibited cleavage of pro-caspase-3 and prevented loss of mitochondrial cytochrome c. These results suggest that caspase-8 mediates caspase-3 activation and cytochrome c release during singlet oxygen-induced apoptosis in HL-60 cells.

Apoptosis↗
PubMed2005

Influence of caspase activity on micronuclei detection: a possible role for caspase-3 in micronucleation.

Aneugenic and clastogenic agents are good inducers of both micronuclei and apoptosis. In its turn, apoptosis may modify the threshold values for the induction of micronuclei. This is of major concern for accurate assessment of hazard related to exposure to mutagens. In the present work we studied the influence of caspases, the key regulators of the apoptotic process, on the induction of micronuclei in the cytokinesis block micronucleus assay. For this, we applied a combined approach in which both human peripheral blood mononucleated cells (PBMC) and the paired human breast carcinoma cell lines MCF-7, which is caspase-3 deficient, and the caspase-3 transfected MCF-7 (MCF-7casp-3) were used to study the influence of caspase activity on micronuclei. When nocodazole induced apoptosis was inhibited by the use of inhibitors of the two main apical caspases-8 and -9 in PBMC, the frequencies of micronucleated binucleates (MNCB) increased with inhibition of these caspases confirming that apoptosis can eliminate micronucleated cells. On the contrary when caspase-3 was inhibited, the frequencies of MNCB was lower, suggesting a role of caspase-3, also in micronuclei formation. To verify this hypothesis, we compared the induction of apoptosis and micronuclei by the aneugen nocodazole, the clastogen methyl methane sulfonate (MMS) and the non-mutagenic apoptogen staurosporin in MCF-7 and MCF-7casp-3 cells. The results showed that when caspase-3 activity was impaired, in the parental MCF-7 cell line or in the MCF-7casp-3 cells in the presence of the caspase-3 inhibitor, the frequencies of nocodazole or MMS induced micronuclei decreased. These results suggest that caspase-3, besides its function as an effector caspase in the apoptotic pathway, is also involved in the formation of micronuclei.

Aneugens↗
PubMed2000

Caspases -2, -3, -6, and -9, but not caspase-1, are activated in sepsis-induced thymocyte apoptosis.

Sepsis induces extensive lymphocyte cell death that may contribute to immune depression and morbidity/mortality in the disorder. bcl-2 is a member of a new class of oncogenes that prevents cell death from an array of noxious stimuli. Transgenic mice that overexpress BCL-2 in T lymphocytes are resistant to sepsis-induced T cell apoptosis, and mortality was decreased in sepsis. The purpose of this study was to identify key initiator and executioner "caspases" involved in sepsis-induced lymphocyte apoptosis and to determine if BCL-2 acts prior to caspase activation. Thymi were removed 5-22 h post-cecal ligation and puncture (CLP) or sham surgery. Apoptosis was evaluated in thymocytes by annexin-V FITC labeling and flow cytometry. Caspase-1 activity was determined by western blot analysis of the procaspase protein and p20 subunit of the activated caspase; activities of caspases -2, -6, and -9 were determined by colorimetric assays using specific substrates conjugated to a color reporter molecule. Caspase-3 activity was determined both by western blot and by a fluorogenic assay in which a fluorescent compound was generated. Thymocytes from CLP mice had markedly increased apoptosis and activation of caspases -2, -3, -6, and -9 in comparison with thymocytes of sham-operated mice. Caspase-1 was not activated. BCL-2 prevented sepsis-induced thymocyte apoptosis and inhibited activation of all caspases. We conclude that sepsis causes activation of multiple caspases and that BCL-2 acts upstream as an inhibitor of caspase activation. The pattern of caspase activation suggests a mitochondrial mediated pathway.

Animals↗
PubMed2006

Caspase-3 deficiency during development increases vulnerability to hypoxic-ischemic injury through caspase-3-independent pathways.

Neonatal hypoxia-ischemia (H-I) is a common cause of perinatal morbidity and mortality leading to prominent activation of caspase-3 in the brain. Previous studies have shown that acute inhibition of caspase-3 can protect against neonatal H-I in rats. In this study, we investigated brain injury following neonatal H-I in mice deficient in caspase-3. Wild-type, caspase-3+/- and caspase-3-/- mice underwent unilateral carotid ligation at postnatal day (P) 7, followed by 45 min of exposure to 8% oxygen. Surprisingly, tissue loss at P14 was significantly higher in caspase-3-/- mice when compared to wild-type littermates. As in rats, we found that acute inhibition of caspase-3 in mice leads to decrease in tissue loss at P14. There was no difference in nuclear morphology, DNA laddering or calpain activation between caspase-3-/-caspase-3+/- and wild-type mice subjected to H-I, and there was no evidence for compensatory activation of other caspases in caspase-3-/- mice. Also, all genotypes showed evidence of mitochondrial dysfunction after H-I, suggesting that this is a critical point in regulation of neuronal cell death following neonatal H-I. Our results suggest that long-term inhibition of caspase-3 during development, unlike acute inhibition, leads to upregulation of caspase-3-independent cell death pathways and increases the vulnerability of the developing brain to neonatal H-I injury.

Animals↗
PubMed2002

Caspase activation and neuroprotection in caspase-3- deficient mice after in vivo cerebral ischemia and in vitro oxygen glucose deprivation.

Caspase-3 is a major cell death effector protease in the adult and neonatal nervous system. We found a greater number and higher density of cells in the cortex of caspase-3(-/-) adult mice, consistent with a defect in developmental cell death. Caspase-3(-/-) mice were also more resistant to ischemic stress both in vivo and in vitro. After 2 h of ischemia and 48 h of reperfusion, cortical infarct volume was reduced by 55%, and the density of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells was decreased by 36% compared with wild type. When subjected to oxygen-glucose deprivation (2 h), cortical neurons cultured from mice deficient in caspase-3 expression were also more resistant to cell death by 59%. Mutant brains showed caspase-specific poly(ADP-ribose) polymerase cleavage product (85-kDa fragment) in vivo and in vitro, suggesting redundant mechanisms and persistence of caspase-mediated cell death. In the present study, we found that caspase-8 mediated poly(ADP-ribose) polymerase cleavage in caspase-3(-/-) neurons in vivo and in vitro. In addition, mutant neurons showed no evidence of compensatory activation by caspase-6 or caspase-7 after ischemia. Taken together, these data extend the pharmacological evidence supporting an important role for caspase-3 and caspase-8 as cell death mediators in mammalian cortex and indicate the potential advantages of targeting more than a single caspase family member to treat ischemic cell injury.

Animals↗
PubMed2006

Cytokine response modifier a inhibition of initiator caspases results in covalent complex formation and dissociation of the caspase tetramer.

Active caspases are generally composed of two catalytic domains, each containing a large (p20) and a small (p10) subunit so that a fully active caspase has the organization (p20-p10)(2). The cowpox serpin crmA suppresses host apoptosis and inflammation by inhibiting endogenous caspases. We report on the mechanism crmA uses to inhibit caspases 1, 6, and 8. Native PAGE showed formation of tight crmA-caspase complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry provided evidence for a covalent crmA-p20 thioester linkage. SDS-PAGE of isolated complexes showed near complete loss of the p10 subunit from initiator caspases 1 and 8 but not from the executioner caspase-6. This was confirmed for caspase-1 by sequencing and Western blotting. Size exclusion chromatography indicated a size of approximately 60 kDa for complexes with caspases 1 and 8, consistent with a crmA.p20 species, suggesting that the p20-p10 interface and possibly the p10-p10 interface had been disrupted. In contrast, crmA.caspase-6 complex behaved as an equilibrium mixture of crmA(2).(p20-p10)(2) and crmA.(p20-p10). Complex deacylation rates were all slow, suggesting effective kinetic trapping of the covalent thioacyl intermediate. These results suggest a novel serpin inhibition mechanism through which crmA down-regulates apoptosis and inflammation. This involves (i) rapid formation of covalent complex with initiator caspases 8 or 1, (ii) very slow deacylation, and (iii) loss of the caspase p10 subunit for initiator but not for executioner caspases, so that any free p20 formed by deacylation of initiator caspases cannot reassociate to active heterotetramer, thus resulting in irreversible inhibition of apoptosis and inflammation.

Biopolymers↗