Caspase structure, proteolytic substrates, and function during apoptotic cell death

Dw, Nicholson

doi:10.1038/sj.cdd.4400598

Cited by 1,400 publications

(1,069 citation statements)

References 197 publications

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“…3 On the molecular level, members of a family of cysteine proteases, designated caspases, are responsible for programmed cell death. 4,5 In response to an apoptotic stimulus a caspase cascade is activated in which initiator caspases are first activated through dimerization and they, in turn, act to cleave and activate downstream effector caspases. 6 Once matured, effector caspases can cleave specific protein substrates within the cell, resulting in the apoptotic phenotype.…”

Section: Introductionmentioning

confidence: 99%

“…6 Once matured, effector caspases can cleave specific protein substrates within the cell, resulting in the apoptotic phenotype. 5,7 Numerous studies have implicated caspase-3 as one of the main effector caspases in mammalian cells. 5 The importance of this gene is exemplified by the phenotype of the caspase-3-null mice that are characterized by severe defects in neuronal development due to a lack of normal programmed cell death.…”

Section: Introductionmentioning

confidence: 99%

“…5,7 Numerous studies have implicated caspase-3 as one of the main effector caspases in mammalian cells. 5 The importance of this gene is exemplified by the phenotype of the caspase-3-null mice that are characterized by severe defects in neuronal development due to a lack of normal programmed cell death. 8 Other studies performed using caspase-3-null MCF-7 breast carcinoma cells demonstrate that caspase-3 is essential for inactivation of ICAD (inhibitor of caspase-activated DNase) and is therefore the primary activator of apoptotic DNA fragmentation.…”

Section: Introductionmentioning

confidence: 99%

“…Maturation is executed via a two-step mechanism. 5 First, the large and the small subunit are generated by proteolytic cleavage mediated via an initiator caspase 12 or through pH-dependent autocatalytic processing. 13 In a second step, the preactivated caspase-3 selfcatalyses removal of its prodomain from the large subunit to generate the p17 polypeptide.…”

Section: Introductionmentioning

confidence: 99%

“…Although in general peptide-based inhibitors show substantial disadvantages, like poor cell penetration and poor metabolic stability in cell-based assays, some classes have proven effective at inhibiting recombinant caspases in vitro, as well as in cellbased systems. The aspartic acid-esterified nonselective tripeptide-fluoromethylketone, Z-VAD(OMe)-CH2F, which is presumably de-esterified and activated upon entry into cells, is efficacious at inhibiting cell death in vivo; 5,17 this is despite the drawbacks of the active counterpart, which include the lack of chemical stability and electrophilic promiscuity, that potentially results in activity against other biological targets. 5 The mechanisms that regulate caspase-3 activation have been extensively studied, but few studies have investigated the fate of caspase-3 once activated or for that matter once inhibited.…”

Section: Introductionmentioning

confidence: 99%

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Catalytic activity of caspase-3 is required for its degradation: stabilization of the active complex by synthetic inhibitors

et al. 2004

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The activation of caspase-3 represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Upon induction of apoptosis, the large (p17) and small (p12) subunits, comprising active caspase-3, are generated via proteolytic processing of a latent proenzyme dimer. Two copies of each individual subunit are generated to form an active heterotetramer. The tetrameric form of caspase-3 cleaves specific protein substrates within the cell, thereby producing the apoptotic phenotype. In contrast to the proenzyme, once activated in HeLa cells, caspase-3 is difficult to detect due to its rapid degradation. Interestingly, however, enzyme stability and therefore detection of active caspase-3 by immunoblot analysis can be restored by treatment of cells with a peptide-based caspase-3 selective inhibitor, suggesting that the active form can be stabilized through protein-inhibitor interaction. The heteromeric active enzyme complex is necessary for its stabilization by inhibitors, as expression of the large subunit alone is not stabilized by the presence of inhibitors. Our results show for the first time, that synthetic caspase inhibitors not only block caspase activity, but may also increase the stability of otherwise rapidly degraded mature caspase complexes. Consistent with these findings, experiments with a catalytically inactive mutant of caspase-3 show that rapid turnover is dependent on the activity of the mature enzyme. Furthermore, turnover of otherwise stable active site mutants of capase-3 is rescued by the presence of the active enzyme suggesting that turnover can be mediated in trans.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Catalytic activity of caspase-3 is required for its degradation: stabilization of the active complex by synthetic inhibitors

et al. 2004

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Detection of caspases activation by fluorochrome-labeled inhibitors: Multiparameter analysis by laser scanning cytometry

et al. 2001

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Background The fluorochrome‐labeled inhibitors of caspases (FLICA) were recently used as markers of activation of these enzymes in live cells during apoptosis (Bedner et al.: Exp Cell Res 259:308–313, 2000). The aims of this study were to (a) explore if FLICA can be used to study intracellular localization of caspases; (b) combine the detection of caspase activation with analysis of the changes with cell morphology detected by microscopy and laser scanning cytometry (LSC); and (c) adapt the assay to fixed cells that would enable correlation, by multiparameter analysis, of caspase activation with the cell attributes that require cell permeabilization in order to be measured. Methods Apoptosis of human MCF‐7, U‐937, or HL‐60 cells was induced by camptothecin (CPT) or tumor necrosis factor‐α (TNF‐α) combined with cycloheximide (CHX). Binding of FLICA to apoptotic versus nonapoptotic cells was studied in live cells as well as following their fixation and counterstaining of DNA. Intensity of cell labeling with FLICA and DNA‐specific fluorochromes was measured by LSC. Results Exposure of live cells to FLICA led to selective labeling of cells that had morphological changes characteristic of apoptosis. The FLICA labeling withstood cell fixation and permeabilization, which made it possible to stain DNA and measure its content for identification of the cell cycle position of labeled cells. When fixed cells were treated with FLICA, both apoptotic and nonapoptotic cells became strongly labeled and the labeling pattern was consistent with the localization of caspases as reported in the literature. A translocation of the FLICA binding targets from mitochondria to cytosol was seen in the MCF‐7 cells treated with CPT. FLICA binding was largely (>90%) prevented by the substrates of the caspases or by the unlabeled caspase inhibitors having the same peptide moiety as the respective FLICA. Conclusions The detection of caspase activation combined with cell permeabilization requires exposure of live cells to FLICA followed by their fixation. Cell reactivity with the respective FLICA, under these conditions, identifies the activated caspases and makes it possible to correlate their activation with the cell cycle position and other cell attributes that can be measured only after cell fixation/permeabilization. FLICA can also be used to study intracellular localization of caspases, including their translocation. Cytometry 44:73–82, 2001. © 2001 Wiley‐Liss, Inc.

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Transient oxidative stress in SH‐SY5Y human neuroblastoma cells results in caspase dependent and independent cell death and tau proteolysis

Krishnamurthy

Mays

Bijur

et al. 2000

J of Neuroscience Research

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The effects of an oxidative insult on cell survival and tau metabolism were investigated in human neuroblastoma SH‐SY5Y cells. In this treatment paradigm cells were exposed to the membrane permeant oxidant tert‐butylhydroperoxide (tBHP) for 40 min, returned to fresh media and cell survival/death was monitored during the post‐treatment period. Cell viability decreased significantly by 6 hr after tBHP exposure, and by 24 hr lactate dehydrogenase (LDH) release was 40.1 ± 8.8% in tBHP treated cells compared to 8.1 ± 4.7% in control cells. This oxidative stress paradigm also resulted in significant activation of caspase‐3 by 2 hr post‐treatment and nuclear apoptotic morphology. Furthermore, tBHP treatment also resulted in delayed tau proteolysis that was first evident 2 hr post‐treatment. Treatment of the cells with the general caspase inhibitor Boc‐Asp(OMe)‐Fluoromethylketone (BAF) completely inhibited caspase‐3 activation in response to tBHP, and delayed, but did not prevent cell death. BAF treatment also decreased tau proteolysis. In vitro, recombinant tau was readily proteolyzed by active recombinant caspase‐3 into a stable breakdown product. Further tau in the cell lysates was cleaved by active recombinant caspase‐3 at a rate, and to an extent similar to that observed for the well‐established caspase‐3 substrate poly(ADP‐ribose)polymerase (PARP). These results suggest that oxidative stress‐induced cell death occurs through both caspase‐dependent and‐independent pathways, and that tau is likely an in situ substrate of caspase‐3. J. Neurosci. Res. 61:515–523, 2000. © 2000 Wiley‐Liss, Inc.

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Caspase structure, proteolytic substrates, and function during apoptotic cell death

Cited by 1,400 publications

References 197 publications

Catalytic activity of caspase-3 is required for its degradation: stabilization of the active complex by synthetic inhibitors

Catalytic activity of caspase-3 is required for its degradation: stabilization of the active complex by synthetic inhibitors

Detection of caspases activation by fluorochrome-labeled inhibitors: Multiparameter analysis by laser scanning cytometry

Transient oxidative stress in SH‐SY5Y human neuroblastoma cells results in caspase dependent and independent cell death and tau proteolysis

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