Interleukin-1beta-converting enzyme (ICE) and related cell death genes ICErel-II and ICErel-III map to the same PAC clone at band 11q22.2-22.3.
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Apoptosis↗
Biomedical subjects
D W Nicholson
Publications and source records attributed to D W Nicholson.
Caspases: killer proteases.
Caspases (cysteinyl aspartate-specific proteinases) mediate highly specific proteolytic cleavage events in dying cells, which collectively manifest the apoptotic phenotype. The key and central role that these enzymes play in a biochemical cell-suicide pathway has been conserved throughout the evolution of multicellular eukaryotes.
Amino Acid Sequence↗
Control of apoptosis by proteases.
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Animals↗
A combinatorial approach for determining protease specificities: application to interleukin-1beta converting enzyme (ICE).
BACKGROUND: Interleukin-1beta converting enzyme (ICE/caspase-1) is the protease responsible for interleukin-1beta (IL-1beta) production in monocytes. It was the first member of a new cysteine protease family to be identified. Members of this family have functions in both inflammation and apoptosis. RESULTS: A novel method for identifying protease specificity, employing a positional-scanning substrate library, was used to determine the amino-acid preferences of ICE. Using this method, the complete specificity of a protease can be mapped in the time required to perform one assay. The results indicate that the optimal tetrapeptide recognition sequence for ICE is WEHD, not YVAD, as previously believed, and this led to the synthesis of an unusually potent aldehyde inhibitor, Ac-WEHD-CHO (Ki = 56 pM). The structural basis for this potent inhibition was determined by X-ray crystallography. CONCLUSIONS: The results presented in this study establish a positional-scanning library as a powerful tool for rapidly and accurately assessing protease specificity. The preferred sequence for ICE (WEHD) differs significantly from that found in human pro-interleukin-1beta (YVHD), which suggests that this protease may have additional endogenous substrates, consistent with evidence linking it to apoptosis and IL-1alpha production.
Caspase 1↗
Disulfiram is a potent inhibitor of proteases of the caspase family.
We have recently shown that dithiocarbamate (DC) disulfides inhibit proteolytic processing of the caspase-3 proenzyme in Jurkat T lymphocytes treated with anti-CD95 (Fas/APO-1) antibody. Because the processing can be accomplished by caspase activity, we investigated the effect of DC disulfides, such as disulfiram (DSF), on active caspases. DSF showed a dose-dependent inhibition was prevented by including dithiothreitol (DTT) in the reaction buffer, thiol-disulfide exchange between inhibitor and target is suggested. Direct interaction of DSF with caspases was confirmed by its inhibition of the purified Ac-DEVD-AMC cleaving protease, caspase-3 (CPP32/apopain). An apparent rate constant (K(app)) for this inhibition was estimated to be 0.45 x 10(3)M(-1)s(-1). DSF was also observed to inhibit the purified Ac-YVAD-AMC cleaving enzyme, caspase-1 (interleukin-1 beta-converting enzyme, ICE), with a K(app) of 2.2 x 10(3) M(-1)s(-1). In this case protein mixed disulfide formation between DSF and caspase-1 was directly demonstrated using 35S-labeled DSF. The physiological disulfide GSSG was also observed to influence the activity of caspases. A glutathione buffer (5 mM) with a GSH:GSSG ratio of 9:1 decreased the Ac-DEVD-AMC cleaving activity in S100 cytosolic extracts by 50% as compared to GSH controls without GSSG. In conclusion, our study shows that caspases are quite sensitive to thiol oxidation and that DSF is a very potent oxidant of caspase protein thiol(s), being 700-fold more potent than glutathione disulfide.
Alcohol Deterrents↗
Interleukin 1 beta converting enzyme-like proteases are essential for p53-mediated transcriptionally dependent apoptosis.
p53-mediated apoptosis in baby rat kidney (BRK) cell lines transformed by E1A and p53(val135) requires a transcriptionally functional p53. Coexpression of the E1B 19K protein in BRK cell lines transformed by E1A and p53(val135) rescues cells from p53-mediated apoptosis, and this is paralleled by the absence of both lamin and poly(ADP-ribose) polymerase cleavage. Therefore, the role of interleukin 1 beta converting enzyme (ICE)-like porteases in p53-mediated, transcriptionally dependent apoptosis was investigated. The ICE-like protease CPP32 was proteolytically activated during p53-mediated apoptosis in BRK cells, and this required a transcriptionally competent p53. Substitution of the p53 transactivation domain with the transactivation domain of herpes simplex virus VP16 (VP16/p53) resulted in accelerated kinetics of both apoptosis and Bax induction. Moreover, apoptosis induced by p53, VP16/p53, and Bax was abrogated by Z-VAD.FMK, an inhibitor of ICE-like proteases. These results indicate that all apoptotic pathways downstream of p53-mediated transcription converge upon the activation of ICE-like proteases.
Adenovirus E1A Proteins↗
Human ICE/CED-3 protease nomenclature.
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Alternative Splicing↗
Cleavage of CPP32 by granzyme B represents a critical role for granzyme B in the induction of target cell DNA fragmentation.
Cytotoxic T lymphocytes (CTLs) are able to recognize and destroy target cells bearing foreign antigen using one of two distinct mechanisms: granule- or Fas-mediated cytotoxicity. The exact mechanisms involved in the induction of apoptotic cell death remain elusive; however, it seems likely that a family of cysteine proteases related to interleukin-1beta converting enzyme are involved. One family member, CPP32, has been identified as an intracellular substrate for granzyme B, a CTL-specific serine protease responsible for the early induction of target cell DNA fragmentation. Here we use cytolytic cells from granzyme B-deficient mice to confirm that cleavage and activation of CPP32 represents a nonredundant role for granzyme B and that this activation plays a role in the induction of DNA fragmentation in target cells, a signature event for apoptotic cell death. A peptide inhibitor of CPP32-like proteases confirmed the function of these enzymes in fragmentation. 51Cr release was not suppressed under these conditions, suggesting that granzyme B cleavage of CPP32 is primarily involved in the induction of DNA fragmentation and not membrane damage during CTL-induced apoptosis.
Animals↗
Regulation of leukotriene-biosynthetic enzymes during differentiation of myelocytic HL-60 cells to eosinophilic or neutrophilic cells.
Leukotrienes (LTs) are potent mediators of bronchial inflammation and are predominantly produced by myeloid cells. As myelocytic cells differentiate towards either eosinophils or neutrophils, the profile of leukotrienes they produce upon stimulation diverges. Eosinophils produce mainly cysteinyl leukotrienes whereas neutrophils predominantly synthesize 5(S), 12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid (LTB delta). The mechanism by which this change in leukotriene composition occurs is unknown. In this study, we investigated the control of leukotriene biosynthetic enzymes during myeloid cell differentiation. Western-blot analyses of myelocytic leukemia cell lines, HL-60#7 and HL-60, differentiated towards eosinophilic or neutrophilic cell types, respectively, demonstrated that as myelocytic cells differentiate towards eosinophils or neutrophils, the protein levels of cytosolic phospholipase A2 (cPLA2) remain constant, whereas 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) levels are simultaneously elevated. As myelocytic cells become more eosinophil-like, 5(S)-hydroxy- 6(R)-S-glutathionyl-7,9-trans-11, 14-cis-eicosatetraenoic acid (LTC delta) synthase activity and expression of both the protein and messenger RNA in the cells are dramatically increased (approximately 75-fold), while the LTC delta synthase level and activity in neutrophil-like cells remain constant at very low levels. In contrast, in neutrophilic cells, the amount of 5,6-oxido-7,9,11,14-eicosatetraenoic acid (LTA delta) hydrolase was elevated approximately 100-fold greater than the increase in LTA delta hydrolase from eosinophilic cells. These results indicate that as a myeloid cell differentiates towards a granulocyte, similar mechanisms of regulation may be applied to the leukotriene biosynthetic pathway up to the point at which the pathway diverges. At the stage in the leukotriene biosynthetic pathway where LTA delta may be converted to either LTC delta or to LTB delta, specific regulators of transcription may become activated as a myelocytic cell differentiates, thereby causing increased LTA delta hydrolase or LTC delta synthase expression.
5-Lipoxygenase-Activating Proteins↗
Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death.
Proteolysis mediated by the interleukin 1 beta-converting enzyme (ICE) homologues is an important mechanism of the apoptotic process. The ICE homologue apopain/CPP-32/Yama (subsequently referred to as apopain) cleaves poly(ADP-ribose)polymerase (PARP) early during apoptosis. Additional apoptosis-specific protein cleavages have been observed in which the direct involvement of ICE-like proteases has been postulated. These substrates include the 70-kD protein component of the U1-ribonucleoprotein (U1-70kD), and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). The present studies demonstrate that U1-70kD and DNA-PKcs are excellent substrates for apopain, with cleavage occurring at sites that are highly similar to the cleavage site within PARP. The fragments generated from isolated protein substrates by apopain are identical to those observed in intact apoptotic cells, in apoptotic cell extracts, and in normal cell extracts to which apopain has been added. Like PARP, cleavage of these substrates in apoptotic cell extracts is abolished by nanomolar concentrations of Ac-DEVD-CHO and micromolar amounts of Ac-YVAD-CHO, confirming the involvement of apopain or an apopain-like activity. We propose that a central function of apopain or similar homologues in apoptosis is the cleavage of nuclear repair proteins, thereby abolishing their critical homeostatic functions.
Amino Acid Sequence↗
Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32.
Interleukin-1 beta converting enzyme (ICE)-like proteases, which are synthesized as inactive precursors, play a key role in the induction of apoptosis. We now demonstrate that benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK), an ICE-like protease inhibitor, inhibits apoptosis by preventing the processing of CPP32 to its active form. These results suggest that novel inhibitors of apoptosis can be developed which prevent processing of proforms of ICE-like proteases.
Amino Acid Chloromethyl Ketones↗
Bcl-2 and adenovirus E1B 19 kDA protein prevent E1A-induced processing of CPP32 and cleavage of poly(ADP-ribose) polymerase.
The E1A oncoproteins of adenovirus type 5 are potent inducers of apoptotic cell death. To manifest growth promoting and transforming properties, therefore, E1A requires the co-expression of a suppressor of apoptosis. During normal viral infection, this function is provided by the E1B 19 kDa protein. However, the cellular suppressor Bcl-2 can substitute for 19K during infection, and both proteins can effectively cooperate with E1A to facilitate transformation of primary cells in culture. How E1A induces apoptosis and at what point(s) on this pathway Bcl-2 and E1B 19K act are not presently known. Here, we demonstrate that E1A-induced apoptosis is accompanied by specific endo-proteolytic cleavage of poly(ADP-ribose) polymerase (PARP), an event that is linked to the Ced-3/ICE apoptotic pathway in other systems. PARP cleavage was also observed in p53-null cells infected with 19K- virus expressing 13S E1A. In addition to PARP cleavage, expression of E1A caused processing of the zymogen form of CPP32, a Ced-3/ICE protease that cleaves PARP and is required for apoptosis in mammalian cells. These events were prevented when E1A was co-expressed with E1B 19K or BCL-2, which places these suppressors of apoptosis either at or upstream of processing of pro-CPP32.
Adenovirus E1A Proteins↗
CPP32/apopain is a key interleukin 1 beta converting enzyme-like protease involved in Fas-mediated apoptosis.
Cysteine proteases of the interleukin 1 beta Converting Enzyme (ICE)/CED-3 family have been implicated in the effector process of apoptosis in several systems, including Fas-mediated apoptosis. We have recently isolated and partially characterized a protease present in extracts from anti-Fas antibody treated Jurkat T cells that promotes apoptotic changes in isolated nuclei (Schlegel, J., Peters, I., and Orrenius, S. (1995) FEBS Lett. 364, 139-142). We now show that this protease cleaves poly-(ADP-ribose) polymerase (PARP) with high efficiency and specificity. Both PARP proteolysis and the proapoptotic effects of the protease are inhibited by nanomolar concentrations of a selective inhibitor of apopain (CPP32), while an inhibitor of IL-1 beta converting enzyme is much less effective, requiring micromolar concentrations for the inhibition of the isolated protease. Kinetic analysis of the isolated protease reveals kinetic constants similar to those reported for apopain. The isolated protease is recognized by antibodies specific for CPP32/apopain but not by an anti-ICE antibody. Furthermore, a selective inhibitor of apopain prevents Fas-induced apoptosis in intact Jurkat T cells. We therefore conclude that CPP32/apopain is activated in Fas-induced apoptosis.
Amino Acid Sequence↗
ICE/CED3-like proteases as therapeutic targets for the control of inappropriate apoptosis.
Excessive or failed apoptosis is a prominent morphological feature of several human diseases. Many of the key biochemical players that contribute to the highly ordered process of apoptotic cell death have recently been identified. These include members of the emerging family of cysteine proteases related to mammalian interleukin-1 beta converting enzyme (ICE) and to CED-3, the product of a gene that is necessary for programmed cell death in the nematode C. elegans. Among a growing number of potential molecular targets for the control of human diseases where inappropriate apoptosis is prominent, ICE/CED-3-like proteases may be an attractive and tangible point for therapeutic intervention.
Amino Acid Sequence↗
Cleavage of huntingtin by apopain, a proapoptotic cysteine protease, is modulated by the polyglutamine tract.
Apoptosis has recently been recognized as a mode of cell death in Huntington disease (HD). Apopain, a human counterpart of the nematode cysteine protease death-gene product, CED-3, has a key role in proteolytic events leading to apoptosis. Here we show that apoptotic extracts and apopain itself specifically cleave the HD gene product, huntingtin. The rate of cleavage increases with the length of the huntingtin polyglutamine tract, providing an explanation for the gain-of-function associated with CAG expansion. Our results show that huntingtin is cleaved by cysteine proteases and suggest that HD might be a disorder of inappropriate apoptosis.
Animals↗
The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis.
Cysteine proteases related to mammalian interleukin-1 beta converting enzyme (ICE) and to its Caenorhabditis elegans homologue, CED-3, play a critical role in the biochemical events that culminate in apoptosis. We have determined the three-dimensional structure of a complex of the human CED-3 homologue CPP32/apopain with a potent tetrapeptide-aldehyde inhibitor. The protein resembles ICE in overall structure, but its S4 subsite is strikingly different in size and chemical composition. These differences account for the variation in specificity between the ICE- and CED-3-related proteases and enable the design of specific inhibitors that can probe the physiological functions of the proteins and disease states with which they are associated.
Amino Acid Sequence↗
Epitope-labeled soluble human interleukin-5 (IL-5) receptors. Affinity cross-link labeling, IL-5 binding, and biological activity.
The human receptor for the potent eosinophilopoietic cytokine interleukin-5 (IL-5) consists of two components: a 60-kDa ligand-binding alpha chain (IL-5 alpha R) and a 130-kDa beta chain (IL-5 beta R). Three ectodomain constructs of the alpha chain (alpha RED) bearing C-terminal epitope tags were engineered and expressed in baculovirus-infected Sf9 cells. Each recombinant alpha chain was secreted into the medium, maximum expression occurring 72 h post-infection. The various soluble alpha chains were shown by affinity cross-link labeling and competition with unlabeled IL-5 to bind recombinant human (rh) 125I-IL-5 specifically with an ED50 of 2-5 nM. The epitope tag provided a simple purification of the receptor from conditioned medium using immunoaffinity chromatography. The purified material had an apparent molecular mass of 43 kDa and was heterogeneously glycosylated. Sedimentation analysis revealed a 1:1 association of the purified epitope-tagged soluble receptor with its ligand, resulting in the formation of a 70-74-kDa complex. Circular dichroism analysis revealed that the soluble alpha chain existed with a significantly ordered structure consisting of 42% beta-sheet and 6% alpha-helix. Such analyses combined with fluorescence spectrometry suggested that ligand-receptor complex formation in solution resulted in minimal conformational changes, consistent with the suggestion that the membrane-associated form of the alpha chain itself has minimal signal transduction capability. Surface plasmon resonance studies of the interaction of the purified alpha RED with immobilized rhIL-5 revealed a specific, competable interaction with a dissociation constant of 9 nM. Preincubation of an IL-5-dependent cell line with the epitope-tagged alpha RED also dose-dependently neutralized rhIL-5-induced proliferation. These data demonstrate that biologically active epitope-tagged recombinant soluble IL-5 receptors are facile to produce in large quantities and may have therapeutic utility in the modulation of IL-5-dependent eosinophilia in man.
Amino Acid Sequence↗
Differential activation of leukotriene biosynthesis by granulocyte-macrophage colony-stimulating factor and interleukin-5 in an eosinophilic substrain of HL-60 cells.
Cytokines can stimulate eosinophils to produce cysteinyl leukotrienes (LTs) in the lung that provoke tissue destruction associated with asthma. Priming of an eosinophilic substrain of HL-60 cells (HL-60#7) with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) before ionophore challenge was found to produce an apparent 45% increase in total LT production in a dose-dependent manner (ED50 = 150 pmol/L) that could be accounted for by a decrease in the time required for maximal formation of LTs. GM-CSF had no effect on the kinetic parameters of LTC4 synthase and therefore probably acts upstream of this catalytic event. Incubation with interleukin-5 (IL-5), however, had no effect on LT biosynthesis. This differential priming ability was not a consequence of different receptor populations or differences in the affinity or stability of the ligand-receptor complexes of GM-CSF and IL-5. GM-CSF and IL-5 each displayed similar populations of high-affinity binding sites and neither GM-CSF nor IL-5 were able to cross-compete for the other's receptor binding sites. Analysis of phosphotyrosine patterns suggest that IL-5 is incapable of transducing a signal in eosinophilic HL-60#7 cells even though IL-5 and GM-CSF receptors mediate signal transduction via a common beta-chain component that is also necessary for high-affinity binding. Overall, this unique system may permit the dissection of distinct events responsible for specific intracellular signals transduced separately by GM-CSF or IL-5.
Butyrates↗