Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.22.36 (caspase-1)
6,285 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) is a novel cysteine protease that cleaves the 31-kD inactive cytoplasmic IL-1 beta precursor into active extracellular 17-kD IL-1 beta. The ICE gene product is a 45-kD proenzyme that requires proteolytic processing to activate ICE. Active ICE is a heterodimer consisting of equal amounts of p20 and p10 subunits. Generation of active ICE is affected by the removal of an 11-kD NH2-terminal precursor domain (p11) and an internal 19-amino acid sequence that separates the 20- and 10-kD subunits. Immuno-electron microscopy was performed on human monocytes with immunoglobulins recognizing the active (p20) or precursor (p11) domains of ICE. Elutriated monocytes were stimulated with 50 pM lipopolysaccharide followed by heat-killed Staphylococcus aureus under conditions that induce maximal rates of IL-1 beta secretion. Ultrathin cryosections were cut from fixed frozen pellets of these monocytes and were immunogold labeled with either antibody. Active and precursor domain ICE epitopes were localized in the cytoplasmic ground substance, but they were not detected within the endoplasmic reticulum, the Golgi apparatus, and secretory granules of activated or inactive monocytes. Importantly, numerous ICE p20 epitopes were also observed on the extracellular surfaces of the cell membrane, and were concentrated on the microvilli. Very similar patterns of ICE localization were obtained with unstimulated blood monocytes. In contrast, ICE p11 epitopes were not detected on the surfaces of these monocytes. Likewise, labeling of fixed ultrathin cryosections of monocytes with a biotinylated irreversible ICE inhibitor [Ac-Tyr-Val-Lys(biotin)-Asp-(acyloxy)-methyl-ketone] showed that the compound localized on the outer cell surface as well, and to a lesser extent, within the cytoplasmic ground substance. Furthermore, antipeptide antibodies specific for either the mature or precursor domains of IL-1 beta were both localized upon the cell membrane after stimulation of IL-1 beta secretion. Lipopolysaccaride-primed monocytes that synthesized, but did not secrete IL-1 beta, exhibited only cytoplasmic staining. The data suggests that mature IL-1 beta is generated via cleavage of the 31-kD inactive cytoplasmic IL-1 beta precursor by ICE after association with the plasma membrane during secretion.
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PMID:The interleukin-1 beta-converting enzyme (ICE) is localized on the external cell surface membranes and in the cytoplasmic ground substance of human monocytes by immuno-electron microscopy. 759 15

Interleukin-1 beta (IL-1 beta) converting enzyme (ICE) processes the precursor of the cytokine IL-1 beta to a mature, biologically active form in monocytes and macrophages. To further understand the role of ICE in regulating IL-1 beta-mediated biological functions, we have isolated several genomic clones encoding the full-length murine ICE gene. Southern blot comparison of murine genomic DNA and the clones indicates that ICE is a compact, single-copy gene 8616 bp in size. We sequenced the entire gene as well as 1.0-kb segment upstream of the coding region and determined that the gene consists of 10 exons whose organization parallels the functional organization of the ICE proenzyme in that the prodomain and p20 and p10 subunits of ICE are encoded by three clusters of exons. Two initiation sites, 37 and 32 nucleotides upstream of the initiator methionine, were identified by primer extension analysis. The 5' region of the ICE gene lacks a TATA box, a CAAT box, and SP1 sites. However, the presence of a completely conserved 14-bp sequence spanning the transcription initiation site of both the murine and the human ICE genes suggests that this sequence plays a role in transcription.
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PMID:The structure and complete nucleotide sequence of the murine gene encoding interleukin-1 beta converting enzyme (ICE). 803 21

We have generated a series of monoclonal antibodies (mAb) using recombinant interleukin (IL)-1 beta-converting enzyme (ICE) p20 and p10 subunits as immunogens. The mAb have been selected for further study based on their reactivity with ICE in transfected COS cells and their lack of cross-reactivity with TX, the closest ICE homolog known to date. Two anti-p20 and one anti-p10 mAb have been used to study ICE expression by Western blotting and immunodetection. In ICE-transfected COS cells, the mAb recognize the p45 ICE precursor and the maturation products (p20 or p10 subunits) for which they are specific. In monocytes and cell lines expressing ICE, only precursor forms are detected and intracellular immunostaining followed by confocal microscopy shows that they are located in the cytoplasm. Quantification experiments show that THP1 cells express approximately 67,000 molecules of ICE precursor per cell, with an estimated precursor to mature ratio of at least 100. In these cells as well as in monocytes, lipopolysaccharide stimulation did not change the pattern of ICE expression, although efficient secretion of mature IL-1 beta was measured. However, upon cell disruption, precursor maturation was observed. Our results, therefore, show that ICE is present in cells as a large pool of intracytoplasmic precursor, and that very limited amounts of mature ICE protein are present, but nevertheless sufficient to allow efficient IL-1 beta cleavage. Altogether, these observations suggest that post-translational maturation of the precursor protein could represent a specific step in the regulation of ICE enzymatic activity.
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PMID:Use of monoclonal antibodies to study interleukin-1 beta-converting enzyme expression: only precursor forms are detected in interleukin-1 beta-secreting cells. 864 64

We report the preparation and characterization of interleukin-1beta converting enzyme (ICE) refolded from its p20 and p10 protein fragments. Refolded ICE heterodimer (p20p10) was catalytically active but unstable, and in size exclusion chromatography eluted at an apparent molecular mass of 30 kDa. The mechanisms of the observed instability were pH-dependent dissociation at low enzyme concentrations, and autolytic degradation of the p10 subunit at high concentrations. Binding and subsequent removal of a high affinity peptidic inhibitor increased the apparent molecular mass to 43 kDa (by size exclusion chromatography), and significantly increased its stability and specific activity. Chemical cross-linking and SDS-polyacrylamide gel electrophoresis analysis of the 43-kDa size exclusion chromatography conformer revealed a 60-kDa species, which was absent in the 30-kDa conformer, suggesting that inhibitor binding caused formation of a (p20p10)2 homodimer. The observation of a reversible equilibrium between ICE (p20p10) and (p20p10)2 suggests that analogous associations, possibly between ICE and ICE homologs, can occur in vivo, resulting in novel oligomeric protease species.
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PMID:Stability and oligomeric equilibria of refolded interleukin-1beta converting enzyme. 870 86

Neuronal apoptosis occurs during nervous system development and after pathological insults to the adult nervous system. Inhibition of CED3/ICE-related proteases has been shown to inhibit neuronal apoptosis in vitro and in vivo, indicating a role for these cysteine proteases in neuronal apoptosis. We have studied the activation of the CED3/ICE-related protease CPP32 in two in vitro models of mouse cerebellar granule neuronal cell death: K+/serum deprivation-induced apoptosis and glutamate-induced necrosis. Pretreatment of granule neurons with a selective, irreversible inhibitor of CED3/ICE family proteases, ZVAD-fluoromethylketone, specifically inhibited granule neuron apoptosis but not necrosis, indicating a selective role for CED3/ICE proteases in granule neuron apoptosis. Extracts prepared from apoptotic, but not necrotic, granule neurons contained a protease activity that cleaved the CPP32 substrate Ac-DEVD-aminomethylcoumarin. Induction of the protease activity was prevented by inhibitors of RNA or protein synthesis or by the CED3/ICE protease inhibitor. Affinity labeling of the protease activity with an irreversible CED3/ICE protease inhibitor, ZVK(biotin)D-fluoromethylketone, identified two putative protease subunits, p20 and p18, that were present in apoptotic but not necrotic granule neuron extracts. Western blotting with antibodies to the C terminus of the large subunit of mouse CPP32 (anti-CPP32) identified p20 and p18 as processed subunits of the CPP32 proenzyme. Anti-CPP32 specifically inhibited the DEVD-amc cleaving activity, verifying the presence of active CPP32 protease in the apoptotic granule neuron extracts. Western blotting demonstrated that the CPP32 proenzyme was expressed in granule neurons before induction of apoptosis. These results demonstrate that the CED3/ICE homolog CPP32 is processed and activated during cerebellar granule neuron apoptosis. CPP32 activation requires macromolecular synthesis and CED3/ICE protease activity. The lack of CPP32 activation during granule neuron necrosis suggests that proteolytic processing and activation of CED3/ICE proteases are specific biochemical markers of apoptosis.
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PMID:Activation of the CED3/ICE-related protease CPP32 in cerebellar granule neurons undergoing apoptosis but not necrosis. 898 78

Proteases of the caspase family, especially caspase-1 (ICE)(-like), caspase-3 (CPP32/Yama/apopain)(-like) and caspase-8 (MACH/FLICE/Mch5) proteases, are implicated in Fas (APO-1/CD95)-mediated apoptosis. Here, we show that the caspase-4 (TX/ICH-2/ICE(rel)II)(-like) protease, another member of the caspase family, is also involved in Fas-mediated apoptosis, based upon the observations: (i) caspase-4 is processed in response to an agonistic anti-Fas antibody treatment, (ii) overexpression of a mutant caspase-4 with active site mutations in both p20 and p10 subunits delays Fas-mediated apoptosis, (iii) microinjected anti-caspase-4 antibodies inhibit Fas-mediated apoptosis. Together with our observations that the mutant caspase-4 inhibits the Fas-mediated activation of caspase-3(-like) proteases and purified caspase-4 cleaves pro-caspase-3 to generate a subunit of active form, these results suggest that Fas-mediated apoptosis is driven by a caspase cascade in which the caspase-4(-like) protease transmits a death signal from caspase-8 to caspase-3(-like) proteases probably through directly cleaving pro-caspase-3(-like) proteases.
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PMID:Involvement of caspase-4(-like) protease in Fas-mediated apoptotic pathway. 923 63

We have identified a human Bcl-2-interacting protein, p28 Bap31. It is a 28-kD (p28) polytopic integral protein of the endoplasmic reticulum whose COOH-terminal cytosolic region contains overlapping predicted leucine zipper and weak death effector homology domains, flanked on either side by identical caspase recognition sites. In cotransfected 293T cells, p28 is part of a complex that includes Bcl-2/Bcl-XL and procaspase-8 (pro-FLICE). Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so. In the absence (but not presence) of elevated Bcl-2 levels, apoptotic signaling by adenovirus E1A oncoproteins promote cleavage of p28 at the two caspase recognition sites. Purified caspase-8 (FLICE/MACH/Mch5) and caspase-1(ICE), but not caspase-3 (CPP32/apopain/ Yama), efficiently catalyze this reaction in vitro. The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells. Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL. This raises the possibility that the p28 complex contributes to the regulation of procaspase-8 or a related caspase in response to E1A, dependent on the status of the Bcl-2 setpoint within the complex.
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PMID:p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum. 933 38

Interleukin-1beta-converting enzyme is a member of a family of human cysteine proteases with specificity for aspartic acid, which have been named caspases. Within this family of enzymes, transcript X (TX) and transcript Y (TY) (caspases 4 and 5, respectively) are very similar to ICE (caspase 1) and form the ICE subfamily. Given the high degree of conservation in the sequences of these proteases (more than 50% amino acid identity in the mature enzymes), it was of interest to examine whether they shared similar substrate specificities. The three enzymes, ICE, TX and TY, were therefore expressed in baculovirus-infected insect cells, as 30-kDa proteins lacking the propeptide. Automaturation into p20 and p10 subunits occurred within the cells. Active ICE, TX and TY were collected in the cell culture supernatants. In addition, their production induced the activation of an endogenous 32-kDa putative cysteine protease (CPP32) like caspase. T7-tagged ICE, TX and TY were purified by immunoaffinity and tested for their catalytic efficiency on YVAD-containing synthetic substrates and on the ICE natural substrate, pro-interleukin-1beta. TX cleaved the same synthetic substrates as ICE (Km of 90 microM and k(cat) of 0.4 s(-1) for Suc-YVAD-NH-Mec, where Suc represents succinyl and NH-Mec represents amino-4-methylcoumarin) and could cleave pro-interleukin-1beta into the same peptides as ICE but less efficiently. On the other hand, TY showed very little efficacy on the different ICE substrates (Km of 860 microM for Suc-YVAD-NH-Mec). These results show that the ICE/TX/TY subfamily has functional heterogeneity and that ICE remains the preferred enzyme for pro-interleukin-1beta cleavage.
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PMID:Enzymatic activity of two caspases related to interleukin-1beta-converting enzyme. 957 63

Members of the tumor necrosis factor receptor (TNFR) superfamily have an important role in the induction of cellular signals resulting in cell growth, differentiation and death. TNFR-1 recruits and assembles a signaling complex containing a number of death domain (DD)-containing proteins, including the adaptor protein TRADD and the serine/threonine kinase RIP, which mediates TNF-induced NF-kappa B activation. RIP also recruits caspase-2 to the TNFR-1 signaling complex via the adaptor protein RAIDD, which contains a DD and a caspase-recruiting domain (CARD). Here, we have identified a RIP-like kinase, termed CARDIAK (for CARD-containing interleukin (IL)-1 beta converting enzyme (ICE) associated kinase), which contains a serine/threonine kinase domain and a carboxy-terminal CARD. Overexpression of CARDIAK induced the activation of both NF-kappa B and Jun N-terminal kinase (JNK). CARDIAK interacted with the TNFR-associated factors TRAF-1 and TRAF-2, and a dominant-negative form of TRAF-2 inhibited CARDIAK-induced NF-kappa B activation. Interestingly, CARDIAK specifically interacted with the CARD of caspase-1 (previously known as ICE), and this interaction correlated with the processing of pro-caspase-1 and the formation of the active p20 subunit of caspase-1. Together, these data suggest that CARDIAK may be involved in NF-kappa B/JNK signaling and in the generation of the proinflammatory cytokine IL-1 beta through activation of caspase-1.
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PMID:Identification of CARDIAK, a RIP-like kinase that associates with caspase-1. 970 38

Caspases are a family of heteromeric (p20/p10) cysteine proteases with important functions in the regulation of apoptosis and inflammation. Up to now, tools to identify new substrates for caspases have mostly been limited to the random screening of in vitro translated proteins that are known, or assumed, to play a role in apoptosis. We describe the use of a yeast three-hybrid approach as a tool that adapts the classical two-hybrid system to the needs of heteromeric caspases for functional dissection of known interactions or screening for physiological substrates and inhibitors. Functional heteromeric caspase-1 was obtained by coexpression of p20(Cys285Ser) and p10 caspase-1 subunits that were each fused to the Gal4 DNA-binding domain. Upon coexpression of a third hybrid of the Gal4 activation domain and the viral caspase-1 pseudosubstrate inhibitors CrmA or p35, or the prototype physiological caspase-1 substrate prointerleukin-1beta, a functional Gal4 transcription factor could be reconstituted. In contrast, no interaction was found between CrmA or p35 and the immature p45 or p30 precursor forms of caspase-1. Therefore, the three-hybrid system might allow screening for new physiological substrates and inhibitors of heteromeric caspases.
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PMID:Use of the yeast three-hybrid system as a tool to study caspases. 975 Jan 44


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