SMART MODE:

NORMAL GENOMIC

• Reddien PW, Cameron S, Horvitz HR
• Phagocytosis promotes programmed cell death in C. elegans.
• Nature. 2001; 412: 198-202
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• In the nematode Caenorhabditis elegans programmed cell death requires the killer genes egl-1, ced-4 and ced-3 (refs 1 and 2), and the engulfment of dying cells requires the genes ced-1, ced-2, ced-5, ced-6, ced-7, ced-10 and ced-12 (refs 3,4,5). Here we show that engulfment promotes programmed cell death. Mutations that cause partial loss of function of killer genes allow the survival of some cells that are programmed to die, and mutations in engulfment genes enhance the frequency of this cell survival. Furthermore, mutations in engulfment genes alone allow the survival and differentiation of some cells that would normally die. Engulfment genes probably act in engulfing cells to promote death, as the expression in engulfing cells of ced-1, which encodes a receptor that recognizes cell corpses, rescues the cell-killing defects of ced-1 mutants. We propose that engulfing cells act to ensure that cells triggered to undergo programmed cell death by the CED-3 caspase die rather than recover after the initial stages of death.

• Parrish J, Li L, Klotz K, Ledwich D, Wang X, Xue D
• Mitochondrial endonuclease G is important for apoptosis in C. elegans.
• Nature. 2001; 412: 90-4
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• Programmed cell death (apoptosis) is a tightly regulated process of cell disassembly in which dying cells and their nuclei shrink and fragment and the chromosomal DNA is degraded into internucleosomal repeats. Here we report the characterization of the cps-6 gene, which appears to function downstream of, or in parallel to, the cell-death protease CED-3 of Caenorhabditis elegans in the DNA degradation process during apoptosis. cps-6 encodes a homologue of human mitochondrial endonuclease G, and its protein product similarly localizes to mitochondria in C. elegans. Reduction of cps-6 activity caused by a genetic mutation or RNA-mediated interference (RNAi) affects normal DNA degradation, as revealed by increased staining in a TUNEL assay, and results in delayed appearance of cell corpses during development in C. elegans. This observation provides in vivo evidence that the DNA degradation process is important for proper progression of apoptosis. CPS-6 is the first mitochondrial protein identified to be involved in programmed cell death in C. elegans, underscoring the conserved and important role of mitochondria in the execution of apoptosis.

• Shibata M et al.
• Prevention of apoptosis of mammalian cells by the CED-3-cleaved form of CED-9.
• Arch Histol Cytol. 2001; 64: 17-28
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• CED-9 prevents apoptosis in embryonic cells of Caenorhabditis elegans but not in mammalian cells. We show here that the prevention of apoptosis in mammalian cells requires a CED-3-cleaved form (68-280) of CED-9 which is localized in the inner mitochondrial membrane. The viability of PC12 and HeLa cells was significantly increased after death stimuli when truncated CED-9 was expressed in these cells but full-length CED-9 did not. The truncated CED-9 expressed in these cells was largely localized to the inner mitochondrial and the endoplasmic reticulum membranes, while full-length CED-9 was detected mainly in endoplasmic reticulum fractions. Moreover, truncated CED-9 in purified mitochondria was resistant to trypsin digestion, but full-length CED-9 was not. These results suggest that the CED-3-cleaved form of CED-9 prevents apoptosis in mammalian cells by localizing to the inner mitochondrial membrane.

• Hoeppner DJ, Hengartner MO, Schnabel R
• Engulfment genes cooperate with ced-3 to promote cell death in Caenorhabditis elegans.
• Nature. 2001; 412: 202-6
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• Genetic studies have identified over a dozen genes that function in programmed cell death (apoptosis) in the nematode Caenorhabditis elegans. Although the ultimate effects on cell survival or engulfment of mutations in each cell death gene have been extensively described, much less is known about how these mutations affect the kinetics of death and engulfment, or the interactions between these two processes. We have used four-dimensional-Nomarski time-lapse video microscopy to follow in detail how cell death genes regulate the extent and kinetics of apoptotic cell death and removal in the early C. elegans embryo. Here we show that blocking engulfment enhances cell survival when cells are subjected to weak pro-apoptotic signals. Thus, genes that mediate corpse removal can also function to actively kill cells.

• Huang DC, Strasser A
• BH3-Only proteins-essential initiators of apoptotic cell death.
• Cell. 2000; 103: 839-42

• Stanfield GM, Horvitz HR
• The ced-8 gene controls the timing of programmed cell deaths in C. elegans.
• Mol Cell. 2000; 5: 423-33
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• Loss-of-function mutations in the gene ced-8 lead to the late appearance of cell corpses during embryonic development in C. elegans. ced-8 functions downstream of or in parallel to-the regulatory cell death gene ced-9 and may function as a cell death effector downstream of the caspase encoded by the programmed cell death killer gene ced-3. In ced-8 mutants, embryonic programmed cell death probably initiates normally but proceeds slowly. ced-8 encodes a transmembrane protein that appears to be localized to the plasma membrane. The CED-8 protein is similar to human XK, a putative membrane transport protein implicated in McLeod Syndrome, a form of hereditary neuroacanthocytosis.

• del Peso L, Gonzalez VM, Inohara N, Ellis RE, Nunez G
• Disruption of the CED-9.CED-4 complex by EGL-1 is a critical step for programmed cell death in Caenorhabditis elegans.
• J Biol Chem. 2000; 275: 27205-11
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• In the nematode Caenorhabditis elegans, the apoptotic machinery is composed of four basic elements: the caspase CED-3, the Apaf-1 homologue CED-4, and the Bcl-2 family members CED-9 and EGL-1. The ced-9(n1950) gain-of-function mutation prevents most, if not all, somatic cell deaths in C. elegans. It encodes a CED-9 protein with a glycine-to-glutamate substitution at position 169, which is located within the highly conserved Bcl-2 homology 1 domain. We performed biochemical analyses with the CED-9G169E protein to gain insight into the mechanism of programmed cell death. We find that CED-9G169E retains the ability to bind both EGL-1 and CED-4, although its affinity for EGL-1 is reduced. In contrast to the behavior of wild-type CED-9, the interaction between CED-9G169E and CED-4 is not disrupted by expression of EGL-1. Furthermore, CED-4 and CED-9G169E co-localizes with EGL-1 to the mitochondria in mammalian cells, and expression of EGL-1 does not induce translocation of CED-4 to the cytosol. Finally, the ability of EGL-1 to promote apoptosis is impaired by the replacement of wild-type CED-9 with CED-9G169E, and this effect is correlated with the inability of EGL-1 to induce the displacement of CED-4 from the CED-9.CED-4 complex. These studies suggest that the release of CED-4 from the CED-9.CED-4 complex is a necessary step for induction of programmed cell death in C. elegans.

• Wang X, Terzyan S, Tang J, Loy JA, Lin X, Zhang XC
• Human plasminogen catalytic domain undergoes an unusual conformational change upon activation.
• J Mol Biol. 2000; 295: 903-14
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• Activation of the serine protease plasmin from its zymogen, plasminogen, is the key step in fibrinolysis leading to blood clot dissolution. It also plays critical roles in cell migration, such as in tumor metastasis. Here, we report the crystal structure of an inactive S741A mutant of human plasminogen catalytic domain at 2.0 A resolution. This structure permits a direct comparison with that of the plasmin catalytic unit. Unique conformational differences are present between these two structures that are not seen in other zymogen-enzyme pairs of the trypsin family. The functional significance of these differences and the structural basis of plasminogen activation is discussed in the light of this new structure.

• Kuan CY, Roth KA, Flavell RA, Rakic P
• Mechanisms of programmed cell death in the developing brain.
• Trends Neurosci. 2000; 23: 291-7
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• Programmed cell death (apoptosis) is an important mechanism that determines the size and shape of the vertebrate nervous system. Recent gene-targeting studies have indicated that homologs of the cell-death pathway in the nematode Caenorhabditis elegans have analogous functions in apoptosis in the developing mammalian brain. However, epistatic genetic analysis has revealed that the apoptosis of progenitor cells during early embryonic development and apoptosis of postmitotic neurons at later stage of brain development have distinct roles and mechanisms. These results provide new insight on the significance and mechanism of neural cell death in mammalian brain development.

• Ou D, Anderson KM, Leslie W, Bonomi P, Harris JE
• Does maximizing programmed cell death necessarily yield an optimum clinical advantage?
• Med Hypotheses. 1999; 52: 235-8
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• Generally it has been believed that a maximum therapeutic induction of programmed cell death in cancer cells is universally desirable. As a corollary, the presence of Bcl-2, a major anti-programmed cell death protein, is considered an unfavorable prognostic sign. The latter is not and the former may not be universally true.

• Canaan S, Roussel A, Verger R, Cambillau C
• Gastric lipase: crystal structure and activity.
• Biochim Biophys Acta. 1999; 1441: 197-204
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• Fat digestion in humans requires not only the classical pancreatic lipase but also gastric lipase, which is stable and active despite the highly acidic stomach environment. We have solved the structure of recombinant human gastric lipase at 3.0 A resolution, the first structure to be described within the mammalian acid lipase family. This globular enzyme (379 residues) consists of a core domain, belonging to the alpha/beta hydrolase fold family, and an extrusion domain. It possesses a classical catalytic triad (Ser 153, His 353, Asp 324) and an oxyanion hole (NH groups of Gln 154 and Leu 67). Four N-glycosylation sites were identified on the electron density maps. The catalytic serine is deeply buried under the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 30 residues, which can be defined as a lid. Its displacement is necessary for the substrates to access the active site. A phosphonate inhibitor was positioned in the active site which clearly suggests the location of the hydrophobic substrate binding site.

• Wardlow S, Penha-Goncalves MN, Argyle DJ, Onions DE, Nicolson L
• Nucleotide sequence of equine caspase-1 cDNA.
• DNA Seq. 1999; 10: 133-7
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• Caspases are a family of cysteine proteases which have important roles in activation of cytokines and in apoptosis. Caspase-1, or interleukin-1 beta converting enzyme (ICE), promotes maturation of interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18) by proteolytic cleavage of precursor forms to generate biologically active peptides. We report the cloning and sequencing of equine caspase-1 cDNA. Equine caspase-1 is 405 amino acids in length and has 72% and 63% identity to human and mouse caspase-1, respectively, at the amino acid level. Sites of proteolytic cleavage and catalytic activity as identified in human caspase-1, are conserved.

• Nechushtan A, Smith CL, Hsu YT, Youle RJ
• Conformation of the Bax C-terminus regulates subcellular location and cell death.
• EMBO J. 1999; 18: 2330-41
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• Bax, a pro-apoptotic member of the Bcl-2 family, translocates from the cytosol to the mitochondria during programmed cell death. We report here that both gain-of-function and loss-of-function mutations can be achieved by altering a single amino acid in the Bax hydrophobic C-terminus. The properly mutated C-terminus of Bax can target a non-relevant protein to the mitochondria, showing that specific conformations of this domain alone allow mitochondrial docking. These data along with N-terminus epitope exposure experiments suggest that the C- and the N-termini interact and that upon triggering of apoptosis, Bax changes conformation, exposing these two domains to insert into the mitochondria and regulate the cell death machinery.

• Shaham S, Reddien PW, Davies B, Horvitz HR
• Mutational analysis of the Caenorhabditis elegans cell-death gene ced-3.
• Genetics. 1999; 153: 1655-71
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• Mutations in the gene ced-3, which encodes a protease similar to interleukin-1beta converting enzyme and related proteins termed caspases, prevent programmed cell death in the nematode Caenorhabditis elegans. We used site-directed mutagenesis to demonstrate that both the presumptive active-site cysteine of the CED-3 protease and the aspartate residues at sites of processing of the CED-3 proprotein are required for programmed cell death in vivo. We characterized the phenotypes caused by and the molecular lesions of 52 ced-3 alleles. These alleles can be ordered in a graded phenotypic series. Of the 30 amino acid sites altered by ced-3 missense mutations, 29 are conserved with at least one other caspase, suggesting that these residues define sites important for the functions of all caspases. Animals homozygous for the ced-3(n2452) allele, which is deleted for the region of the ced-3 gene that encodes the protease domain, seemed to be incompletely blocked in programmed cell death, suggesting that some programmed cell death can occur independently of CED-3 protease activity.

• Tortorella MD et al.
• Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.
• Science. 1999; 284: 1664-6
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• We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.

• Watt W, Koeplinger KA, Mildner AM, Heinrikson RL, Tomasselli AG, Watenpaugh KD
• The atomic-resolution structure of human caspase-8, a key activator of apoptosis.
• Structure Fold Des. 1999; 7: 1135-43
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• BACKGROUND: Caspases are a family of cysteine proteases that have important intracellular roles in inflammation and apoptosis. Caspase-8 activates downstream caspases which are unable to carry out autocatalytic processing and activation. Caspase-8 is designated as an initiator caspase and is believed to sit at the apex of the Fas- or TNF-mediated apoptotic cascade. In view of this role, the enzyme is an attractive target for the design of inhibitors aimed at blocking the undesirable cell death associated with a range of degenerative disorders. RESULTS: The structure of recombinant human caspase-8, covalently modified with the inhibitor acetyl-Ile-Glu-Thr-Asp-aldehyde, has been determined by X-ray crystallography to 1.2 A resolution. The asymmetric unit contains the p18-p11 heterodimer; the biologically important molecule contains two dimers. The overall fold is very similar to that of caspase-1 and caspase-3, but significant differences exist in the substrate-binding region. The structure answers questions about the enzyme-inhibitor complex that could not be explained from earlier caspase structures solved at lower resolution. CONCLUSIONS: The catalytic triad in caspase-8 comprises Cys360, His317 and the backbone carbonyl oxygen atom of Arg258, which points towards the Nepsilon atom of His317. The oxygen atom attached to the tetrahedral carbon in the thiohemiacetal group of the inhibitor is hydrogen bonded to Ndelta of His317, and is not in a region characteristic of a classical 'oxyanion hole'. The N-acetyl group of the inhibitor is in the trans configuration. The caspase-8-inhibitor structure provides the basis for understanding structure/function relationships in this important initiator of the proteolytic cascade that leads to programmed cell death.

• Karanewsky DS et al.
• Conformationally constrained inhibitors of caspase-1 (interleukin-1 beta converting enzyme) and of the human CED-3 homologue caspase-3 (CPP32, apopain).
• Bioorg Med Chem Lett. 1998; 8: 2757-62
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• A systematic study of interleukin-1 beta converting enzyme (ICE, caspase-1) and caspase-3 (CPP32, apopain) inhibitors incorporating a P2-P3 conformationally constrained dipeptide mimetic is reported. Depending on the nature of the P4 substituent, highly selective inhibitors of both Csp-1 or Csp-3 were obtained.

• Moldoveanu E, Stoian I, Voinea L, Marta D, Popescu LM
• BCL-2--general considerations.
• Haematologia (Budap). 1998; 29: 167-80
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• BCL-2 protein is a oncoprotein considered to have an important role in the regulation of programmed cell death--apoptosis. The intracellular localization, the relation with other oncoproteins, the antioxidant function and the pathological implications are discussed.

• del Peso L, Gonzalez VM, Nunez G
• Caenorhabditis elegans EGL-1 disrupts the interaction of CED-9 with CED-4 and promotes CED-3 activation.
• J Biol Chem. 1998; 273: 33495-500
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• In the nematode Caenorhabditis elegans, programmed cell death is implemented by the protease CED-3 whose activity is inhibited by CED-9 through physical associations with the regulator CED-4. The product of a recently described gene, egl-1, binds to and inhibits CED-9. In the present studies, we have addressed the molecular mechanism by which EGL-1 regulates CED-9 function and promotes cell death. Expression of CED-4 and CED-3 resulted in decreased survival and apoptosis of mammalian cells, activities that could be inhibited by CED-9. Importantly, this protective effect of CED-9 was antagonized by EGL-1. Immunoprecipitation analysis showed that EGL-1 binding to CED-9 disrupts the association between CED-4 and CED-9, an activity that required the BH3 motif of EGL-1. Consistent with these results, expression of EGL-1 promoted CED-4-dependent processing of CED-3, and this activity of EGL-1 was mediated through inhibition of CED-9. In mammalian cells, CED-9 is known to target the subcellular localization of CED-4 from the cytosol to intracellular membranes. Expression of EGL-1 resulted in redistribution of CED-4 from intracellular membranes, where it co-localized with CED-9, to the cytoplasm, providing further evidence that EGL-1 regulates CED-4 through CED-9. Finally, the levels of EGL-1 were greatly enhanced by co-expression of CED-9 in both mammalian cells and in a cell-free system, suggesting a role for CED-9 in the expression and/or stabilization of EGL-1. These studies provide a mechanism for how EGL-1 functions to antagonize pro-survival of CED-9 and to promote CED-3 activation and programmed cell death.

• Sorenson CM
• Life, death and kidneys: regulation of renal programmed cell death.
• Curr Opin Nephrol Hypertens. 1998; 7: 5-12
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• Apoptosis plays an integral role during nephrogenesis and is tightly regulated by bcl-2. Transgenic mice manifesting a loss of bcl-2 expression demonstrate fulminant apoptosis of the metanephric blastema during kidney formation leading to renal hypoplasia at birth and multicystic renal disease later in life. In adult kidneys, the rate of apoptosis and level of bcl-2 expression are relatively low. Renal disease can alter the rate of apoptosis and/or elevate bcl-2 expression. The implications of such alterations are discussed.

• Brady HJ, Gil-Gomez G
• Bax. The pro-apoptotic Bcl-2 family member, Bax.
• Int J Biochem Cell Biol. 1998; 30: 647-50
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• Bax is a pro-apoptotic member of the Bcl-2 family of genes which regulate programmed cell death. The Bax protein shares highly conserved domains with Bcl-2, some of which are required for the formation of Bax-Bcl-2 heterodimers. Bax expression is elevated in certain tissues after apoptotic stimuli and can be directly regulated by p53. Bax -/- mice have increased numbers of lymphoid cells and bax -/- neurons survive in culture following nerve growth factor deprivation. Bax can accelerate cell cycle entry in T-cells and has recently been shown to have a tumour suppressor function as well as carrying mutations in certain cancers. Bax can form ion-conducting channels in planar lipid bilayers which may be the biochemical mechanism through which it exerts its multiple effects. Pharmacological manipulation of Bax has implications for many diseases involving apoptosis such as cancer or neurodegenerative disorders.

• Bajusz S, Fauszt I, Nemeth K, Barabas E, Juhasz A, Patthy M
• Peptidyl beta-homo-aspartals: specific inhibitors of interleukin-1 beta converting enzyme and its homologues (caspases).
• Bioorg Med Chem Lett. 1998; 8: 1477-82
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• Inhibition of interleukin-1 beta converting enzyme (ICE), apopain, papain, thrombin and trypsin with substrate like peptidyl L- and D-alpha-aldehydes and their L-beta-homo-aldehyde analogues was investigated. The L-beta-homo-aspartals appear to be specific inhibitors for ICE and its homologues; the other enzymes were not inhibited with such L-beta-homo aldehydes. Papain shows tolerance for D-residues at P1 depending on their chiral stability.

• Brattsand M, Egelrud T
• Purification and characterization of interleukin 1 beta from human plantar stratum corneum. Evidence of interleukin 1 beta processing in vivo not involving interleukin 1 beta convertase.
• Cytokine. 1998; 10: 506-13
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• The major interleukin 1 beta (IL-1 beta) species from human plantar stratum corneum was purified and found to have an N-terminal amino acid sequence homologous to a stretch of the human IL-1 beta precursor, starting with His115. Whereas SDS-polyacrylamide gel electrophoresis followed by immunoblotting revealed only one component in plantar stratum corneum with IL-1 beta-like immunoreactivity, and with an apparent molecular mass around 18 kDa, isoelectric focusing under non-denaturing conditions showed one major component with isoelectric point around 6.1 and two minor components isoelectric at pH 6.3 and 6.9, respectively. Digestion of recombinant human IL-1 beta precursor with chymotrypsin, producing a C-terminal fragment with N-terminal Yal114, yielded a component with IL-1 beta-like immunoreactivity isoelectric at pH 6.3. Recombinant bacterial variants of human IL-1 beta with N-terminal amino acids corresponding to Val114, His115 and Ala117 were isoelectric at pH 6.3, 6.1 and 6.9, respectively. Cloning and subsequent nucleotide sequencing of IL-1 beta precursor cDNA from a human keratinocyte line showed total identify with the sequence previously published for the human monocyte IL-1 beta precursor. The authors conclude that the IL-1 beta species present in plantar stratum corneum have isoelectric points determined by their respective amino acid sequences, and that there is a mechanism for IL-1 beta activation in human epidermis not involving interleukin 1 beta convertase.

• Conradt B, Horvitz HR
• The C. elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl-2-like protein CED-9.
• Cell. 1998; 93: 519-29
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• Gain-of-function mutations in the Caenorhabditis elegans gene egl-1 cause the HSN neurons to undergo programmed cell death. By contrast, a loss-of-function egl-1 mutation prevents most if not all somatic programmed cell deaths. The egl-1 gene negatively regulates the ced-9 gene, which protects against cell death and is a member of the bcl-2 family. The EGL-1 protein contains a nine amino acid region similar to the Bcl-2 homology region 3 (BH3) domain but does not contain a BH1, BH2, or BH4 domain, suggesting that EGL-1 may be a member of a family of cell death activators that includes the mammalian proteins Bik, Bid, Harakiri, and Bad. The EGL-1 and CED-9 proteins interact physically. We propose that EGL-1 activates programmed cell death by binding to and directly inhibiting the activity of CED-9, perhaps by releasing the cell death activator CED-4 from a CED-9/CED-4-containing protein complex.

• Semple G et al.
• Peptidomimetic aminomethylene ketone inhibitors of interleukin-1 beta-converting enzyme (ICE).
• Bioorg Med Chem Lett. 1998; 8: 959-64
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• Pyridone-based peptidomimetic inhibitors of recombinant human Interleukin-1 beta-converting enzyme (ICE, caspase-1) with an aminomethylene ketone activating group in the P1' position are described. Several analogues with sub-nanomolar Ki's versus ICE and improved aqueous solubility are reported.

• Lincz LF
• Deciphering the apoptotic pathway: all roads lead to death.
• Immunol Cell Biol. 1998; 76: 1-19
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• Research into apoptosis is proceeding at such a fast and ferocious pace that anyone who is not completely engrossed in the field has difficulty keeping track of the constant stream of newly identified proteins involved in the process. Apart from being an enticing concept, the process of cell suicide is an important function with wide-reaching implications. Virologists, biologists, immunologists, physiologists and oncologists alike have had to incorporate this phenomenon into their disciplines. The purpose of this article is to provide a solid background on which to further review recent advances in this exciting field. The Bcl-2 and caspase family homologues are discussed in detail and various models are proposed to explain how they function to regulate and execute the death programme. Finally, the importance of programmed cell death with respect to immune function is explored, emphasizing the targets of viral inhibitors of apoptosis.

• Silke J, Vaux DL
• Cell death: shadow baxing.
• Curr Biol. 1998; 8: 52831-52831
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• Bcl-2, one of a family of key regulators of apoptosis, was the first cell-death machinery component to be identified, but how the family functions is still not clear. Mammalian Bax, a pro-apoptotic family member, can cause yeast cells to die, and two recent yeast genetic screens shed light on how Bax might function.

• Liao H, Chen X, Ye Y
• [Identification of interleukin-1 beta converting enzyme gene expression in human kidney]
• Zhonghua Yi Xue Za Zhi. 1998; 78: 218-21
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• OBJECTIVE: To confirm that Interleukin-1 beta-converting enzyme(ICE) is a first identified apoptosis related gene in mammals. METHODS: The expressions of ICE mRNA in human kidney tissue, different segment of nephron and glomerular resident cells were measured by using the method of microdissection, reverse transcription-polymerase chain reaction(RT-PCR) and Northern hybridization. RESULTS: Compared with peripheral blood mononuclear lymphocyte weak expressions of ICE mRNA were noted not only in glomeruli and tubule but also in glomerular mesangial cells, glomerular endothelial cells and tubular epithelial cells of the human kidney. CONCLUSION: There is a widely distributed expression of ICE mRNA in the human kidney, which may provide a theoretical basis for the study of ICE gene in apoptosis of kidney.

• Clem RJ et al.
• Modulation of cell death by Bcl-XL through caspase interaction.
• Proc Natl Acad Sci U S A. 1998; 95: 554-9
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• The caspases are cysteine proteases that have been implicated in the execution of programmed cell death in organisms ranging from nematodes to humans. Many members of the Bcl-2 family, including Bcl-XL, are potent inhibitors of programmed cell death and inhibit activation of caspases in cells. Here, we report a direct interaction between caspases and Bcl-XL. The loop domain of Bcl-XL is cleaved by caspases in vitro and in cells induced to undergo apoptotic death after Sindbis virus infection or interleukin 3 withdrawal. Mutation of the caspase cleavage site in Bcl-XL in conjunction with a mutation in the BH1 homology domain impairs the death-inhibitory activity of Bcl-XL, suggesting that interaction of Bcl-XL with caspases may be an important mechanism of inhibiting cell death. However, once Bcl-XL is cleaved, the C-terminal fragment of Bcl-XL potently induces apoptosis. Taken together, these findings indicate that the recognition/cleavage site of Bcl-XL may facilitate protection against cell death by acting at the level of caspase activation and that cleavage of Bcl-XL during the execution phase of cell death converts Bcl-XL from a protective to a lethal protein.

• Gjertsen BT, Logothetis CJ, McDonnell TJ
• Molecular regulation of cell death and therapeutic strategies for cell death induction in prostate carcinoma.
• Cancer Metastasis Rev. 1998; 17: 345-51
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• Many of the common molecular alterations associated with prostate cancer progression involve genes known to regulate cell death susceptibility. The significance of these molecular events is discussed in the context of developing and implementing new strategies designed to restore cell death susceptibility in prostate cancer cells and overcome therapeutic resistance.

• Howard RD, McIlwraith CW, Trotter GW, Nyborg JK
• CLoning of equine interleukin 1 alpha and equine interleukin 1 beta and determination of their full-length cDNA sequences.
• Am J Vet Res. 1998; 59: 704-11
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• OBJECTIVES: To clone equine interleukin 1 alpha (IL-1 alpha) and equine interleukin 1 beta (IL-1 beta) and determine their full-length cDNA sequences. PROCEDURES: The mRNA isolated from lipopolysaccharide-stimulated cultured equine monocytes was reverse transcribed, and a cDNA library was constructed in a lambda phage. The cDNA library was screened by means of plaque hybridization with radiolabeled human IL-1 alpha and IL-1 beta cDNA probes. The cDNA nucleotide sequences for equine IL-1 alpha and equine IL-1 beta were determined by use of the dideoxy chain termination technique. The cDNA sequences were analyzed, using computer software, for sequence characteristics and compared with sequences reported for other species. RESULTS: The cDNA for equine IL-1 alpha was 1,728 base pairs in length with an ORF encoding a peptide of 270 amino acids with a predicted molecular mass of 30.823 kd. The cDNA for equine IL-1 beta was 1,473 base pairs in length with an ORF encoding a peptide of 268 amino acids with a predicted molecular mass of 30.342 kd. Similarity between amino acid sequence of equine IL-1 alpha and sequences for IL-1 alpha of other species ranged from 62.5 to 82.2%; similarity between amino acid sequence of equine IL-1 beta and sequences for IL-1 beta of other species ranged from 62.5 to 82.2%; similarity between amino acid sequences of equine IL-1 alpha and equine IL-1 beta was 26%. CONCLUSIONS AND CLINICAL RELEVANCE: Results establish a basis for studying the roles of interleukin 1 in healthy and diseased joints in horses.

• Wang HG, Reed JC
• Mechanisms of Bcl-2 protein function.
• Histol Histopathol. 1998; 13: 521-30
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• The Bcl-2 protein blocks a distal step in an evolutionarily conserved pathway for programmed cell death and apoptosis. The gene encoding this protein was first discovered because of its involvement in the t(14;18) chromosomal translocations commonly found in B-cell lymphomas. Overexpression of Bcl-2 also occurs in many other types of human cancers, and prevents cell death induced by nearly all anticancer drugs and radiation. Since the discovery of Bcl-2 over ten years ago, several cellular and viral homologs have been identified, some of which suppress cell death and others which promote apoptosis. Many of these proteins can interact with each other through a complex network of homo- and heterodimers. Though functionally important, dimerization events still do not explain in a broader sense how these proteins actually control cell life and death. Recent findings that Bcl-2 can function both as an ion channel and as an adapter or docking protein however are beginning to provide insights into the molecular mechanisms through which these proteins regulate the programmed cell death pathway in normalcy and disease.

• Kelekar A, Thompson CB
• Bcl-2-family proteins: the role of the BH3 domain in apoptosis.
• Trends Cell Biol. 1998; 8: 324-30
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• Bcl-2-related proteins have come to occupy a prominent position in the realm of programmed cell death. Members of this fast-growing family are highly related in one or more specific regions, commonly referred to as Bcl-2 homology (BH) domains. BH domains contribute at multiple levels to the function of these proteins in cell death and survival. Particularly intriguing is the emergence of the BH3 domain as a potent 'death domain' and of a growing subclass of pro-apoptotic proteins with no similarity to Bcl-2 beyond their BH3 homology. Here, the authors classify proteins of the Bcl-2 family on the basis of function and domain organization, discuss the importance of the BH3 domain in protein-protein interactions and in cell death and provide possible explanations for the perceived redundancy in the expression of this subclass of death promoters.

• Juan TS et al.
• Identification and mapping of Casp7, a cysteine protease resembling CPP32 beta, interleukin-1 beta converting enzyme, and CED-3.
• Genomics. 1997; 40: 86-93
• Display abstract

• Cloning of interleukin-1 beta converting enzyme (ICE) and Caenorhabditis elegans death protein CED-3 revealed the structural and functional homology between these two proteases. It also suggested the involvement of ICE-like cysteine proteases in apoptosis. Several CED-3- and ICE-like cysteine proteases have been described, including Nedd2/Ich-1, CPP32 beta, Tx, ICErel3, and Mch2. We have previously described a mouse ortholog of cysteine protease CPP32 beta that shares strong homology with ICE and CED-3. Here, we describe the cloning of mouse and human Casp7, another member of this family of cysteine proteases. Mouse Casp7 encodes a putative 340-amino-acid polypeptide that contains all the known conserved residues required for protease function, including the QACRG sequence, aspartic acid residues for internal cleavage sites, and the residues required for substrate binding. Three RNA variants of human Casp7 were also cloned. Amino acid sequence analysis indicated that Casp7 shared high homology with CPP32 beta/Casp3 and Mch2/Casp6. Northern blot analysis demonstrated that a 2.6-kb Casp7 mRNA was expressed in various tissues except brain. Mouse interspecific backcross mapping allowed localization of Casp7 to the distal region of mouse chromosome 19, linked to Mxi1, Adra2a, and Aop1.

• Seshagiri S, Miller LK
• Caenorhabditis elegans CED-4 stimulates CED-3 processing and CED-3-induced apoptosis.
• Curr Biol. 1997; 7: 455-60
• Display abstract

• BACKGROUND: Programmed cell death or apoptosis is a key feature of normal development, tissue homeostasis and disease progression in metazoans. Genetic studies in the nematode C. elegans have identified three key genes involved in apoptosis, ced-3, ced-4 and ced-9. Expression of ced-3 and ced-4 is required for the induction of cell death, whereas expression of ced-9 is necessary to inhibit cell death. The precise mechanism by which these genes influence the life or death decision of a cell is not known. In this study, we have expressed the genes in an insect cell line to explore their role in the apoptotic pathway. RESULTS: Co-expression of ced-4 with ced-3 in insect cells stimulated both the induction and the level of CED-3-mediated apoptosis. Stimulation of CED-3-dependent apoptosis by CED-4 was accompanied by accelerated processing of CED-3, which was dependent on the presence of a wild-type CED-3 prodomain and a conserved lysine residue within a putative ATP/GTP-binding motif of CED-4. Co-expression of ced-9 with ced-4 and ced-3 inhibited the ability of CED-4 to stimulate CED-3 processing and CED-3-dependent apoptosis. Although a temperature-sensitive CED-9 mutant was unable to block CED-4 activity and failed to associate with CED-4, a deletion mutant of CED-9 lacking the carboxy-terminal hydrophobic domain could associate with CED-4 and block CED-4 activity. CONCLUSIONS: Our results establish a role for CED-4 in the processing of CED-3 and the stimulation of CED-3-induced apoptosis. Furthermore, we show that CED-9 achieves its anti-apoptotic effect by associating with CED-4 and blocking the ability of CED-4 to process CED-3.

• Spector MS, Desnoyers S, Hoeppner DJ, Hengartner MO
• Interaction between the C. elegans cell-death regulators CED-9 and CED-4.
• Nature. 1997; 385: 653-6
• Display abstract

• Programmed cell death (apoptosis) is an evolutionarily conserved process used by multicellular organisms to eliminate cells that are not needed or are potentially detrimental to the organism. Members of the Bcl-2 family of mammalian proteins are intimately involved in the regulation of apoptosis, but, their precise mechanism of action remains unresolved. In Caenorhabditis elegans, the Bcl-2 homologue CED-9 prevents cell death by antagonizing the death-promoting activities of CED-3, a member of the Caspase family of death proteases, and of CED-4, a protein with no known mammalian homologue. Here we show that CED-9 interacts physically with CED-4. Mutations that reduce or eliminate CED-9 activity also disrupt its ability to bind CED-4, suggesting that this interaction is important for CED-9 function. Thus, CED-9 might control C. elegans cell death by binding to and regulating CED-4 activity. We propose that mammalian Bcl-2 family members might control apoptosis in a similar way through interaction and regulation of CED-4 homologues or analogues.

• Wu D, Wallen HD, Inohara N, Nunez G
• Interaction and regulation of the Caenorhabditis elegans death protease CED-3 by CED-4 and CED-9.
• J Biol Chem. 1997; 272: 21449-54
• Display abstract

• In the nematode Caenorhabditis elegans, three genes, ced-3, ced-4, and ced-9, play critical roles in the induction and execution of the death pathway. Genetic studies have suggested that ced-9 controls programmed cell death by regulating ced-4 and ced-3. However, the mechanism by which CED-9 controls the activities of CED-4 and the cysteine protease CED-3, the effector arm of the cell-death pathway, remains poorly understood. Immunoprecipitation analysis demonstrates that CED-9 forms a multimeric protein complex with CED-4 and CED-3 in vivo. Expression of wild-type CED-4 promotes the ability of CED-3 to induce apoptosis in mammalian cells, which is inhibited by CED-9. The pro-apoptotic activity of CED-4 requires the expression of a functional CED-3 protease. Significantly, loss-of-function CED-4 mutants are impaired in their ability to promote CED-3-mediated apoptosis. Expression of CED-4 enhances the proteolytic activation of CED-3. We also show that CED-9 inhibits the formation of p13 and p15, two cleavage products of CED-3 associated with its proteolytic activation in vivo. Moreover, CED-9 inhibits the enzymatic activity of CED-3 promoted by CED-4. Thus, these results provide evidence that CED-4 and CED-9 regulate the activity of CED-3 through physical interactions, which may provide a molecular basis for the control of programmed cell death in C. elegans.

• Tocci MJ
• Structure and function of interleukin-1 beta converting enzyme.
• Vitam Horm. 1997; 53: 27-63
• Display abstract

• An overwhelming body of evidence has shown that IL-1 beta is a major mediator of inflammatory disease (Tocci and Schmidt, 1996). The discovery of ICE, a unique processing enzyme involved in the production of active IL-1 beta, has provided a new approach to specifically block the production of this potent cytokine. Consequently, the discovery and development of inhibitors against the enzyme could hold great promise therapeutically. Potent inhibitors of the enzyme would be useful in the treatment of a number of important inflammatory diseases and potentially in the management of leukemia (Arend, 1993b; Estrov and Talpaz, 1996). A number of key questions must be answered before the therapeutic potential of such inhibitors can be realized. The development of a pharmaceutically acceptable cysteine proteinase inhibitor will almost certainly involve new chemical strategies gauged at safely inactivating the enzyme. For such inhibitors, it will be necessary to achieve selectivity for ICE from among the growing number of ICE family members while retaining potency. It will also be important to establish the level of inhibition of IL-1 beta required to achieve therapeutic efficacy. The studies comparing IL-1 beta- and ICE-deficient mice suggest that complete abrogation of IL-1 beta is required to achieve efficacy in models of inflammation. It is not known if this is the case in humans. Understanding the source of the residual IL-1 beta produced in ICE-deficient mice will be important in order to ascertain if a similar mechanism could generate active IL-1 beta in patients receiving if a ICE inhibitor. As for ICE itself, a number of formidable questions remain regarding its regulation and mechanism of activation. Answering these questions experimentally will present a major challenge due to the extremely low levels of enzyme present in cells. Studies on other family members may provide easier access to some of these questions and provide clues that can be applied to ICE. The components of the pathway involved in IL-1 trafficking and secretion are unknown, as are the mechanisms of ICE activation and regulation. Clearly other cellular proteins that have yet to be discovered will be involved in each of these processes, opening up new avenues of research in this field.

• Margolin N et al.
• Substrate and inhibitor specificity of interleukin-1 beta-converting enzyme and related caspases.
• J Biol Chem. 1997; 272: 7223-8
• Display abstract

• Interleukin-1beta-converting enzyme (ICE) is a novel cysteine protease responsible for the cleavage of pre-interleukin-1beta (pre-IL-1beta) to the mature cytokine and a member of a family of related proteases (the caspases) that includes the Caenorhabditis elegans cell death gene product, CED-3. In addition to their sequence homology, these cysteine proteases display an unusual substrate specificity for peptidyl sequences with a P1 aspartate residue. We have examined the kinetics of processing pre-IL-1beta to the mature form by ICE and three of its homologs, TX, CPP-32, and CMH-1. Of the ICE homologs, only TX processes pre-IL-1beta, albeit with a catalytic efficiency 250-fold less than ICE itself. We also investigated the ability of these four proteases to process poly(ADP-ribose) polymerase, a DNA repair enzyme that is cleaved within minutes of the onset of apoptosis. Every caspase examined cleaves PARP, with catalytic efficiencies ranging from 2.3 x 10(6) M-1 s-1 for CPP32 to 1.0 x 10(3) M-1 s-1 for TX. In addition, we report kinetic constants for several reversible inhibitors and irreversible inactivators, which have been used to implicate one or more caspases in the apoptotic proteolysis cascade. Ac-Asp-Glu-Val-Asp aldehyde (DEVD-CHO) is a potent inhibitor of CPP-32 with a Ki value of 0.5 nM, but is also potent as inhibitor of CMH-1 (Ki = 35 nM) and ICE (Ki = 15 nM). The x-ray crystal structure of DEVD-CHO complexed to ICE presented here reveals electrostatic interactions not present in the Ac-YVAD-CHO co-complex structure (Wilson, K. P., Black, J.-A. F., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. A., Murcko, M. A., Chambers, S. P., Aldape, R. A., Raybuck, S. A., and Livingston, D. J. (1994) Nature 370, 270-275), accounting for the surprising potency of this inhibitor against ICE.

• Xue D, Horvitz HR
• Caenorhabditis elegans CED-9 protein is a bifunctional cell-death inhibitor.
• Nature. 1997; 390: 305-8
• Display abstract

• The Caenorhabditis elegans gene ced-9 prevents cells from undergoing programmed cell death and encodes a protein similar to the mammalian cell-death inhibitor Bcl-2. We show here that the CED-9 protein is a substrate for the C. elegans cell-death protease CED-3, which is a member of a family of cysteine proteases first defined by CED-3 and human interleukin-1beta converting enzyme (ICE). CED-9 can be cleaved by CED-3 at two sites near its amino terminus, and the presence of at least one of these sites is important for complete protection by CED-9 against cell death. Cleavage of CED-9 by CED-3 generates a carboxy-terminal product that resembles Bcl-2 in sequence and in function. Bcl-2 and the baculovirus protein p35, which inhibits cell death in different species through a mechanism that depends on the presence of its cleavage site for the CED-3/ICE family of proteases, inhibit cell death additively in C. elegans. Our results indicate that CED-9 prevents programmed cell death in C. elegans through two distinct mechanisms: first, CED-9 may, by analogy with p35, directly inhibit the CED-3 protease by an interaction involving the CED-3 cleavage sites in CED-9; second, CED-9 may directly or indirectly inhibit CED-3 by means of a protective mechanism similar to that used by mammalian Bcl-2.

• Bossy-Wetzel E, Bakiri L, Yaniv M
• Induction of apoptosis by the transcription factor c-Jun.
• EMBO J. 1997; 16: 1695-709
• Display abstract

• c-Jun, a signal-transducing transcription factor of the AP-1 family, normally implicated in cell cycle progression, differentiation and cell transformation, recently has also been linked to apoptosis. To explore further the functional roles of c-Jun, a conditional allele was generated by fusion of c-Jun with the hormone-binding domain of the human estrogen receptor (ER). Here we demonstrate that increased c-Jun activity is sufficient to trigger apoptotic cell death in NIH 3T3 fibroblasts. c-Jun-induced apoptosis is evident at high serum levels, but is enhanced further in factor-deprived fibroblasts. Furthermore, apoptosis by c-Jun is not accompanied by an increase in DNA synthesis. Constitutive overexpression of the apoptosis inhibitor protein Bcl-2 delays the c-Jun-mediated cell death. The regions of c-Jun necessary for apoptosis induction include the amino-terminal transactivation and the carboxy-terminal leucine zipper domain, suggesting that c-Jun may activate cell death by acting as a transcriptional regulator. We further show that alpha-fodrin, a substrate of the interleukin 1beta-converting enzyme (ICE) and CED-3 family of cysteine proteases, becomes proteolytically cleaved in cells undergoing cell death by increased c-Jun activity. Moreover, cell-permeable irreversible peptide inhibitors of the ICE/CED-3 family of cysteine proteases prevented the cell death.

• Estrov Z, Talpaz M
• Role of interleukin-1 beta converting enzyme (ICE) in acute myelogenous leukemia cell proliferation and programmed cell death.
• Leuk Lymphoma. 1997; 24: 379-91
• Display abstract

• The proinflammatory cytokine interleukin (IL)-1 has been shown to play a pivotal role in stimulating acute myelogenous leukemia (AML) cell proliferation. The gene for its prominent IL-1 beta form produces a 31-kDa precursor protein (pro-IL-1 beta) that is biologically inactive unless cleaved to its mature form by a cytoplasmic cysteine protease termed Il- 1 beta converting enzyme (ICE). Although ICE was first thought to be a unique enzyme with a single biologic activity, several investigators have demonstrated that ICE shares sequence homology with the protein product of ced-3, the gene for cell death of the nematode Caenorhabditis elegans, and induces apoptosis in different experimental models. It was therefore hypothesized that ICE may either augment the production of mature IL-1 beta and stimulate the proliferation of cells, in which IL-1 beta acts as an autocrine growth factor, or induce apoptosis. Recent data indicate that ICE is a member of an increasingly recognized family of cysteine proteases. Unlike ICE, the other members of this family do not cleave pro-IL-1 beta but are effective inducers of apoptotic cell death, whereas ICE acts primarily as an IL-1 beta converting enzyme. Because IL-1 beta serves as either an autocrine or paracrine growth factor in AML, we recently investigated the effect of ICE inhibition on AML colony growth and found that ICE inhibition reduced the production of mature IL-1 beta and suppressed AML progenitor proliferation. Our data suggest that ICE does not function as an apoptosis gene in AML but rather increases mature Il-1 beta production and AML cell proliferation. It is possible, therefore, that ICE inhibitors may be beneficial in AML therapy.

• Golstein P
• Controlling cell death.
• Science. 1997; 275: 1081-2

• Wallach D, Boldin M, Varfolomeev E, Beyaert R, Vandenabeele P, Fiers W
• Cell death induction by receptors of the TNF family: towards a molecular understanding.
• FEBS Lett. 1997; 410: 96-106

• Schreuder H et al.
• A new cytokine-receptor binding mode revealed by the crystal structure of the IL-1 receptor with an antagonist.
• Nature. 1997; 386: 194-200
• Display abstract

• Inflammation, regardless of whether it is provoked by infection or by tissue damage, starts with the activation of macrophages which initiate a cascade of inflammatory responses by producing the cytokines interleukin-1 (IL-1) and tumour necrosis factor-alpha (ref. 1). Three naturally occurring ligands for the IL-1 receptor (IL1R) exist: the agonists IL-1alpha and IL-1beta and the IL-1-receptor antagonist IL1RA (ref. 2). IL-1 is the only cytokine for which a naturally occurring antagonist is known. Here we describe the crystal structure at 2.7 A resolution of the soluble extracellular part of type-I IL1R complexed with IL1RA. The receptor consists of three immunoglobulin-like domains. Domains 1 and 2 are tightly linked, but domain three is completely separate and connected by a flexible linker. Residues of all three domains contact the antagonist and include the five critical IL1RA residues which were identified by site-directed mutagenesis. A region that is important for biological function in IL-1beta, the 'receptor trigger site' is not in direct contact with the receptor in the IL1RA complex. Modelling studies suggest that this IL-1beta trigger site might induce a movement of domain 3.

• Wendt KU, Poralla K, Schulz GE
• Structure and function of a squalene cyclase.
• Science. 1997; 277: 1811-5
• Display abstract

• The crystal structure of squalene-hopene cyclase from Alicyclobacillus acidocaldarius was determined at 2.9 angstrom resolution. The mechanism and sequence of this cyclase are closely related to those of 2,3-oxidosqualene cyclases that catalyze the cyclization step in cholesterol biosynthesis. The structure reveals a membrane protein with membrane-binding characteristics similar to those of prostaglandin-H2 synthase, the only other reported protein of this type. The active site of the enzyme is located in a large central cavity that is of suitable size to bind squalene in its required conformation and that is lined by aromatic residues. The structure supports a mechanism in which the acid starting the reaction by protonating a carbon-carbon double bond is an aspartate that is coupled to a histidine. Numerous surface alpha helices are connected by characteristic QW-motifs (Q is glutamine and W is tryptophan) that tighten the protein structure, possibly for absorbing the reaction energy without structural damage.

• Eguchi Y, Shimizu S, Tsujimoto Y
• [Molecular biology of apoptosis]
• Rinsho Byori. 1997; 45: 470-6
• Display abstract

• Apoptosis, a mechanism involving programmed cell death, is important for normal development and maintenance of tissue homeostasis of multicellular organisms. Apoptotic cells are defined by their fragmented nuclei with condensed chromatin, fragmented or condensed cytoplasm and formation of apoptotic bodies. The apoptotic signal transducing pathways activated by a variety of stimuli, including depletion of growth factors, heat shock, cytokines, DNA damaging reagents and crosslinking of Fas receptor, finally converge into the phylogenically conserved apoptotic main machinery, consisting of death-driving ICE-family proteases and anti-cell death protein Bcl-2. Recently, we noted that necrotic cell death induced by chemical hypoxia shares at least some part of the apoptotic main machinery. Using this system, we have shown that Bcl-2 prevents the loss of the mitochondrial membrane potential observed in both apoptotic and necrotic cell death. We also showed that the ICE protease cascade operates in apoptosis and that Bcl-2 functions upstream of the ICE prolease cascade. Here, we review the signal transducing pathway of the apoptotic main machinery.

• Rudin CM, Thompson CB
• Apoptosis and disease: regulation and clinical relevance of programmed cell death.
• Annu Rev Med. 1997; 48: 267-81
• Display abstract

• Regulation of the homeostatic balance between cell proliferation and cell death is essential for development and maintenance of multicellular organisms. Physiologic, or programmed, cell death is dependent on a genetically encoded and evolutionarily conserved pathway that induces a form of cellular suicide known as apoptosis. In the past decade, it has become clear that the regulatory mechanisms controlling programmed cell death are as fundamental, and as complex, as those regulating cell proliferation. Perturbation of the signaling cascades regulating apoptosis, whether by extracellular triggers, acquired or germline genetic mutations, or viral mimicry of signaling molecules, can result in a wide variety of human diseases. Analysis of these regulatory pathways has led to a better understanding of the etiology and pathogenesis of many human diseases, notably cancers, infectious diseases including AIDS, autoimmune diseases, and neurodegenerative/neurodevelopmental diseases. Our understanding of the regulation of programmed cell death in health and disease is far from complete, and the challenge of converting that understanding into new therapeutic modalities has only begun to be approached.

• Yuan J
• Genetic control of cellular suicide.
• Reprod Toxicol. 1997; 11: 377-84
• Display abstract

• Genetic analysis of programmed cell death in C. elegans has led to the identification of two genes, ced-9, a cell death suppressor, and ced-3, a cell death inducer, that play critical roles in regulating programmed cell death. The ced-9 and ced-3 genes were found to encode proteins that share structural and functional similarities with the mammalian proto-oncogene product Bcl-2 and interleukin-1 beta converting enzyme (ICE), respectively. Multiple members of the Bcl-2 family and the ICE family have been identified in vertebrates. These results suggest that the mechanism of apoptosis in vertebrates may be evolved from a much simpler version of a similar pathway in primitive organisms.

• Thornberry NA
• The caspase family of cysteine proteases.
• Br Med Bull. 1997; 53: 478-90
• Display abstract

• The discovery that CED-3, the product of a gene necessary for programmed cell death in the nematode Caenorhabditis elegans, is related to the mammalian cysteine protease interleukin-1 beta converting enzyme (ICE/caspase-1) has led to intense interest in the role of proteases in apoptosis. It is now clear that at least some members of the caspase (ICE/CED-3) family, which at present includes ten homologues of human origin, are essential components of an evolutionarily conserved pathway of apoptosis. These enzymes appear to be involved in both the initial signaling events and the downstream proteolytic cleavages that result in the apoptotic phenotype. Selective macromolecular and peptide-based inhibitors attenuate apoptosis in whole cells, suggesting that one or more of these enzymes will be suitable targets for therapeutic intervention in diseases resulting from inappropriate cell death.

• Cheng EH et al.
• Conversion of Bcl-2 to a Bax-like death effector by caspases.
• Science. 1997; 278: 1966-8
• Display abstract

• Caspases are a family of cysteine proteases implicated in the biochemical and morphological changes that occur during apoptosis (programmed cell death). The loop domain of Bcl-2 is cleaved at Asp34 by caspase-3 (CPP32) in vitro, in cells overexpressing caspase-3, and after induction of apoptosis by Fas ligation and interleukin-3 withdrawal. The carboxyl-terminal Bcl-2 cleavage product triggered cell death and accelerated Sindbis virus-induced apoptosis, which was dependent on the BH3 homology and transmembrane domains of Bcl-2. Inhibitor studies indicated that cleavage of Bcl-2 may further activate downstream caspases and contribute to amplification of the caspase cascade. Cleavage-resistant mutants of Bcl-2 had increased protection from interleukin-3 withdrawal and Sindbis virus-induced apoptosis. Thus, cleavage of Bcl-2 by caspases may ensure the inevitability of cell death.

• Petit F, Bertagnoli S, Gelfi J, Fassy F, Boucraut-Baralon C, Milon A
• Characterization of a myxoma virus-encoded serpin-like protein with activity against interleukin-1 beta-converting enzyme.
• J Virol. 1996; 70: 5860-6
• Display abstract

• A genomic library of myxoma virus (MV) DNA, a leporipoxvirus that causes myxomatosis, was constructed and screened by in vitro transcription-translation. A clone was selected on the basis of its strong reactivity with MV antiserum. Analysis of the corresponding DNA sequence and the deduced amino acid sequence revealed an open reading frame coding for a 34-kDa protein with strong homologies to members of the serpin superfamily. The gene encoding this new protein, called serp2, was localized on the MV genome. Interestingly, this gene is deleted in an attenuated strain. We constructed a baculovirus vector to produce recombinant Serp2 protein and raised specific antisera that allowed the characterization of Serp2 expression during the MV cycle. The biological relevance of this new serpin from MV was monitored, and it was shown that Serp2 could inhibit human interleukin-1 beta-converting enzyme activity.

• Petit PX, Susin SA, Zamzami N, Mignotte B, Kroemer G
• Mitochondria and programmed cell death: back to the future.
• FEBS Lett. 1996; 396: 7-13
• Display abstract

• Programmed cell death, or apoptosis, has in the past few years undoubtedly become one of the most intensively investigated biological processes. However, fundamental questions concerning the molecular and biochemical mechanisms remain to be elucidated. The central question concerns the biochemical steps shared by the numerous death induction pathways elicited by different stimuli. Heterogeneous death signals precede a common effector phase during which cells pass a threshold of 'no return' and are engaged in a degradation phase where they acquire the typical onset of late apoptosis. Alterations in mitochondrial permeability transition linked to membrane potential disruption precede nuclear and plasma membrane changes. In vitro induction of permeability transition in isolated mitochondria provokes the release of a protein factor capable of inducing nuclear chromatin condensation and fragmentation. This permeability transition is regulated by multiple endogenous effectors, including members of the bcl-2 gene family. Inhibition of these effects prevents apoptosis.

• Hugunin M, Quintal LJ, Mankovich JA, Ghayur T
• Protease activity of in vitro transcribed and translated Caenorhabditis elegans cell death gene (ced-3) product.
• J Biol Chem. 1996; 271: 3517-22
• Display abstract

• The Caenorhabditis elegans cell death gene, ced-3, encodes one of the two proteins required for apoptosis in this organism. The primary sequence similarities between Ced-3 and the mammalian interleukin-1beta converting enzyme (ICE) suggest that these two proteins may have functionally similar active sites and that Ced-3 may function as a cysteine protease. Here we report that in vitro transcribed and translated Ced-3 protein (p56) underwent rapid processing to smaller fragments. Replacement of the predicted active site cysteine of Ced-3 with serine (C364S) prevented the generation of smaller proteolytic fragments, suggesting that the processing might be an autocatalytic process. Peptide aldehydes with aspartic acid at the P1 position blocked Ced-3 autocatalysis. Furthermore, the protease inhibition profile of Ced-3 was similar to the profile reported for ICE. These functional data demonstrate that Ced-3 is an Asp-dependent cysteine protease with substrate specificity similar to that of ICE. Aurintricarboxylic acid, an inhibitor of apoptosis in mammalian cells, blocked Ced-3 autocatalytic activity, suggesting that an aurintricarboxylic acid-sensitive Ced-3/ICE-related protease might be involved in the apoptosis pathway(s) in mammalian cells.

• Hoeppner DJ, Hengartner MO, Fisher DE
• Programmed cell death: from development to disease. Meeting report.
• Biochim Biophys Acta. 1996; 1242: 217-20

• Zhivotovsky B, Burgess DH, Orrenius S
• Proteases in apoptosis.
• Experientia. 1996; 52: 968-78
• Display abstract

• The interleukin-1 beta-converting enzyme (ICE)-like family proteases have recently been identified as key enzymes in apoptotic cell death. Among these proteases one can identify specific activities which may be involved in cytokine production or in resident protein cleavage. Several factors influence the constitutive apoptotic mechanism and may provide insight into the role of protease(s) in apoptosis. Although it appears that ICE family members play a most important role in promoting apoptotic cell death, evidence has been advanced that other proteases are also involved in sequential or parallel steps of apoptosis. Activation of a particular protease can lead to processing molecules either of the same or different proteases, leading to an activation of a protease cascade. Here we attempt to summarize the current thinking concerning these proteases and their involvement in apoptosis.

• Shaham S, Horvitz HR
• Developing Caenorhabditis elegans neurons may contain both cell-death protective and killer activities.
• Genes Dev. 1996; 10: 578-91
• Display abstract

• We developed a method for examining the effects of overexpressing cell-death-related genes in specific Caenorhabditis elegans neurons that normally live. Using this method, we demonstrated that the cell-death genes ced-3, ced-4, and ced-9 all can act cell autonomously to control programmed cell death. Our observations indicate further that not only the protective activity of ced-9 but also the killer activities of ced-3 and ced-4 are likely to be present in cells that normally live. We propose that both in C. elegans and in other organisms a competition between antagonistic protective and killer activities determines whether specific cells will live or die. Our results suggest a genetic pathway for programmed cell death in C. elegans in which ced-4 acts upstream of or in parallel to ced-3 and ced-9 negatively regulates the activity of ced-4.

• Yuan J
• Evolutionary conservation of a genetic pathway of programmed cell death.
• J Cell Biochem. 1996; 60: 4-11
• Display abstract

• Genetic analysis of programmed cell death in Caenorhabditis elegans has led to the identification of 13 genes that constitute a developmental pathway of programmed cell death. Two of the three key genes in this pathway, ced-9, a cell death suppressor, and ced-3, a cell death inducer, were found to encode proteins that share structural and functional similarities with the mammalian proto-oncogene product Bcl-2 and interleukin-1 beta converting enzyme, respectively. These results suggest that the genetic pathway of programmed cell death may be evolutionarily conserved from worms to mammals.

• Rotonda J et al.
• The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis.
• Nat Struct Biol. 1996; 3: 619-25
• Display abstract

• 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.

• Nicholson DW
• ICE/CED3-like proteases as therapeutic targets for the control of inappropriate apoptosis.
• Nat Biotechnol. 1996; 14: 297-301
• Display abstract

• 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.

• Miura M, Yuan J
• Regulation of programmed cell death by interleukin-1 beta-converting enzyme family of proteases.
• Adv Exp Med Biol. 1996; 389: 165-72

• Martinou JC, Sadoul R
• ICE-like proteases execute the neuronal death program.
• Curr Opin Neurobiol. 1996; 6: 609-14
• Display abstract

• The past year has witnessed significant advances in our understanding of the mechanisms that kill neurons during programmed cell death. The executioners are members of a family of proteases founded by ced-3, the product of a gene that is required for programmed cell death in the nematode Caenorhabditis elegans, and by mammalian interleukin-1 beta-converting enzyme. These proteases represent interesting novel targets for the therapy of acute and chronic pathologies of the nervous system associated with neuronal death.

• Hale AJ et al.
• Apoptosis: molecular regulation of cell death.
• Eur J Biochem. 1996; 236: 1-26
• Display abstract

• The field of apoptosis is unusual in several respects. Firstly, its general importance has been widely recognised only in the past few years and its surprising significance is still being evaluated in a number of areas of biology. Secondly, although apoptosis is now accepted as a critical element in the repertoire of potential cellular responses, the picture of the intra-cellular processes involved is probably still incomplete, not just in its details, but also in the basic outline of the process as a whole. It is therefore a very interesting and active area at present and is likely to progress rapidly in the next two or three years. This review emphasises recent work on the molecular mechanisms of apoptosis and, in particular, on the intracellular interactions which control this process. This latter area is of crucial importance since dysfunction of the normal control machinery is likely to have serious pathological consequences, probably including oncogenesis, autoimmunity and degenerative disease. The genetic analysis of programmed cell death during the development of the nematode Caenorhabditis elegans has proved very useful in identifying important events in the cell death programme. Recently defined genetic connections between C. elegans cell death and mammalian apoptosis have emphasised the value of this system as a model for cell death in mammalian cells, which, inevitably, is more complex. The signals inducing apoptosis are very varied and the same signals can induce differentiation and proliferation in other situations. However, some pathways appear to be of particular significance in the control of cell death; recent analysis of the apoptosis induced through the cell-surface Fas receptor has been especially important for immunology. Two gene families are dealt with in particular detail because of their likely importance in apoptosis control. These are, first, the genes encoding the interleukin-1 beta-converting enzyme family of cysteine proteases and, second, those related to the proto-oncogene bcl-2. Both of these families are homologous to cell death genes in C. elegans. In mammalian cells the number of members of both families which have been identified is growing rapidly and considerable effort is being directed towards establishing the roles played by each member and the ways in which they interact to regulate apoptosis. Other genes with established roles in the regulation of proliferation and differentiation are also important in controlling apoptosis. Several of these are known proto-oncogenes, e.g. c-myc, or tumour suppressors, e.g. p53, an observation which is consistent with the importance of defective apoptosis in the development of cancer. Viral manipulation of the apoptosis of host cells frequently involves interactions with these cellular proteins. Finally, the biochemistry of the closely controlled cellular self-destruction which ensues when the apoptosis programme has been engaged is also very important. The biochemical changes involved in inducing phagocytosis of the apoptotic cell, for example, allow the process to be neatly integrated within the tissues, under physiological conditions. Molecular defects in this area too may have important pathological consequences.

• Faucheu C, Blanchet AM, Collard-Dutilleul V, Lalanne JL, Diu-Hercend A
• Identification of a cysteine protease closely related to interleukin-1 beta-converting enzyme.
• Eur J Biochem. 1996; 236: 207-13
• Display abstract

• The present study describes the identification and molecular cloning of a new member of the interleukin-1 beta-converting enzyme (ICE) family denoted transcript Y (TY). TY is very closely related to both ICE (51% amino acid identity) and a protein named transcript X (TX) (75% amino acid identity) that we recently identified [Faucheu, C., Diu, A., Chan, A.W.E., Blanchet, A.-M., Miossec, C., Herve, F.,Collard-Dutilleul, V., Gu, Y., Aldape, R., Lippke, J., Rocher, C., Su, M.S.-S., Livingston, D.J., Hercend, T. & Lalanne, J.-L. (1995) EMBO J. 14, 1914-1922]. The amino acids that are implicated in both the ICE catalytic site and in the PI aspartate-binding pocket are conserved in TY. Within the ICE gene family, TY belongs to a subfamily of proteins closely related to the prototype ICE protein. Using transfection experiments into mammalian cells, we demonstrate that TY has protease activity on its own precursor and that this activity is dependent on the presence of a cysteine residue at position 245. However, despite the close similarity between TY and ICE active sites, TY fails to process the interleukin-1 beta precursor. In addition, as already observed for ICE and TX, TY is able to induce apoptosis when overexpressed in COS cells. TY therefore represents a new member of the growing family of apoptosis-inducing ICE-related cysteine proteases.

• Tsukuda E et al.
• EI-1507-1 and -2, novel interleukin-1 beta converting enzyme inhibitors produced by Streptomyces sp. E-1507.
• J Antibiot (Tokyo). 1996; 49: 333-9
• Display abstract

• EI-1507-1 and -2, novel interleukin-1 beta converting enzyme (ICE) inhibitors, were isolated from the culture broths of Streptomyces sp. E-1507. EI-1507-1 and EI-1507-2 selectively inhibited the recombinant human ICE activity with IC50 values of 0.23 and 0.42 microM, respectively. EI-1507-1 and EI-1507-2 also inhibited mature interleukin-1 beta secretion from THP-1 cells with IC50 values of 1.1 and 1.4 microM, respectively.

• Miossec C, Decoen MC, Durand L, Fassy F, Diu-Hercend A
• Use of monoclonal antibodies to study interleukin-1 beta-converting enzyme expression: only precursor forms are detected in interleukin-1 beta-secreting cells.
• Eur J Immunol. 1996; 26: 1032-42
• Display abstract

• 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.

• Gura T
• Structure of gene-tag protein solved.
• Science. 1996; 273: 1336-1336

• Lemaigre-Dubreuil Y, Brugg B, Chianale C, Delhaye-Bouchaud N, Mariani J
• Over-expression of interleukin-1 beta-converting enzyme mRNA in staggerer cerebellum.
• Neuroreport. 1996; 7: 1777-80
• Display abstract

• Interleukin-1 beta-converting enzyme (ICE), involved in the maturation process of interleukin-1 beta (IL-1 beta, is a homologue of ced-3, a protease required for programmed cell death in Caenorhabditis elegans. Over-expression of ICE induces programmed cell death in certain mammalian cell types, whereas in neurones of the central nervous system such a role has yet to be established. We show that ICE mRNA expression is increased 4-fold in the cerebellum of homozygous staggerer mice, where IL-1 beta mRNA is overexpressed and programmed neuronal cell death occurs. Intraperitoneal injection of endotoxin (LPS) induced a strong phasic increase in IL mRNA levels in the cerebellum, whereas the ICE mRNA level increased only moderately. Involvement of ICE in neuronal cell death in the cerebellum of staggerer mice is suspected.

• Estrov Z, Talpaz M
• Role of interleukin-1 beta converting enzyme (ICE) in leukemia.
• Cytokines Mol Ther. 1996; 2: 1-11
• Display abstract

• Interleukin (IL)-1 is a proinflammatory cytokine that plays a pivotal role in driving the in vitro proliferation of leukemic cells through autocrine or paracrine pathways. Both IL-1 genes, IL-1 alpha and the prominent IL-1 beta, produce 31 kDa proteins. Whereas the precursor (pro) 31 kDa form of IL-1 alpha is biologically active, pro-IL-1 beta is inactive unless cleaved to its mature form by a cytoplasmic cysteine protease termed IL-1 beta converting enzyme (ICE). Although ICE was first thought to be a unique enzyme with a single biologic activity, several investigators have demonstrated that ICE shares sequence homology with the protein product of ced-3, the gene for cell death of the nematode Caenorhabditis elegans, and can induce apoptosis in different cellular systems. However, recent data indicate that ICE is a member of an increasingly recognized family of ICE-related molecules whose other members, such as CPP32, do not cleave pro-IL-1 beta but rather are effective inducers of apoptotic cell death. We recently investigated the effect of ICE inhibition on acute myelogenous leukemia (AML) colony growth. We found that inhibition of ICE reduced the production of mature IL-1 beta and suppressed the proliferation of AML colony-forming units, confirming the central role of IL-1 beta in AML progenitor proliferation. These data suggest that the primary role of ICE in AML cells is cleavage of pro-IL-1 beta rather than induction of apoptosis and that the antileukemic activity of specific ICE inhibitors warrants further exploitation.

• Juan TS, McNiece IK, Jenkins NA, Gilbert DJ, Copeland NG, Fletcher FA
• Molecular characterization of mouse and rat CPP32 beta gene encoding a cysteine protease resembling interleukin-1 beta converting enzyme and CED-3.
• Oncogene. 1996; 13: 749-55
• Display abstract

• Interleukin-1 beta converting enzyme (ICE) defines a new class of mammalian cysteine protease that shares strong homology with the Caenorhabditis elegans death gene ced-3. Both ICE and CED-3, when introduced into cultured cells, induce apoptosis, indicating that this type of cysteine protease may play an important role in the process of programmed cell death. Here, we report the cloning of a mouse and rat gene encoding a novel cysteine protease. The putative proteins encoded by these cDNAs contain the conserved sequence (QACRG) necessary for covalent linkage to the substrate as well as the three amino acids responsible for substrate binding and catalysis in ICE. Amino acid sequence analysis indicates that this rodent cysteine protease is the homolog of human CPP32 beta. Mouse CPP32 beta mRNA is highly expressed in spleen, and to a lesser degree in brain, lung, liver, and kidney. The mouse CPP32 beta genomic locus spans a region of approximately 20 kb, including seven exons and six introns. Mouse interspecific backcross mapping allowed localization of CPP32 beta to the central region of mouse chromosome 8, linked to Scvr, Lpl, Jund1 and Mlr.

• Monney L et al.
• Bcl-2 overexpression blocks activation of the death protease CPP32/Yama/apopain.
• Biochem Biophys Res Commun. 1996; 221: 340-5
• Display abstract

• The C. elegans gene product ced-9 inhibits programmed cell death by negatively regulating the death-mediating protease ced-3. The mammalian homolog of ced-9 is the oncoprotein Bcl-2. Overexpression of Bcl-2 spares mammalian and nematodal cells from dying and prevents ectopic cell death in ced-9 loss-of-function mutants. Although Bcl-2 has been shown to act as an antioxidant under certain conditions, additional functions have emerged from studies under low oxygen pressure. Here we show that Bcl-2 overexpression impairs activation of the interleukin-1beta converting enzyme-related death protease CPP32/Yama/apopain, the mammalian homolog of ced-3. When U937 monocytes undergo programmed cell death in response to tumor necrosis factor alpha, the inactive CPP32 precursor is cleaved into its active forms. As a consequence poly(ADP ribose) polymerase, a major substrate of CPP32, is faithfully cleaved into a 85 kD fragment. Bcl-2 overexpressing cells are protected from tumor necrosis factor alpha-induced death and display neither CPP32 maturation nor PARP cleavage. The inhibitory effect of Bcl-2 on CPP32 activation is indirect since no physical interaction between the two proteins could be detected. These results indicate that Bcl-2 neutralizes an unknown cellular activator of CPP32 to save cells from programmed cell death.

• Kato H et al.
• Identification of an alternatively spliced transcript of equine interleukin-1 beta.
• Gene. 1996; 177: 11-6
• Display abstract

• Using lipopolysaccharide (LPS)-stimulated equine peripheral blood mononuclear cell (PBMC) cDNA as a template, we performed polymerase chain reaction (PCR) amplification with equine interleukin-1 beta (IL-1 beta) specific primers. Electrophoresis of the PCR product on agarose gel revealed an additional smaller fragment that hybridized with an equine IL-1 beta cDNA probe. Sequencing of this fragment demonstrated that it was shorter than normal equine IL-1 beta cDNA by 162 nucleotides, which corresponded to exon 5 of the human and murine IL-1 beta genes. The deletion of 162 nucleotides did not result in a frame shift but spliced out the putative exon 5 of the IL-1 beta gene which includes the cleavage site for the IL-1 beta converting enzyme (ICE) in human and murine IL-1 beta. Expression of the alternatively spliced IL-1 beta transcript in PBMC was also detected after stimulation with other compounds. These results clearly indicate the existence of an alternatively spliced IL-1 beta transcript in equine PBMC.

• Dolle RE et al.
• First examples of peptidomimetic inhibitors of interleukin-1 beta converting enzyme.
• J Med Chem. 1996; 39: 2438-40

• White E
• Life, death, and the pursuit of apoptosis.
• Genes Dev. 1996; 10: 1-15

• Faucheu C et al.
• A novel human protease similar to the interleukin-1 beta converting enzyme induces apoptosis in transfected cells.
• EMBO J. 1995; 14: 1914-22
• Display abstract

• We have identified a novel cDNA encoding a protein (named TX) with > 50% overall sequence identity with the interleukin-1 beta converting enzyme (ICE) and approximately 30% sequence identity with the ICE homologs NEDD-2/ICH-1L and CED-3. A computer homology model of TX was constructed based on the X-ray coordinates of the ICE crystal recently published. This model suggests that TX is a cysteine protease, with the P1 aspartic acid substrate specificity retained. Transfection experiments demonstrate that TX is a protease which is able to cleave itself and the p30 ICE precursor, but not to generate mature IL-1 beta from pro-IL-1 beta. In addition, this protein induces apoptosis in transfected COS cells. TX therefore delineates a new member of the growing Ice/ced-3 gene family coding for proteases with cytokine processing activity or involved in programmed cell death.

• Irmler M et al.
• Granzyme A is an interleukin 1 beta-converting enzyme.
• J Exp Med. 1995; 181: 1917-22
• Display abstract

• Apoptosis is critically dependent on the presence of the ced-3 gene in Caenorhabditis elegans, which encodes a protein homologous to the mammalian interleukin (IL)-1 beta-converting enzyme (ICE). Overexpression of ICE or ced-3 promotes apoptosis. Cytotoxic T lymphocyte-mediated rapid apoptosis is induced by the proteases granzyme A and B. ICE and granzyme B share the rare substrate site of aspartic acid, after which amino acid cleavage of precursor IL-1 beta (pIL-1 beta) occurs. Here we show that granzyme A, but not granzyme B, converts pIL-1 beta to its 17-kD mature form. Major cleavage occurs at Arg120, four amino acids downstream of the authentic processing site, Asp116. IL-1 beta generated by granzyme A is biologically active. When pIL-1 beta processing is monitored in lipopolysaccharide-activated macrophage target cells attacked by cytotoxic T lymphocytes, intracellular conversion precedes lysis. Prior granzyme inactivation blocks this processing. We conclude that the apoptosis-inducing granzyme A and ICE share at least one downstream target substrate, i.e., pIL-1 beta. This suggests that lymphocytes, by means of their own converting enzyme, could initiate a local inflammatory response independent of the presence of ICE.

• Yuan J
• Molecular control of life and death.
• Curr Opin Cell Biol. 1995; 7: 211-4
• Display abstract

• Recent evidence has shown that two of the three key genes in the programmed cell death pathway of Caenorhabditis elegans, ced-9 (a cell death suppressor) and ced-3 (a cell death inducer), encode proteins that share structural and functional similarities with the mammalian proto-oncogene product Bcl-2 and interleukin-1 beta converting enzyme, respectively. These findings reveal key molecules that control life and death decisions in vertebrates.

• Kamens J et al.
• Identification and characterization of ICH-2, a novel member of the interleukin-1 beta-converting enzyme family of cysteine proteases.
• J Biol Chem. 1995; 270: 15250-6
• Display abstract

• Interleukin-1 beta converting enzyme (ICE) is a cytoplasmic cysteine protease required for generating the bioactive form of the interleukin-1 beta cytokine from its inactive precursor. We report the identification of ICH-2, a novel human gene encoding a member of the ICE cysteine protease family, and characterization of its protein product. ICH-2 mRNA is widely expressed in human tissues in a pattern similar to, but distinct from, that of ICE. Overexpression of ICH-2 in insect cells induces apoptosis. Purified ICH-2 is functional as a protease in vitro. A comparison of the inhibitor profiles and substrate cleavage by ICH-2 and ICE shows that the enzymes share catalytic properties but may differ in substrate specificities, suggesting that the two enzymes have different functions in vivo.

• Munday NA et al.
• Molecular cloning and pro-apoptotic activity of ICErelII and ICErelIII, members of the ICE/CED-3 family of cysteine proteases.
• J Biol Chem. 1995; 270: 15870-6
• Display abstract

• Cysteine proteases related to mammalian interleukin-1 beta-converting enzyme (ICE) and the nematode cell death abnormal ced-3 gene product have been implicated in the effector mechanism of apoptotic cell death. Two novel members of this new family of ICE/CED-3-related proteases, designated ICErel-II and ICErel-III, were cloned from human monocytic cells. Both were highly homologous to human ICE (52% identical) and CED-3 (25% identical) and both contained the absolutely conserved pentapeptide sequence Gln-Ala-Cys-Arg-Asp containing the catalytic cysteine residue. Other structural motifs that were comparable with ICE suggest that ICErel-II and ICErel-III are also synthesized as larger proenzymes which are proteolytically processed to form heterodimeric active enzymes. Pro-interleukin-1 beta processing activity could not be detected in cells transfected with ICErel-II or ICErel-III, but pro-domain-less truncated forms of ICErel-II and ICErel-III were capable of effectively inducing fibroblast apoptosis. ICErel-II and ICErel-III may, therefore, participate in proteolytic events culminating in the apoptotic death of human cells.

• Gu Y et al.
• Interleukin-1 beta converting enzyme requires oligomerization for activity of processed forms in vivo.
• EMBO J. 1995; 14: 1923-31
• Display abstract

• Interleukin-1 beta converting enzyme (ICE) is composed of 10' (p10) and 20 kDa (p20) subunits, which are derived from a common 45 kDa precursor. Recent crystallographic studies have shown that ICE exists as a tetramer (p20/p10)2 in the crystal lattice. We provide evidence that the p10 and p20 subunits of ICE associate as oligomers in transfected COS cells. Using intragenic complementation, we show that the activity of a p10/p10 interface mutant defective in autoprocessing can be restored by co-expression with active site ICE mutants. Different active site mutants can also complement each other by oligomerization to form active ICE. These studies indicate that ICE precursor polypeptides may associate in different quaternary structures and that oligomerization is required for autoprocessing. Furthermore, integenic complementation of active site mutants of ICE and an ICE homolog restores autoprocessing activity, suggesting that hetero-oligomerization occurs between ICE homologs.

• Howard AD et al.
• Human IL-1 beta processing and secretion in recombinant baculovirus-infected Sf9 cells is blocked by the cowpox virus serpin crmA.
• J Immunol. 1995; 154: 2321-32
• Display abstract

• Biologically active, mature IL-1 beta (mIL-1 beta) is released from activated monocytes after proteolytic processing from an inactive precursor (pIL-1 beta). IL-1 beta converting enzyme (ICE), the first member of a newly discovered family of cysteine proteinases, is required for this processing event. The cleaved cytokine is released from monocytes by an unknown mechanism which does not employ a standard hydrophobic signal sequence. As in mammalian fibroblasts, insect Sf9 cells do not normally process or secrete human IL-1 beta. The expression of active ICE enables Sf9 cells to process 31-kDa pIL-1 beta correctly at Asp27 and Asp116, and to export 17.5-kDa mIL-1 beta. The recombinant heterodimeric human enzyme purified from Sf9 cells possesses a sp. act. of 2.9 +/- 0.5 x 10(6) U/mg and is indistinguishable from native ICE with regard to its subunit composition and catalytic properties. In this system, co-expression of the cowpox virus crmA gene, an extremely potent serpin inhibitor of ICE (Ki < 7 pM), inhibits ICE activation completely and blocks pIL-1 beta processing and mIL-1 beta secretion by approximately 95%. The results indicate that ICE, in addition to its processing function, facilitates the transport of IL-1 beta across the plasma membrane.

• Nalin CM
• Apoptosis research enters the ICE age.
• Structure. 1995; 3: 143-5
• Display abstract

• Recent elucidation of the structure of interleukin-1 beta-converting enzyme (ICE), a protease with sequence homology to a nematode protein associated with programmed cell death, opens a new chapter in the study of how proteases may control cellular suicide.

• Fernandes-Alnemri T, Litwack G, Alnemri ES
• Mch2, a new member of the apoptotic Ced-3/Ice cysteine protease gene family.
• Cancer Res. 1995; 55: 2737-42
• Display abstract

• We have developed a PCR approach to clone new apoptotic Ced-3/Ice-like cysteine protease genes. This approach uses degenerate oligonucleotides encoding the highly conserved pentapeptides QACRG and GSWFI that are present in all known apoptotic cysteine proteases. Using this approach, we have cloned a novel apoptotic gene from human Jurkat T lymphocytes. The new gene encodes a approximately 34-kilodalton protein that is highly homologous to human CPP32, Caenorhabditis elegans cell death protein CED-3, mammalian Ich-1 (Nedd2), and mammalian interleukin-1 beta converting enzyme. Because of its high homology to the C. elegans Ced-3 gene, we named the new gene mammalian Ced-3 homologue Mch2. Two Mch2 transcripts (Mch2 alpha, 1.7 kb; Mch2 beta, 1.4 kb) were detected in Jurkat T lymphocytes and other cell lines. We believe that the Mch2 alpha transcript encodes the full-length Mch2, whereas the Mch2 beta transcript encodes a shorter Mch2 isoform, probably as a result of alternative splicing. Like interleukin-1 beta converting enzyme and CPP32, recombinant Mch2 alpha, but not Mch2 beta, possesses protease activity, as determined by its ability to cleave the fluorogenic peptide DEVD-AMC. CPP32 and Mch2 alpha can also cleave poly(ADP-ribose) polymerase in vitro, suggesting that these enzymes participate in poly(ADP-ribose) polymerase cleavage observed during cellular apoptosis. In addition, overexpression of recombinant Mch2 alpha, but not Mch2 beta, induces apoptosis in Sf9 insect cells. Our data suggest that Mch2 is a Ced-3/interleukin-1 beta converting enzyme-like cysteine protease and could be another important mediator of apoptosis in mammalian cells.

• Los M et al.
• Requirement of an ICE/CED-3 protease for Fas/APO-1-mediated apoptosis.
• Nature. 1995; 375: 81-3
• Display abstract

• The Fas/APO-1 receptor is one of the major regulators of apoptosis. We report here that Fas/APO-1-mediated apoptosis requires the activation of a new class of cysteine proteases, including interleukin-1 beta-converting enzyme (ICE), which are homologous to the product of the Caenorhabditis elegans cell-death gene ced-3 (refs 11, 12). Triggering of Fas/APO-1 rapidly stimulated the proteolytic activity of ICE. Overexpression of ICE, achieved by electroporation and microinjection, strongly potentiated Fas/APO-1-mediated cell death. In addition, inhibition of ICE activity by protease inhibitors, as well as by transient expression of the pox virus-derived serpin inhibitor CrmA or an antisense ICE construct, substantially suppressed Fas/APO-1-triggered cell death. We conclude that activation of ICE or an ICE-related protease is a critical event in Fas/APO-1-mediated cell death.

• Kondo S et al.
• Interleukin-1 beta-converting enzyme mediates cisplatin-induced apoptosis in malignant glioma cells.
• Cancer Res. 1995; 55: 6166-71
• Display abstract

• Increasing the susceptibility of tumor cells to apoptotic cell death following chemotherapy is of importance to the outcome of cancer treatment. Although the tumor suppressor gene p53 is required for efficient induction of apoptosis by chemotherapeutic agents, it is not the only apoptosis mediator gene. The molecular mechanisms mediating apoptosis following chemotherapy via p53-dependent or p53-independent pathways remain unclear. We show here that cis-diamminedichloroplatinum (cisplatin) induces the expression of interleukin-1 beta-converting enzyme (ICE), a mammalian homologue of the Caenorhabiditis elegans cell death gene ced-3, in murine and human malignant glioma cells during apoptosis regardless of their p53 status. Furthermore, overexpression of the murine ICE gene induces apoptosis in these tumor cells. The apoptosis induced by cisplatin treatment or murine ICE overexpression can be suppressed by the tetrapeptide ICE inhibitor Ac-YVAD-CMK or the apoptosis inhibitors bcl-2 or bcl-2-related bcl-XL gene. These findings suggest that ICE may mediate apoptosis induced by chemotherapy, and its induction could represent a novel approach for the effective treatment of malignant glioma.

• Alnemri ES, Fernandes-Alnemri T, Litwack G
• Cloning and expression of four novel isoforms of human interleukin-1 beta converting enzyme with different apoptotic activities.
• J Biol Chem. 1995; 270: 4312-7
• Display abstract

• To understand the mechanism of interleukin-1 beta converting enzyme (ICE) activation in apoptosis, we analyzed the expression of ICE mRNA in two human cell lines by reverse transcription-polymerase chain reaction technique. This resulted in the identification and cloning of four alternatively spliced ICE mRNA isoforms. Although all the alternative splicing events were within the coding sequence of ICE, the four ICE isoforms maintained open reading frames and were designated as ICE beta, gamma, delta, and epsilon. In ICE gamma, most of the propeptide (amino acids 20-112) is deleted, which suggests that it may function as a catalyst for ICE autoprocessing in vivo. In ICE delta, amino acids 288-335, which contain the cleavage sites between the p20 and p10 subunits of ICE, are deleted thus resulting in its inactivation. Intriguingly, in ICE epsilon amino acids 20-335, which encompass most of the propeptide and the p20 subunit, are deleted resulting in the formation of a molecule that is homologous to the p10 subunit. Examination of the ability of these four ICE isoforms to cause apoptosis revealed that only the parental ICE alpha and isoforms beta and gamma, but not isoforms delta and epsilon, can induce apoptosis when overexpressed in Sf9 insect cells. In addition, coexpression of the p20 and p10 but not the p20 and ICE epsilon in Sf9 cells results in apoptosis. Interestingly, expression of ICE epsilon and to a lesser degree ICE delta resulted in extension of the survival of baculovirus-infected cells in a manner similar to expression of BCL2. The ability of ICE epsilon to extend the survival of Sf9 cells suggests that baculovirus-induced apoptosis in these cells is mediated by an ICE-like protease. We show that ICE epsilon can bind to the p20 subunit of ICE and potentially may compete with the p10 subunit to form an inactive ICE complex. Therefore, by acting as a dominant inhibitor of ICE activity, ICE epsilon may regulate ICE activation in vivo.

• Thornberry NA, Molineaux SM
• Interleukin-1 beta converting enzyme: a novel cysteine protease required for IL-1 beta production and implicated in programmed cell death.
• Protein Sci. 1995; 4: 3-12
• Display abstract

• Interleukin-1 beta converting enzyme is the first member of a new class of cysteine proteases. The most distinguishing feature of this family is a nearly absolute specificity for cleavage at aspartic acid. This enzyme has been the subject of intense research because of its role in the production of IL-1 beta, a key mediator of inflammation. These studies have culminated in the design of potent inhibitors and determination of its crystal structure. The structure secures the relationship of the enzyme to CED-3, the product of a gene required for programmed cell death in Caenorhabditis elegans, suggesting that members of this family function in cell death in vertebrates.

• Lin X, Denmeade SR, Isaacs JT
• The genetics of programmed (apoptotic) cell death.
• Cancer Surv. 1995; 25: 173-94
• Display abstract

• The genetic pathway for the activation and completion of programmed death of cells is as complex and well regulated as the pathway for cell proliferation. The identification of both the genetic elements in the signal transduction pathway involved with the initiation of programmed cell death, as well as the cellular machinery involved with DNA and cellular fragmentation into apoptotic bodies, is developing rapidly. Attempts at understanding how these elements are co-ordinated into a temporally discrete series of metabolic steps is only just beginning. This research not only will be fruitful from a basic science standpoint, but should also identify new approaches for cancer chemoprevention and therapy.

• Malinowski JJ et al.
• Production, purification, and crystallization of human interleukin-1 beta converting enzyme derived from an Escherichia coli expression system.
• Protein Sci. 1995; 4: 2149-55
• Display abstract

• Interleukin-1 beta converting enzyme (ICE) is a cysteine protease that catalyzes the conversion of the inactive precursor form of IL-1 beta to an active mature form. The mature form of IL-1 beta is involved in mediating inflammatory responses and in the progression of autoimmune diseases. We recently reported on the production of active human ICE in insect cells using the baculovirus expression system (Wang XM et al., 1994, Gene 145:273-277). Because the levels of expression achieved with this system were limiting for the purpose of performing detailed biochemical and biophysical studies, we examined the production of ICE in Escherichia coli. By using a tac promoter-based expression system and fusion to thioredoxin we were able to recover high levels of active ICE protein. The expressed protein, which was distributed between the soluble and insoluble fractions, was purified to homogeneity from both fractions using a combination of classical and affinity chromatography. Comparisons of ICE derived from both fractions indicated that they were comparable in their specific activities, subunit composition, and sensitivities to specific ICE inhibitors. The combined yields of ICE obtained from the soluble and insoluble fractions was close to 1 mg/L of induced culture. Recombinant human ICE was crystallized in the presence of a specific ICE inhibitor in a form suitable for X-ray crystallographic analysis. This readily available source of ICE will facilitate the further characterization of this novel and important protease.

• Kuida K et al.
• Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme.
• Science. 1995; 267: 2000-3
• Display abstract

• The interleukin-1 beta (IL-1 beta) converting enzyme (ICE) processes the inactive IL-1 beta precursor to the proinflammatory cytokine. Adherent monocytes from mice harboring a disrupted ICE gene (ICE-/-) did not export IL-1 beta or interleukin-1 alpha (IL-1 alpha) after stimulation with lipopolysaccharide. Export of tumor necrosis factor-alpha and interleukin-6 (IL-6) from these cells was also diminished. Thymocytes from ICE-/- mice were sensitive to apoptosis induced by dexamethasone or ionizing radiation, but were resistant to apoptosis induced by Fas antibody. Despite this defect in apoptosis, ICE-/- mice proceed normally through development.

• Flaws JA et al.
• Interleukin-1 beta-converting enzyme-related proteases (IRPs) and mammalian cell death: dissociation of IRP-induced oligonucleosomal endonuclease activity from morphological apoptosis in granulosa cells of the ovarian follicle.
• Endocrinology. 1995; 136: 5042-53
• Display abstract

• The Caenorhabditis elegans death susceptibility gene, ced-3, has a number of homologs in vertebrate species, including interleukin-1 beta (IL-1 beta)-converting enzyme (ICE), Ich-1long, and CPP32. These genes, which encode a family of related proteases, have been shown to induce apoptosis when transfected into eukaryotic cells. However, it remains to be determined whether these proteases are involved in apoptotic cell death under physiological conditions. The purpose of these studies was to examine the role of ICE-related proteases (IRPs) in apoptosis using a physiologically relevant model system, the ovarian follicle. Somatic granulosa cells within ovarian follicles undergo apoptosis during follicular atresia, a process responsible for the depletion of greater than 95% of the follicles established in the postnatal ovary. To accomplish these studies, we cloned partial rat complementary DNAs encoding ICE, Ich-1, and CPP32 and used these complementary DNAs to examine the gonadotropin regulation of ICE, Ich-1, and CPP32 gene expression in the immature rat ovary. We also examined levels of ICE activity in healthy and atretic rat follicles by monitoring the conversion of exogenous pro-IL-1 beta to the active cytokine, and then evaluated the actions of recombinant IL-1 beta on apoptosis in follicles incubated in vitro. Finally, we tested the requirement for IRP activity in granulosa cell apoptosis and follicular atresia by incubating follicles without and with IRP inhibitors. Northern blot analysis of total RNA samples indicated that gonadotropin-promoted follicular survival was associated with reduced ovarian expression of messenger RNAs encoding Ich-1 and CPP32. In contrast, ICE messenger RNA levels were extremely low and were not affected by gonadotropin treatment. We were also unable to detect ICE activity in proteins extracted from either healthy or atretic rat follicles, collectively suggesting that ICE per se may not function in granulosa cell death. As another approach to determine whether ICE is involved in atresia, healthy antral follicles were isolated from ovaries of gonadotropin-primed immature rats and incubated for 24 h in the absence or presence of 100 ng/ml transforming growth factor-alpha (TGF alpha) without and with 100 ng/ml IL-1 beta. Granulosa cells within follicles incubated in medium alone exhibited extensive levels of apoptosis, and this onset of apoptosis was prevented by the inclusion of TGF alpha. Addition of IL-1 beta did not alter basal levels of apoptosis nor did the cytokine antagonize TGF-alpha-promoted follicle survival, providing additional evidence that ICE activity is not required for atresia to occur.(ABSTRACT TRUNCATED AT 400 WORDS)

• Singer II et al.
• 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.
• J Exp Med. 1995; 182: 1447-59
• Display abstract

• 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.

• Kato H et al.
• Molecular cloning of equine interleukin-1 alpha and -beta cDNAs.
• Vet Immunol Immunopathol. 1995; 48: 221-31
• Display abstract

• Equine interleukin-1 alpha (IL-1 alpha) and IL-1 beta were molecularly cloned to establish a basis for research on inflammatory and immune responses in the horse. Equine peripheral blood mononuclear cells (PBMC) were stimulated with lipopolysaccharide (LPS), and cDNA clones of equine IL-1 alpha and IL-1 beta covering the whole coding sequences were isolated from them. These equine IL-1 alpha and IL-1 beta clones contained open reading frames encoding 271 and 269 amino acids, respectively. The deduced amino acid sequence of equine IL-1 alpha showed 71.6% and 60.2% similarity with that of human and murine IL-1 alpha, respectively. Similarly, the amino acid sequence of equine IL-1 beta showed 66.7% and 61.8% similarity with that of human and murine IL-1 beta, respectively. In both equine IL-1 alpha and IL-1 beta, amino acids at miristoylation sites were well conserved. Dot blot analysis indicated that the expression of IL-1 beta was predominant to that of IL-1 alpha in equine PBMC stimulated with LPS or phorbol myristate acetate.

• Xue D, Horvitz HR
• Inhibition of the Caenorhabditis elegans cell-death protease CED-3 by a CED-3 cleavage site in baculovirus p35 protein.
• Nature. 1995; 377: 248-51
• Display abstract

• The baculovirus protein p35 inhibits programmed cell death in such diverse animals as insects, nematodes and mammals. Here we show that p35 protein is a substrate for and inhibitor of the Caenorhabditis elegans cell-death protease CED-3 (refs 6, 7) and a substrate for four CED-3-like vertebrate cysteine protease activities implicated in apoptosis in mammals. A p35 mutation that greatly reduced p35 activity in vitro as a CED-3 substrate and inhibitor abolished p35 activity in vivo in protecting against cell death in C. elegans. Introduction of the CED-3 cleavage site in p35 into the cowpox virus protein crmA, which inhibits mammalian apoptosis but not programmed cell death in C. elegans, caused crmA to block CED-3-mediated cell death. These observations suggest that p35 may prevent programmed cell death in C. elegans and other species by acting as a competitive inhibitor of cysteine proteases.

• Gu Y, Sarnecki C, Aldape RA, Livingston DJ, Su MS
• Cleavage of poly(ADP-ribose) polymerase by interleukin-1 beta converting enzyme and its homologs TX and Nedd-2.
• J Biol Chem. 1995; 270: 18715-8
• Display abstract

• The proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) is an early biochemical event, which occurs during apoptosis. A recent study suggested that PARP cleavage can be mediated by a novel cytosolic protease (prICE) that resembles interleukin-1 beta converting enzyme (ICE), but cannot be mediated by ICE itself (Lazebnik, Y.A., Kaufmann, S.H., Desnoyers, S., Poirier, G.G., and Earnshaw, W.C. (1994) Nature 371, 346-347). We have used a COS cell co-transfection assay to investigate if ICE or any known ICE-like protease is active in PARP cleavage within the cell. Here we report that co-expression of human PARP with human ICE, or the ICE homologs TX and Nedd-2, resulted in a cleavage of PARP identical to that observed in apoptotic cells. Experiments with purified recombinant human ICE indicated that PARP polypeptide can be specifically cleaved in vitro by ICE in a time- and enzyme concentration-dependent manner. PARP cleavage, however, requires a 50-100-fold higher ICE concentration than does processing of the interleukin-1 beta precursor at an equivalent substrate concentration. The abilities of ICE, TX, and Nedd-2, when expressed at high intracellular concentrations, to cleave PARP are consistent with their induction of apoptosis in transfected cells.

• Ariizumi K, Kitajima T, Bergstresser OR, Takashima A
• Interleukin-1 beta converting enzyme in murine Langerhans cells and epidermal-derived dendritic cell lines.
• Eur J Immunol. 1995; 25: 2137-41
• Display abstract

• Interleukin (IL)-1 beta plays an essential role in the induction of T cell-mediated immune responses in skin. Langerhans cells (LC), which constitutively express IL-1 beta mRNA, have been assumed to be the primary source of IL-1 beta in murine epidermis. The purpose of this study was to determine whether LC express mRNA for the IL-1 beta converting enzyme (ICE), a protease that is required for processing pro-IL-1 beta into an active form. Here, we report that both IL-1 beta and ICE mRNA are expressed by the Ia+ population (i.e. LC) in murine epidermis. Moreover, murine epidermal-derived DC lines (XS series) also express both IL-1 beta and ICE mRNA, and they secrete relatively large amounts of IL-1 beta following lipopolysaccharide (LPS) stimulation. Finally, LPS-triggered IL-1 beta secretion by XS cells is blocked almost completely by the ICE inhibitor acetyl-Tyr-Val-Ala-Asp-CH2OC(O)-[2,6-(CF3)2]Ph. These results demonstrate that LC are the primary source of IL-1 beta within the epidermis, and suggest that the proinflammatory role of IL-1 beta may be regulated pharmacologically by ICE inhibitors in vivo.

• Ramage P et al.
• Expression, refolding, and autocatalytic proteolytic processing of the interleukin-1 beta-converting enzyme precursor.
• J Biol Chem. 1995; 270: 9378-83
• Display abstract

• The interleukin-1 beta-converting enzyme is a heterodimeric cysteine protease that is produced as a 45-kDa precursor. The full-length precursor form of the enzyme was expressed in Escherichia coli as insoluble inclusion bodies. Following solubilization and refolding of the 45-kDa protein, autoproteolytic conversion to a heterodimeric form containing 10- and 20-kDa subunits was observed. This enzyme had catalytic activity against both natural (interleukin-1 beta precursor) and synthetic peptide substrates. The inclusion of a specific inhibitor (SDZ 223-941) of the converting enzyme in the refolding mixture prevented proteolytic processing to the 10-/20-kDa form. Similarly, refolding under nonreducing conditions also prevented processing. Time course experiments showed that the 10-kDa subunit was released from the 45-kDa precursor before the 20-kDa subunit, implying that the N-terminal portion of the precursor is released last and may play a regulatory role.

• Wang XM et al.
• Production of active human interleukin-1 beta-converting enzyme in a baculovirus expression system.
• Gene. 1994; 145: 273-7
• Display abstract

• The cDNA coding for the precursor form of human interleukin-1 beta-converting enzyme (proICE) was expressed in Spodoptera frugiperda (Sf9) insect cells using a baculovirus expression system. The 45-kDa recombinant protein was further processed to several smaller forms of 32, 24, 20, 13 and 10 kDa. Active recombinant ICE derived from the baculovirus expression system (bvICE) was found to be present in soluble lysates of insect cells as an associated heterodimer consisting of 10- and 20-kDa subunits. The activity of bvICE was determined by conversion of precursor interleukin-1 beta (preIL-1 beta) to the mature form (mIL-1 beta) and via site-specific cleavage of a decapeptide which spans the ICE cleavage site in preIL-1 beta. The bvICE system was inhibited by an ICE inhibitor to the same extent as native ICE from the monocytic cell line THP-1. Expression of an active-site mutant (Cys285 to Ser) of proICE in insect cells resulted in the accumulation of partially processed (32-kDa) ICE. The availability of a facile expression system will permit further characterization of the biochemical properties and processing pathway of this unique protease.

• Jacobson MD, Evan GI
• Apoptosis. Breaking the ICE.
• Curr Biol. 1994; 4: 337-40
• Display abstract

• Structural and functional similarities have been discovered between two mammalian proteins. Bcl-2 ang interleukin 1 beta-converting enzyme, and proteins encoded by nematode cell-death genes.

• Rubin LL, Gatchalian CL, Rimon G, Brooks SF
• The molecular mechanisms of neuronal apoptosis.
• Curr Opin Neurobiol. 1994; 4: 696-702
• Display abstract

• During the past year, apoptosis has been recognized as a process that is perpetually poised to be initiated--often from the cytoplasm rather than from the nucleus--unless it is suppressed by survival factors. Suspected mediators of apoptosis that have recently been investigated include the cysteine protease interleukin-1 beta-converting enzyme, free radicals and cell cycle kinases. Known inhibitors of programmed cell death, such as Bcl-2 and its homologues, have been further studied, and the results suggest that cell death may be regulated by multiple pathways. With the recent identification of the Drosophila gene reaper, which appears to play a role in the initiation of apoptosis, another genetic system for studying cell death has become available.

• Isaacs JT
• Advances and controversies in the study of programmed cell death/apoptosis in the development of and therapy for cancer.
• Curr Opin Oncol. 1994; 6: 82-9
• Display abstract

• Whether normal or malignant, cells possess within their repertoire of epigenetic programs the ability to undergo a process of cellular suicide, termed programmed cell death. This programmed cell death process involves an epigenetic reprogramming of the cell that results in an energy-dependent cascade of biochemical and morphologic changes within the cell (also termed apoptosis), resulting in its death and elimination. Activation of programmed cell death is controlled by a series of endogenous cell-type-specific signals. In addition, various exogenous cell-damaging treatments (eg, radiation, chemicals, and viruses) can activate this pathway if sufficient injury to the cell occurs.

• Pennington MW, Thornberry NA
• Synthesis of a fluorogenic interleukin-1 beta converting enzyme substrate based on resonance energy transfer.
• Pept Res. 1994; 7: 72-6
• Display abstract

• Interleukin 1 beta converting enzyme (ICE) is responsible for processing an inactive 31-kDa precursor to the active, mature 17-kDa Il-1 beta with cleavage occurring between the Asp116-Ala117 amide bond. We have prepared a peptide substrate that contains the protease cleavage site situated between two fluorophores located at the termini of the molecule. Upon cleavage of DABCYL-Tyr-Val-Ala-Asp-Ala-Pro-Val-EDANS (DABCYL-ICE-EDANS), an increase in fluorescence is observed at the EDANS emission wavelength of 490 nm, permitting a continuous assay of ICE that is useful in the screening of inhibitory compounds. The Km and kcat results for hydrolysis of DABCYL-ICE-EDANS by ICE were 11.4 +/- 1.6 microM and 0.79 +/- 0.4 s-1. The second order rate constant for hydrolysis of this substrate (kcat/Km = 7.0 +/- 1.3 x 10(4) M-1 s-1) is comparable to that for the cleavage of the previously described fluorogenic substrate, Ac-Tyr-Val-Ala-Asp-AMC (6.4 x 10(4) M-1 s-1).

• Miura M
• [Genetic pathways of programmed cell death in mammal and in the nematode C. elegans]
• Tanpakushitsu Kakusan Koso. 1994; 39: 1549-59

• Fernandes-Alnemri T, Litwack G, Alnemri ES
• CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta-converting enzyme.
• J Biol Chem. 1994; 269: 30761-4
• Display abstract

• We have cloned a novel apoptotic gene from human Jurkat T-lymphocytes. The new gene encodes a 32-kDa putative cysteine protease (CPP32) with significant homology to Caenorhabditis elegans cell death protein Ced-3, mammalian interleukin-1 beta-converting enzyme (ICE), and the product of the mouse nedd2 gene. The CPP32 transcript is highly expressed and most abundant in cell lines of lymphocytic origin. Overexpression of CPP32 or ICE in Sf9 insect cells resulted in apoptosis. In addition, coexpression of recombinant p20 and p11 derived from the parental full-length CPP32 sequence resulted in apoptosis in Sf9 cells. Our data suggest that similar to ICE, CPP32 is made of two subunits, p20 and p11, which form the active CPP32 complex. The apoptotic activity of CPP32 and its high expression in lymphocytes suggest that CPP32 is an important mediator of apoptosis in the immune system.

• Cerretti DP et al.
• Molecular characterization of the gene for human interleukin-1 beta converting enzyme (IL1BC).
• Genomics. 1994; 20: 468-73
• Display abstract

• Interleukin-1 beta (IL-1 beta) mediates a wide range of immune and inflammatory responses. The active cytokine is generated by proteolytic cleavage of an inactive precursor by a protease called the IL-1 beta converting enzyme (ICE). A cDNA encoding this protease was recently isolated. A human genomic clone containing the ICE gene (IL1BC) was isolated using the cDNA as a probe. The gene consists of 10 exons spanning at least 10.6 kb. 5'-anchored polymerase chain reaction indicated a single transcription start site approximately 33 bp upstream of the initiator Met codon. The 5'-flanking region does not have an apparent TATA box but may contain an initiator (Inr) promoter element. However, transcriptional activity could not be detected with a fusion gene containing the 5'-flanking region linked to the bacterial chloramphenicol acetyltransferase gene (CAT) when transfected into the human acute monocytic leukemia cell line THP-1. Using the genomic IL1BC clone, we have confirmed the localization of the gene to chromosome 11 band q22.2-q22.3 by fluorescence in situ hybridization.

• Casano FJ, Rolando AM, Mudgett JS, Molineaux SM
• The structure and complete nucleotide sequence of the murine gene encoding interleukin-1 beta converting enzyme (ICE).
• Genomics. 1994; 20: 474-81
• Display abstract

• 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.

• Hengartner MO, Horvitz HR
• The ins and outs of programmed cell death during C. elegans development.
• Philos Trans R Soc Lond B Biol Sci. 1994; 345: 243-6
• Display abstract

• During the development of the C. elegans hermaphrodite, 131 of the 1090 cells generated undergo programmed cell death. Genetic studies have identified mutations in 14 genes that specifically affect this process. These genes define a genetic pathway for programmed cell death in C. elegans. Two genes, ced-3 and ced-4, are required for cells to undergo programmed cell death, while a third gene, ced-9, protects cells that should live from undergoing programmed cell death. The proteins encoded by ced-3 and ced-9 show significant similarity to proteins that affect programmed cell death in vertebrates, suggesting that the molecular cell death pathway in which ced-3, ced-4, and ced-9 act has been conserved between nematodes and vertebrates.

• Kumar S, Kinoshita M, Noda M, Copeland NG, Jenkins NA
• Induction of apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3 and the mammalian IL-1 beta-converting enzyme.
• Genes Dev. 1994; 8: 1613-26
• Display abstract

• By subtraction cloning we previously identified a set of mouse genes (named Nedd1 through Nedd10) with developmentally down-regulated expression in brain. We now show that one such gene, Nedd2, encodes a protein similar to the mammalian interleukin-1 beta-converting enzyme (ICE) and the product of the Caenorhabditis elegans cell death gene ced-3 (CED-3). Both ICE and CED-3 are known to encode putative cysteine proteases and induce apoptosis when overexpressed in cultured cells. Overexpression of Nedd2 in cultured fibroblast and neuroblastoma cells also resulted in cell death by apoptosis, which was suppressed by the expression of the human bcl-2 gene, indicating that Nedd2 is functionally similar to the ced-3 gene in C. elegans. We also show that during embryonic development, Nedd2 is highly expressed in several types of mouse tissue undergoing high rates of programmed cell death such as central nervous system and kidney. Our data suggest that Nedd2 is an important component of the mammalian programmed cell death machinery.

• Dolle RE et al.
• P1 aspartate-based peptide alpha-((2,6-dichlorobenzoyl)oxy)methyl ketones as potent time-dependent inhibitors of interleukin-1 beta-converting enzyme.
• J Med Chem. 1994; 37: 563-4

• Wells TN et al.
• The three-dimensional structure of human interleukin-5 at 2.4-angstroms resolution: implication for the structures of other cytokines.
• Ann N Y Acad Sci. 1994; 725: 118-27

• Thornberry NA, Peterson EP, Zhao JJ, Howard AD, Griffin PR, Chapman KT
• Inactivation of interleukin-1 beta converting enzyme by peptide (acyloxy)methyl ketones.
• Biochemistry. 1994; 33: 3934-40
• Display abstract

• Interleukin-1 beta converting enzyme (ICE) is a cysteine protease in monocytes that is essential for the proteolytic activation of interleukin-1 beta, an important mediator of inflammation. Peptide (acyloxy)methyl ketones designed with the appropriate peptide recognition sequence (Ac-Tyr-Val-Ala-Asp-CH2-OC(O)Ar) are potent, competitive, irreversible inhibitors. Mass spectrometry and sequence analysis indicate that inactivation proceeds through expulsion of the carboxylate leaving group to form a thiomethyl ketone with the active site Cys285. The second-order inactivation rate is independent of leaving group pKa, with an approximate value of 1 x 10(6) M-1 s-1. This rate constant is directly proportional to the reaction macroviscosity, indicating that the rate-limiting step in inactivation is association of enzyme and inhibitor, rather than any bond-forming reactions. Affinity labeling of THP.1 monocytic cell cytosol with a biotinylated tetrapeptide (acyloxy)methyl ketone for 28 half-lives resulted in labeling of only ICE, demonstrating the selectivity of these inhibitors. These inhibitors are relatively inert toward other bionucleophiles such as glutathione (< 5 x 10(-4) M-1 s-1), making them excellent candidates for in vivo studies of enzyme inhibition.

• Salvatore MJ et al.
• L-741,494, a fungal metabolite that is an inhibitor of interleukin-1 beta converting enzyme.
• J Nat Prod. 1994; 57: 755-60
• Display abstract

• gamma-Pyrone-3-acetic acid (L-741,494) is a novel metabolite produced by a culture of the fungal genus Xylaria. This substance is a water-soluble, competitive, irreversible inhibitor of Interleukin-1 beta Converting Enzyme that is inactive against papain and trypsin. It has a mol wt of 154 and an empirical formula of C7H6O4. We propose the name xylaric acid for this compound.

• Hengartner MO, Horvitz HR
• Programmed cell death in Caenorhabditis elegans.
• Curr Opin Genet Dev. 1994; 4: 581-6
• Display abstract

• Programmed cell death in the nematode Caenorhabditis elegans requires the activities of the genes ced-3 and ced-4 and is antagonized by the activity of the gene ced-9. Cloning of these C. elegans genes has shown that two of them encode proteins with similarity to vertebrate cell death genes and has revealed that nematodes and mammals share a common pathway for programmed cell death.

• Darmon AJ, Ehrman N, Caputo A, Fujinaga J, Bleackley RC
• The cytotoxic T cell proteinase granzyme B does not activate interleukin-1 beta-converting enzyme.
• J Biol Chem. 1994; 269: 32043-6
• Display abstract

• Murine granzyme B (cytotoxic cell proteinase-1 (CCP1)) is a member of a family of seven serine proteases found in cytoplasmic granules of cytotoxic T lymphocytes (CTLs). Evidence has suggested that it is involved in target cell DNA fragmentation during CTL-mediated cytotoxicity, although intracellular substrates for granzyme B have not yet been identified. The substrate specificity of granzyme B, requiring an aspartic acid residue at site P1, is unique among eukaryotic serine proteases and is shared with only one other known eukaryotic protease, interleukin-1 beta-converting enzyme (ICE). ICE is responsible for processing pro-interleukin-1 beta to produce biologically active interleukin-1 beta and is itself synthesized as an inactive precursor. Recent evidence has suggested a role for ICE in programmed cell death, which led to a model for CTL-mediated cytotoxicity. In this proposal granzyme B activates ICE in the target cell by proteolytically processing the ICE precursor, resulting in active ICE heterodimer that induces apoptosis in the target cell. We have isolated the cDNA encoding murine ICE and generated in vitro translated ICE precursor. Using lysates from COS cells expressing granzyme B we show that ICE precursor is not a substrate for granzyme B and propose an alternate mechanism for CTL-mediated cytotoxicity.

• Sugimoto A, Friesen PD, Rothman JH
• Baculovirus p35 prevents developmentally programmed cell death and rescues a ced-9 mutant in the nematode Caenorhabditis elegans.
• EMBO J. 1994; 13: 2023-8
• Display abstract

• Programmed cell death, or apoptosis, occurs throughout the course of normal development in most animals and can also be elicited by a number of stimuli such as growth factor deprivation and viral infection. Certain morphological and biochemical characteristics of programmed cell death are similar among different tissues and species. During development of the nematode Caenorhabditis elegans, a single genetic pathway promotes the death of selected cells in a lineally fixed pattern. This pathway appears to be conserved among animal species. The baculovirus p35-encoding gene (p35) is an inhibitor of virus-induced apoptosis in insect cells. Here we demonstrate that expression of p35 in C. elegans prevents death of cells normally programmed to die. This suppression of developmentally programmed cell death results in appearance of extra surviving cells. Expression of p35 can rescue the embryonic lethality of a mutation in ced-9, an endogenous gene homologous to the mammalian apoptotic suppressor bcl-2, whose absence leads to ectopic cell deaths. These results support the hypothesis that viral infection can activate the same cell death pathway as is used during normal development and suggest that baculovirus p35 may act downstream or independently of ced-9 in this pathway.

• Wang L, Miura M, Bergeron L, Zhu H, Yuan J
• Ich-1, an Ice/ced-3-related gene, encodes both positive and negative regulators of programmed cell death.
• Cell. 1994; 78: 739-50
• Display abstract

• We report here the isolation and characterization of Ich-1, a gene related to the C. elegans cell death gene ced-3 and the mammalian homolog of ced-3, interleukin-1 beta-converting enzyme (ICE). Alternative splicing results in two distinct Ich-1 mRNA species. One mRNA species encodes a protein product of 435 amino acids (ICH-1L) that is homologous to both the P20 and P10 subunits of ICE (27% identity) and the entire CED-3 protein (28% identity). The other mRNA encodes a 312 amino acid truncated version of ICH-1L protein (ICH-1S). Overexpression of IchL induces programmed cell death, suggesting that Ich-1 is also a mammalian programmed cell death gene. More interestingly, overexpression of the Ich-1S suppresses Rat-1 cell death induced by serum deprivation. These observations suggest that Ich-1 plays an important role in both positive and negative regulation of programmed cell death in vertebrate animals.

• Reiter LA
• Peptidic p-nitroanilide substrates of interleukin-1 beta-converting enzyme.
• Int J Pept Protein Res. 1994; 43: 87-6
• Display abstract

• Peptidic p-nitroanilides are useful colorimetric substrates for enzymes. With the aim of developing a convenient, quantitative assay for inhibitors of interleukin-1 beta-converting enzyme (ICE), we have explored three approaches to the synthesis of peptidic p-nitroanilides relevant to this enzyme. The first approach involved a late stage oxidation of a p-aminoanilide such as CbzValAlaAsp(beta- tert-butyl)-p-(t-Boc-amino)anilide. The second and third approaches used the preformed amino acid p-nitroanilides HAsp-p-nitroanilide hydrochloride and HAsp(beta-tert-butyl)-p-nitroanilide which were coupled iteratively with preactivated amino acid derivatives or with an appropriate peptide, respectively. While each approach had it merits and limitations, all three produced p-nitroanilides that were substrates for ICE.

• Graybill TL, Dolle RE, Helaszek CT, Miller RE, Ator MA
• Preparation and evaluation of peptidic aspartyl hemiacetals as reversible inhibitors of interleukin-1 beta converting enzyme (ICE).
• Int J Pept Protein Res. 1994; 44: 173-82
• Display abstract

• Aspartyl aldehyde, Ac-Tyr-Val-Ala-Asp-H 1 (L-709,049), has been reported to be a potent, reversible inhibitor of interleukin-1 beta converting enzyme (ICE) [Thornberry, N.A. et al. (1992) Nature (London) 356, 768-774]. In the context of our own work, we have developed a general synthetic approach to peptidic aspartyl aldehydes. Semicarbazone derivative, H-Asp(Ot-Bu)-Sc 4, was identified as a stable, masked aspartyl aldehyde equivalent. We have used 4 to synthesize a series of mono-, di- and tripeptide aldehydes, and multigram quantities of Ac-Tyr-Val-Ala-Asp-H 1, Ac-Tyr-Val-Lys-Asp-Sc 21 and Ac-Tyr-Val-Lys-Asp-H 2. Biological evaluation of these aspartyl aldehydes and derivatives suggests that the tripeptide scaffold, Z-Val-Ala-Asp, is a peptide scaffold that retains good potency and selectivity for ICE.

• Borner C et al.
• The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis.
• J Cell Biol. 1994; 126: 1059-68
• Display abstract

• Bcl-2 is a mitochondrial- and perinuclear-associated protein that prolongs the lifespan of a variety of cell types by interfering with programmed cell death (apoptosis). Bcl-2 seems to function in an antioxidant pathway, and it is believed that membrane attachment mediated by a COOH-terminal hydrophobic tail is required for its full activity. To identify critical regions in bcl-2 alpha for subcellular localization, activity, and/or interaction with other proteins, we created, by site-directed mutagenesis, various deletion, truncation, and point mutations. We show here that membrane attachment is not required for the survival activity of bcl-2 alpha. A truncation mutant of bcl-2 alpha lacking the last 33 amino acids (T3.1) including the hydrophobic COOH terminus shows full activity in blocking apoptosis of nerve growth factor-deprived sympathetic neurons or TNF-alpha-treated L929 fibroblasts. Confocal microscopy reveals that the T3 mutant departs into the extremities of neurites in neurons and filopodias in fibroblasts. Consistently, T3 is predominantly detected in the soluble fraction by Western blotting, and is not inserted into microsomes after in vitro transcription/translation. We further provide evidence for motifs (S-N and S-II) at the NH2 and COOH terminus of bcl-2, which are crucial for its activity.

• Schwartz LM, Osborne BA
• Ced-3/ICE: evolutionarily conserved regulation of cell death.
• Bioessays. 1994; 16: 387-9

• Ayala JM, Yamin TT, Egger LA, Chin J, Kostura MJ, Miller DK
• IL-1 beta-converting enzyme is present in monocytic cells as an inactive 45-kDa precursor.
• J Immunol. 1994; 153: 2592-9
• Display abstract

• The major form of IL-1 beta-converting enzyme (ICE) identified in THP.1 monocytic cells and human monocytes is the 45-kDa precursor protein (p45), which is found in the cytoplasm. Cytoplasmic extracts of these cells show no pIL-1 beta cleavage activity, indicating that the p45 has no detectable catalytic activity. pIL-1 beta cleavage activity can only be observed after incubation in vitro when p45 breaks down to the active p20 form of the enzyme. LPS stimulation of human monocytes or THP.1 monocytic cells results in no change in the amount of p45 or its activity and no detectable appearance of p20 ICE. Immunoprecipitation of [35S]Met-labeled LPS-stimulated monocyte extracts revealed only p45 with no other co-precipitating protein. The inability to identify active ICE in stimulated monocytic cells was probably a reflection of the very low levels of active ICE present.

• Thornberry NA
• Inflammation. Key mediator takes shape.
• Nature. 1994; 370: 251-2

• Hacker G, Vaux DL
• Expression of candidate cell death genes in cell lines during apoptosis.
• Biochem Cell Biol. 1994; 72: 451-4
• Display abstract

• We have cloned mouse candidate cell death genes RP-2 and nedd2 and used Northern blot analysis to study their expression in a growth-factor-dependent cell line (FDC-P1) that can be induced to undergo apoptosis by growth factor withdrawal and in a thymoma line (ST4) that undergoes apoptosis when irradiated. RP-2 was expressed in FDC-P1 cells even when not undergoing apoptosis, and mRNA levels did not increase when apoptosis was induced by growth factor withdrawal. FDC-P1 cells expressed two genes that are similar to the Caenorhabditis elegans cell death gene ced-3: a 3.7 kilobase (kb) nedd2 message and low levels of a 5-kb message for interleukin-1 beta converting enzyme. Levels of these messages did not change with the induction of cell death, but transfection of fibroblasts with constructs expressing nedd2 caused them to undergo apoptosis. These results suggest that expression of RP-2 and nedd2 is not sufficient for apoptosis, and if the products of these genes are involved in apoptosis of FDC-P1 cells, they are regulated by posttranslational mechanisms.

• Hengartner MO, Horvitz HR
• C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto-oncogene bcl-2.
• Cell. 1994; 76: 665-76
• Display abstract

• The activity of the C. elegans gene ced-9 is required to protect cells that normally survive from undergoing programmed cell death. Here we describe the cloning and molecular characterization of this gene. ced-9 is an element of a polycistronic locus that also contains the gene cyt-1, which encodes a protein similar to cytochrome b560 of complex II of the mitochondrial respiratory chain. ced-9 encodes a 280 amino acid protein showing sequence and structural similarities to the mammalian proto-oncogene bcl-2. Overexpression of bcl-2 can mimic the protective effect of ced-9 on C. elegans cell death and can prevent the ectopic cell deaths that occur in ced-9 loss-of-function mutants. These results suggest that ced-9 and bcl-2 are homologs and that the molecular mechanism of programmed cell death has been conserved from nematodes to mammals.

• Thornberry NA
• Interleukin-1 beta converting enzyme.
• Methods Enzymol. 1994; 244: 615-31

• Gu L, Fausnaugh J
• Stability and characterization of human interleukin-1 beta.
• Pharm Biotechnol. 1993; 5: 221-48

• Molineaux SM et al.
• Interleukin 1 beta (IL-1 beta) processing in murine macrophages requires a structurally conserved homologue of human IL-1 beta converting enzyme.
• Proc Natl Acad Sci U S A. 1993; 90: 1809-13
• Display abstract

• Murine interleukin 1 beta (IL-1 beta) convertase (mICE) was identified in cytosolic extracts of peritoneal exudate cells (PECs) and macrophage cell lines. mICE cleaves both the human and mouse IL-1 beta precursors (pIL-1 beta) at sites 1 and 2 but fails to cleave a human pIL-1 beta (Asp116 to Ala) mutant at site 2, indicating that Asp is required to the left of the scissile bond. Ac-Tyr-Val-Ala-Asp-amino-4-methyl coumarin, patterned after site 2 of human pIL-1 beta, is a fluorogenic substrate for mICE, while the tetrapeptide aldehyde Ac-Tyr-Val-Ala-Asp-CHO is a potent inhibitor (Ki = 3 nM) that prevents generation and release of mature IL-1 beta by PECs (IC50 = 7 microM). Cloning of a full-length 1.4-kb cDNA shows that mICE is encoded as a 402-aa proenzyme (p45) that can be divided into a prodomain (Met1-Asp122), followed by a p20 subunit (Gly123-Asp296), a connecting peptide (Ser297-Asp314), and a p10 subunit (Gly315-His402). At the amino acid level, p45, p20, and p10 are 62%, 60%, and 81% identical with human IL-1 beta convertase (hICE). The active site Cys284 lies within a completely conserved stretch of 18 residues; however, Ser289 in hICE, which aligns with the catalytic region of serine and viral cysteinyl proteases, is absent from mICE. Expression in Escherichia coli of a truncated cDNA encoding Asn119-His402 generated active enzyme, which was autocatalytically processed at three internal Asp-Xaa bonds to generate a p20 subunit (Asn119-Asp296) complexed with either p11 (Ala309-His402) or p10. Recombinant mICE cleaves murine pIL-1 beta accurately at the Asp117-Val118 bond. The striking similarities of the human and murine enzymes will make it possible to assess the therapeutic potential of hICE inhibitors in murine models of disease.

• Miller DK et al.
• Purification and characterization of active human interleukin-1 beta-converting enzyme from THP.1 monocytic cells.
• J Biol Chem. 1993; 268: 18062-9
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• Interleukin-1 beta-converting enzyme (ICE) was purified from dialyzed cytoplasmic extracts of THP.1 human monocytic cells by a combination of DEAE-5PW and SP-5PW ion exchange and C4 reverse phase high performance liquid chromatography. Sequence information from tryptic and Asp.N peptides on the isolated 20-kDa (p20) and a 10-kDa (p10) proteins enabled the subsequent cloning of ICE (Thornberry, N. A., Bull, H. G., Calaycay, J. R., Chapman, K. T., Howard, A. D., Kostura, M. J., Miller, D. K., Molineaux, S. M., Weidner, J. R., Aunins, J., Elliston, K. O., Ayala, J. M., Casano, F. J., Chin, J., Ding, G. J.-F., Egger, L. A., Gaffney, E. P., Limjuco, G., Palyha, O. C., Raju, S. M., Rolando, A. M., Salley, J. P., Yamin, T.-T., Lee, T. D., Shively, J. E., MacCross, M., Mumford, R. A., Schmidt, J. A., and Tocci, M. J. (1992) Nature 356, 768-774) and localized the active site Cys. Immunoblots with ICE specific antibodies and NH2-terminal sequencing indicated that ICE active column fractions contained in addition to p20 and p10 an alternatively processed form of the p20 protein (p22) containing an extra 16 amino acids NH2-terminal to the p20. Furthermore, immunoblot analysis of the ion exchange column effluent showed that p20 and p22 were found together in three separate fractions distinguished by differences in p10: an intact p10 with complete ICE activity, a COOH-terminally truncated form of p10 with decreased ICE activity, and an absence of p10 with no ICE activity. These results indicate that the p10 protein is essential for ICE activity and that the ICE holoenzyme contains an intact p10 subunit paired with a p20 or p22 catalytic subunit.

• Miller DK et al.
• The IL-1 beta converting enzyme as a therapeutic target.
• Ann N Y Acad Sci. 1993; 696: 133-48

• Ray CA et al.
• Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme.
• Cell. 1992; 69: 597-604
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• Cowpox virus effectively inhibits inflammatory responses against viral infection in the chick embryo. This study demonstrates that one of the viral genes necessary for this inhibition, the crmA gene (a cytokine response modifier gene), encodes a serpin that is a specific inhibitor of the interleukin-1 beta converting enzyme. This serpin can prevent the proteolytic activation of interleukin-1 beta, thereby suppressing an interleukin-1 beta response to infection. However, the modification of this single cytokine response is not sufficient to inhibit inflammatory responses. This suggests that cowpox virus encodes several cytokine response modifiers that act together to inhibit the release of pro-inflammatory cytokines in response to infection. These viral countermeasures to host defenses against infection may contribute significantly to the pathology associated with poxvirus infections.

• Kronheim SR et al.
• Purification of interleukin-1 beta converting enzyme, the protease that cleaves the interleukin-1 beta precursor.
• Arch Biochem Biophys. 1992; 296: 698-703
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• We have purified the IL-1 beta converting enzyme from the THP-1 cell line using standard chromatographic techniques and obtained the N-terminal amino acid sequence of this novel protein. After stimulation of THP-1 cells with lipopolysaccharide, hydroxyurea, and silica, the protease was solubilized by multiple freeze/thawing. The protein was purified by ion-exchange chromatography, affinity chromatography on blue agarose, gel filtration, and chromatofocusing. The molecular weight of the protein is approximately 22,000 Da and the pI is between 7.1 and 6.8. The overall yield for this procedure was 16% of the activity found in the initial cell lysates. An antiserum raised against a peptide based on the N-terminus was used to precipitate the protease, confirming our identification of the 22,000-Da protein as the IL-1 beta converting enzyme.

• Black R et al.
• The proteolytic activation of interleukin-1 beta.
• Agents Actions Suppl. 1991; 35: 85-9
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• Interleukin-1 beta (IL-1 beta) is released from an inactive precursor by a proteolytic cleavage. A monocytic protease has been identified that appears to be involved in the physiological activation of this cytokine. Two situations have been found in which precursor IL-1 beta exists without the monocytic processing enzyme, and in these cases other proteases, such as neutrophil elastase, cathepsin G and cathepsin L, may be involved in generating the active cytokine.

• van Oostrum J, Priestle JP, Grutter MG, Schmitz A
• The structure of murine interleukin-1 beta at 2.8 A resolution.
• J Struct Biol. 1991; 107: 189-95
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• The three-dimensional structure of recombinant murine interleukin-1 beta has been solved by X-ray crystallographic techniques to 2.8 A resolution and refined to a crystallographic R factor of 0.192. Although murine interleukin-1 beta crystallizes in the same space group as human interleukin-1 beta with almost identical unit cell dimensions, the packing of the molecules is quite different. The murine interleukin-1 beta structure was solved by molecular replacement using the refined structure of human interleukin-1 beta as trial structure, and found to be related to the human structure by a nearly perfect twofold rotation about the crystallographic y-axis and a 14 degrees rotation about the z-axis, with no translation. The folding of murine interleukin-1 beta is similar to that found for the human variant, consisting of 12 beta strands wrapped around a core of hydrophobic side chains in a tetrahedron-like fashion. Significant differences with respect to the human structure are seen at the N terminus and in 4 of the 11 loops connecting the 12 beta strands.

• Howard AD et al.
• Probing the role of interleukin-1 beta convertase in interleukin-1 beta secretion.
• Agents Actions Suppl. 1991; 35: 77-83
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• Interleukin-1 beta must be processed from its precursor form of 31.5 kDa to its mature form of 17 kDa in order to elaborate its wide array of bioactivities. The recent identification of a monocyte-specific endoprotease, termed interleukin-1 beta-converting enzyme (ICE), capable of generating authentic, bioactive 17 kDa IL-1 beta suggests that this protease may serve a specific role in the processing and subsequent secretion of IL-1 beta. To test this hypothesis, we describe initial attempts to establish a monocytic cell-based system to test if mutant preIL-1 beta molecules which are poor substrates for ICE in vitro will be processed and secreted by monocytic cells.

• Yuan JY, Horvitz HR
• The Caenorhabditis elegans genes ced-3 and ced-4 act cell autonomously to cause programmed cell death.
• Dev Biol. 1990; 138: 33-41
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• Mutations in the genes ced-3 and ced-4 prevent almost all of the programmed cell deaths that occur during Caenorhabditis elegans development. To determine the sites of action of these two genes, we performed genetic mosaic analyses. We generated C. elegans animals that carried a free chromosomal duplication bearing either ced-3(+) or ced-4(+) in an otherwise homozygous ced-3 or ced-4 genetic background. We used other genes on the duplication as markers to identify genetic mosaic animals in which the duplication was present in some but not all cells. The patterns of cell death survivors in these mosaic animals indicated that the products of both ced-3 and ced-4 function within dying cells to cause cell death.

• Fiskerstrand C, Sargan D
• Nucleotide sequence of ovine interleukin-1 beta.
• Nucleic Acids Res. 1990; 18: 7165-7165

• Joseph-Liauzun E, Leplatois P, Legoux R, Guerveno V, Marchese E, Ferrara P
• Human recombinant interleukin-1 beta isolated from Escherichia coli by simple osmotic shock.
• Gene. 1990; 86: 291-5
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• A synthetic gene coding for the C-terminal 153 amino acids of the human interleukin-1 beta (IL-1 beta) was used to produce large quantities of recombinant IL-1 beta in Escherichia coli. The expression of the synthetic gene was under the control of an inducible promoter. The recombinant protein was released from the cells by an osmotic shock. This procedure did not lyse the cells. The IL-1 beta that represented 90% of the total extracted protein was purified to homogeneity by a single chromatographic step. Sequence analysis revealed a heterogeneous N-terminal sequence resulting from the cleavage of the N-terminal methionine in 50% of the molecules and of both the N-terminal methionine and alanine in the other 50%. This recombinant IL-1 beta had a specific activity of 1.3 x 10(8) international units per mg.

• Dalboge H, Bayne S, Christensen T, Hejnaes KR
• Cloning and expression of an interleukin-1 beta precursor and its conversion to interleukin-1 beta.
• FEBS Lett. 1989; 246: 89-93
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• A gene coding for a N-terminal precursor of interleukin-1 beta (IL-1 beta) was cloned and expressed in E. coli. The isolated Met-Glu-Ala-Glu-IL-1 beta precursor was enzymatically converted to IL-1 beta by means of dipeptidylaminopeptidase (DAP I). This method ensured a correct N-terminal residue and the often observed expression of Met-IL-1 beta was thus avoided. The pure and physically homogeneous product exhibited the characteristic properties of natural IL-1 beta. The in vitro biological activity was measured in the lymphocyte-activating factor assay and was compared to that of natural IL-1 beta isolated from stimulated monocyte culture using exactly the same purification procedure. The specific biological activity of both products was 2 x 10(-8) U/mg indicating that the recombinant product exhibits full biological activity.

• Young PR, Sylvester D
• Cloning of rabbit interleukin-1 beta: differential evolution of IL-1 alpha and IL-1 beta proteins.
• Protein Eng. 1989; 2: 545-51
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• We have cloned the rabbit IL-1 beta cDNA, which encodes a 268 amino acid precursor similar in length to other sequenced IL-1 precursors. Comparison of all published IL-1 alpha and IL-1 beta sequences respectively indicates that the IL-1 alpha gene family is evolving faster than the IL-1 beta family, and that the two genes diverged approximately 270 million years ago. Surprisingly, there are differences in the regions preferentially conserved within the two families. The IL-1 alpha family is most conserved at the amino terminus whereas the IL-1 beta family is most conserved in the carboxy-terminal half. This is despite the fact that the carboxy-terminal half encodes the active portion of both molecules and would be expected to adopt a similar beta-sheet structure in IL-1 alpha as in the published X-ray structure of mature IL-1 beta. These findings suggest that differences in the function and properties of the IL-1 alpha and IL-1 beta precursor molecules may have been conserved. These differences may therefore provide an explanation for the existence of two IL-1 molecules.

• Priestle JP, Schar HP, Grutter MG
• Crystal structure of the cytokine interleukin-1 beta.
• EMBO J. 1988; 7: 339-43
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• The crystal structure of human recombinant interleukin-1 beta has been determined at 3.0 A resolution by the isomorphous replacement method in conjunction with solvent flattening techniques. The model prior to refinement has a crystallographic R-factor of 42.3%. The structure is composed of 12 beta-strands forming a complex network of hydrogen bonds. The core of the structure can best be described as a tetrahedron whose edges are each formed by two antiparallel beta-strands. The interior of this structure is filled with hydrophobic side chains. There is a 3-fold repeat in the folding of the polypeptide chain. Although this folding pattern suggests gene triplication, no strong internal sequence homology between topologically corresponding residues exists. The folding topology of interleukin-1 beta is very similar to that described by McLachlan (1979) J. Mol. Biol., 133, 557-563, for soybean trypsin inhibitor.

• Priestle JP, Schar HP, Grutter MG
• The three-dimensional structure of interleukin-1 beta.
• Biochem Soc Trans. 1988; 16: 949-53
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• The three-dimensional structure of human recombinant interleukin-1 beta has been determined at 0.24 nm resolution by X-ray crystallographic techniques. The partially refined model has a crystallographic R-factor of just under 19%. The structure is composed of 12 beta-strands forming a complex network of hydrogen bonds. The core of the structure can best be described as a tetrahedron whose edges are each formed by two antiparallel beta-strands. The interior of this structure is filled with hydrophobic side-chains. There is a 3-fold repeat in the folding of the polypeptide chain. Although this folding pattern suggests gene triplication, no significant internal sequence homology between topologically corresponding residues exists. The folding topology of interleukin-1 beta is very similar to that described by A. D. McLachlan [(1979) J. Mol. Biol. 133, 557-563] for soybean trypsin inhibitor.

• Maliszewski CR et al.
• Cloning, sequence and expression of bovine interleukin 1 alpha and interleukin 1 beta complementary DNAs.
• Mol Immunol. 1988; 25: 429-37
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• Interleukin 1 (IL-1) is a cytokine which mediates a variety of immunoregulatory and inflammatory activities. Using human IL-1 alpha and IL-1 beta probes, cDNAs for the corresponding bovine genes were isolated from an alveolar macrophage library. The open reading frames of the bovine IL-1 alpha and IL-1 beta cDNAs encode proteins of 268 and 266 amino acids, respectively, each with a predicted mol. wt of approx. 31,000. Both forms of bovine IL-1 exhibit a high degree of sequence homology with IL-1 gene products from other mammalian species. Based upon comparisons with human IL-1 amino acid sequences, the post-translationally processed, mature forms of bovine IL-1 would occur as 17-18,000 mol. wt proteins. Sequences encoding mature bovine IL-1 alpha and IL-1 beta were inserted into E. coli expression plasmids and biologically active proteins were synthesized as judged by the ability of the recombinant proteins to induce proliferation of bovine thymocytes. Both IL-1 alpha and IL-1 beta exist as single genomic copies. In addition, bovine IL-1 beta mRNA is approx. 10-fold more abundant than IL-1 alpha mRNA in stimulated alveolar macrophages.

• Black RA et al.
• Generation of biologically active interleukin-1 beta by proteolytic cleavage of the inactive precursor.
• J Biol Chem. 1988; 263: 9437-42
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• Interleukin-1 beta (IL-1 beta) is derived from an inactive precursor by proteolytic cleavage. To study IL-1 beta processing, we expressed the precursor in Escherichia coli, partially purified it, and used it as a substrate for various potentially relevant protease preparations. The precursor alone was virtually inactive, but incubation with membranes from human monocytes or myeloid cell lines yielded a 500-fold increase in IL-1 bioactivity. Western blot analysis of the incubated material showed that the 31,000-Da precursor is broken down to three major products, ranging from 17,400 to about 19,000 Da. The most active of these products is the smallest one, and it co-migrates during electrophoresis with mature IL-1 beta. Four purified known proteases were also tested for their effect on precursor IL-1 beta, and none of these products co-migrated with the mature protein. Chymotrypsin and Staphylococcus aureus protease yielded slightly larger products, which were highly active. Elastase and trypsin yielded substantially larger products, and these had little IL-1 activity. The products of three of the known proteases were identified by NH2-terminal sequencing. These results show conclusively that proteolysis of precursor IL-1 beta generates biological activity and that the cleavage must occur close to the mature NH2 terminus.

• Mori S et al.
• Cloning and sequence analysis of a cDNA for lymphocyte proliferation potentiating factor of rabbit polymorphonuclear leukocytes: identification rabbit interleukin 1 beta.
• Biochem Biophys Res Commun. 1988; 150: 1237-43
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• Complementary DNA for a rabbit PMN-derived lymphocyte proliferation potentiating factor (PMN factor) was cloned from a cDNA library constructed from poly(A)+RNA of early inflammatory exudate PMN. Oligodeoxyribonucleotides as probes for cloning were synthesized according to the previously reported amino acid sequence of the purified PMN factor. This cDNA encodes a 268-residue protein which is homologous to the established structures of human IL 1 beta (74%0 or murine IL 1 beta (71%). The NH2-terminal amino acid sequence of the endogenously produced PMN factor indicates that the mature molecule is made up of carboxy terminal 152 amino acids of the precursor molecule. Taking into consideration the previous studies on biological activities, this cloned PMN factor is therefore considered to be rabbit IL 1 beta.

• Gray PW, Glaister D, Chen E, Goeddel DV, Pennica D
• Two interleukin 1 genes in the mouse: cloning and expression of the cDNA for murine interleukin 1 beta.
• J Immunol. 1986; 137: 3644-8
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• A human interleukin 1 beta (IL 1 beta) cDNA probe was utilized to identify a homologous murine cDNA clone. The murine cDNA encodes a 269-residue protein which is 67% homologous to human IL 1 beta. The murine sequence was engineered for expression in mammalian cells and directs the synthesis of biologically active IL 1. This protein, termed murine IL 1 beta, is only 22% homologous with the previously described murine IL 1 sequence. Both IL 1 alpha and IL 1 beta are encoded by single genes, but IL 1 beta mRNA is about fivefold more abundant in a stimulated macrophage cell line.

• Ellis HM, Horvitz HR
• Genetic control of programmed cell death in the nematode C. elegans.
• Cell. 1986; 44: 817-29
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• The wild-type functions of the genes ced-3 and ced-4 are required for the initiation of programmed cell deaths in the nematode Caenorhabditis elegans. The reduction or loss of ced-3 or ced-4 function results in a transformation in the fates of cells that normally die; in ced-3 or ced-4 mutants, such cells instead survive and differentiate, adopting fates that in the wild type and associated with other cells. ced-3 and ced-4 mutants appear grossly normal in morphology and behavior, indicating that programmed cell death is not an essential aspect of nematode development. The genes ced-3 and ced-4 define the first known step of a developmental pathway for programmed cell death, suggesting that these genes may be involved in determining which cells die during C. elegans development.

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