Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1997-02-10
2000-08-15
Myers, Carla J.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435226, 435212, 435 23, 530350, 530351, 536 232, 536 235, C12N 950, C12N 964, C07H 2104
Patent
active
061035130
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
Interleukin-1.beta. (IL-1.beta.) is a major mediator of chronic and acute inflammation. Along with IL-1.beta., human monocytes produce two additional members of the IL-1 gene family; interleukin-1.alpha. (IL-1.alpha.) and IL-1 receptor antagonist (IL-RA). All three proteins bind to the membrane-anchored forms of the type 1 and type 2 IL-1 receptors (IL1R) on target cells. IL-1.alpha. and IL-1.beta. elicit virtually identical biological responses whereas IL-1RA blocks these effects. Both IL-1.alpha. and IL1.beta. are synthesized as 31 kDa primary translation products which lack functional hydrophobic signal sequences. The 31 kDa form of IL-1.alpha. is fully active without further processing but does not appear to be actively released from cells. IL-1.beta., the predominant form of IL-1 released by activated monocytes, is synthesized as an inactive 31 kDa precursor (pIL-1.beta.) that is processed to its mature 17.5 kDa form (mIL-1.beta.) by interleukin-1.beta. converting enzyme (ICE), a novel cysteine proteinase. ICE generates fully active mIL-1.beta. by cleaving pIL-1.beta. between Asp.sub.116 and Ala.sub.117, a unique site for prohormone processing. The sequence around this cleavage site, -Tyr-Val-His-Asp-Ala-, is evolutionarily conserved in all known pIL-1.beta. polypeptides.
Active human ICE as shown by conventional HPLC and affinity purification techniques is a heterodimer consisting of a 1:1 stoichiometric complex of 19,866 Da (p20) and 10,244 Da (p10) subunits. Cloned cDNAs have revealed that ICE is constituitively expressed as a 45 kDa proenzyme (p45) composed of a 14 kDa prodomain, followed by p20 which contains the active site Cys.sub.285, a 19 residue connecting peptide that is not present in the mature enzyme, and p10, a required component of the active enzyme. The mature subunits are flanked by Asp-X sequences. Mutational analysis of these sites and expression in heterologous systems indicates that the generation of active enzyme is autocatalytic. Murine and rat ICE have also been cloned and show a high degree of sequence similarity including these structural motifs.
Recently, a family of ICE-like genes has begun to emerge, including the nematode cell death abnormal gene (CED-3) of Caenorhabiditis elegans, Caenorhabiditis briggsae and Caenorhabiditis vulgaris, and the murine neuronal precursor cell embroyonic developmentally downregulated (NEDD-2) gene. The predicted polypeptide sequences of these genes exhibit 29% and 27% sequence identity with human ICE, respectively. The sequence identity of CED-3 with ICE is higher in the regions corresponding to the p20 and p10 subunits of mature human ICE. All known sequences for ICE and for CED-3 contain the pentapeptide sequence -Gln-Ala-Cys-Arg-Gly- surrounding the catalytic cysteine of ICE or its equivalent in CED-3.
Both CED-3 and murine ICE, when expressed by transfection in fibroblast cell lines or by microinjection into neuronal cells, cause programmed cell death (apoptosis) to occur. The pro-apoptotic effects of CED-3 or ICE can be prevented by co-transfection with either bcl-2, a mammalian proto-oncogene which appears to function as a cell death suppressor gene, or with the cytokine response modifier A (crmA) gene product, a serpin-like inhibitor of ICE.
SUMMARY OF THE INVENTION
A novel human thiol proteinase termed ICE.sub.rel -II (interleukin-1.beta. converting enzyme--related cysteine proteinase II) has been isolated and purified. A DNA molecule encoding the full length precursor form of the ICE.sub.rel -II protein has been isolated, purified and the nucleotide sequence determined. The ICE.sub.rel -II encoding DNA has been cloned for expression in recombinant hosts. The DNA clones produce recombinant full-length ICE.sub.rel -II and the individual subunits of the mature form of the enzyme. Recombinant ICE.sub.rel -II is useful for identifying modulators of ICE.sub.rel -II activity and hence modifiers of pathological conditions related to the pro-inflammatory or pro-apoptotic effects of ICE.sub.rel -II. ICE.sub.re
REFERENCES:
patent: 5416013 (1995-05-01), Black et al.
Cerretti et al., Science, vol. 256, Apr. 3, 1992, pp. 97-100.
Ali Ambereen
Munday Neil A.
Nicholson Donald W.
Vaillancourt John P.
Coppola Joseph A.
Merck Frosst Canada & Co.
Myers Carla J.
Tribble Jack L.
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