Relatedness of human interleukin-1.beta. convertase gene to a C. elegans cell death gene, inhibitory portions of these genes and uses therefor

#Cell death is a fundamental aspect of animal development. Many cells die during the normal development of both vertebrates (Glucksmann, Biol. Rev. Cambridge Philos. Soc. 26:59-86 (1951)) and invertebrates (Truman, Ann. Rev. Neurosci. 7:171-188 (1984)). These deaths appear to function in morphogenesis, metamorphosis and tissue homeostasis, as well as in the generation of neuronal specificity and sexual dimorphism (reviewed by Ellis et al., Ann. Rev. Cell Biol. 7:663-698 (1991)). An understanding of the mechanisms that cause cells to die and that specify which cells are to live and which cells are to die is essential for an understanding of animal development.

#The nematode Caenorhabditis elegans is an appropriate organism for analyzing naturally-occurring or programmed cell death (Horvitz et al., Neurosci. Comment. 1:56-65 (1982)). The generation of the 959 somatic cells of the adult C. elegans hermaphrodite is accompanied by the generation and subsequent deaths of an additional 131 cells (Sulston and Horvitz, Dev. Biol. 82:110-156 (1977); Sulston et al., Dev. Biol. 100:64-119 (1982)). The morphology of cells undergoing programmed cell death in C. elegans has been described at both the light and electron microscopic levels (Sulston and Horvitz, Dev. Biol. 82:100-156 (1977); Robertson and Thomson, J. Embryol. Exp. Morph. 67:89-100 (1982)).

#Many genes that affect C. elegans programmed cell death have been identified (reviewed by Ellis et al., Ann. Rev. Cell Biol. 7:663-698 (1991)). The activities of two of these genes, ced-3 and ced-4, are required for the onset of almost all C. elegans programmed cell deaths (Ellis and Horvitz, Cell 44:817-829 (1986)). When the activity of either ced-3 or ced-4 is eliminated, cells that would normally die instead survive and can differentiate into recognizable cell types and even function (Ellis and Horvitz, Cell 44:817-829 (1986); Avery and Horvitz, Cell 51:1071-1078 (1987); White et al., Phil. Trans. R. Soc. Lond. B. 331:263-271 (1991)). Genetic mosaic analyses have indicated that the ced-3 and ced-4 genes most likely act in a cell autonomous manner within dying cells, suggesting that the products of these genes are expressed within dying cells and either are cytotoxic molecules or control the activities of cytotoxic molecules (Yuan and Horvitz, Dev. Biol. 138:33-41 (1990)).

#FIG. 1 shows the physical and genetic maps of the ced-3 region on chromosome IV.

#FIG. 2 summarizes the experiments to localize ced-3 within C48D1. Restriction sites of plasmid C48D1 and subclone plasmids are shown. Ced-3 activity was scored as the number of cell corpses in the head of L1 young animals. ++, the number of cell corpses above 10. +, the number of cell corpses below 10 but above 2. -, the number of cell corpses below 2.

#FIG. 3 shows the nucleotide sequence (SEQ ID NO: 1) of ced-3 and deduced amino acid sequence (SEQ ID NO: 2). The genomic sequence of the ced-3 region was obtained from plasmid pJ107. The introns and the positions of 12 ced-3 mutations are indicated. The likely translation initiation site is indicated by a solid arrowhead. The SL1 splice acceptor of the RNA is boxed. Repetitive elements are indicated as arrows above the relevant sequences. Numbers on the sides indicate nucleotide positions. Numbers under the amino acid sequence indicate codon positions.

#This invention is based on the discovery that the human enzyme interleukin-1.beta. convertase (ICE) has significant structural similarity to the protein product of the C. elegans cell death gene, ced-3. The activities of ced-3 and another cell death gene, ced-4, have been shown to be required for almost all the cell deaths which occur during the development of the nematode. ICE is a cysteine protease whose physiological significance has been thought to be related to its role in the maturation of one form of interleukin-1 (IL-1), a major mediator of the immune and inflammatory response (Fuhlbrigge et al., in: The Year in Immunology, Cruse and Lewis (eds.), Karger, Basel, 1989, pp. 21-37). There are two distantly related forms of IL-1, .alpha. and .beta., of which the .beta. form is the predominant species. ICE selectively converts pro-interleukin-1.beta. to the active cytokine, IL-1.beta.. The production of active IL-1.beta. has been implicated in acute and chronic inflammatory diseases, septic shock, and other physiological processes, including wound healing and resistance to viral infection (Ray et al., Cell 69:597-604 (1992)). As a result of this discovery, an enzyme which has been known to be involved in the inflammatory response and inflammatory diseases is implicated as having a role in cell death processes. This discovery is consistent with the notion that cell death genes equivalent to the nematode ced-3 gene function in a variety of organisms. The structural similarity between their gene products suggests that the ICE gene is a human equivalent of the ced-3 cell death gene. As further described below, the conservation of certain features of ICE in the Ced-3 protein further suggests that Ced-3 is a protease with a substrate-specificity similar to that of ICE.

#Furthermore, the identification of ced-3 and ICE as structurally related genes (i.e., genes whose encoded products, or which themselves, are structurally similar) presents the possibility that a family of structurally related genes exists and provides probes to identify additional members of this ced-3/ICE gene family. Comparison of the genes within this family could indicate functionally important features of the genes or their gene products, and thus, provide information for designing drugs which are useful for treating conditions characterized by cell deaths and/or inflammatory disease.

#This discovery provides novel drugs based on the ced-3, ICE and other ced-3/ICE genes and encoded products that inhibit the production of IL-1.beta. and are useful for treatment (preventive and therapeutic) of acute and chronic inflammatory disease, as well as drugs which reduce cell deaths and are useful for treatment of diseases and conditions involving cell deaths (such as myocardial infarction, stroke, traumatic brain injury, viral and other types of pathogenic infection, degenerative diseases, aging, and hair loss). These drugs may also be useful for treating leukemias in which IL-1.beta. has been implicated.