The identification of downstream substrates for the interleukin 1beta-converting enzyme (CE) family, whose aggregated cleavage alters critical cell structures and functions, thereby generating apoptosis, remains a major challenge. The broad, long-term objectives of this proposal are to define the proteolytic routes to cell death, thereby highlighting potential therapeutic targets in a variety of diseases in which inappropriate apoptosis is prominent (including malignancy, autoimmunity, and neurodegenerative diseases).
The specific aims of the proposal are (1) to use the lupus autoantibodies that recognize antigens cleaved early during apoptosis to identify the actual molecules cleaved; (2) to identify the protease(s) responsible for cleaving each molecule; (3) to elucidate the sites at which these cleavages occurs; and (4) to determine the physiologic functions of fragments generated by cleavage, thereby addressing whether the fragments themselves are important transducers of the apoptotix signal. Autoantibodies will be used to identify substrates by screening human cDNA expression libraries. Cleavage sites will be determined by peptide sequencing studies, and novel ICE-like cleavage sites identified will be used to fashion appropriate tetrapeptide inhibitors as tools to more selectively define the other ICE-like proteases. The catalytic efficiency of cleavage of the different purified substrates by purified recombinant ICE-like proteases (including ICE, ICE-2, ICE-3, Ich-1, CPP32, Mch2, Mch-3, FLICE/MACH and Ced-3) will be determined, as well the IC50 values for inhibition of different substrates by a panel of tetrapeptide aldehyde inhibitors in apoptotic extracts. This quantitative data will indicate which ICE-like activity(ies) are likely to be responsible for cleaving particular substrates in the apoptotic cell. Transient and stable transfections of cDNAs encoding the fragments generated by ICE-like protease cleavage will be used to address the effects of these fragments in mammalian cells.
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