The caspase family represents a new class of intracellular cysteine proteases with known or suspected roles in cytokine maturation and apoptosis. These enzymes display a preference for Asp in the P1 position of substrates. To clarify differences in the biological roles of the interleukin-1␤ converting enzyme (ICE) family proteases, we have examined in detail the specificities beyond the P1 position of caspase-1, -2, -3, -4, -6, and -7 toward minimal length peptide substrates in vitro. We find differences and similarities between the enzymes that suggest a functional subgrouping of the family different from that based on overall sequence alignment. The primary specificities of ICE homologs explain many observed enzyme preferences for macromolecular substrates and can be used to support predictions of their natural function(s). The results also suggest the design of optimal peptidic substrates and inhibitors.A growing body of evidence supports important roles for the interleukin-1␤ converting enzyme (ICE) 1 (1, 2) and its homologs (recently renamed caspases (3)) in cytokine maturation and apoptosis. The caspase gene family, defined by protein sequence homology but also characterized by conservation of key catalytic and substrate-recognition amino acids, includes caspase-2 (4), caspase-3 (5-7), caspase-4 (8 -10), caspase-5 (10), caspase-6 (11), caspase-7 (12-14), caspase-8 (15-17), caspase-9 (18, 19), and caspase-10 (17). Each is an intracellular cysteine protease that shares with the serine protease granzyme B specificity for Asp in the P1 position of substrates. The specific biological roles and interrelationships of these enzymes are for the most part unknown and are areas of active investigation in many laboratories.A role for caspase-1 in inflammation is supported by several lines of evidence. Caspase-1-deficient mice, and cells derived from those animals, are deficient in IL-1␤ maturation and are resistant to endotoxic shock (20,21). Peptidic inhibitors of caspase-1 can be effective in blocking maturation and release of IL-1␤ by cultured cells (1) and in whole animals (22, 23) and of inflammation in animal models (24,25). The selectivity of the inhibitors employed in these studies among the caspases has not been demonstrated, and so the precise role of each caspase in inflammation is uncertain. Nevertheless the results uphold the promise of caspase-1 and/or its homologs as targets for anti-inflammatory drug discovery.Caspases play important roles in apoptosis signaling and effector mechanisms. Sequence alignments reveal homology with Ced-3 (26), a nematode cysteine protease (27, 28) that is required for cell death. The viral proteins CrmA and p35 are antiapoptotic and act by inhibition of caspases (29,30). A bacterial invasin induces apoptosis by binding to and activating caspase-1 specifically (31). Caspase-3 is necessary and sufficient for apoptosis in one acellular model (6); however, in mice the essential function of this enzyme is limited to apoptosis in the brain (32). A hallmark of apoptosis is the pr...

The baculovirus antiapoptotic protein p35 inhibited the proteolytic activity of human interleukin-1 beta converting enzyme (ICE) and three of its homologs in enzymatic assays. Coexpression of p35 prevented the autoproteolytic activation of ICE from its precursor form and blocked ICE-induced apoptosis. Inhibition of enzymatic activity correlated with the cleavage of p35 and the formation of a stable ICE-p35 complex. The ability of p35 to block apoptosis in different pathways and in distantly related organisms suggests a central and conserved role for ICE-like proteases in the induction of apoptosis.

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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.

Although the available evidence suggests that whereas the caspase family plays a major role in apoptosis, they are not the sole stimulators of death. A random yeast two-hybrid screen of a lymphocyte cDNA library (using caspase-3 as the bait) found an interaction between caspase-3 and the regulatory subunit A␣ of protein phosphatase 2A. This protein was found to be a substrate for caspase-3, but not caspase-1, and could compete effectively against either a protein or synthetic peptide substrate.In Jurkat cells induced to undergo apoptosis with anti-Fas antibody, protein phosphatase 2A (PP2A) activity increased 4.5-fold after 6 h. By 12 h, the regulatory A␣ subunit could no longer be detected in cell lysates. There was no change in the amount of the catalytic subunit. The effects on PP2A could be prevented by the caspase family inhibitors acetyl-Asp-Glu-Val-Asp (DEVD) aldehyde or Ac-DEVD fluoromethyl ketone. The mitogen-activated protein (MAP) kinase pathway is regulated by PP2A. At 12 h after the addition of anti-Fas antibody, a decrease in the amount of the phosphorylated forms of MAP kinase was observed. Again, this loss of activated MAP kinase could be prevented by the addition of DEVD-cho or DEVD-fmk. These data are consistent with a pathway whereby induction of apoptosis activates caspase-3. This enzyme then cleaves the regulatory A␣ subunit of PP2A, increasing its activity. These data show that the activated PP2A will then effect a change in the phosphorylation state of the cell. These data provide a link between the caspases and signal transduction pathways.