The protease responsible for the cleavage of poly(ADP-ribose) polymerase and necessary for apoptosis has been purified and characterized. This enzyme, named apopain, is composed of two subunits of relative molecular mass (M(r)) 17K and 12K that are derived from a common proenzyme identified as CPP32. This proenzyme is related to interleukin-1 beta-converting enzyme (ICE) and CED-3, the product of a gene required for programmed cell death in Caenorhabditis elegans. A potent peptide aldehyde inhibitor has been developed and shown to prevent apoptotic events in vitro, suggesting that apopain/CPP32 is important for the initiation of apoptotic cell death.

Interleukin-1 beta (IL-1 beta)-converting enzyme cleaves the IL-1 beta precursor to mature IL-1 beta, an important mediator of inflammation. The identification of the enzyme as a unique cysteine protease and the design of potent peptide aldehyde inhibitors are described. Purification and cloning of the complementary DNA indicates that IL-1 beta-converting enzyme is composed of two nonidentical subunits that are derived from a single proenzyme, possibly by autoproteolysis. Selective inhibition of the enzyme in human blood monocytes blocks production of mature IL-1 beta, indicating that it is a potential therapeutic target.

Cysteine proteases of the interleukin 1 beta Converting Enzyme (ICE)/CED-3 family have been implicated in the effector process of apoptosis in several systems, including Fas-mediated apoptosis. We have recently isolated and partially characterized a protease present in extracts from anti-Fas antibody treated Jurkat T cells that promotes apoptotic changes in isolated nuclei (Schlegel, J., Peters, I., and Orrenius, S. (1995) FEBS Lett. 364, 139-142). We now show that this protease cleaves poly-(ADP-ribose) polymerase (PARP) with high efficiency and specificity. Both PARP proteolysis and the proapoptotic effects of the protease are inhibited by nanomolar concentrations of a selective inhibitor of apopain (CPP32), while an inhibitor of IL-1 beta converting enzyme is much less effective, requiring micromolar concentrations for the inhibition of the isolated protease. Kinetic analysis of the isolated protease reveals kinetic constants similar to those reported for apopain. The isolated protease is recognized by antibodies specific for CPP32/apopain but not by an anti-ICE antibody. Furthermore, a selective inhibitor of apopain prevents Fas-induced apoptosis in intact Jurkat T cells. We therefore conclude that CPP32/apopain is activated in Fas-induced apoptosis.

IL-11 is a master cytokine responsible for the induction of a number of proteins associated with inflammation, such as metalloproteinases, cyclooxygenase, nitric-oxide synthetase, and adhesion proteins (1). Many of these responses are activated by the rapid activation of the transcription factor NF-B following signal transduction by IL-1␣ or IL-1␤ bound to the type I IL-1 receptor (IL-1R1) (see Ref. 2 for review). Activation of the IL-1R1 leads to the activation of at least two kinases resulting in the phosphorylation of IB␣ and the activation of NF-B. Following IL-1 activation, a distinctive Ser/Thr kinase activity binds to and immunoprecipitates with the IL-1R1 (3, 4). This purified IL-1 receptor-associated kinase (IRAK) is highly homologous to the Drosophila kinase Pelle but not to other mammalian Ser/Thr kinases, and it has only a limited ability to phosphorylate IB␣ (5, 6). A second Ser/Thr kinase has been identified that is dependent upon ubiquitinylation for its subsequent phosphorylation of IB␣ (7). Presumably this latter kinase is activated directly by IRAK or indirectly by as yet unidentified intermediate kinases as well as the TNF receptor associated kinases (see Refs. 8 -11). The specific phosphorylation of IB␣ on Ser-32 and Ser-36 by this ubiquitin-dependent kinase leads to the recognition and destruction of the IB␣ by proteasomes, which then frees the NF-B to translocate into the nucleus and begin transcription (8,(12)(13)(14)(15)(16).A similar signal transduction sequence has been observed in Drosophila. In Drosophila Toll activates a several-step signal transduction pathway leading to the nuclear localization and activity of the transcription factor Dorsal, which is highly homologous to mammalian NF-B (see Refs. 17 and 18)). The IL-1R1 cytoplasmic domain is homologous to that of the Drosophila receptor Toll, which provides the essential signal for embryo ventralization following fertilization (19 -21). These homologous cytoplasmic regions of Toll and the IL-1R1 can be interchanged so that a chimera of the Toll cytoplasmic domain and the IL-1R1 extracellular domain is active when stimulated by IL-1. 2 The activity of Dorsal is controlled by the inhibitor protein Cactus, which like its homologous counterpart IB␣, is phosphorylated at discreet N-terminal sites, and is then destroyed by proteasomes, freeing Dorsal to migrate to the nucleus and begin transcription (22,23). Key to the phosphorylation of Cactus in Drosophila is the unique Ser/Thr kinase Pelle, which is activated by its interaction with the membrane bound adapter protein Tube via death domains found on the N terminus of both proteins (24 -26). While it appears that Tube recruits Pelle to the Toll receptor following activation, the molecular details of how this recruitment occurs and how Pelle is subsequently activated are currently unclear.Cao et al. (5) have shown in IL-1R1-transfected HEK cells that IRAK is rapidly translocated to the IL-1R1 and becomes multiphosphorylated following IL-1 stimulation. In the present paper we show that a...

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.