We report here the inactivation of a member of the Ice/Ced-3 (caspase) family of cell death genes, casp-11, by gene targeting. Like Ice-deficient mice, casp-11 mutant mice are resistant to endotoxic shock induced by lipopolysaccharide. Production of both IL-1alpha and IL-1beta after lipopolysaccharide stimulation, a crucial event during septic shock and an indication of ICE activation, is blocked in casp-11 mutant mice. casp-11 mutant embryonic fibroblast cells are resistant to apoptosis induced by overexpression of ICE. Furthermore, we found that pro-caspase-11 physically interacts with pro-ICE in cells, and the expression of casp-11 is essential for activation of ICE. Our data suggest that caspase-11 is a component of ICE complex and is required for the activation of ICE.

Survivin, an apoptosis inhibitor/cell-cycle regulator, is critically required for suppression of apoptosis and ensuring normal cell division in the G2/M phase of the cell cycle. It is highly expressed in a cell cycle-regulated manner and localizes together with caspase-3 on microtubules within centrosomes. Whether survivin is a physiologically relevant caspase inhibitor has been unclear due to the difficulties with obtaining correctly folded survivin and finding the right conditions for inhibition assay. In this study, recombinant, active human survivin was expressed in Escherichia coli and purified to homogeneity. The protein, existing as a homodimer in solution, binds caspase-3 and -7 tightly with dissociation constants of 20.9 and 11.5 nM, respectively, when evaluated by surface plasmon resonance spectroscopy. Consistently, survivin potently inhibits the cleavage of a physiological substrate poly(ADP-ribose) polymerase and an artificial tetrapeptide by caspase-3 and -7 in vitro with apparent inhibition constants of 36.0 and 16.5 nM, respectively. The data suggest that sequestering caspase-3 and -7 in inhibited states on microtubules is at least one mechanism of survivin in the suppression of default apoptosis in the G2/M phase. The localization of survivin on microtubules, which is essential for its function, should increase the protective activity at the action site.

We report here the isolation and characterization of a new member of the ice/ced-3 family of cell death genes, named ich-3. The predicted amino acid sequence of Ich-3 protein shares 54% identity with murine interleukin-1␤ converting enzyme (ICE). Overexpression of ich-3 in Rat-1 and HeLa cells induces apoptosis, which can be inhibited by CrmA and Bcl-2. The mRNA and proteins of ich-3 are dramatically induced in vivo upon stimulation with lipopolysaccharide, an inducer of septic shock. The ich-3 gene product can be cleaved by cytotoxic T cells granule serine protease granzyme B, suggesting that Ich-3 may mediate apoptosis induced by granzyme B. Ich-3 does not process proIL-1␤ directly but does promote proIL-1␤ processing by ICE. These results suggest that Ich-3 may play a very important role in apoptosis and inflammatory responses and may be an upstream regulator of ICE. Interleukin-1␤ converting enzyme (ICE)1 family is a growing family of cysteine proteases involved in cytokine maturation and apoptosis (1). ICE is a cytoplasmic cysteine protease responsible for proteolytically processing pro-interleukin-1␤ (31 kDa) into active form (17 kDa) (2). The amino acid sequence of ICE shares 29% identity with Caenorhabditis elegans cell death gene product Ced-3 (3). Expression of ice in a number of mammalian cell lines induces apoptosis (4, 5). Microinjection of an expression vector of crmA, a cowpox virus gene encoding a serpin that is a specific inhibitor of ICE, prevents death of neurons of dorsal root ganglia and ciliary ganglia induced by trophic factor deprivation (6, 7). Expression of crmA can also suppress apoptosis induced by . These experiments suggest that the members of the ICE family play important roles in controlling mammalian apoptosis.Cytotoxic T lymphocytes (CTL) are important players in host cell-mediated immunity (12). Granzyme B (GraB) is a serine protease that plays a major role in apoptosis induced by CTLs because mice that are deficient for GraB generated by gene targeting technique are severely defective in CTL-induced apoptosis (13). GraB can induce apoptosis of many if not all cell types in the presence of pore forming protein perforin (14, 15). A recent report showed that CPP32, a member of the ICE family, is activated by cytotoxic T-cell-derived GraB, suggesting that CPP32 is important for CTL killing (16). CPP32, however, cannot be the only ICE family activated by CTL because CrmA is a very poor inhibitor of CPP32 (17). Tewari et al. (18) showed that expression of crmA completely blocks the Ca 2ϩ -independent component of CTL killing (i.e. Fas-mediated); if CPP32 were the only ICE family member responsible for CTL cytotoxicity, expression of crmA should not suppress CTL killing. We predict that there are additional members of the ICE family that play an important role in CTL-induced apoptosis. The amino acid sequence of GraB is not homologous with ICE; however, GraB and ICE share many enzymatic similarities. Like ICE, GraB requires Asp at P1 position for cleavage. Inhibitors of ICE or the ...

Many transcription coactivators interact with nuclear receptors in a ligand-and C-terminal transactivation function (AF2)-dependent manner. We isolated a nuclear factor (designated ASC-2) with such properties by using the ligand-binding domain of retinoid X receptor as a bait in a yeast two-hybrid screening. ASC-2 also interacted with other nuclear receptors, including retinoic acid receptor, thyroid hormone receptor, estrogen receptor ␣, and glucocorticoid receptor, basal factors TFIIA and TBP, and transcription integrators CBP/p300 and SRC-1. In transient cotransfections, ASC-2, either alone or in conjunction with CBP/p300 and SRC-1, stimulated ligand-dependent transactivation by wild type nuclear receptors but not mutant receptors lacking the AF2 domain. Consistent with an idea that ASC-2 is essential for the nuclear receptor function in vivo, microinjection of anti-ASC-2 antibody abrogated the liganddependent transactivation of retinoic acid receptor, and this repression was fully relieved by coinjection of ASC-2-expression vector. Surprisingly, ASC-2 was identical to a gene previously identified during a search for genes amplified and overexpressed in breast and other human cancers. From these results, we concluded that ASC-2 is a bona fide transcription coactivator molecule of nuclear receptors, and its altered expression may contribute to the development of cancers.The nuclear receptor superfamily is a group of ligand-dependent transcriptional regulatory proteins that function by binding to specific DNA sequences named hormone response elements in the promoters of target genes (for a review, see Ref.1). The superfamily includes receptors for a variety of small hydrophobic ligands such as steroids, T3, 1 and retinoids as well as a large number of related proteins that do not have known ligands, referred to as orphan nuclear receptors (reviewed in Ref.2). Functional analysis of nuclear receptors has shown that there are two major activation domains. The activation function-2 (AF-2) at the extreme C-terminal region of the ligandbinding domain (LBD) exhibits ligand-dependent transactivation, whereas the N-terminal activation function-1 contains a ligand-independent transactivation domain. The AF-2 region is conserved among nuclear receptors, and deletion or point mutations in this region impair transcriptional activation without changing ligand and DNA binding affinities. X-ray crystallographic studies of the LBD of nuclear receptors revealed that the ligand binding induces a major conformational change in the AF-2 region (3-7), suggesting that this region may play a critical role in mediating transactivation by a ligand-dependent interaction with coactivators. As expected, many coactivators fail to interact with AF-2 mutants of nuclear receptors (8 -10). Transcriptional activation of most nuclear receptors involves at least two separate processes as follows: derepression and activation. Repression is mediated in part by interaction of unliganded receptors with corepressors such as N-CoR (11) and SMRT (12). H...