Substrate and Inhibitor Specificity of Interleukin-1β-converting Enzyme and Related Caspases

Abstract

Interleukin-1␤-converting enzyme (ICE) is a novel cysteine protease responsible for the cleavage of pre-interleukin-1␤ (pre-IL-1␤) to the mature cytokine and a member of a family of related proteases (the caspases) that includes the Caenorhabditis elegans cell death gene product, CED-3. In addition to their sequence homology, these cysteine proteases display an unusual substrate specificity for peptidyl sequences with a P 1 aspartate residue. We have examined the kinetics of processing pre-IL-1␤ to the mature form by ICE and three of its homologs, TX, CPP-32, and CMH-1. Of the ICE homologs, only TX processes pre-IL-1␤, albeit with a catalytic efficiency 250-fold less than ICE itself. We also investigated the ability of these four proteases to process poly(ADPribose) polymerase, a DNA repair enzyme that is cleaved within minutes of the onset of apoptosis. Every caspase examined cleaves PARP, with catalytic efficiencies ranging from 2.3 ؋ 10 ICE 1 is the prototypical member of a new family of mammalian cysteine proteases (the caspases) 2 that is distinct from cysteine proteases in the papain superfamily (1-3). The mutagenesis experiments and crystal structure reported by Wilson et al. (4) revealed a different active site geometry and catalytic mechanism for ICE than observed for papain. The structure of the ICE active site contains a Cys-His catalytic diad, and two Arg residues that confer high selectivity for peptidyl substrates with Asp residues at the P 1 position (Nterminal to the scissile bond) (4, 5). Although ICE has recently been reported to cleave other proteins in vitro (6, 7), it was identified from its essential role in processing the inactive 31-kDa precursor of interleukin-1␤ (pre-IL-1␤) to the mature 17-kDa cytokine (8).In 1993, Yuan et al. (9) reported the sequence of the Caenorhabditis elegans programmed cell death gene ced-3. This gene is 29% identical to human ICE. Due to the central role of the CED-3 protein in C. elegans apoptosis, Yuan and colleagues deduced that ICE or ICE homologs might play a similar role in mammalian apoptosis. Overexpression of ICE in rat fibroblast, mammalian COS cells, and neuronal cell lines demonstrated that this protease can indeed induce apoptosis (6, 10, 11). Subsequently, Kuida et al. (12) confirmed an in vivo role for ICE in Fas-mediated apoptosis by disruption of the murine ICE gene.A family of ICE-related proteases (the caspases) was discovered by searching human cDNA libraries for sequences homologous to 14). At present, at least 10 human homologs of ICE possessing cysteine protease activity have been identified. These homologs can be grouped by sequence similarity into three subfamilies. The most closely related homologs to ICE are TX (caspase-4, also denoted ICH-2 or ICErel II) (15-17) and TY (caspase-5, also denoted ICE rel III) (17, 18), which are 58 and 57% identical to ICE at the amino acid level. Another homolog, ICH-1 (caspase-2, corresponding to the murine Nedd-2 gene) (19,20), is 20% identical to ICE and belongs to a distinct subfamily. A third gr...

Apoptotic and Antiapoptotic Activity of L Protein of Theiler's Murine Encephalomyelitis Virus

“…Interestingly, we found that the level of cleaved PARP was also similar in the HeLa cells infected with either DA or GDVII wt viruses (Fig. 5B), perhaps because a number of caspases can cleave PARP, including caspase-9 (15).…”

Novel synergistic mechanism for sst2 somatostatin and TNFα receptors to induce apoptosis: crosstalk between NF-κB and JNK pathways

“…After 5-h cell serum starvation, apoptosis in sst2-expressing cells was 3.6-71.2-fold higher than in mock cells (5.670.8 versus 1.570.5% of apoptotic cells, respectively) ( Figure 1a). Cell treatment with the specific executioner caspase competitive inhibitor DEVD-CHO (uncleavable substrate) 13 abrogated sst2-mediated apoptosis, and reverted apoptosis levels in NIH3T3/sst2 to those observed in mock cells (NIH3T3/mock) ( Figure 1a). By using a cell viability assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)), we showed as well that sst2 decreased cell viability in NIH3T3/ sst2 to 7271.3% of mock cells (Figure 1b).…”

IL-1β Converting Enzyme Is a Target for Nitric Oxide-Releasing Aspirin: New Insights in the Antiinflammatory Mechanism of Nitric Oxide-Releasing Nonsteroidal Antiinflammatory Drugs

“…The kinetic of NO generation from NCX-4016, however, is different from that of SNAP. In fact, the NO moiety of NCX-4016 dissociates slowly from aspirin, resulting in a lower, but sustained, release of NO; third, further supporting the view that the inhibitory activity of NCX-4016 on cytokine release was due to its NO moiety, we demonstrated that substituting the nitrooxymethyl-phenyl ester group, the NO moiety, with an hydroxymethyl-phenyl group resulted in a compound, the NCX-4017, highly cytotoxic, devoid of any pharmacological activity; fourth, NCX-4016 activates NOdependent pathways as demonstrated by the finding that incubating the cells with ODQ, to prevent guanosilyl cyclase activation, almost completely inhibited changes in [cGMP] i , although it had no effect on cytokines release (17); fifth, incubating monocytes with LPS resulted in a time-dependent activation of ICE-like endoproteases, an effect that was almost completely prevented by cotreating cells with NCX-4016, but not with aspirin or NCX-4017; sixth, inhibition of LPS-induced YVAD cleaving activities by NCX-4016 was reverted by DTT, an agent that effectively removes thiolbound NO groups from proteins (17,19,20,27,32); seventh, exposure to HgCl 2 , an agent that bind to thiol group, displaced NO from lysates obtained from NCX-4016-treated monocytes and resulted in a DTT-reversible inhibition of YVADase activity, indicating that the NO removed by HgCl 2 was bound to a cysteine group (17,19,20,27,32); and, finally, incubating LPS-challenged monocytes with NCX-4016, but not with aspirin, prevented ICE activation as measured by assessing the release of the p20 subunit (23,33). Taken together, these data demonstrated that incubating human monocytes with NCX-4016 results in intracellular NO formation and S-nitrosation/inhibition of ICE-like cysteine proteases involved in pro-IL-1␤ and pro-IL-18 processing (10 -12).…”