Stability and Oligomeric Equilibria of Refolded Interleukin-1β Converting Enzyme

Abstract

We report the preparation and characterization of interleukin-1␤ converting enzyme (ICE) refolded from its p20 and p10 protein fragments. Refolded ICE heterodimer (p20p10) was catalytically active but unstable, and in size exclusion chromatography eluted at an apparent molecular mass of 30 kDa. The mechanisms of the observed instability were pH-dependent dissociation at low enzyme concentrations, and autolytic degradation of the p10 subunit at high concentrations. Binding and subsequent removal of a high affinity peptidic inhibitor increased the apparent molecular mass to 43 kDa (by size exclusion chromatography), and significantly increased its stability and specific activity. Chemical cross-linking and SDS-polyacrylamide gel electrophoresis analysis of the 43-kDa size exclusion chromatography conformer revealed a 60-kDa species, which was absent in the 30-kDa conformer, suggesting that inhibitor binding caused formation of a (p20p10) 2 homodimer. The observation of a reversible equilibrium between ICE (p20p10) and (p20p10) 2 suggests that analogous associations, possibly between ICE and ICE homologs, can occur in vivo, resulting in novel oligomeric protease species.Interleukin-1␤ converting enzyme (ICE) 1 (1, 2) is an intracellular cysteine protease that activates the proinflammatory cytokine interleukin-1␤ (IL-1␤) by cleavage at Asp 116 -Ala 117 (3-5). Several lines of evidence suggest that ICE activity is required for IL-1␤ activation and that this is a crucial step in inflammation. IL-1␤ activation is effectively blocked by CrmA, a cowpox virus serpin that binds and inhibits ICE (6). The tetrapeptide ICE inhibitor acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO) (2) is also effective in blocking IL-1␤ activation (2, 7-9). Mice lacking functional copies of the murine ICE gene (10, 11), and cells derived from those animals, are deficient in IL-1␤ maturation. ICE-deficient mice are also resistant to endotoxic shock (10). These results suggest that inhibition of IL-1␤ activation by ICE is sufficient to block inflamation, and encourage efforts to develop ICE inhibitors as antiinflammatory drugs (12, 13).Several human genes encoding proteins homologous to ICE have been discovered, and elucidation of the biological functions of these proteins is currently an active area of research. These include Ich-1 (14), TX/Ich-2/ICE rel II (15-17), CPP32 (18), ICE rel III (17), Mch2 (19), and Mch3/ICE-LAP3/CMH-1 (20 -22). A clue to the function of ICE homologs, and possibly a second function of ICE itself, is provided by ced-3, a Caenorhabditis elegans protease that is highly homologous to ICE and is required for apoptosis (23,24). The hypothesis that ICE or ICE homologs participate in apoptosis is supported by the antiapoptotic effects of CrmA and the baculovirus protein p35 (25, 26), which is also an inhibitor of ICE and ICE homologs, and by the observation that transient expression of antisense-ICE cDNA blocks Fas-induced apoptosis (27). Overexpression of ICE or many of its homologs in cultured cells causes apoptosis. Biochemical ...

Crystal Structure of Caspase-2, Apical Initiator of the Intrinsic Apoptotic Pathway

“…1a). This observation came as a surprise, because an equilibrium between the monomeric and the dimeric form was reported for all examined long prodomain caspases; an equilibrium that depends on the ligand binding status (36,(41)(42)(43). The presence of a covalent linkage between the two monomers in caspase-2, however, is not compatible with such a monomer-dimer equilibrium.…”

“…The existence of this central disulfide bridge was also proven by mass spectrometry: a dimer of p12 was detected, but not of p19, and as expected, addition of a reducing agent to the denatured p12 subunit resulted in complete separation of the two chains. In conclusion, caspase-2 revealed properties in solution that are clearly different from all other investigated long prodomain caspases, the death effector domain-containing caspase-8 (42,43) and the CARDcaspases-9 (36) and -1 (41), which are all predominantly monomers in the absence of a ligand and dimerize upon inhibitor binding.…”

“…These findings are contrasted by caspases-9, -8, and -1, which all occur predominantly as monomers in the absence of an inhibitor. In these cases, dimer formation is either driven by ligand binding or by increasing the protein concentration to non-physiologically high values, such as that used in crystallization (36,(41)(42)(43).…”

Cytokine Response Modifier A Inhibition of Initiator Caspases Results in Covalent Complex Formation and Dissociation of the Caspase Tetramer

“…a procaspase-8 monomer and a dissociated p10 subunit (31). In the case of caspase-6 such heterodimers would still be activable, and so there would be no advantage to their formation and, hence, no advantage in inducing loss of p10 from the crmA-caspase-6 complex.…”

Role of caspases in the regulation of apoptotic pancreatic islet beta‐cells death

“…These two heterodimers associate with each other primarily through the interaction between the p10 subunits. Each caspase tetramer has two cavity-shaped active sites formed by amino acids from both the p20 and p10 subunits and these two active sites are likely to function independently (Walker et al, 1994;Talanian et al, 1996;Kumar, 1999;Zimmermann et al, 2001).…”