Enzymatic activity of two caspases related to interleukin‐1β‐converting enzyme

Abstract

Interleukin-1β-converting enzyme is a member of a family of human cysteine proteases with specificity for aspartic acid, which have been named caspases. Within this family of enzymes, transcript X (TX) and transcript Y (TY) (caspases 4 and 5, respectively) are very similar to ICE (caspase 1) and form the ICE subfamily. Given the high degree of conservation in the sequences of these proteases (more than 50% amino acid identity in the mature enzymes), it was of interest to examine whether they shared similar substrate specificities. The three enzymes, ICE, TX and TY, were therefore expressed in baculovirus-infected insect cells, as 30-kDa proteins lacking the propeptide. Automaturation into p20 and p10 subunits occured within the cells. Active ICE, TX and TY were collected in the cell culture supernatants. In addition, their production induced the activation of an endogenous 32-kDa putative cysteine protease (CPP32) like caspase. T7-tagged ICE, TX and TY were purified by immunoaffinity and tested for their catalytic efficiency on YVAD-containing synthetic substrates and on the ICE natural substrate, pro-interleukin-1β. TX cleaved the same synthetic substrates as ICE (K m of 90 µM and k cat of 0.4 s Ϫ1 for Suc-YVAD-NH-Mec, where Suc represents succinyl and NH-Mec represents amino-4-methylcoumarin) and could cleave pro-interleukin-1β into the same peptides as ICE but less efficiently. On the other hand, TY showed very little efficacy on the different ICE substrates (K m of 860 µM for Suc-YVAD-NH-Mec). These results show that the ICE/TX/TY subfamily has functional heterogeneity and that ICE remains the preferred enzyme for pro-interleukin-1β cleavage.Keywords : caspase ; cysteine protease ; interleukin-1β-converting enzyme homolog; interleukin-1β.Interleukin-1β (IL-1β)-converting enzyme (ICE) is a cyste-tein with death-domain-like ICE2) [3, 4]. A second subfamily is ine protease responsible for the cleavage of the inflammatory composed of caspase 1 (ICE), caspase 4 [transcript X (TX)/ICE cytokine proIL-1β to its biologically active form [ [7,8]. The protein implicated in apoptosis during the development of third group has only one member, caspase 2 (ICH1) [3]. All the Caenorhabditis elegans. Since then, numerous homologs of ICE proteins of the family share characteristic properties based on and Ced3 have been identified. They are designated the caspase sequence comparisons: conserved QACXG motif around the family to indicate that they are all cysteine proteases and cleave active-site cysteine ; specificity for aspartic acid at P1; and a at the C-terminus of an aspartic acid residue [3]. The human heterodimeric structure after activation by cleavage of a precurenzymes are classified into three groups according to similarities sor at aspartic acid residues. Automaturation has been demonin amino acid sequences. One group, related to Ced3, is called strated in vitro for ICE p45 zymogen [9, 10] but its physiological the 32-kDa putative cysteine protease (CCP32) subfamily and relevance remains to be established [11]. Sequ...

Noncanonical Inflammasome Activation of Caspase-4/Caspase-11 Mediates Epithelial Defenses against Enteric Bacterial Pathogens

“…By contrast, caspase-4 dependent IL-18 processing and secretion appears to be independent of caspase-1 in human IECs. Caspase-4 is able to cleave IL-18 (Fassy et al, 1998), at the same processing site as human caspase-1 (Gu et al, 1997), which might account for our findings. These distinctive features suggest there are mechanistic differences in non-canonical inflammasome activation between myeloid-derived cells and epithelial cells.…”

Differential roles of caspase-1 and caspase-11 in infection and inflammation

“…Caspase-1 is unequivocally required for the proteolytic processing of pro-IL-1β and pro-IL-18 and for pyroptosis in response to canonical inflammasome activators23458910. Caspase-11 cannot directly proteolytically process pro-IL-1β and pro-IL-1861, although previous studies suggest that one of the caspase-11 homologs in humans, caspase-4, could cleave pro-IL-1β and pro-IL-186263. Caspase-11 itself is capable of driving pyroptosis in a caspase-1–independent manner in response to non-canonical activation of the inflammasome, that is, in response to transfection of LPS or during infection by certain Gram-negative bacteria, including C. rodentium, E. coli and Vibrio cholerae 16.…”

Converging roles of caspases in inflammasome activation, cell death and innate immunity

“…Further studies have demonstrated that caspases 4, 5 and 11 activate the NLRP3 inflammasome in response to Gram-negative bacteria and intracellularly-delivered lipopolysaccharide (LPS), placing caspases 4, 5 and 11 as activators of caspase 1 to promote caspase 1-dependent cleavage of IL-1β and IL-18 (Refs 29, 32-41 ). Direct cleavage of IL-1β and IL-18 by caspase 4 has been proposed 42, 43 , but further study is required to confirm this observation. Activation of caspases 4, 5 and 11 also leads to pyroptosis, and the release of IL-1α and the alarmin high-mobility group protein 1 (HMGB1) independently of caspase 1 (Refs 29, 32-39, 44, 45 ).…”