Substrate Specificities of Caspase Family Proteases

Abstract

The caspase family represents a new class of intracellular cysteine proteases with known or suspected roles in cytokine maturation and apoptosis. These enzymes display a preference for Asp in the P1 position of substrates. To clarify differences in the biological roles of the interleukin-1␤ converting enzyme (ICE) family proteases, we have examined in detail the specificities beyond the P1 position of caspase-1, -2, -3, -4, -6, and -7 toward minimal length peptide substrates in vitro. We find differences and similarities between the enzymes that suggest a functional subgrouping of the family different from that based on overall sequence alignment. The primary specificities of ICE homologs explain many observed enzyme preferences for macromolecular substrates and can be used to support predictions of their natural function(s). The results also suggest the design of optimal peptidic substrates and inhibitors.A growing body of evidence supports important roles for the interleukin-1␤ converting enzyme (ICE) 1 (1, 2) and its homologs (recently renamed caspases (3)) in cytokine maturation and apoptosis. The caspase gene family, defined by protein sequence homology but also characterized by conservation of key catalytic and substrate-recognition amino acids, includes caspase-2 (4), caspase-3 (5-7), caspase-4 (8 -10), caspase-5 (10), caspase-6 (11), caspase-7 (12-14), caspase-8 (15-17), caspase-9 (18, 19), and caspase-10 (17). Each is an intracellular cysteine protease that shares with the serine protease granzyme B specificity for Asp in the P1 position of substrates. The specific biological roles and interrelationships of these enzymes are for the most part unknown and are areas of active investigation in many laboratories.A role for caspase-1 in inflammation is supported by several lines of evidence. Caspase-1-deficient mice, and cells derived from those animals, are deficient in IL-1␤ maturation and are resistant to endotoxic shock (20,21). Peptidic inhibitors of caspase-1 can be effective in blocking maturation and release of IL-1␤ by cultured cells (1) and in whole animals (22, 23) and of inflammation in animal models (24,25). The selectivity of the inhibitors employed in these studies among the caspases has not been demonstrated, and so the precise role of each caspase in inflammation is uncertain. Nevertheless the results uphold the promise of caspase-1 and/or its homologs as targets for anti-inflammatory drug discovery.Caspases play important roles in apoptosis signaling and effector mechanisms. Sequence alignments reveal homology with Ced-3 (26), a nematode cysteine protease (27, 28) that is required for cell death. The viral proteins CrmA and p35 are antiapoptotic and act by inhibition of caspases (29,30). A bacterial invasin induces apoptosis by binding to and activating caspase-1 specifically (31). Caspase-3 is necessary and sufficient for apoptosis in one acellular model (6); however, in mice the essential function of this enzyme is limited to apoptosis in the brain (32). A hallmark of apoptosis is the pr...

VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation

“…34,35 The pattern of relative cleavage rate of different peptides by spruce cell extracts in vitro is highly stable (Figure 1) and similar to the one specific for mammalian caspase-6, which shows favorable interaction with peptides containing bbranched amino acids in P4 position. 26 Therefore, we further conclude that there is no other caspase-like protease activated during spruce embryo pattern formation.…”

“…The similar pattern of relative cleavage rates of synthetic peptides, with the strongest preference for Val in P4 position (as in VEID) and tolerance for Ile in the same position (as in IETD), is a distinguishing characteristic of mammalian caspase-6. 26 In general, caspase-like activities cannot be inhibited by protease inhibitors other than caspase-specific ones. 8,27 Accordingly, we observed no inhibitory effect of a range of serine and cysteine protease inhibitors (calpain inhibitor I, aprotinin, leupeptin, phenylmethylsulphonyl fluoride (PMSF) and L-1-chloro-3-(4-tosylamido)-7-amino-2-heptanone (TLCK)), or of pepstatin (inhibitor for aspartic acid proteases) or lactacystine (inhibitor for proteasome), on the cleavage of VEID-AMC by cell extracts of Norway spruce (Figure 2a).…”

Caspase substrates

“…The first tells us a lot about caspase catalysis and is much easier to investigate than the second. The seminal work of Thornberry et al 8 using positional scanning synthetic peptide-based libraries and Talanian et al 9 using sets of individual peptide substrates, later refined by others, led to our current understanding of the inherent substrate preferences summarized in Figure 2. From this came the idea of caspase-specific consensus recognition sequences that could be applied to predicting natural substrates.…”

Evidence for a p23 caspase-cleaved form of p27[KIP1] involved in G1 growth arrest

“…However these cells express constitutive levels of p27 and therefore do not allow the approach of speci®c biological function of p23. Based upon the results obtained with the caspase inhibitors ( Figure 5), and given the presence of a 136 DXXD 139 consensus caspase site in p27 sequence (Levkau et al, 1998;Polyak et al, 1994b;Talanian et al, 1997), we have constructed a mammalian expression vector containing the cDNA sequence of the ®rst 408 base pairs of human p27, coding for the 23 kDa fragment comprised between Met 1 and Asp 139 (Figure 7 panel a). This construction was used to express p23 into the osteosarcoma cell line SaOS-2 that does not exhibit endogenously p27 (Toyoshima and Hunter, 1994).…”