A Combinatorial Approach Defines Specificities of Members of the Caspase Family and Granzyme B

Abstract

There is compelling evidence that members of the caspase (interleukin-1␤ converting enzyme/CED-3) family of cysteine proteases and the cytotoxic lymphocytederived serine protease granzyme B play essential roles in mammalian apoptosis. Here we use a novel method employing a positional scanning substrate combinatorial library to rigorously define their individual specificities. The results divide these proteases into three distinct groups and suggest that several have redundant functions. The specificity of caspases 2, 3, and 7 and Caenorhabditis elegans CED-3 (DEXD) suggests that all of these enzymes function to incapacitate essential homeostatic pathways during the effector phase of apoptosis. In contrast, the optimal sequence for caspases 6, 8, and 9 and granzyme B ((I/L/V)EXD) resembles activation sites in effector caspase proenzymes, consistent with a role for these enzymes as upstream components in a proteolytic cascade that amplifies the death signal.

Changes in nuclear morphology during apoptosis correlate with vimentin cleavage by different caspases located either upstream or downstream of Bcl‐2 action

“…Although the sequences of Leu79-Asp82 and Val254/Asp257 were not altered in the D85N and D259N mutants, respectively, either the cleavage product of 47 kDa (D85N) or that of 28 kDa (D259N) was absent from cell extracts containing these mutant vimentin, suggesting that both Asp82 and Asp257 are required for cleavage but not for cleavage at these sites. The observed effect of the Asp82 and Asp257 mutations is thus likely due to alteration of either the P4 or the P3 residue, both of which affect the cleavage efficiency of caspases (Thornberry et al 1997). Taken together with the fact that the vimentin cleavage is efficiently suppressed by Z-VAD-fmk in Jurkat cells, we concluded that the vimentin cleavage occurs at Asp85 and Asp259 in vivo.…”

“…Ile at P4 (Thornberry et al 1997). According to the current model, only caspase-8 among these caspases can function upstream of the Bcl-2 inhibitable step.…”

True

“…Recently, caspases involved in apoptosis have been subgrouped into two groups according to cleavage specificities of tetrapeptide substrates. 47 The DExD peptide recognition motif has been found to be optimal for caspases-2, -3, -7 (group II), and the (I/L/V)ExD sequence for caspases-6, -8, -9 (group III). 47,48 The observed DEVD, IETD and LEHD cleavage activities in cell lysates from HHT-treated cells as well as inhibition of apoptosis by these tetrapeptides indicate an involvement of caspases from both groups in HHT-induced cell death.…”

In vitro model of “wound healing” analyzed by laser scanning cytometry: Accelerated healing of epithelial cell monolayers in the presence of hyaluronate

“…Its covalent binding to the cysteine at the active enzyme center occurs only after enzyme activation (12). This reagent, which has a threeamino acid recognition peptide moiety (VAD), reacts with all caspases, possibly with the exception of caspase-2 (15,16). As shown in Figure 1, the LSC analysis made it possible to simultaneously measure red (DNA-bound PI) and green (FAM-VAD-FMK) fluorescence of individual cells in relation to their location vis-à-vis the scratch.…”