Erin P. Peterson

A Combinatorial Approach Defines Specificities of Members of the Caspase Family and Granzyme B FUNCTIONAL RELATIONSHIPS ESTABLISHED FOR KEY MEDIATORS OF APOPTOSIS

There is compelling evidence that members of the caspase (interleukin-1β converting enzyme/CED-3) family of cysteine proteases and the cytotoxic lymphocyte-derived 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 andCaenorhabditis 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.

PURIFICATION AND CATALYTIC PROPERTIES OF HUMAN CASPASE FAMILY MEMBERS

Members of the caspase family of cysteine proteases are known to be key mediators of mammalian inflammation and apoptosis. To better understand the catalytic properties of these enzymes, and to facilitate the identification of selective inhibitors, we have systematically purified and biochemically characterized ten homologues of human origin (caspases 1–10). The method used for production of most of these enzymes involves folding of active enzymes from their constituent subunits which are expressed separately in E. coli, followed by ion exchange chromatography. In cases where it was not possible to use this method (caspase-6 and -10), the enzymes were instead expressed as soluble proteins in E. coli, and partially purified by ion exchange chromatography. Based on the optimal tetrapeptide recognition motif for each enzyme, substrates with the general structure Ac-XEXD-AMC were used to develop continuous fluorometric assays. In some cases, enzymes with virtually identical tetrapeptide specificities have kcat/Km values for fluorogenic substrates that differ by more than 1000-fold. Using these assays, we have investigated the effects of a variety of environmental factors (e.g. pH, NaCl, Ca2+) on the activities of these enzymes. Some of these variables have a profound effect on the rate of catalysis, a finding that may have important biological implications.

A combinatorial approach for determining protease specificities: application to interleukin-1beta converting enzyme (ICE).

BACKGROUND Interleukin-1beta converting enzyme (ICE/caspase-1) is the protease responsible for interleukin-1beta (IL-1beta) production in monocytes. It was the first member of a new cysteine protease family to be identified. Members of this family have functions in both inflammation and apoptosis. RESULTS A novel method for identifying protease specificity, employing a positional-scanning substrate library, was used to determine the amino-acid preferences of ICE. Using this method, the complete specificity of a protease can be mapped in the time required to perform one assay. The results indicate that the optimal tetrapeptide recognition sequence for ICE is WEHD, not YVAD, as previously believed, and this led to the synthesis of an unusually potent aldehyde inhibitor, Ac-WEHD-CHO (Ki = 56 pM). The structural basis for this potent inhibition was determined by X-ray crystallography. CONCLUSIONS The results presented in this study establish a positional-scanning library as a powerful tool for rapidly and accurately assessing protease specificity. The preferred sequence for ICE (WEHD) differs significantly from that found in human pro-interleukin-1beta (YVHD), which suggests that this protease may have additional endogenous substrates, consistent with evidence linking it to apoptosis and IL-1alpha production.

Biochemical and Genetic Interactions between Drosophila Caspases and the Proapoptotic Genes rpr, hid, and grim

ABSTRACT In Drosophila melanogaster, the induction of apoptosis requires three closely linked genes, reaper(rpr), head involution defective(hid), and grim. The products of these genes induce apoptosis by activating a caspase pathway. Two very similarDrosophila caspases, DCP-1 and drICE, have been previously identified. We now show that DCP-1 has a substrate specificity that is remarkably similar to those of human caspase 3 and Caenorhabditis elegans CED-3, suggesting that DCP-1 is a death effector caspase. drICE and DCP-1 have similar yet different enzymatic specificities. Although expression of either in cultured cells induces apoptosis, neither protein was able to induce DNA fragmentation inDrosophila SL2 cells. Ectopic expression of a truncated form of dcp-1 (ΔN-dcp-1) in the developingDrosophila retina under an eye-specific promoter resulted in a small and rough eye phenotype, whereas expression of the full-length dcp-1 (fl-dcp-1) had little effect. On the other hand, expression of either full-length drICE(fl-drICE) or truncated drICE(ΔN-drICE) in the retina showed no obvious eye phenotype. Although active DCP-1 protein cleaves full-length DCP-1 and full-length drICE in vitro, GMR-ΔN-dcp-1 did not enhance the eye phenotype of GMR-fl-dcp-1 or GMR-fl-drICEflies. Significantly, GMR-rpr and GMR-grim, but not GMR-hid, dramatically enhanced the eye phenotype of GMR-fl-dcp-1 flies. These results indicate that Reaper and Grim, but not HID, can activate DCP-1 in vivo.

Inhibition of interleukin-1β converting enzyme by N-Acyl-aspartic acid ketones

Abstract N-Acyl aspartic acid ketones ( 3a-3n ) were prepared from the corresponding bromomethyl ketone. The inhibition of interleukin-1β converting enzyme (ICE) by these single amino acid ketones is reported. The best compound had K i of 3.5 μM versus ICE.

Inhibition of interleukin-1β converting enzyme by N-acyl-aspartyl aryloxymethyl ketones

Abstract The synthesis of N-allyloxy-aspartyl aryloxymethyl ketones and their evaluation as inhibitors of interleukin-1β converting enzyme (ICE) are described. 3-Substituted 2-naphthyloxymethyl ketones were found to have Kis of 90–500 nM. The SAR suggests that the substituents on the aryloxy group may have a specific binding interaction in the active site of ICE.

Activated ketones as potent reversible inhibitors of interleukin-Iβ converting enzyme

Abstract Replacement of the β′-carbon of the side chain of the phenylalkyl ketones 4 with a heteroatom or electron-withdrawing group resulted in significant improvement in inhibitory activity against the cysteine proteinase Interleukin1-β Converting Enzyme(ICE).