A COMBINATORIAL APPROACH FOR DETERMINING PROTEASE SPECIFICITIES - APPLICATION TO INTERLEUKIN-1-BETA CONVERTING-ENZYME (ICE)

Background: Interleukin-1 beta converting enzyme (ICE/caspase-1) is th e protease responsible for interleukin-1 beta (IL-1 beta) production i n monocytes. It was the first member of a new cysteine protease family to be identified Members of this family have functions in both inflam mation and apoptosis. Results: A novel method for identifying protease specificity, employing a positional-scanning substrate library, was u sed to determine the amino-acid preferences of ICE, Using this method, the complete specificity of a protease can be mapped in the time requ ired to perform one assay. The results indicate that the optimal tetra peptide recognition sequence for ICE is WEHD, not YVAD, as previously believed, and this led to the synthesis of an unusually potent aldehyd e inhibitor, Ac-WEHD-CHO (K-i=56 pM). The structural basis for this po tent inhibition was determined by X-ray crystallography. Conclusions: The results presented in this study establish a positional-scanning li brary as a powerful tool for rapidly and accurately assessing protease specificity, The preferred sequence for ICE (WEHD) differs significan tly from that found in human pro-interleukin-beta (YVHD), which sugges ts that this protease may have additional endogenous substrates, consi stent with evidence linking it to apoptosis and IL-1 alpha production.