Activation of the native 45-kDa precursor form of interleukin-1-converting enzyme

Abstract

Active interleukin-1beta-converting enzyme (ICE) is composed of 20- and 10-kDa polypeptides (p20 and p10) derived from the processing of a cytosolic 45-kDa precursor protein (p45). The cleavage and activation of the native p45 ICE precursor have been characterized by use of specific inhibitors and antibodies recognizing various regions of ICE. The processing of p45 in vitro in THP.1 monocytic cell cytoplasmic extracts is inhibited only by protease inhibitors that inhibit ICE and not by inhibitors of other protease classes. The addition of L-742,395, a biotinylated irreversible ICE inhibitor, to these extracts labels only p45 and simultaneously inhibits p45 processing, demonstrating that the p45 has catalytic activity. Following a cleavage of p45 at a site that becomes the COOH terminus of p20, a more active intermediate is formed which migrates on SDS-polyacrylamide gel electrophoresis with an molecular mass of 35 kDa (ED50 of approximately 0.1 microM L-742,395 labeling versus 5 microM for p45). This new more active ICE form serves both as an intermediate enzyme to cleave p45 as well as a substrate for the formation of the final active ICE (ED50 of 1 nM L-742,395 labeling of p20 and for p22, an NH2-terminally extended form of p20). While initial cleavage of p45 can be found at the sites corresponding to both the NH2 termini of p22 and p20, these fragments cannot be labeled by L-742,395 and are hence inactive. p45 is not processed at the site corresponding to the NH2 terminus of the p10. Less than 50% of the p45 is cleaved down to active p20 or p22 ICE as determined by band shift on SDS-polyacrylamide gel electrophoresis of the biotinylated fragments, indicating that the in vitro activation is highly inefficient. The ICE fragmentation occurs by an intermolecular process and is highly dilution sensitive. Cleavage of p45 by exogenous p20/p10 ICE differs from that of the endogenous p45 cleavage activity in that the p20/p10 activity is more salt sensitive, and it produces a different pattern of cleavage fragments, principally 35- and 12-kDa fragments. These results indicate that the nature of the ICE activity changes as p45 is processed down to the p20/p10 form of the enzyme.