PROTEASE ACTIVITY OF IN-VITRO TRANSCRIBED AND TRANSLATED CAENORHABDITIS-ELEGANS CELL-DEATH GENE (CED-3) PRODUCT

The Caenorhabditis elegans cell death gene, ced-3, encodes one of the two proteins required for apoptosis in this organism. The primary sequ ence similarities between Ced-3 and the mammalian interleukin-1 beta c onverting enzyme (ICE) suggest that these two proteins may have functi onally similar active sites and that Ced-3 may function as a cysteine protease, Here we report that in vitro transcribed and translated Ced- S protein (p56) underwent rapid processing to smaller fragments. Repla cement of the predicted active site cysteine of Ced-3 with serine (C36 4S) prevented the generation of smaller proteolytic fragments, suggest ing that the processing might be an autocatalytic process. Peptide ald ehydes with aspartic acid at the P1 position blocked Ced-3 autocatalys is. Furthermore, the protease inhibition profile of Ced-3 was similar to the profile reported for ICE. These functional data demonstrate tha t Ced-3 is an Asp-dependent cysteine protease with substrate specifici ty similar to that of ICE. Aurintricarboxylic acid, an inhibitor of ap optosis in mammalian cells, blocked Ced-3 autocatalytic activity, sugg esting that an aurintricarboxylic acid-sensitive Ced-3/ICE-related pro tease might be involved in the apoptosis pathway(s) in mammalian cells .