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 sequence similarities between Ced-3 and the mammalian interleukin-1beta converting enzyme (ICE) suggest that these two proteins may have functionally similar active sites and that Ced-3 may function as a cysteine protease. Here we report that in vitro transcribed and translated Ced-3 protein (p56) underwent rapid processing to smaller fragments. Replacement of the predicted active site cysteine of Ced-3 with serine (C364S) prevented the generation of smaller proteolytic fragments, suggesting that the processing might be an autocatalytic process. Peptide aldehydes with aspartic acid at the P1 position blocked Ced-3 autocatalysis. Furthermore, the protease inhibition profile of Ced-3 was similar to the profile reported for ICE. These functional data demonstrate that Ced-3 is an Asp-dependent cysteine protease with substrate specificity similar to that of ICE. Aurintricarboxylic acid, an inhibitor of apoptosis in mammalian cells, blocked Ced-3 autocatalytic activity, suggesting that an aurintricarboxylic acid-sensitive Ced-3/ICE-related protease might be involved in the apoptosis pathway(s) in mammalian cells.
Hugunin
M
M
BASF Bioresearch Corporation, Worcester, Massachusetts, 01605-4314, USA.
Quintal
L J
LJ
Mankovich
J A
JA
Ghayur
T
T
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins; D020170:Caspase 1; D020169:Caspases; D003545:Cysteine; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D015801:Helminth Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine; D014158:Transcription, Genetic
D029742:Caenorhabditis elegans Proteins; D017931:DNA Primers; D015801:Helminth Proteins; D011994:Recombinant Proteins; D001312:Aurintricarboxylic Acid; D012694:Serine; D020169:Caspases; D003546:Cysteine Endopeptidases; C089223:ced-3 protein, C elegans; D020170:Caspase 1; D003545:Cysteine