Catalytic activity of caspase-3 is required for its degradation: stabilization of the active complex by synthetic inhibitors.
The activation of caspase-3 represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Upon induction of apoptosis, the large (p17) and small (p12) subunits, comprising active caspase-3, are generated via proteolytic processing of a latent proenzyme dimer. Two copies of each individual subunit are generated to form an active heterotetramer. The tetrameric form of caspase-3 cleaves specific protein substrates within the cell, thereby producing the apoptotic phenotype. In contrast to the proenzyme, once activated in HeLa cells, caspase-3 is difficult to detect due to its rapid degradation. Interestingly, however, enzyme stability and therefore detection of active caspase-3 by immunoblot analysis can be restored by treatment of cells with a peptide-based caspase-3 selective inhibitor, suggesting that the active form can be stabilized through protein-inhibitor interaction. The heteromeric active enzyme complex is necessary for its stabilization by inhibitors, as expression of the large subunit alone is not stabilized by the presence of inhibitors. Our results show for the first time, that synthetic caspase inhibitors not only block caspase activity, but may also increase the stability of otherwise rapidly degraded mature caspase complexes. Consistent with these findings, experiments with a catalytically inactive mutant of caspase-3 show that rapid turnover is dependent on the activity of the mature enzyme. Furthermore, turnover of otherwise stable active site mutants of capase-3 is rescued by the presence of the active enzyme suggesting that turnover can be mediated in trans.
Tawa
P
P
Department of Biochemistry & Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada.
Hell
K
K
Giroux
A
A
Grimm
E
E
Han
Y
Y
Nicholson
D W
DW
Xanthoudakis
S
S
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002384:Catalysis; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D015394:Molecular Structure; D014407:Tumor Cells, Cultured
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases