Proteolytic processing of the caspase-9 zymogen is required for apoptosome-mediated activation of caspase-9.

Maturation of the single-chain caspase-9 zymogen through autoproteolytic processing is mediated by the Apaf-1 apoptosome at the onset of apoptosis. Processed caspase-9 and the apoptosome form a holoenzyme with robust proteolytic activity that is 2-3 orders of magnitude higher than that of free processed caspase-9. An unresolved important question is the role of caspase-9 processing, with some experimental data suggesting its dispensability. In this study, we demonstrate that, in contrast to wild-type caspase-9, the unprocessed single-chain caspase-9 triple mutant E306A/D315A/D330A (Casp9-TM) could no longer be adequately activated by the apoptosome. Compared with the protease activity of wild-type caspase-9, that of Casp9-TM in the presence of the apoptosome was drastically reduced. The crippled protease activity of Casp9-TM in the presence of the apoptosome is likely attributable to a markedly reduced ability of Casp9-TM to form homodimers. These data identify an essential role for the autoproteolytic processing of caspase-9 in its activation.