GENERATION OF CONSTITUTIVELY ACTIVE RECOMBINANT CASPASES-3 AND CASPASES-6 BY REARRANGEMENT OF THEIR SUBUNITS

Caspases play a major role in the transduction of the apoptotic signal and execution of apoptosis in mammalian cells. Ectopic overexpression of the short prodomain caspases-3 and -6 precursors in mammalian cell s does not induce apoptosis. This is due to their inability to undergo autocatalytic processing/activation and suggests that they depend on the long prodomain caspases for activation. To investigate directly th e apoptotic activity of these two caspases in vivo, we engineered cons titutively active recombinant caspases-3 and -6 precursors. This was a chieved by making contiguous precursor caspases-3 and -6 molecules, wh ich have their small subunits preceding their large subunits. Unlike t heir wild type counterparts, these recombinant molecules were capable of autocatalytic processing in an in vitro translation reaction, sugge sting that they are catalytically active. They were also capable of au toprocessing and inducing apoptosis in vivo independent of the upstrea m caspases. Furthermore, their autocatalytic and apoptotic activities were inhibited by the pancaspase inhibitor z-VAD-fluoromethylketone, b ut not by CrmA or Bcl-2, thus directly demonstrating that the targets of inhibition of apoptosis by CrmA and Bcl-2 are upstream of caspases- 3 and -6. Since caspases-3 and -6 are the most downstream executioners of apoptosis, the constitutively active versions of these caspases co uld be used at very low concentrations in gene therapy model systems t o induce apoptosis in target tissues or tumors.