A CLONING METHOD FOR CASPASE SUBSTRATES THAT USES THE YEAST 2-HYBRID SYSTEM - CLONING OF THE ANTIAPOPTOTIC GENE GELSOLIN

Caspase-mediated proteolysis is a critical and central element of the apoptotic process; therefore, it is important to identify the downstre am molecular targets of caspases. We established a method for cloning the genes of caspase substrates by two major modifications of the yeas t two-hybrid system: (i) both large and small subunits of active caspa ses were expressed in past under ADH1 promoters and the small subunit was fused to the LexA DNA-binding domain; and (ii) a point mutation wa s introduced that substituted serine for the active site cysteine and thereby prevented proteolytic cleavage of the substrates, possibly sta bilizing the enzyme-substrate complexes in yeast. After screening a mo use embryo cDNA expression library by using the bait plasmid for caspa se-3, we obtained 13 clones that encoded proteins binding to caspase-3 , and showed that 10 clones including gelsolin, an actin-regulatory pr otein implicated in apoptosis, were cleaved by recombinant caspase-3 i n vitro. Using the same bait, we also isolated human gelsolin cDNA fro m a human thymus cDNA expression library. We showed that human gelsoli n was cleaved during Fas-mediated apoptosis in vivo and that the caspa se-3 cleavage site of human gelsolin was at D-352 Of DQTD(352)G, findi ngs consistent with previous observations on murine gelsolin, In addit ion, we ascribed the antiapoptotic activity of gelsolin (which we prev iously reported) to prevention of a step leading to cytochrome c relea se from the mitochondria into the cytosol, Our results indicate that t his cloning method is useful for identification of the substrates of c aspases and possibly also of other enzymes.