[W206R]-procaspase 3: An inactivatable substrate for caspase 8

We report here the cloning and high-level expression of a soluble preform o f human caspase 3 (Ser(24)-H-277) engineered to contain a short stretch of N-terminal sequence (MTISDSPREQD) from the prosegment of procaspase 8 and a C-terminal heptahistidine tag. The precursor protein isolated from extract s of recombinant Escherichia coli by immobilized metal-ion affinity chromat ography was predominantly unprocessed and migrated as a 32-kDa-polypeptide on sodium dodecyl sulfate-polyacrylamide gels. Incubation of this protein w ith recombinant human caspase 8 produced fragments characteristic of the pr operly processed caspase 3, but the product was inactive. Amino-terminal se quence analysis of the caspase 3 polypeptides proved that caspase 8 had spe cifically cleaved the ASP(175)-Ser(176) bond to yield the expected p18 and p12 subunits, with partial cleavage at the Asp(28)-Ser(29) bond to release the prosegment. The lack of caspase 3 activity was found to be the result o f a fortuitous mutation in which Trp(206) in the S4 subsite was replaced by arginine (W206R). This mutant procaspase 3, which we call m-pro3, serves a s a useful reagent with which to test the efficacy of caspase 8 inhibitors in blocking processing of the natural polypeptide substrate of this enzyme and may be valuable as a source of "proenzyme" for crystallographic analysi s. (C) 2001 Academic Press.