Participation of a cathepsin L-type protease in the activation of caspase-3

A previous paper from this laboratory reported the activation of a caspase- 3-like protease by a digitonin-treated lysosomal fraction [FEBS Lett, 435, 233-236, 1998]. In this study, we examined the effects of specific inhibito rs of lysosomal cysteine proteases, such as cathepsins B, S, and L, on the activation of caspase-3 to find out which cathepsin is responsible for the activation. Pro-caspase-3 in the cytosol was cleaved by a lysosomal proteas e(s) contained in the supernatant of a digitonin-treated crude mitochondria l fraction containing lysosomes (ML) and the cleaved product was detected b y Western blotting using anti-caspase-3 antibody. The activation of caspase -3 by the lysosomal protease(s) was pH dependent and the optimum pH for act ivation was pH 6.6-6.8, This activation was not inhibited by CA-074, a spec ific inhibitor of cathepsin B, but was strongly inhibited by CLIK-066 and C LIK-181, specific inhibitors of cathepsin L, The inhibitory effect of CLIK- 060, a specific inhibitor of cathepsin S, was very weak, Furthermore, the a ctivation of caspase-3 was enhanced by addition of purified cathepsin L onl y in the presence of the supernatant of the digitonin-treated ML, These res ults suggested that a cathepsin L-type protease activity might participate in the activation mechanism of caspase-3 in the presence of the supernatnat from the ML.