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 inhibitors 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 protease(s) contained in the supernatant of a digitonin-treated crude mitochondrial fraction containing lysosomes (ML) and the cleaved product was detected by 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 activation was pH 6.6-6.8. This activation was not inhibited by CA-074, a specific inhibitor of cathepsin B, but was strongly inhibited by CLIK-066 and CLIK-181, specific inhibitors of cathepsin L. The inhibitory effect of CLIK-060, a specific inhibitor of cathepsin S, was very weak. Furthermore, the activation of caspase-3 was enhanced by addition of purified cathepsin L only in the presence of the supernatant of the digitonin-treated ML. These results 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.
Ishisaka
R
R
Institute of Medical Science, Kurashiki Medical Center, Japan.
Utsumi
T
T
Kanno
T
T
Arita
K
K
Katunuma
N
N
Akiyama
J
J
Utsumi
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism*; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D003546:Cysteine Endopeptidases; D003574:Cytochrome c Group / Q000378:metabolism; D010450:Endopeptidases; D008247:Lysosomes / Q000201:enzymology; D051381:Rats
D003574:Cytochrome c Group; D000255:Adenosine Triphosphate; D002403:Cathepsins; D010450:Endopeptidases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534280:Ctsl protein, rat