Participation of a Cathepsin L-Type Protease in the Activation of Caspase-3.

Abstract

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.

Lysosomal membrane permeabilization in cell death

“…The most common cathepsins implicated in cell death are CB and CD, although CL can also induce caspase-3 activation (Ishisaka et al, 1999;Hishita et al, 2001). In addition, CL has an important function in the cell death associated with the involution of the mammary gland after weaning, as CL inhibitors reduce cell death in this system (Burke et al, 2003).…”

Selective suppression of cathepsin L by antisense cDNA impairs human brain tumor cell invasion in vitro and promotes apoptosis

“…17,42 -45 It has been proposed that lysosomal dysfunction and destabilization induced by various factors, result in the release of cathepsins, particularly cathepsin L, which may then activate caspase -3. 16,46,47 However, the ability of cathepsins to activate /up -regulate caspase -3 activity may be dependant on cell type. It has been demonstrated on the human neuronal cell line NT2 that cysteine cathepsins did not activate caspase-3, although total lysosomal extract from these cells was effective in stimulating apoptosis by cleaving the proapoptotic protein Bid.…”

Apoptosis caused by cathepsins does not require Bid signaling in an in vivo model of progressive myoclonus epilepsy (EPM1)

“…13,[15][16][17][18][19] Similarly, cathepsin inhibitor treatment blocked this apoptosis. 11,14,[18][19][20][21][22] Theoretically, the cathepsin proteases could induce apoptosis by a variety of different mechanisms. 23 One possibility is that cathepsins could nonspecifically degrade important cellular proteins, thereby causing the cell to initiate apoptosis.…”

“…In support of the former mechanism, it has been shown that cathepsin B can activate the inflammatory caspases 1 and 11, 17,26 and that cathepsin L may activate caspase-3. 20 The final possible mode of cathepsin action places cathepsins far upstream in the apoptotic cascade, cleaving the proapoptotic Bcl-2 family member Bid to initiate mitochondrial release of cytochrome c. This last hypothesis received in vitro confirmation when it was shown that lysosomal extracts containing cathepsins were able to cleave purified Bid in a physiologically relevant manner that supported apoptosis. 27 Cytosolic extracts prepared from Bid-deficient mice resulted in significantly less apoptosis, demonstrating the dependence of this pathway on Bid.…”