Lysosomal protease pathways to apoptosis - Cleavage of Bid, not pro-caspases, is the most likely route

We investigated the mechanism of lysosome-mediated cell death using purifie d recombinant pro-apoptotic proteins, and cell-free extracts from the human neuronal progenitor cell line NT2, Potential effectors were either isolate d lysosomes or purified lysosomal proteases. Purified lysosomal cathepsins B, H, K, L, S, and X or an extract of mouse lysosomes did not directly acti vate either recombinant caspase zymogens or caspase zymogens present in an NT2 cytosolic extract to any significant extent. In contrast, a cathepsin L -related protease from the protozoan parasite Trypanosana cruzi, cruzipain, showed a measurable caspase activation rate. This demonstrated that member s of the papain family can directly activate caspases but that mammalian ly sosomal members of this family may have been negatively selected for caspas e activation to prevent inappropriate induction of apoptosis, Given the lac k of evidence for a direct role in caspase activation by lysosomal protease s, we hypothesized that an indirect mode of caspase activation may involve the Bcl-2 family member Bid. In support of this, Bid was cleaved in the pre sence of lysosomal extracts, at a site six residues downstream from that se en for pathways involving capase 8, Incubation of mitochondria with Bid tha t had been cleaved by lysosomal extracts resulted in cytochrome c release. Thus, cleavage of Bid may represent a mechanism by which proteases that hav e leaked from the lysosomes can precipitate cytochrome c release and subseq uent caspase activation. This is supported by the finding that cytosolic ex tracts from mice ablated in the bid gene are impaired in the ability to rel ease cytochrome c in response to lysosome extracts, Together these data sug gest that Bid represents a sensor that allows cells to initiate apoptosis i n response to widespread adventitious proteolysis.