Triggering of apoptosis by cathepsins

Two recent papers have expanded the role of the proteolytic mediators of apoptotic cell death beyond caspases. The newly discovered proteases capable of triggering pro-apoptotic changes in mitochondria are in fact old-fashioned proteins confined to the lysosomes and known for their involvement in non-specific cellular waste disposal, i.e. the cathepsins. `Knife and fork are evolutionary optimized tools to dine in a neat and efficient way'. Using this analogy of dining for cell demise and cutlery for caspases, two evident questions arise: are knife and fork sufficient tools in all cases ± and, are they always necessary? The first question brings to mind an array of other sophisticated tools developed for eating specific foods like e.g. lobster or fondue. In the cell death world such additional gadgets could correspond to other proteases like the above mentioned cathepsins. Formerly, cathepsins were thought to lead to cellular autolysis and damage of neighboring cells during necrosis (Figure 1). Accordingly, lysosomes were regarded as `suicide bags' that would release unspecific digestive enzymes after rupturing during uncontrolled cellular stress. Recent in vitro ± 6 as well as in vivo data suggest, however, that cathepsins may also act as mediators of programmed cell death. The fact that the frequently used caspase inhibitor zVAD-fmk efficiently blocks cysteine cathepsins like cathepsin-B (cathB), further emphasizes the need for a re-evaluation of the existing literature on the relative roles of caspases and cathepsins in a number of apoptosis paradigms. How is it possible that a specific cell death program can be triggered by the rather unspecific digestive power of lysosomal proteases? It appears as if a specific translocation process could be a key to the understanding of this phenomenon. For instance the selective translocation of cathB from lysosomes to cytosol and nucleus is well documented for bile salt-induced hepatic apoptosis. Similarly, there is clear evidence of early cathepsin-D translocation from secondary lysosomes to the cytosol under conditions of oxidative stress induced apoptosis. A second possibility is a quantitative relationship between the amount of lysosomal rupture and the mode of cell death. According to this model, low stress intensities trigger a limited release of lysosomal enzymes to the cytoplasm followed by apoptotic death, while high intensity stresses would lead to generalized lysosomal rupture and rapid cellular necrosis. A causal association between a limited lysosomal rupture and apoptosis has been supported by experiments showing cytoprotection by membrane stabilizing agents as well as triggering of cell death by selective lysosomal disrupters. The work by Guicciardi et al. from G. Gores' laboratory now provides some powerful evidence for the causal involvement of cathB in the caspase-8-dependent apoptotic signaling cascade triggered by TNF in vivo. The main conclusions rely on the comparisons of cathB-deficient mice with wild-type animals. For in vitro or in vivo experiments, hepatocytes were rendered sensitive to the apoptosis-inducing TNF signals by adenoviral transfer of an IkB super-repressor. When treated afterwards with TNF, liver damage in cathB-deficient mice was reduced, survival was increased and apoptotic markers of cultured hepatocytes from such mice were reduced compared to the similarly treated wild-type controls. Additional mechanistic studies revealed a release of cathB to the cytosol following TNF exposure. Here, the question arises whether cathB activity is sufficiently stable at the neutral intracellular pH to trigger downstream apoptotic processes in the cytosol. CathB is inactivated rapidly in neutral buffers in vitro, and, consistent with its normal localization within an acidic organelle, its dipeptidylpeptidase activity has an optimum at around pH 5. However, biological fluids seem to have a stabilizing effect on cathB even at neutral pH, and, in fact, cathB's endopeptidase activity has its optimum at pH 7.4 (M. Leist and M. JaÈ aÈ ttelaÈ , unpublished observations; reviewed in). Accordingly, Guicciardi et al. demonstrated that cathB can trigger cytochrome c release from mitochondria in an in vitro system, providing that a cytosolic extract was also added. This places cathB within the TNF-triggered apoptotic cascade as an enhancer acting upstream of mitochondria. Consistent with this, further experiments in a cell free system showed a release of cathB from lysosomes during incubation with caspase-8. The question on whether this mechanism is relevant within cells awaits further elucidation, since cathB does not seem to take a major role in another caspase-8-dependent pathway within the same hepatocytes, i.e. CD95-triggered apoptosis (M. Guicciardi, unpublished observations). An elegant study by Stoka et al. coordinated by G. Salvesen complements the above in vivo data by addressing the potential biochemical mechanism of cathepsin-triggered apoptosis. First, in vitro cleavage experiments showed that the major human apoptosisrelevant caspases are poor direct targets for lysomal extracts or a number of cysteine cathepsins. A possibility for a direct caspase activation was, however, indicated by data showing that a trypanosomal cysteine cathepsin (cruzipain, related to human cathL) can activate caspases-3/7. This corroborates previous observations of direct caspase-3 activation by a human cathL-like Cell Death and Differentiation (2001) 8, 324 ± 326 ã 2001 Nature Publishing Group All rights reserved 1350-9047/01 $15.00