Evidence that inhibition of cathepsin-B contributes to the neuroprotectiveproperties of caspase inhibitor Tyr-Val-Ala-Asp-chloromethyl ketone

During the use of tetrapeptide and other proprietary caspase inhibitors in the study of neurodegeneration, we had concluded that mechanisms other than the inhibition of caspases contributed to the protective effects of certai n caspase inhibitors. Here we report our studies to identify a target for a nd hence a mechanism by which the tetrapeptide inhibitor tyrosine-valine-al anine-aspartate-chloromethyl ketone (Ac-YVAD-cmk) is able to rescue neurona l cell cultures from cell death. Ac-YVAD-cmk rescued neuronal cells from ce ll death in response to oxidative stress and oxygen/glucose deprivation. Af finity labeling with biotinylated YVAD-cmk demonstrated distinct binding pr oteins for the inhibitor in cells from the central nervous system versus Ju rkat cells. Binding to the novel target protein was displaced by class-spec ific protease inhibitors and suggested that the target is a cysteine protea se. Affinity purification and sequencing identified the target as cathepsin -B. Cathepsin-B inhibitors competed with biotinylated YVAD-cmk for the targ et protein. The availability of the target for binding was reduced in cells that had been rescued by unlabeled inhibitor. Cathepsin-D inhibitors rescu e hippocampal slices from cell death induced by oxygen/glucose deprivation. These data provide evidence to support a role for cathepsin-B in neuronal cell death, particularly that following ischemia.