Caspase-1 and caspase-8 cleave and inactivate cellular parkin.

Lesions in the parkin gene cause early onset Parkinson's disease by a loss of dopaminergic neurons, thus demonstrating a vital role for parkin in the survival of these neurons. Parkin is inactivated by caspase cleavage, and the major cleavage site is after Asp126. Caspases responsible for parkin cleavage were identified by several experimental paradigms. Transient coexpression of caspases and wild type parkin in HEK-293 cells identified caspase-1, -3, and -8 as efficient inducers of parkin cleavage whereas caspase-2, -7, -9, and -11 did not induce cleavage. A D126A parkin mutation abrogates cleavage induced by caspase-1 and -8, but not by caspase-3. In anti-Fas-treated Jurkat T cells, parkin cleavage was inhibited by caspase inhibitors hFlip and CrmA (but not by X-linked inhibitor of apoptosis (XIAP)), indicating that caspase-8 (but not caspase-3) is responsible for the parkin cleavage in this model. Moreover, induction of apoptosis in caspase-3-deficient MCF7 cells, either by caspase-1 or -8 overexpression or by tumor necrosis factor-alpha treatment, led to parkin cleavage. These results demonstrate that caspase-1 and -8 can directly cleave parkin and suggest that death receptor activation and inflammatory stress can cause loss of the ubiquitin ligase activity of parkin, thus causing accumulation of toxic parkin substrates and triggering dopaminergic cell death.

Caspase-mediated parkin cleavage in apoptotic cell death.

The parkin protein is important for the survival of the neurons that degenerate in Parkinson's disease as demonstrated by disease-causing lesions in the parkin gene. The Chinese hamster ovary and the SH-SY5Y cell line stably expressing recombinant human parkin combined with epitope-specific parkin antibodies were used to investigate the proteolytic processing of human parkin during apoptosis by immunoblotting. Parkin is cleaved during apoptosis induced by okadaic acid, staurosporine, and camptothecin, thereby generating a 38-kDa C-terminal fragment and a 12-kDa N-terminal fragment. The cleavage was not significantly affected by the disease-causing mutations K161N, G328E, T415N, and G430D and the polymorphism R366W. Parkin and its 38-kDa proteolytic fragment is preferentially associated with vesicles, thereby indicating that cleavage is a membrane-associated event. The proteolysis is sensitive to inhibitors of caspases. The cleavage site was mapped by site-directed mutagenesis of potential aspartic residues and revealed that mutation of Asp-126 alone abrogated the parkin cleavage. The tetrapeptide aldehyde LHTD-CHO, representing the amino acid sequence N-terminal to the putative cleavage site was an efficient inhibitor of parkin cleavage. This suggests that parkin function is compromised in neuropathological states associated with an increased caspase activation, thereby further adding to the cellular stress.