Caspase-mediated Parkin Cleavage in Apoptotic Cell Death

Abstract

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.

Herp Stabilizes Neuronal Ca2+ Homeostasis and Mitochondrial Function during Endoplasmic Reticulum Stress

“…Although the present study did not address possible cytotoxic properties of the released Herp fragments, it is more likely that cleavage reduces its intrinsic anti-apoptotic function at the ER. In this regard, it is noteworthy that the N-terminal ubiquitin-like domain of Parkin, a cytosolic protein, which also confers cytoprotection toward ER stress (50), is similarly cleaved and removed by caspases (51). As Herp has no demonstrable E3 ubiquitin-protein ligase activity, it is unlikely that Herp plays a direct role in protein substrate ubiquitination.…”

Caspase-1 and Caspase-8 Cleave and Inactivate Cellular Parkin

“…DNA Constructs-Plasmids used for the expression of parkin variants were pcDNA3.1-parkin WT and D126A (15). For transient expression of different caspases, we used pCAGGS-caspase-1, -2, -3, -7, -11, (16), pcDNA3.1-caspase-8 (17), pEF1A-caspase-9, and caspase-9s (18).…”

“…Electrophoresis, immunoblotting, and the T160 antibody specific for parkin was prepared as described previously (15). The antibody recognizing poly(ADP-ribose) polymerase (PARP) was from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA), and the antibody against caspase-8 was from BioSource Europe, S.A. (Nivelles, Belgium).…”

“…We have previously shown that parkin is cleaved by hitherto unidentified caspases during apoptotic cell death (15). The aim of the current study was to identify the caspases that cleave parkin.…”

Alterations in the common fragile site gene Parkin in ovarian and other cancers

“…Mutational analysis of ARJP patients has revealed the presence of various intragenic deletions/duplications and point mutations in Parkin Lu¨cking et al, 2000;Hedrich et al, 2001Hedrich et al, , 2002Hoenicka et al, 2002;Kann et al, 2002;Nichols et al, 2002;West et al, 2002). Subsequently, these mutations have been demonstrated to be responsible for the manifestation of juvenile, earlyonset, and sometimes late-onset Parkinson's disease (Imai et al, 2000;Tanaka et al, 2001;Kahns et al, 2002).…”