No abstract

Loss of parkin function is the major cause of autosomal recessive Parkinson's disease (ARPD). A wide variety of parkin mutations have been identified in patients; however, the pathophysiological mechanisms leading to the inactivation of mutant parkin are poorly understood. In this study we characterized pathogenic C-and N-terminal parkin mutants and found distinct pathways of parkin inactivation. Deletion of the C terminus abrogated the association of parkin with cellular membranes and induced rapid misfolding and aggregation. Four N-terminal missense mutations, located within the ubiquitin-like domain (UBL), decrease the stability of parkin; as a consequence, these mutants are rapidly degraded by the proteasome. Furthermore, we present evidence that a smaller parkin species of 42 kDa, which is present in extracts prepared from human brain and cultured cells, originates from an internal start site and lacks the N-terminal UBL domain.

Loss of parkin function is linked to autosomal recessive juvenile parkinsonism. Here we show that proteotoxic stress and short C-terminal truncations induce misfolding of parkin. As a consequence, wild-type parkin was depleted from a high molecular weight complex and inactivated by aggregation. Similarly, the pathogenic parkin mutant W453Stop, characterized by a Cterminal deletion of 13 amino acids, spontaneously adopted a misfolded conformation. Mutational analysis indicated that C-terminal truncations exceeding 3 amino acids abolished formation of detergent-soluble parkin. In the cytosol scattered aggregates of misfolded parkin contained the molecular chaperone Hsp70. Moreover, increased expression of chaperones prevented aggregation of wild-type parkin and promoted folding of the W453Stop mutant. Analyzing parkin folding in vitro indicated that parkin is aggregation-prone and that its folding is dependent on chaperones. Our study demonstrates that C-terminal truncations impede parkin folding and reveal a new mechanism for inactivation of parkin.Autosomal recessive juvenile parkinsonism (AR-JP), 1 the major cause of early onset parkinsonism, is characterized by mutations within the parkin gene. Parkin, a 465-amino acid protein, shows homology to ubiquitin at the N terminus and harbors a RING box near the C terminus, consisting of two RING finger motifs that flank a cysteine-rich domain (in-between RING fingers domain) (1, 2). Functional studies established that parkin acts as a ubiquitin-protein isopeptide ligase and that pathogenic mutations compromise this activity (3-6). As a consequence, substrates destined for proteasomal degradation via parkin might accumulate in parkin-deficient cells. Indeed, recent studies with cell culture models provide experimental evidence for such a scenario. It was shown that disease-related mutations in the parkin gene impair protein interactions of parkin with either a parkin substrate or another component of the ubiquitin ligase complex. Accumulation of Pael-R, one of the identified parkin substrates, causes endoplasmic reticulum (ER) stress, indicating that parkin has the potential to suppress unfolded protein stress-induced cell death (3, 4). Recent studies (7) revealed that parkin deficiency potentiates the accumulation of cyclin E and promotes apoptosis in neuronal cells exposed to excitotoxic stress. Interestingly, parkin is a significant component of Lewy bodies, the histopathologic hallmark of PD (5,8,9). Furthermore, it has been shown that parkin is protective against the toxic effects of proteasomal dysfunction and mutant ␣-synuclein (10), implying that the impact of parkin function and dysfunction might not be restricted to the entity of AR-JP.It still remains enigmatic why dopaminergic neurons in the substantia nigra are highly vulnerable in AR-JP, as parkin as well as its substrates identified so far are not selectively expressed in these cells. However, an inherent feature of dopaminergic neurons is an elevated level of reactive oxygen and nitrogen species due to...

Parkinson disease (PD)2 is the second most common neurodegenerative disease after Alzheimer disease. Although most PD cases occur sporadically, familial variants share important features with sporadic PD, most notably the demise of dopaminergic neurons in the substantia nigra pars compacta. Consequently, insight into the function of PD-associated genes might promote our understanding of pathogenic mechanisms not only in familial, but also in sporadic PD. Five genes have unambiguously been linked to PD over the past decade, the genes encoding ␣-synuclein and LRRK2 for autosomal dominant PD, and those encoding Parkin, PINK1, and DJ-1 for autosomal recessive PD (reviewed in Refs. 1-3). So far, over a hundred different pathogenic mutations in the parkin gene have been identified, which account for the majority of autosomal recessive PD cases. Parkin is a member of the RBR (ring between ring fingers) protein family, characterized by the presence of two RING domains (really interesting new gene), which flank a cysteine-rich in-between RINGs (IBR) domain. Similarly to other RBR proteins, Parkin has an E3 ubiquitin ligase activity, mediating the attachment of ubiquitin to substrate proteins (4 -6). Parkin can obviously mediate different modes of ubiquitylation, including monoubiquitylation, multiple monoubiquitylation, and polyubiquitylation both via lysine 48 and lysine 63, depending on the experimental conditions and the putative Parkin substrate analyzed (reviewed in Refs. 7 and 8). Importantly, the neuroprotective activity of Parkin seems to be associated with its ability to promote degradation-independent ubiquitylation (9, 10).Different lines of evidence indicate that pathogenic parkin mutations result in a loss of Parkin function. Our initial studies revealed that misfolding and aggregation is characteristic for C-terminal deletion mutants of Parkin based on the following biochemical features specific for mutant Parkin: 1) insolubility in non-ionic and ionic detergents; 2) sedimentation in a sucrose step gradient; 3) resistance to a limited proteolytic digestion; 4) loss of membrane association; and 5) formation of scattered aggregates in cells determined by immunocytochemistry (11,12). Alterations in the detergent solubility of Parkin and formation of Parkin aggregates/inclusion bodies have also been reported for various Parkin missense mutants (13-18). We also observed that even wild-type Parkin is prone to misfolding under severe oxidative stress (12). Remarkably, insoluble, catechol-modified Parkin could be detected in the substantia nigra of patients suffering from sporadic PD, suggesting a more general role of Parkin in the pathogenesis of PD (19). In support of this concept, the E3 ligase activity of Parkin has been shown to be impaired by nitrosative stress, and there is indeed evidence for the presence of S-nitrosylated Parkin in the brains of PD patients (20,21).Based on our finding that the deletion of C-terminal amino acids results in misfolding and aggregation of Parkin, we performed a comparative an...