It is widely accepted that the familial Parkinson's disease (PD)-linked gene product, parkin, functions as a ubiquitin ligase involved in protein turnover via the ubiquitin-proteasome system. Substrates ubiquitinated by parkin are hence thought to be destined for proteasomal degradation. Because we demonstrated previously that parkin interacts with and ubiquitinates synphilin-1, we initially expected synphilin-1 degradation to be enhanced in the presence of parkin. Contrary to our expectation, we found that synphilin-1 is normally ubiquitinated by parkin in a nonclassical, proteasomal-independent manner that involves lysine 63 (K63)-linked polyubiquitin chain formation. Parkin-mediated degradation of synphilin-1 occurs appreciably only at an unusually high parkin to synphilin-1 expression ratio or when primed for lysine 48 (K48)-linked ubiquitination. In addition we found that parkin-mediated ubiquitination of proteins within Lewy-body-like inclusions formed by the coexpression of synphilin-1, ␣-synuclein, and parkin occurs predominantly via K63 linkages and that the formation of these inclusions is enhanced by K63-linked ubiquitination. Our results suggest that parkin is a dual-function ubiquitin ligase and that K63-linked ubiquitination of synphilin-1 by parkin may be involved in the formation of Lewy body inclusions associated with PD.

Mutations in parkin are currently recognized as the most common cause of familial Parkinsonism. Emerging evidence also suggests that parkin expression variability may confer a risk for the development of the more common, sporadic form of Parkinson's disease (PD). Supporting this, we have recently demonstrated that parkin solubility in the human brain becomes altered with age. As parkin apparently functions as a broad-spectrum neuroprotectant, the resulting decrease in the availability of soluble parkin with age may underlie the progressive susceptibility of the brain to stress. Interestingly, we also observed that many familial-PD mutations of parkin alter its solubility in a manner that is highly reminiscent of our observations with the aged brain. The converging effects on parkin brought about by aging and PD-causing mutations are probably not trivial and suggest that environmental modulators affecting parkin solubility would increase an individual's risk of developing PD. Using both cell culture and in vivo models, we demonstrate here that several PD-linked stressors, including neurotoxins (MPP+, rotenone, 6-hydroxydopamine), paraquat, NO, dopamine and iron, induce alterations in parkin solubility and result in its intracellular aggregation. Furthermore, the depletion of soluble, functional forms of parkin is associated with reduced proteasomal activities and increased cell death. Our results suggest that exogenously introduced stress as well as endogenous dopamine could affect the native structure of parkin, promote its misfolding, and concomitantly compromise its protective functions. Mechanistically, our results provide a link between the influence of environmental and intrinsic factors and genetic susceptibilities in PD pathogenesis.

Parkinson disease (PD), a prevalent neurodegenerative motor disorder, is characterized by the rather selective loss of dopaminergic neurons and the presence of ␣-synuclein-enriched Lewy body inclusions in the substantia nigra of the midbrain. Although the etiology of PD remains incompletely understood, emerging evidence suggests that dysregulated iron homeostasis may be involved. Notably, nigral dopaminergic neurons are enriched in iron, the uptake of which is facilitated by the divalent metal ion transporter DMT1. To clarify the role of iron in PD, we generated SH-SY5Y cells stably expressing DMT1 either singly or in combination with wild type or mutant ␣-synuclein. We found that DMT1 overexpression dramatically enhances Fe 2؉ uptake, which concomitantly promotes cell death. This Fe 2؉ -mediated toxicity is aggravated by the presence of mutant ␣-synuclein expression, resulting in increased oxidative stress and DNA damage. Curiously, Fe 2؉ -mediated cell death does not appear to involve apoptosis. Instead, the phenomenon seems to occur as a result of excessive autophagic activity. Accordingly, pharmacological inhibition of autophagy reverses cell death mediated by Fe 2؉ overloading. Taken together, our results suggest a role for iron in PD pathogenesis and provide a mechanism underlying Fe 2؉ -mediated cell death.Parkinson disease (PD) 3 is the most common motor neurodegenerative disorder, affecting 1-2% of the population over the age of 65. Pathologically, it is characterized by selective dopaminergic neuron loss and the presence of Lewy bodies immunoreactive for ␣-synuclein in the substantia nigra pars compacta. To date, the leading causes for the sporadic form of the disease remain unclear, although there is accumulating evidence implicating oxidative stress (1), including the finding that PD brains have increased levels of oxidative damage to DNA, proteins, and lipids (2-4). One potential player contributing to increased oxidative stress is iron, which can convert hydrogen peroxide to highly reactive hydroxyl radicals via the Fenton reaction. Indeed, increased deposition of iron was found in microglia, astrocytes, oligodendrocytes, and dopaminergic neurons of the substantia nigra pars compacta of post-mortem PD brains (5, 6). The total iron content was found to be significantly higher in the substantia nigra pars compacta of PD patients together with a corresponding increase in divalent metal transporter-1 (DMT1) transcripts in the same region (7). This suggests a close association among DMT1 expression, iron overload, and PD.Mutations in a number of genes have also been implicated in the pathogenesis of PD (8) of which the first to be discovered was ␣-synuclein. Besides the A53T, A30P, and E46K missense mutations (9 -11), duplication (12, 13) and triplication (14) of the ␣-synuclein gene have also been linked to familial forms of PD. It has been suggested that the tendency of ␣-synuclein to undergo misfolding and aggregation may underlie its involvement in Lewy body formation and hence PD (15). Given that i...

BackgroundMutations in the parkin gene, which encodes a ubiquitin ligase (E3), are a major cause of autosomal recessive parkinsonism. Although parkin-mediated ubiquitination was initially linked to protein degradation, accumulating evidence suggests that the enzyme is capable of catalyzing multiple forms of ubiquitin modifications including monoubiquitination, K48- and K63-linked polyubiquitination. In this study, we sought to understand how a single enzyme could exhibit such multifunctional catalytic properties.Methods and FindingsBy means of in vitro ubiquitination assays coupled with mass spectrometry analysis, we were surprised to find that parkin is apparently capable of mediating E2-independent protein ubiquitination in vitro, an unprecedented activity exhibited by an E3 member. Interestingly, whereas full length parkin catalyzes solely monoubiquitination regardless of the presence or absence of E2, a truncated parkin mutant containing only the catalytic moiety supports both E2-independent and E2-dependent assembly of ubiquitin chains.ConclusionsOur results here suggest a complex regulation of parkin's activity and may help to explain how a single enzyme like parkin could mediate diverse forms of ubiquitination.

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