Loss-of-function DJ-1 mutations can cause early-onset Parkinson's disease. The function of DJ-1 is unknown, but an acidic isoform accumulates after oxidative stress, leading to the suggestion that DJ-1 is protective under these conditions. We addressed whether this represents a posttranslational modification at cysteine residues by systematically mutating cysteine residues in human DJ-1. WT or C53A DJ-1 was readily oxidized in cultured cells, generating a pI 5.8 isoform, but an artificial C106A mutant was not. We observed a cysteine-sulfinic acid at C106 in crystalline DJ-1 but no modification of C53 or C46. Oxidation of DJ-1 was promoted by the crystallization procedure. In addition, oxidation-induced mitochondrial relocalization of DJ-1 and protection against cell death were abrogated in C106A but not C53A or C46A. We suggest that DJ-1 protects against neuronal death, and that this is signaled by acidification of the key cysteine residue, C106.

No abstract

Several mutations in PTEN-induced putative kinase 1 (PINK1) gene have been reported to be associated with recessive parkinsonism. The encoded protein is predicted to be a Ser͞Thr protein kinase targeted to mitochondria. In this study, we have investigated the effects of mutations on PINK1 kinase activity in vitro and on expression levels and localization in mammalian cells. We chose to examine two point mutations: G309D, which was originally reported to be stable and properly localized in cells and L347P, which is of interest because it is present at an appreciable carrier frequency in the Philippines. We were able to confirm kinase activity and produce artificial ''kinase-dead'' mutants that are stable but lack activity. The L347P mutation grossly destabilizes PINK1 and drastically reduces kinase activity, whereas G309D has much more modest effects on these parameters in vitro. This finding is in line with predictions based on homology modeling. We also examined the localization of PINK1 in transfected mammalian cells by using constructs that were tagged with myc or GFP at either end of the protein. These results show that PINK1 is processed at the N terminus in a manner consistent with mitochondrial import, but the mature protein also exists in the cytosol. The physiological relevance of this observation is not yet clear, but it implies that a portion of PINK1 may be exported after processing in the mitochondria. mitochondria ͉ PARK6 ͉ Parkinson's disease S everal genes have now been identified that are causally associated with recessive parkinsonism. Mutations in parkin (1) are a relatively frequent cause of parkinsonism with onset before the age of 50, having a varied but generally mild phenotype. DJ-1 mutations are less common than parkin mutations, but the phenotype is broadly similar (2). Given that recessive mutations in either of these two genes cause parkinsonism, it is likely that mutations in either parkin or DJ-1 lead to loss of dopaminergic neurons in the substantia nigra that project to the striatum. Positron-emission tomography demonstrates a loss of dopaminergic function in parkin (e.g., 3-6) and DJ-1 (7, 8) patients, supporting this idea. Therefore, although detailed pathology of these two genetic forms of parkinsonism is not available, there are clear phenotypic overlaps. This conclusion argues that there are relationships between parkin and DJ-1, but the normal functions of the two wild-type proteins are not obviously linked. Parkin is an E3 protein-ubiquitin ligase (9), whereas DJ-1 may have a number of roles (10) but can affect the ability of neurons to survive oxidative stress generated as a result of mitochondrial damage (11).Recently, mutations in the PTEN-induced putative kinase 1 (PINK1) gene have been described that are also associated with recessive parkinsonism. Initially, three pedigrees were described with identified mutations: a G309D point substitution in one family and a truncation mutation (W437X) in two additional families (12). Subsequently, several studies have described co...

The formation of cysteine-sulfinic acid has recently become appreciated as a modification that links protein function to cellular oxidative status. Human DJ-1, a protein associated with inherited parkinsonism, readily forms cysteine-sulfinic acid at a conserved cysteine residue (Cys 106 in human DJ-1). Mutation of Cys 106 causes the protein to lose its normal protective function in cell culture and model organisms. However, it is unknown whether the loss of DJ-1 protective function in these mutants is due to the absence of Cys 106 oxidation or the absence of the cysteine residue itself. To address this question, we designed a series of substitutions at a proximal glutamic acid residue (Glu 18 ) in human DJ-1 that alter the oxidative propensity of Cys 106 through changes in hydrogen bonding. We show that two mutations, E18N and E18Q, allow Cys 106 to be oxidized to Cys 106 -sulfinic acid under mild conditions. In contrast, the E18D mutation stabilizes a cysteine-sulfenic acid that is readily reduced to the thiol in solution and in vivo. We show that E18N and E18Q can both partially substitute for wild-type DJ-1 using mitochondrial fission and cell viability assays. In contrast, the oxidatively impaired E18D mutant behaves as an inactive C106A mutant and fails to protect cells. We therefore conclude that formation of Cys 106 -sulfinic acid is a key modification that regulates the protective function of DJ-1.Reactive cysteine residues are susceptible to a variety of covalent modifications that are increasingly recognized as a major means of regulating the activities of many proteins (1). Cysteine forms three different species by the direct addition of oxygen; cysteine-sulfenic (-SOH), -sulfinic (-SO 2 H), and -sulfonic (-SO 3 H) acid. Because cysteine can be oxidized to three distinct species, each with different structural and chemical properties, cysteine oxidation is a versatile way for reactive oxygen species (ROS) 4 to alter the activity of a protein.Of the three oxidation products of cysteine, only cysteine-sulfenic acid is readily reduced to the thiol under physiological conditions. However, enzymes that catalyze the ATP-dependent reduction of overoxidized peroxiredoxins containing cysteine-sulfinic acid to cysteine have been discovered and characterized (2, 3). With reversibility comes the potential for cysteine-sulfinic acid modifications to modulate the function of various target proteins in a redox-dependent manner. Therefore, at least in some proteins, cysteine-sulfinic acid should be regarded as a post-translational modification rather than simply a type of protein damage.As expected, many of the proteins that are modified by cysteine oxidation are involved in the oxidative stress response or in the maintenance of cellular redox homeostasis. Of these proteins, DJ-1 has special importance in understanding the role of regulatory cysteine oxidation in neuronal survival. Loss of function mutations in DJ-1 are a rare cause of early onset recessive parkinsonism (4, 5), although the exact function of DJ-1 i...

PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.

Mutations in a gene on chromosome 1, DJ-1, have been reported recently to be associated with recessive, earlyonset Parkinson's disease. While one mutation is a large deletion that is predicted to produce an effective knockout of the gene, the second is a point mutation, L166P, whose precise effects on protein function are unclear. In the present study, we show that L166P destabilizes DJ-1 protein and promotes its degradation through the ubiquitin-proteasome system. A double mutant (K130R, L166P) was more stable than L166P, suggesting that this lysine residue contributes to stability of the protein. Subcellular localization was broadly similar for both wild type and L166P forms of the protein, indicating that the effect of the mutation is predominantly on protein stability. These observations are reminiscent of other recessive gene mutations that produce an effective loss of function. The L166P mutation has the simple effect of promoting DJ-1 degradation, thereby reducing net DJ-1 protein within the cell.

Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanisms involved in the degeneration of neurons remain unclear. DJ-1 mutations are one cause of recessive parkinsonism, but this gene is also reported to be involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of DJ-1 is unknown, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here, we show, using four independent methods, that DJ-1 associates with RNA targets in cells and the brain, including mitochondrial genes, genes involved in glutathione metabolism, and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that DJ-1 is sufficient for RNA binding at nanomolar concentrations. Further, we show that DJ-1 binds RNA but dissociates after oxidative stress. These data implicate a single mechanism for the pleiotropic effects of DJ-1 in different model systems, namely that the protein binds multiple RNA targets in an oxidation-dependent manner.gene expression ͉ oxidative stress ͉ Parkinson's disease ͉ translation M utations in any of three genes cause a recessively inherited early-onset movement disorder reminiscent of Parkinson's disease (PD). Parkin is an E3 protein-ubiquitin ligase and PINK1 is a mitochondrial kinase (1). Results from Drosophila models suggest that PINK1 and parkin define a single pathway that, when disrupted, leads to mitochondrial damage (2, 3). The third, and rarest, gene for recessive parkinsonism is DJ-1. The DJ-1 protein responds to oxidative stress evidenced by a pI shift in sporadic PD (4, 5) and in cell (6, 7) and animal (8) models. DJ-1 knockout models also show increased sensitivity to toxins that cause mitochondrial dysfunction or oxidative stress (9-13). Cys-106 of DJ-1, which is oxidized to form a cysteine-sulfinic acid, is critically required for DJ-1 to protect against these types of damage both in vitro (6) and in vivo (12). However, the molecular function of DJ-1 is unclear. DJ-1 is part of the ThiJ/PfPI superfamily but the proteins most similar to DJ-1 form a distinct clade away from other members with known function (14, 15), implying a novel activity. As well as effects on oxidation and mitochondrial function, DJ-1 enhances Ras-mediated oncogenesis (16), modulates the PTEN/Akt survival pathway (17,18), suppresses Ask1-mediated apoptosis (19), and increases glutathione (GSH) synthesis, Hsp70 (20) and tyrosine hydroxylase (21, 22) expression. DJ-1 is a small, dimeric, single-domain protein (23), so if all of these effects are true then either the protein has multiple functions or there is a single biochemical activity that explains all of them.One of the original descriptions of the cloning of DJ-1 was as RS, a regulatory component of an RNA binding complex (24). Therefore, we examined whether DJ-1 associates with R...

Abnormal accumulation of a-synuclein in Lewy bodies is a neuropathological hallmark of both sporadic and familial Parkinson's disease (PD). Although mutations in a-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic cell death occurs remains unknown. We investigated transcriptional changes in neuroblastoma cell lines transfected with either normal or mutant (A30P or A53T) a-synuclein using microarrays, with confirmation of selected genes by quantitative RT-PCR. Gene products whose expression was found to be significantly altered included members of diverse functional groups such as stress response, transcription regulators, apoptosis-inducing molecules, transcription factors and membrane-bound proteins. We also found evidence of altered expression of dihydropteridine reductase, which indirectly regulates the synthesis of dopamine. Because of the importance of dopamine in PD, we investigated the expression of all the known genes in dopamine synthesis. We found co-ordinated downregulation of mRNA for GTP cyclohydrolase, sepiapterin reductase (SR), tyrosine hydroxylase (TH) and aromatic acid decarboxylase by wild-type but not mutant a-synuclein. These were confirmed at the protein level for SR and TH. Reduced expression of the orphan nuclear receptor Nurr1 was also noted, suggesting that the co-ordinate regulation of dopamine synthesis is regulated through this transcription factor.