Epidermal Growth Factor-dependent Activation of the Extracellular Signal-regulated Kinase Pathway by DJ-1 Protein through Its Direct Binding to c-Raf Protein

DJ-1 is an oncogene and also a causative gene for familial Parkinson disease. DJ-1 has various functions, and the oxidative status of cysteine at position 106 (Cys-106) is crucial for determination of the activation level of DJ-1. Although DJ-1 requires activated Ras for its oncogenic activity and although it activates the extracellular signal-regulated kinase (ERK) pathway, a cell growth pathway downstream of Ras, the precise mechanism underlying activation of the ERK pathway by DJ-1 is still not known. In this study, we found that DJ-1 directly bound to the kinase domain of c-Raf but not to Ras and that Cys-106 mutant DJ-1 bound to c-Raf more weakly than did wild-type DJ-1. Co-localization of DJ-1 with c-Raf in the cytoplasm was enhanced in epidermal growth factor (EGF)-treated cells. Knockdown of DJ-1 expression attenuated the phosphorylation level of c-Raf in EGF-treated cells, resulting in reduced activation of MEK and ERK1/2. Although EGF-treated DJ-1 knock-out cells also showed attenuated c-Raf activation, reintroduction of wild-type DJ-1, but not C106S DJ-1, into DJ-1 knock-out cells restored c-Raf activation in a DJ-1 binding activity in a c-Raf-dependent manner. DJ-1 was not responsible for activation of c-Raf in phorbol myristate acetate-treated cells. Furthermore, DJ-1 stimulated self-phosphorylation activity of c-Raf in vitro, but DJ-1 was not a target for Raf kinase. Oxidation of Cys-106 in DJ-1 was not affected by EGF treatment. These findings showed that DJ-1 is a positive regulator of the EGF/Ras/ERK pathway through targeting c-Raf.     

The reversible low-temperature instability of human DJ-1 oxidative states

DJ-1 is a homodimeric protein that is centrally involved in various human diseases including Parkinson disease (PD). DJ-1 protects against oxidative damage and mitochondrial dysfunction through a homeostatic control of reactive oxygen species (ROS). DJ-1 pathology results from a loss of function, where ROS readily oxidizes a highly conserved and functionally essential cysteine (C106). The over-oxidation of DJ-1 C106 leads to a dynamically destabilized and biologically inactivated protein. An analysis of the structural stability of DJ-1 as a function of oxidative state and temperature may provide further insights into the role the protein plays in PD progression. NMR spectroscopy, circular dichroism, analytical ultracentrifugation sedimentation equilibrium, and molecular dynamics simulations were utilized to investigate the structure and dynamics of the reduced, oxidized (C106-SO₂⁻), and over-oxidized (C106-SO₃⁻) forms of DJ-1 for temperatures ranging from 5°C to 37°C. The three oxidative states of DJ-1 exhibited distinct temperature-dependent structural changes. A cold-induced aggregation occurred for the three DJ-1 oxidative states by 5°C, where the over-oxidized state aggregated at significantly higher temperatures than both the oxidized and reduced forms. Only the oxidized and over-oxidized forms of DJ-1 exhibited a mix state containing both folded and partially denatured protein that likely preserved secondary structure content. The relative amount of this denatured form of DJ-1 increased as the temperature was lowered, consistent with a cold-denaturation. Notably, the cold-induced aggregation and denaturation for the DJ-1 oxidative states were completely reversible. The dramatic changes in the structural stability of DJ-1 as a function of oxidative state and temperature are relevant to its role in PD and its functional response to oxidative stress.     

Cysteine pKa depression by a protonated glutamic acid in human DJ-1

Human DJ-1, a disease-associated protein that protects cells from oxidative stress, contains an oxidation-sensitive cysteine (C106) that is essential for its cytoprotective activity. The origin of C106 reactivity is obscure, due in part to the absence of an experimentally determined p K a value for this residue. We have used atomic-resolution X-ray crystallography and UV spectroscopy to show that C106 has a depressed p K a of 5.4 +/- 0.1 and that the C106 thiolate accepts a hydrogen bond from a protonated glutamic acid side chain (E18). X-ray crystal structures and cysteine p K a analysis of several site-directed substitutions at residue 18 demonstrate that the protonated carboxylic acid side chain of E18 is required for the maximal stabilization of the C106 thiolate. A nearby arginine residue (R48) participates in a guanidinium stacking interaction with R28 from the other monomer in the DJ-1 dimer and elevates the p K a of C106 by binding an anion that electrostatically suppresses thiol ionization. Our results show that the ionizable residues (E18, R48, and R28) surrounding C106 affect its p K a in a way that is contrary to expectations based on the typical ionization behavior of glutamic acid and arginine. Lastly, a search of the Protein Data Bank (PDB) produces several candidate hydrogen-bonded aspartic/glutamic acid-cysteine interactions, which we propose are particularly common in the DJ-1 superfamily.     

BRSK2 is regulated by ER stress in protein level and involved in ER stress-induced apoptosis

Loss-of-functional mutation in the DJ-1 gene causes a subset of familial Parkinson's disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. Dopamine is synthesized by two enzymes and then packed into synaptic vesicles by vesicular monoamine transporter 2 (VMAT2). In this study, we found that knockdown of DJ-1 expression reduced the levels of mRNA and protein of VMAT2, resulting in reduced VMAT2 activity. Co-immunoprecipitation and pull-down experiments revealed that DJ-1 directly bound to VMAT2, and DJ-1 was co-localized with VMAT2 in cells. Furthermore, ectopic expression of wild-type DJ-1, but not that of L166P, M26I and C106S mutants of DJ-1, increased mRNA and protein levels of VMAT2 and VMAT2 activity. Since VMAT2 and a portion of DJ-1 are localized in the synaptic membrane, these results suggest that DJ-1, but not pathogenically mutated DJ-1, stimulates VMAT2 activity in the synapse by transactivation of the VMAT gene and by direct binding to VMAT2 and that cysteine 106 is necessary for the stimulating activity of DJ-1 toward VMAT2.     

Cysteine-106 of DJ-1 is the most sensitive cysteine residue to hydrogen peroxide-mediated oxidation in vivo in human umbilical vein endothelial cells

Mutation in DJ-1 gene is the cause of autosomal recessive Parkinson's disease, however, its physiological function remains unclear. The isoelectric point of DJ-1 shows an acidic shift after cells are treated with hydrogen peroxide. This suggests that DJ-1 is modified in response to oxidative stress. Here we report the structural characterization of an acidic isoform of DJ-1 using a proteomic approach with nanospray interface liquid chromatography-electrospray ionization/linear ion trap mass spectrometer. When human umbilical vein endothelial cells were exposed to hydrogen peroxide, all three cysteines in DJ-1 were oxidized to cysteine sulphonic acid. Although a small part of the Cys-46 and Cys-53 were oxidized, Cys-106 was oxidized completely at any hydrogen peroxide concentration used here. These results suggest that Cys-106 is the most sensitive among three cysteine residues to oxidative stress, and that DJ-1 function is regulated, in terms of the intracellular redox state, by oxidation of Cys-106.     

Crystal structure of human DJ-1, a protein associated with early onset Parkinson's disease

We report the crystal structure at 1.8-A resolution of human DJ-1, which has been linked to early onset Parkinson's disease. The monomer of DJ-1 contains the alpha/beta-fold that is conserved among members of the DJ-1/ThiJ/PfpI superfamily. However, the structure also contains an extra helix at the C terminus, which mediates a novel mode of dimerization for the DJ-1 proteins. A putative active site has been identified near the dimer interface, and the residues Cys-106, His-126, and Glu-18 may play important roles in the catalysis by this protein. Studies with the disease-causing L166P mutant suggest that the mutation has disrupted the C-terminal region and the dimerization of the protein. The DJ-1 proteins may function only as dimers. The Lys to Arg mutation at residue 130, the site of sumoylation of DJ-1, has minimal impact on the structure of the protein.     

Effect of single amino acid substitution on oxidative modifications of the Parkinson's disease-related protein, DJ-1

Mutations in the gene encoding DJ-1 have been identified in patients with familial Parkinson's disease (PD) and are thought to inactivate a neuroprotective function. Oxidation of the sulfhydryl group to a sulfinic acid on cysteine residue C106 of DJ-1 yields the "2O " form, a variant of the protein with enhanced neuroprotective function. We hypothesized that some familial mutations disrupt DJ-1 activity by interfering with conversion of the protein to the 2O form. To address this hypothesis, we developed a novel quantitative mass spectrometry approach to measure relative changes in oxidation at specific sites in mutant DJ-1 as compared with the wild-type protein. Treatment of recombinant wild-type DJ-1 with a 10-fold molar excess of H(2)O(2) resulted in a robust oxidation of C106 to the sulfinic acid, whereas this modification was not detected in a sample of the familial PD mutant M26I exposed to identical conditions. Methionine oxidized isoforms of wild-type DJ-1 were depleted, presumably as a result of misfolding and aggregation, under conditions that normally promote conversion of the protein to the 2O form. These data suggest that the M26I familial substitution and methionine oxidation characteristic of sporadic PD may disrupt DJ-1 function by disfavoring a site-specific modification required for optimal neuroprotective activity. Our findings indicate that a single amino acid substitution can markedly alter a protein's ability to undergo oxidative modification, and they imply that stimulating the conversion of DJ-1 to the 2O form may be therapeutically beneficial in familial or sporadic PD.     

Differential effects of Parkinson's disease-associated mutations on stability and folding of DJ-1

Mutations in the PARK7/DJ-1 gene cause autosomal-recessive Parkinson's disease. In some patients the gene is deleted. The molecular basis of disease in patients with point mutations is less obvious. We have investigated the molecular properties of [L166P]DJ-1 and the novel variant [E64D]DJ-1. When transfected into non-neuronal and neuronal cell lines, steady-state expression levels of [L166P]DJ-1 were dramatically lower than wild-type [WT]DJ-1 and [E64D]DJ-1. Cycloheximide and pulse-chase experiments revealed that the decreased expression levels of [L166P]DJ-1 were because of accelerated protein turnover. Proteasomal degradation was not the major pathway of DJ-1 breakdown because treatment with the proteasome inhibitor MG-132 caused only minimal accumulation of DJ-1, even of the very unstable [L166P]DJ-1 mutant. Because of the structural resemblance of DJ-1 with bacterial cysteine proteases, we considered an autoproteolytic mechanism. However, neither pharmacological inhibition nor site-directed mutagenesis of the putative active site residue Cys-106 stabilized DJ-1. To gain further insight into the structural defects of DJ-1 mutants, human [WT]DJ-1 and both mutants were expressed in Escherichia coli. As in eukaryotic cells, expression levels of [L166P]DJ-1 were dramatically reduced compared with [WT]DJ-1 and [E64D]DJ-1. Circular dichroism spectrometry revealed that the solution structures of [WT]DJ-1 and [E64D]DJ-1 are rich in beta-strand and alpha-helix conformation. Alpha-helices were more susceptible to thermal denaturation than the beta-sheet, and [WT]DJ-1 was more flexible in this regard than [E64D]DJ-1. Thus, structural defects of [E64D]DJ-1 only become apparent upon denaturing conditions, whereas the L166P mutation causes a drastic defect that leads to excessive degradation.     

Oxidized DJ-1 interacts with the mitochondrial protein BCL-XL

Parkinson disease (PD)- and cancer-associated protein, DJ-1, mediates cellular protection via many signaling pathways. Deletions or mutations in the DJ-1 gene are directly linked to autosomal recessive early-onset PD. DJ-1 has potential roles in mitochondria. Here, we show that DJ-1 increases its mitochondrial distribution in response to ultraviolet B (UVB) irradiation and binds to Bcl-X(L). The interactions between DJ-1 and Bcl-X(L) are oxidation-dependent. DJ-1(C106A), a mutant form of DJ-1 that is unable to be oxidized, binds Bcl-X(L) much less than DJ-1 does. Moreover, DJ-1 stabilizes Bcl-X(L) protein level by inhibiting its ubiquitination and degradation through ubiquitin proteasome system (UPS) in response to UVB irradiation. Furthermore, under UVB irradiation, knockdown of DJ-1 leads to increases of Bcl-X(L) ubiquitination and degradation upon UVB irradiation, thereby increasing mitochondrial Bax, caspase-3 activation and PARP cleavage. These data suggest that DJ-1 protects cells against UVB-induced cell death dependent on its oxidation and its association with mitochondrial Bcl-X(L).     

Destabilization of DJ-1 by familial substitution and oxidative modifications: implications for Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder characterized by oxidative stress and protein aggregation. Both toxic phenomena are mitigated by DJ-1, a homodimeric protein with proposed antioxidant and chaperone activities. The neuroprotective function of DJ-1 is modulated by oxidation of cysteine 106, a residue that may act as an oxidative stress sensor. Loss-of-function mutations in the DJ-1 gene have been linked to early onset PD, and age-dependent over-oxidation of DJ-1 is thought to contribute to sporadic PD. The familial mutant L166P fails to dimerize and is rapidly degraded, suggesting that protein destabilization accounts for the dysfunction of this mutant. In this study, we investigated how the structure and stability of DJ-1 are impacted by two other pathogenic substitutions (M26I and E64D) and by over-oxidation with H2O2. Whereas the recombinant wild-type protein and E64D both adopted a stable dimeric structure, M26I showed an increased propensity to aggregate and decreased secondary structure. Similar to M26I, over-oxidized wild-type DJ-1 exhibited reduced secondary structure, and this property correlated with destabilization of the dimer. The engineered mutant C106A had a greater thermodynamic stability and was more resistant to oxidation-induced destabilization than the wild-type protein. These results suggest that (i) the M26I substitution and over-oxidation destabilize dimeric DJ-1, and (ii) the oxidation of cysteine 106 contributes to DJ-1 destabilization. Our findings provide a structural basis for DJ-1 dysfunction in familial and sporadic PD, and they suggest that dimer stabilization is a reasonable therapeutic strategy to treat both forms of this disorder.     

Oxidation of DJ-1 induced by 6-hydroxydopamine decreasing intracellular glutathione

DJ-1, the causative gene of a familial form of Parkinson's disease (PD), has been reported to undergo preferential oxidation of the cysteine residue at position 106 (Cys-106) under oxidative stress; however, details of the molecular mechanisms are not well known. In the present study, mechanisms of DJ-1 oxidation induced by 6-hydroxydopamine (6-OHDA) were investigated by using SH-SY5Y cells. The treatment of these cells with 6-OHDA caused an obvious acidic spot sift of DJ-1 due to its oxidation. However, when catalase, which is an hydrogen peroxide (H(2)O(2))-removing enzyme, was added during the treatment, it failed to prevent the oxidation induced by 6-OHDA, suggesting that electrophilic p-quinone formed from 6-OHDA, but not H(2)O(2), was responsible for the DJ-1 oxidation. Benzoquinone, another electrophilic p-quinone, also induced DJ-1 oxidation. The intracellular glutathione (GSH) levels were significantly decreased by 6-OHDA, irrespective of the presence or absence of catalase. The inhibition of GSH synthesis by buthionine sulfoximine resulted in a decrease in GSH levels and enhancement of DJ-1 oxidation. The pretreatment of cells with N-acetyl-cysteine prevented the loss of intracellular GSH and subsequently DJ-1 oxidation induced by 6-OHDA. Collectively, these results suggest that electrophilic p-quinone formed from 6-OHDA induces DJ-1 oxidation by decreasing intracellular GSH.     

Roles of distinct cysteine residues in S-nitrosylation and dimerization of DJ-1

A significant proportion of early onset parkinsonism is inherited as an autosomal-recessive trait (AR-EP). DJ-1 was identified as one of the causative genes for AR-EP (PARK7), and DJ-1 protein has been implicated in oxidative stress response through oxidation of one of the three cysteine residues (i.e., Cys106). However, the individual roles of these cysteine residues remained unclear. We show by a systematic mutagenesis analysis that Cys46 and Cys53 of DJ-1, but not Cys106, are susceptible to S-nitrosylation in vitro as well as in cultured cells. Furthermore, alanine substitution of Cys46 diminished dimerization of DJ-1, a fundamental feature of this protein. These results indicate that distinct cysteine residues of DJ-1 harbor differential roles in relation to its structure and function.     

Choice of biological source material supersedes oxidative stress in its influence on DJ-1 in vivo interactions with Hsp90

DJ-1 is a small but relatively abundant protein of unknown function that may undergo stress-dependent cellular translocation and has been implicated in both neurodegenerative diseases and cancer. As such, DJ-1 may be an excellent study object to elucidate the relative influence of the cellular context on its interactome and for exploring whether acute exposure to oxidative stressors alters its molecular environment. Using quantitative mass spectrometry, we conducted comparative DJ-1 interactome analyses from in vivo cross-linked brains or livers and from hydrogen peroxide-treated or na?ve embryonic stem cells. The analysis identified a subset of glycolytic enzymes, heat shock proteins 70 and 90, and peroxiredoxins as interactors of DJ-1. Consistent with a role of DJ-1 in Hsp90 chaperone biology, we document destabilization of Hsp90 clients in DJ-1 knockout cells. We further demonstrate the existence of a C106 sulfinic acid modification within DJ-1 and thereby establish that this previously inferred modification also exists in vivo. Our data suggest that caution has to be exerted in interpreting interactome data obtained from a single biological source material and identify a role of DJ-1 as an oxidative stress sensor and partner of a molecular machinery notorious for its involvement in cell fate decisions.     

Conservation of oxidative protein stabilization in an insect homologue of parkinsonism-associated protein DJ-1

DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1α and DJ-1β) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1β. The structure of D. melanogaster DJ-1β is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1β. His126 in human DJ-1 is substituted with a tyrosine in DJ-1β, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO(2)(-)) results in considerable thermal stabilization of both Drosophila DJ-1β and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.