Structural effects of Parkinson's disease linked DJ-1 mutations

Mutations in the protein DJ-1 are associated with familial forms of Parkinson's disease, indicating that DJ-1 may be involved in pathways related to the etiology of this disorder. Here we have used solution state NMR and circular dichroism spectroscopies to evaluate the extent of structural perturbations associated with five different Parkinson's disease linked DJ-1mutations: L166P, E64D, M26I, A104T, and D149A. Comparison of the data with those obtained for the wild-type protein shows that the L166P mutation leads to severe and global destabilization and unfolding of the protein structure, while the structure of the E64D mutation, as expected, is nearly unperturbed. Interestingly, the remaining three mutants all show different degrees of structural perturbation, which are accompanied by a reduction in the thermodynamic stability of the protein. The observed structural and thermodynamic differences are likely to underlie any functional variations between these mutants and the wild type, which in turn are likely responsible for the pathogenicity of these mutations.     

Reduced protein stability of human DJ-1/PARK7 L166P, linked to autosomal recessive Parkinson disease, is due to direct endoproteolytic cleavage by the proteasome

Parkinson's disease (PD) is characterized by dopaminergic dysfunction and degeneration. DJ-1/PARK7 mutations have been linked with a familial form of early onset PD. In this study, we found that human DJ-1 wild type and the missense mutants M26I, R98Q, A104T and D149A were stable proteins in cells, only the L166P mutant was unstable. In parallel, the former were not degraded and the L166P mutant was directly degraded in vitro by proteasome-mediated endoproteolytic cleavage. Furthermore, genetic evidence in fission yeast showed the direct involvement of proteasome in the degradation of human DJ-1 L166P and the corresponding L169P mutant of SPAC22E12.03c, the human orthologue of DJ-1 in Schizosaccharomyces Pombe, as their protein levels were increased at restrictive temperature in fission yeast (mts4 and pts1-732) harboring temperature sensitive mutations in proteasomal subunits. In total, our results provide evidence that direct proteasomal endoproteolytic cleavage of DJ-1 L166P is the mechanism of degradation contributing to the loss-of-function of the mutant protein, a property not shared by other DJ-1 missense mutants associated with PD.     

Structural determinants of the C-terminal helix-kink-helix motif essential for protein stability and survival promoting activity of DJ-1

Mutations in the PARK7 gene encoding DJ-1 cause autosomal recessive Parkinson disease. The most deleterious point mutation is the L166P substitution, which resides in a structure motif comprising two alpha-helices (G and H) separated by a kink. Here we subjected the C-terminal helix-kink-helix motif to systematic site-directed mutagenesis, introducing helix-incompatible proline residues as well as conservative substitutions into the helical interface. Furthermore, we generated deletion mutants lacking the H-helix, the kink, and the entire C terminus. When transfected into neural and nonneural cell lines, steady-state levels of G-helix breaking and kink deletion mutants were dramatically lower than wild-type DJ-1. The effects of H-helix breakers were comparably smaller, and the non-helix breaking mutants only slightly destabilized DJ-1. The decreased steady-state levels were due to accelerated protein degradation involving in part the proteasome. G-helix breaking DJ-1 mutations abolished dimer formation. These structural perturbations had functional consequences on the cytoprotective activities of DJ-1. The destabilizing mutations conferred reduced cytoprotection against H(2)O(2) in transiently retransfected DJ-1 knock-out mouse embryonic fibroblasts. The loss of survival promoting activity of the DJ-1 mutants with destabilizing C-terminal mutations correlated with impaired anti-apoptotic signaling. We found that wild-type, but not mutant DJ-1 facilitated the Akt pathway and simultaneously blocked the apoptosis signal-regulating kinase 1, with which DJ-1 interacted in a redox-dependent manner. Thus, the G-helix and kink are critical determinants of the C-terminal helix-kink-helix motif, which is absolutely required for stability and the regulation of survival-promoting redox signaling of the Parkinson disease-associated protein DJ-1.     

Parkinson's disease-associated DJ-1 mutations impair mitochondrial dynamics and cause mitochondrial dysfunction

Mitochondrial dysfunction represents a critical event during the pathogenesis of Parkinson's disease (PD) and expanding evidences demonstrate that an altered balance in mitochondrial fission/fusion is likely an important mechanism leading to mitochondrial and neuronal dysfunction/degeneration. In this study, we investigated whether DJ-1 is involved in the regulation of mitochondrial dynamics and function in neuronal cells. Confocal and electron microscopic analysis demonstrated that M17 human neuroblastoma cells over-expressing wild-type DJ-1 (WT DJ-1 cells) displayed elongated mitochondria while M17 cells over-expressing PD-associated DJ-1 mutants (R98Q, D149A and L166P) (mutant DJ-1 cells) showed significant increase of fragmented mitochondria. Similar mitochondrial fragmentation was also noted in primary hippocampal neurons over-expressing PD-associated mutant forms of DJ-1. Functional analysis revealed that over-expression of PD-associated DJ-1 mutants resulted in mitochondria dysfunction and increased neuronal vulnerability to oxidative stress (H(2) O(2)) or neurotoxin. Further immunoblot studies demonstrated that levels of dynamin-like protein (DLP1), also known as Drp1, a regulator of mitochondrial fission, was significantly decreased in WT DJ-1 cells but increased in mutant DJ-1 cells. Importantly, DLP1 knockdown in these mutant DJ-1 cells rescued the abnormal mitochondria morphology and all associated mitochondria/neuronal dysfunction. Taken together, these studies suggest that DJ-1 is involved in the regulation of mitochondrial dynamics through modulation of DLP1 expression and PD-associated DJ-1 mutations may cause PD by impairing mitochondrial dynamics and function.     

A Two-step Protein Quality Control Pathway for a Misfolded DJ-1 Variant in Fission Yeast

A mutation, L166P, in the cytosolic protein, PARK7/DJ-1, causes protein misfolding and is linked to Parkinson disease. Here, we identify the fission yeast protein Sdj1 as the orthologue of DJ-1 and calculate by in silico saturation mutagenesis the effects of point mutants on its structural stability. We also map the degradation pathways for Sdj1-L169P, the fission yeast orthologue of the disease-causing DJ-1 L166P protein. Sdj1-L169P forms inclusions, which are enriched for the Hsp104 disaggregase. Hsp104 and Hsp70-type chaperones are required for efficient degradation of Sdj1-L169P. This also depends on the ribosome-associated E3 ligase Ltn1 and its co-factor Rqc1. Although Hsp104 is absolutely required for proteasomal degradation of Sdj1-L169P aggregates, the degradation of already aggregated Sdj1-L169P occurs independently of Ltn1 and Rqc1. Thus, our data point to soluble Sdj1-L169P being targeted early by Ltn1 and Rqc1. The fraction of Sdj1-L169P that escapes this first inspection then forms aggregates that are subsequently cleared via an Hsp104- and proteasome-dependent pathway.     

Molecular basis for the structural instability of human DJ-1 induced by the L166P mutation associated with Parkinson's disease

DJ-1 is a dimeric protein of unknown function in vivo. A mutation in the human DJ-1 gene causing substitution of proline for leucine at residue 166 (L166P) has been linked to early onset Parkinson's disease. Lack of structural stability has precluded experimental determination of atomic-resolution structures of the L166P DJ-1 polymorph. We have performed multiple molecular dynamics (MD) simulations ( approximately 1/3 mus) of the wild-type and L166P DJ-1 polymorph at physiological temperature to predict specific structural effects of the L166P substitution. L166P disrupted helices alpha1, alpha5, alpha6 and alpha8 with alpha8 undergoing particularly severe disruption. Secondary structural elements critical for protein stability and dimerization were significantly disrupted across the entire dimer interface, as were extended hydrophobic surfaces involved in dimer formation. Relative to wild-type DJ-1, L166P DJ-1 populated a broader ensemble of structures, many of which corresponded to distorted conformations. In a L166P dimer model the substitution significantly destabilized the dimer interface, interrupting >100 intermolecular contacts that are important for dimer formation. The L166P substitution also led to major perturbations in the region of a highly conserved cysteine residue (Cys-106) that participates in dimerization and that is critical for a proposed chaperone function of DJ-1. Cys-106 is located approximately 16 A from the substitution site, demonstrating that structural disruptions propagate throughout the whole protein. Furthermore, L166P DJ-1 showed a significant increase in hydrophobic surface area relative to wild-type protein, possibly explaining the tendency of the mutant protein to aggregate. These simulations provide details about specific structural disturbances throughout L166P DJ-1 that previous studies have not revealed.     

Structural impact of three Parkinsonism-associated missense mutations on human DJ-1

A number of missense mutations in the oxidative stress response protein DJ-1 are implicated in rare forms of familial Parkinsonism. The best-characterized Parkinsonian DJ-1 missense mutation, L166P, disrupts homodimerization and results in a poorly folded protein. The molecular basis by which the other Parkinsonism-associated mutations disrupt the function of DJ-1, however, is incompletely understood. In this study we show that three different Parkinsonism-associated DJ-1 missense mutations (A104T, E163K, and M26I) reduce the thermal stability of DJ-1 in solution by subtly perturbing the structure of DJ-1 without causing major folding defects or loss of dimerization. Atomic resolution X-ray crystallography shows that the A104T substitution introduces water and a discretely disordered residue into the core of the protein, E163K disrupts a key salt bridge with R145, and M26I causes packing defects in the core of the dimer. The deleterious effect of each Parkinsonism-associated mutation on DJ-1 is dissected by analysis of engineered substitutions (M26L, A104V, and E163K/R145E) that partially alleviate each of the defects introduced by the A104T, E163K and M26I mutations. In total, our results suggest that the protective function of DJ-1 can be compromised by diverse perturbations in its structural integrity, particularly near the junctions of secondary structural elements.     

DJ-1~（L166P）与DJ-1~（M26I）基因对NIH3T3细胞增殖和凋亡的比较

目的在细胞学水平比较DJ、DJ-1M26 I、DJ-1L166 P基因对NIH 3T3细胞增殖速率与凋亡的关系,为建立转基因动物模型及帕金森疾病发病机制研究奠定基础。方法分别将pcDNA3.1/myc-His-DJ-1、pcDNA3.1/myc-His-DJ-1L166 P和pcDNA3.1/myc-His-DJ-1M26 I重组质粒脂质体方法转染NIH 3T3细胞,500μg/ml G418压力筛选稳定克隆,对3种转染细胞在DNA水平、RNA水平和蛋白质水平进行鉴定,采用MTT染色方法和Annexin V-FITC试剂盒进行转染阳性克隆细胞的细胞活力与细胞凋亡检测。结果 pcDNA3.1/myc-His-DJ-1、pcDNA3.1/myc-His-DJ-1L166 P和pcDNA3.1/myc-His-DJ-1M26 I重组质粒转染NIH 3T3细胞经G418筛选后,PCR方法检测分别获得1个、4个、3个阳性细胞克隆,RT-PCR及Western blot方法进行DJ-1-His基因表达检测,结果均证明外源插入基因的表达,Caspase-3 RNA水平检测DJ-1L166 P和DJ-1M26 I组表达高于正常NIH 3T3细胞组,而DJ-1组caspase-3转录水平相对最低,MTT实验结果初步证明转染DJ-1L166 P和DJ-1M26 I基因的NIH3T3阳性细胞组细胞增殖速率均低于DJ-1组和正常NIH 3T3细胞组（P〈0.05）,转染DJ-1基因的NIH 3T3阳性细胞增殖速率与正常NIH 3T3细胞相比无明显差别;细胞凋亡检测表明转染DJ-1L166 P和DJ-1M26 I基因的NIH3T3阳性细胞凋亡率均高于正常NIH 3T3细胞,转染DJ-1基因的NIH 3T3阳性细胞凋亡率低于正常NIH 3T3细胞（P〈0.05）。结论 DJ-1L166 P和DJ-1M26 I基因突变均降低NIH3T3细胞增殖速率,DJ-1L166 P和DJ-1M26 I基因突变更易导致NIH 3T3细胞的凋亡,DJ-1L166 P和DJ-1M26 I基因突变对NIH3T3细胞增殖速率和细胞凋亡影响是相似的。     

Elevated expression of DJ-1 (encoded by the human PARK7 gene) protects neuronal cells from sevoflurane-induced neurotoxicity

Sevoflurane, an inhaled ether general anesthetic agent, exerts a variety of neurotoxic effects, including oxidative stress, mitochondrial dysfunction, and neuronal apoptosis. However, the underlying molecular mechanisms remain to be elucidated. DJ-1 is a protein that exerts neuroprotective effects against different kinds of stress through multiple pathways. This study aimed to investigate the neuroprotective effects of DJ-1 against sevoflurane-induced neurotoxicity. Here, we found that sevoflurane treatment significantly increased DJ-1 expression in human neuroblastoma M17 cells in a dose-dependent manner at both the mRNA and protein levels. Interestingly, we found that overexpression of wild-type (WT) DJ-1 prevented sevoflurane-induced generation of reactive oxygen species (ROS) and nitric oxide (NO), deletion of reduced GSH, reduction of adenosine triphosphate (ATP), and mitochondrial membrane potential. Interestingly, we found that WT DJ-1 could inhibit sevoflurane-induced apoptosis by modulating the mitochondrial pathway. However, its “loss of function” mutation DJ-1(L166P) exacerbated sevoflurane-induced neurotoxicity in M17 cells. Our findings suggest that WT DJ-1 protects neuronal cells against sevoflurane-induced neurotoxicity.     

A missense mutation (L166P) in DJ-1, linked to familial Parkinson's disease, confers reduced protein stability and impairs homo-oligomerization

The identification of genetic mutations responsible for rare familial forms of Parkinson's disease (PD) have provided tremendous insight into the molecular pathogenesis of this disorder. Mutations in the DJ-1 gene cause autosomal recessive early onset PD in two European families. A Dutch kindred displays a large homozygous genomic deletion encompassing exons 1-5 of the DJ-1 gene, whereas an Italian kindred harbors a single homozygous L166P missense mutation. A homozygous M26I missense mutation was also recently reported in an Ashkenazi Jewish patient with early onset PD. Mutations in DJ-1 are predicted to be loss of function. The recent determination of the crystal structure of human DJ-1 demonstrates that it exists in a homo-dimeric form in vitro, whereas the L166P mutant exists only as a monomer. Here, we examine the in vivo effects of the pathogenic L166P and M26I mutations on the properties of DJ-1 in cell culture. We report that the L166P mutation confers markedly reduced protein stability to DJ-1, which results from enhanced degradation by the 20S/26S proteasome but not from a loss of mRNA expression. Furthermore, the L166P mutant protein exhibits an impaired ability to self-interact to form homo-oligomers. In contrast, the M26I mutation does not appear to adversely affect either protein stability, turnover by the proteasome, or the capacity of DJ-1 to form homo-oligomers. These properties of the L166P mutation may contribute to the loss of normal DJ-1 function and are likely to be the underlying cause of early onset PD in affected members of the Italian kindred.     

L166P mutant DJ-1 promotes cell death by dissociating Bax from mitochondrial Bcl-XL

ABSTRACT: BACKGROUND: Mutations or deletions in DJ-1/PARK7 gene are causative for recessive forms of early onset Parkinson's disease (PD). Wild-type DJ-1 has cytoprotective roles against cell death through multiple pathways. The most commonly studied mutant DJ-1(L166P) shifts its subcellular distribution to mitochondria and renders cells more susceptible to cell death under stress stimuli. We previously reported that wild-type DJ-1 binds to Bcl-XL and stabilizes it against ultraviolet B (UVB) irradiation-induced rapid degradation. However, the mechanisms by which mitochondrial DJ-1(L166P) promotes cell death under death stimuli are largely unknown. RESULTS: We show that DJ-1(L166P) is more prone to localize in mitochondria and it binds to Bcl-XL more strongly than wild-type DJ-1. In addition, UVB irradiation significantly promotes DJ-1(L166P) translocation to mitochondria and binding to Bcl-XL. DJ-1(L166P) but not wild-type DJ-1 dissociates Bax from Bcl-XL, thereby leading to Bax enrichment at outer mitochondrial membrane and promoting mitochondrial apoptosis pathway in response to UVB irradiation. CONCLUSION: Our findings suggest that wild-type DJ-1 protects cells and DJ-1(L166P) impairs cells by differentially regulating mitochondrial Bax/Bcl-XL functions.     

L166P mutant DJ-1, causative for recessive Parkinson's disease,is degraded through the ubiquitin-proteasome system

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.     

MicroRNA-452 promotes tumorigenesis in hepatocellular carcinoma by targeting cyclin-dependent kinase inhibitor 1B

Rare genetic mutations in the DJ-1 and Parkin genes cause recessive Parkinsonism, however, the relationship between these two genes is not fully elucidated. Current emerging evidence suggests that these genes are involved in mitochondrial homeostasis, and that a deficiency in either of these two genes is associated with damages in mitochondrial function and morphology. In this study, we demonstrated that knockdown of DJ-1 expression or the overexpression of the DJ-1 L166P mutation results in a damaged phenotype in mitochondria and a hypersensitivity to H₂O₂-induced cell apoptosis. These phenotypes result from increased levels of endogenous oxidative stress. However, overexpression of wild-type Parkin rescued the phenotypes observed in the mitochondria of DJ-1 knockdown and DJ-1 L166P mutant cells. We also determined that there were differences between the two cell models. Furthermore, both H₂O₂ treatment and the DJ-1 L166P mutation weakened the interaction between DJ-1 and Parkin. Taken together, these findings suggested that DJ-1 and Parkin were linked through oxidative stress, and that overexpression of Parkin protects DJ-1 protein-deficient and DJ-1 L166P mutant-expressing cells via inhibition of oxidative stress.     

On the oligomeric state of DJ-1 protein and its mutants associated with Parkinson Disease. A combined computational and in vitro study

Mutations in the DJ-1 protein are present in patients suffering from familial Parkinson disease. Here we use computational methods and biological assays to investigate the relationship between DJ-1 missense mutations and the protein oligomeric state. Molecular dynamics calculations suggest that: (i) the structure of DJ-1 wild type (WT) in aqueous solution, in both oxidized and reduced forms, is similar to the crystal structure of the reduced form; (ii) the Parkinson disease-causing M26I variant is structurally similar to the WT, consistent with the experimental evidence showing the protein is a dimer as WT; (iii) R98Q is structurally similar to the WT, consistent with the fact that this is a physiological variant; and (iv) the L166P monomer rapidly evolves toward a conformation significantly different from WT, suggesting a change in its ability to oligomerize. Our combined computational and experimental approach is next used to identify a mutant (R28A) that, in contrast to L166P, destabilizes the dimer subunit-subunit interface without significantly changing secondary structure elements.     

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.     

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.     

Parkinson's disease DJ-1 L166P alters rRNA biogenesis by exclusion of TTRAP from the nucleolus and sequestration into cytoplasmic aggregates via TRAF6

Mutations in PARK7/DJ-1 gene are associated to autosomal recessive early onset forms of Parkinson's disease (PD). Although large gene deletions have been linked to a loss-of-function phenotype, the pathogenic mechanism of missense mutations is less clear. The L166P mutation causes misfolding of DJ-1 protein and its degradation. L166P protein may also accumulate into insoluble cytoplasmic aggregates with a mechanism facilitated by the E3 ligase TNF receptor associated factor 6 (TRAF6). Upon proteasome impairment L166P activates the JNK/p38 MAPK apoptotic pathway by its interaction with TRAF and TNF Receptor Associated Protein (TTRAP). When proteasome activity is blocked in the presence of wild-type DJ-1, TTRAP forms aggregates that are localized to the cytoplasm or associated to nucleolar cavities, where it is required for a correct rRNA biogenesis. In this study we show that in post-mortem brains of sporadic PD patients TTRAP is associated to the nucleolus and to Lewy Bodies, cytoplasmic aggregates considered the hallmark of the disease. In SH-SY5Y neuroblastoma cells, misfolded mutant DJ-1 L166P alters rRNA biogenesis inhibiting TTRAP localization to the nucleolus and enhancing its recruitment into cytoplasmic aggregates with a mechanism that depends in part on TRAF6 activity. This work suggests that TTRAP plays a role in the molecular mechanisms of both sporadic and familial PD. Furthermore, it unveils the existence of an interplay between cytoplasmic and nucleolar aggregates that impacts rRNA biogenesis and involves TRAF6.     

Involvement of ERK1/2 signaling pathway in DJ-1-induced neuroprotection against oxidative stress

Parkinson’s disease (PD) is a progressive neurodegenerative disorder. Although the precise mechanism remains unclear, mounting evidence suggests that oxidative stress plays an important role in the pathogenesis of PD. DJ-1 gene is associated with oxidative stress and mutations in DJ-1 are involved in an autosomal recessive, early onset familial form of PD. The ERK1/2 signaling pathway contributes to neuroprotection during oxidative stress. However, the correlation between DJ-1 and the ERK1/2 signaling pathway remains unknown. To test for an association of DJ-1 with the ERK1/2 signaling pathway, we transfected wild-type and L166P mutated DJ-1 into COS-7 and MN9D cells. The results showed that over-expression of WT-DJ-1 dramatically enhanced the phosphorylation of ERK1/2 and its upstream kinase MEK1/2. Meanwhile, WT-DJ-1, but not L166P-DJ-1 inhibited the expression of protein phosphatase 2A (PP2A), an inhibitor of the ERK1/2 signaling pathway. Moreover, over-expression of WT-DJ-1 increased cell viability and decreased cell sensitivity to H₂O₂-induced neurotoxicity. Inhibition of the ERK1/2 signaling pathway with a MEK1/2 inhibitor reversed these changes. We conclude that DJ-1 does affect the ERK1/2 signaling pathway and change the susceptibility of cells to oxidative stress.