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.     

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.     

BAG1 restores formation of functional DJ-1 L166P dimers and DJ-1 chaperone activity

Mutations in the gene coding for DJ-1 protein lead to early-onset recessive forms of Parkinson’s disease. It is believed that loss of DJ-1 function is causative for disease, although the function of DJ-1 still remains a matter of controversy. We show that DJ-1 is localized in the cytosol and is associated with membranes and organelles in the form of homodimers. The disease-related mutation L166P shifts its subcellular distribution to the nucleus and decreases its ability to dimerize, impairing cell survival. Using an intracellular foldase biosensor, we found that wild-type DJ-1 possesses chaperone activity, which is abolished by the L166P mutation. We observed that this aberrant phenotype can be reversed by the expression of the cochaperone BAG1 (Bcl-2–associated athanogene 1), restoring DJ-1 subcellular distribution, dimer formation, and chaperone activity and ameliorating cell survival.     

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.     

DJ-1 up-regulates glutathione synthesis during oxidative stress and inhibits A53T alpha-synuclein toxicity

DJ-1 is the third gene that has been linked to Parkinson disease. Mutations in the DJ-1 gene cause early onset PD with autosomal recessive inheritance. To clarify the mechanism of DJ-1 protection, we have overexpressed the gene in cultured dopaminergic cells that were then subjected to chemical stress. In the rat dopaminergic cell line, N27, and in primary dopamine neurons, overexpression of wild type DJ-1 protected cells from death induced by hydrogen peroxide and 6-hydroxydopamine. Overexpressing the L166P mutant DJ-1 had no protective effect. By contrast, knocking down endogenous DJ-1 with antisense DJ-1 rendered cells more susceptible to oxidative damage. We have found that DJ-1 improves survival by increasing cellular glutathione levels through an increase in the rate-limiting enzyme glutamate cysteine ligase. Blocking glutathione synthesis eliminated the beneficial effect of DJ-1. Protection could be restored by adding exogenous glutathione. Wild type DJ-1 reduced cellular reactive oxygen species and reduced the levels of protein oxidation caused by oxidative stress. By a separate mechanism, overexpressing wild type DJ-1 inhibited the protein aggregation and cytotoxicity usually caused by A53T human alpha-synuclein. Under these circumstances, DJ-1 increased the level of heat shock protein 70 but did not change the glutathione level. Our data indicate that DJ-1 protects dopaminergic neurons from oxidative stress through up-regulation of glutathione synthesis and from the toxic consequences of mutant humanalpha-synuclein through increased expression of heat shock protein 70. We conclude that DJ-1 has multiple specific mechanisms for protecting dopamine neurons from cell death.     

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.     

Familial Parkinson's disease-associated L166P mutation disrupts DJ-1 protein folding and function

Mutations in DJ-1, a protein of unknown function, were recently identified as the cause for an autosomal recessive, early onset form of familial Parkinson's disease. Here we report that DJ-1 is a dimeric protein that exhibits protease activity but no chaperone activity. The protease activity was abolished by mutation of Cys-106 to Ala, suggesting that DJ-1 functions as a cysteine protease. Our studies revealed that the Parkinson's disease-linked L166P mutation impaired the intrinsic folding propensity of DJ-1 protein, resulting in a spontaneously unfolded structure that was incapable of forming a homodimer with itself or a heterodimer with wild-type DJ-1. Correlating with the disruption of DJ-1 structure, the L166P mutation abolished the catalytic function of DJ-1. Furthermore, as a result of protein misfolding, the L166P mutant DJ-1 was selectively polyubiquitinated and rapidly degraded by the proteasome. Together these findings provide insights into the molecular mechanism by which loss-of-function mutations in DJ-1 lead to Parkinson's disease.     

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.     

Hypoxic preconditioning up-regulates DJ-1 protein expression in rat heart-derived H9c2 cells through the activation of extracellular-regulated kinase 1/2 pathway

Myocardial preconditioning is a powerful phenomenon that can attenuate ischemia/reperfusion-induced oxidant stress and elicit delayed cardioprotection. Its mechanisms involve activation of intracellular signaling pathways and up-regulation of the protective antioxidant proteins. DJ-1 protein, as a multifunctional intracellular protein, plays an important role in attenuating oxidant stress and promoting cell survival. In the present study, we investigated whether DJ-1 is up-regulated during the late phase of hypoxic preconditioning (HP) and the up-regulation of DJ-1 is mediated by extracellular-regulated kinase 1/2 (ERK1/2) signaling pathway. Rat heart-derived H9c2 cells were exposed to HP. Twenty-four hours later cells were subjected to hypoxia/reoxygenation (H/R) and then cell viability, lactate dehydrogenase (LDH), intracellular reactive oxygen species (ROS), ERK1/2 phosphorylation, and DJ-1 protein were measured appropriately. The results showed that HP efficiently attenuated H/R-induced viability loss and LDH leakage. In addition, HP promoted ERK1/2 activation, up-regulated DJ-1 protein expression, inhibited H/R induced the elevation of ROS. However, when ERK1/2 phosphorylation was specifically inhibited by U0126, the increase in DJ-1 expression occurring during HP was almost completely abolished and, as a result, the delayed cardioprotection induced by HP was abolished, and the inhibitory effect of HP on H/R-induced oxidant stress was also reversed. Furthermore, knocking down DJ-1 by siRNA attenuated the delayed cardioprotection induced by HP. Our data indicate that HP can up-regulate DJ-1 protein expression through the ERK1/2-dependent signaling pathway. Importantly, DJ-1 might be involved in the delayed cardioprotective effect of HP against H/R injury.     

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.     

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.     

Down regulation of DJ-1 enhances cell death by oxidative stress, ER stress, and proteasome inhibition

Mutations in DJ-1 gene have been linked to autosomal recessive early onset parkinsonism (AR-EOP). Although the mechanism of neuronal cell death due to DJ-1 mutation has not been fully elucidated, loss of DJ-1 function was considered to cause the phenotype. Here, we demonstrated that the down regulation of endogenous DJ-1 of the neuronal cell line by siRNA enhanced the cell death which was induced by oxidative stress, ER stress, and proteasome inhibition, but not by pro-apoptotic stimulus. The cell death with hydrogen peroxide was dramatically rescued by over-expression of wild-type DJ-1, but not by that of L166P mutant DJ-1. Furthermore, DJ-1 rescued the cell death caused by over-expression of Pael receptor, which was a substrate of Parkin, another gene product for autosomal recessive juvenile parkinsonism. These results suggest that loss of protective activity of DJ-1 from neuro-toxicity induced by these stresses contributes to neuronal cell death in AR-EOP with mutant DJ-1.     

Tyrosine kinase-independent activation of extracellular-regulated kinase (ERK) 1/2 by the insulin-like growth factor-1 receptor

The extracellular-regulated kinase (ERK1/2) is a key conduit for transduction of signals from growth factor receptors to the nucleus. Previous work has shown that ERK1/2 activation in response to IGF-1 may require the participation of G proteins, but the role of the receptor tyrosine kinase in this process has not been clearly resolved. This investigation of IGF-1 receptor function was therefore designed to examine the contribution of the receptor tyrosine kinase to ERK1/2 activation. Phosphorylation of ERK1/2 in smooth muscle cells following treatment with IGF-1 was not blocked by pretreatment with AG1024 or picropodophylin, inhibitors of the IGF-1 receptor tyrosine kinase. Likewise, IGF-1 activated ERK1/2 in cells expressing a kinase-dead mutant of the IGF-1 receptor. ERK1/2 activation was unaffected by the phosphatidylinositol 3-kinase inhibitor LY-294002, but was sensitive to inhibitors of Src kinase, phospholipase C and Gβγ subunit signalling. Treatment with αIR-3, a neutralizing monoclonal antibody, also stimulated ERK1/2 phosphorylation without concomitant activation of the receptor tyrosine kinase. Phosphoprotein mapping of IGF-1 and αIR-3 treated cells confirmed that antibody-induced ERK1/2 phosphorylation occurred in the absence of tyrosine kinase phosphorylation, and enabled extension of these findings to p38 MAPK. These results suggest that stimulation of ERK1/2 phosphorylation by IGF-1 does not require activation of the receptor tyrosine kinase.     

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.     

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。     

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.     

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.