DJ-1基因与帕金森病

帕金森病是仅次于阿尔茨海默病的第二大神经退行性疾病。DJ-1基因的突变可以引起常染色体隐性遗传性帕金森病。该文将从DJ-1基因及DJ-1蛋白的结构,组织分布,及其在帕金森病发病机制中的功能等方面进行阐述,为研制开发帕金森病的治疗药物提供新的思路。     

Hsp31, a member of the DJ-1 superfamily,is a multitasking stress responder with chaperone activity

Among different types of protein aggregation, amyloids are a biochemically well characterized state of protein aggregation that are associated with a large number of neurodegenerative diseases including Parkinson's disease, Alzheimer and Creutzfeldt-Jakob disease. Yeast, Saccharomyces cerevisiae is an insightful model to understand the underlying mechanism of protein aggregation. Many yeast molecular chaperones can modulate aggregation and misfolding of proteins including α-Syn and the Sup35 prion. Hsp31 is a homodimeric protein structurally similar to human DJ-1, a Parkinson's disease-linked protein, and both are members of the DJ-1/ThiJ/PfpI superfamily. An emerging view is that Hsp31 and its associated superfamily members each have divergent multitasking functions that have the common theme of responding and managing various types of cellular stress. Hsp31 has several biochemical activities including chaperone and detoxifying enzyme activities that modulate at various points of a stress pathway such as toxicity associated with protein misfolding. However, we have shown the protective role of Hsp31's chaperone activity can operate independent of detoxifying enzyme activities in preventing the early stages of protein aggregate formation and associated cellular toxicities. We provide additional data that collectively supports the multiple functional roles that can be accomplished independent of each other. We present data indicating Hsp31 purified from yeast is more active compared to expression and purification from E. coli suggesting that posttranslational modifications could be important for Hsp31 to be fully active. We also compare the similarities and differences in activities among paralogs of Hsp31 supporting a model in which this protein family has overlapping but diverging roles in responding to various sources of cellular stresses.     

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.     

The atomic resolution crystal structure of the YajL (ThiJ) protein from Escherichia coli: a close prokaryotic homologue of the Parkinsonism-associated protein DJ-1

The Escherichia coli protein YajL (ThiJ) is a member of the DJ-1 superfamily with close homologues in many prokaryotes. YajL also shares 40% sequence identity with human DJ-1, an oncogene and neuroprotective protein whose loss-of-function mutants are associated with certain types of familial, autosomal recessive Parkinsonism. We report the 1.1 angstroms resolution crystal structure of YajL in a crystal form with two molecules in the asymmetric unit. The structure of YajL is remarkably similar to that of human DJ-1 (0.9 angstroms C(alpha) RMSD) and both proteins adopt the same dimeric structure. The conserved cysteine residue located in the "nucleophile elbow" is oxidized to either cysteine sulfenic or sulfinic acid in the two molecules in the asymmetric unit, and a mechanism for this oxidation is proposed that may be valid for other proteins in the DJ-1 superfamily as well. Rosenfield difference matrix analysis of the refined anisotropic displacement parameters in the YajL structure reveals significant differences in the intramolecular flexibility of the two non-crystallographic symmetry-related molecules in the asymmetric unit. Lastly, a comparison of the crystal structures of the four different E.coli members of the DJ-1 superfamily indicates that the variable oligomerization in this superfamily is due to a combination of protein-specific insertions into the core fold that form specific interfaces while occluding others plus optimization of residues in the structurally invariant regions of the core fold that facilitate protein-protein interactions.     

Localization of DJ-1 protein in the murine brain

Mutations in the DJ-1 gene have been identified to cause Parkinson's disease. In humans, nonmutated DJ-1 is expressed in specific brain areas but seems to be expressed by astrocytes rather than by neurons. In contrast, DJ-1 mRNA is mainly found in neurons in the mouse brain. We have investigated the distribution of DJ-1 protein in the mouse brain and found that DJ-1 protein is predominantly expressed by neurons but can also be detected in astrocytes. Consistent with a global role of DJ-1 in the brain, we found immunoreactivity, for example, in cortical areas, hippocampus, basolateral amygdala, the reticular nucleus of the thalamus, zona incerta, and locus coeruleus. Within the substantia nigra, however, DJ-1 is localized in both neuronal and nonneuronal cells, suggesting a distinct role in this area.     

Expression and role of DJ-1 in leukemia

DJ-1 is a multifunctional protein that has been implicated in pathogenesis of some solid tumors. In this study, we found that DJ-1 was overexpressed in acute leukemia (AL) patient samples and leukemia cell lines, which gave the first clue that DJ-1 overexpression might be involved in leukemogenesis and/or disease progression of AL. Inactivation of DJ-1 by RNA-mediated interference (RNAi) in leukemia cell lines K562 and HL60 resulted in inhibition of the proliferation potential and enhancement of the sensitivity of leukemia cells to chemotherapeutic drug etoposide. Further investigation of DJ-1 activity revealed that phosphatase and tensin homolog (PTEN), as well as some proliferation and apoptosis-related genes, was regulated by DJ-1. Thus, DJ-1 might be involved in leukemogesis through regulating cell growth, proliferation, and apoptosis. It could be a potential therapeutic target for leukemia.     

A new evolutionary paradigm for the Parkinson disease gene DJ-1

The DJ-1 gene is extensively studied because of its involvement in familial Parkinson disease. DJ-1 belongs to a complex superfamily of genes that includes both prokaryotic and eukaryotic representatives. We determine that many prokaryotic groups, such as proteobacteria, cyanobacteria, spirochaetes, firmicutes, or fusobacteria, have genes, often incorrectly called "Thij," that are very close relatives of DJ-1, to the point that they cannot be clearly separated from the eukaryotic DJ-1 genes by phylogenetic analyses of their sequences. In addition, and contrary to a previous study that suggested that DJ-1 genes were animal specific, we show that DJ-1 genes are found in at least 5 of the 6 main eukaryotic groups: opisthokonta (both animals and fungi), plantae, chromalveolata, excavata, and amoebozoa. Our results thus provide strong evidence for DJ-1 genes originating before the origin of eukaryotes. Interestingly, we found that some fungal species, among them the model yeast Schizosaccharomyces pombe, have DJ-1-like genes, most likely orthologous to the animal genes. This finding opens new ways for the analysis of the functions of this group of genes.     

Silencing DJ-1 reveals its contribution in paraquat-induced autophagy

The role of autophagy as a survival strategy of cells constitutes an emerging topic in the study of the pathogenesis of several diseases with autophagic changes being described in a number of age-related neurodegenerative disorders, including Parkinson's disease (PD). Although the etiology of PD is still unknown, both environmental (for example, paraquat exposure) and genetic factors have been investigated as putative causes of the disease. In the latter case, mutations or changes in the protein DJ-1 have been reported to be associated with autosomal recessive, early-onset parkinsonism. In this paper we established a model system to study the involvement of the DJ-1 protein in paraquat-induced autophagy. When human neuroblastoma SH-SY5Y cells were transfected with DJ-1-specific small interfering RNAs and exposed to paraquat, we observed (i) sensitization additive with paraquat-induced apoptotic cell death, (ii) inhibition of the cytoplasmic accumulation of autophagic vacuoles as well as the recruitment of LC3 fusion protein to the vacuoles, (iii) exacerbation of apoptotic cell death in the presence of the autophagy inhibitor 3-methyladenine, and (iv) an increase in mammalian target of rapamycin phosphorylation. Taken together, these findings suggest an active role for DJ-1 in the autophagic response produced by paraquat, providing evidence for the role of PD-related proteins in the autophagic degradation pathway, a factor that should be considered in the design of potential therapies for the treatment of the disease.     

Increased level of DJ-1 in the cerebrospinal fluids of sporadic Parkinson's disease

DJ-1 is an antioxidant protein whose loss of function by gene mutations has been linked to familial Parkinson's disease (PD). The main objective of the present study was to determine if this molecule was also involved in the pathogenesis of sporadic PD. For this purpose, quantitative immunoblot assays were performed to evaluate DJ-1 in cerebrospinal fluids (CSF) collected from sporadic PD patients (n=40) and non-PD controls (n=38). The results showed that the CSF DJ-1 levels in PD were significantly higher than those in non-PD controls. Especially, upregulation of CSF DJ-1 in the early stage of PD (Yahr I-II) were distinct compared to those in the advanced stage of PD (Yahr III-IV) and non-PD controls (p<0.001 by ANOVA with post hoc Bonferroni's test), suggesting a protective role of DJ-1 against oxidative stress during the early stage. Thus, we propose that CSF DJ-1 could be a possible biomarker for early sporadic PD.     

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.     

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.     

Free radicals impair the anti-oxidative stress activity of DJ-1 through the formation of SDS-resistant dimer

DJ-1 is a causative gene for familial Parkinson’s disease (PD). Loss-of-function of DJ-1 protein is suggested to contribute to the onset of PD, but the causes of DJ-1 dysfunction remain insufficiently elucidated. In this study, we found that the SDS-resistant irreversible dimer of DJ-1 protein was formed in human dopaminergic neuroblastoma SH-SY5Y cells when the cells were exposed to massive superoxide inducers such as paraquat and diquat. The dimer was also formed in vitro by superoxide in PQ redox cycling system and hydroxyl radical produced in Fenton reaction. We, thus, found a novel phenomenon that free radicals directly affect DJ-1 to form SDS-resistant dimers. Moreover, the formation of the SDS-resistant dimer impaired anti-oxidative stress activity of DJ-1 both in cell viability assay and H₂O₂-elimination assay in vitro. Similar SDS-resistant dimers were steadily formed with several mutants of DJ-1 found in familial PD patients. These findings suggest that DJ-1 is impaired due to the formation of SDS-resistant dimer when the protein is directly attacked by free radicals yielded by external and internal stresses and that the DJ-1 impairment is one of the causes of sporadic PD.     

Mechanisms of DJ-1 neuroprotection in a cellular model of Parkinson's disease

Mitochondrial dysfunction, proteasome inhibition, and α-synuclein aggregation are thought to play important roles in the pathogenesis of Parkinson's disease (PD). Rare cases of early-onset PD have been linked to mutations in the gene encoding DJ-1, a protein with antioxidant and chaperone functions. In this study, we examined whether DJ-1 protects against various stresses involved in PD, and we investigated the underlying mechanisms. Expression of wild-type DJ-1 rescued primary dopaminergic neurons from toxicity elicited by rotenone, proteasome inhibitors, and mutant α-synuclein. Neurons with reduced levels of endogenous DJ-1 were sensitized to each of these insults, and DJ-1 mutants involved in familial PD exhibited decreased neuroprotective activity. DJ-1 alleviated rotenone toxicity by up-regulating total intracellular glutathione. In contrast, inhibition of α-synuclein toxicity by DJ-1 correlated with up-regulation of the stress-inducible form of Hsp70. RNA interference studies revealed that this increase in Hsp70 levels was necessary for DJ-1-mediated suppression of α-synuclein aggregation, but not toxicity. Our findings suggest that DJ-1 acts as a versatile pro-survival factor in dopaminergic neurons, activating different protective mechanisms in response to a diverse range of PD-related insults.     

The oxidation state of DJ-1 regulates its chaperone activity toward alpha-synuclein

DJ-1 has been reported to have chaperone activity by preventing the aggregation of some proteins, and by structural analogy to Hsp31. The L166P mutation has been linked to a familial early onset form of Parkinson's disease (PD). Since the aggregation of alpha-synuclein is believed to be a critical step in the etiology of PD, we have investigated the interaction of wild-type DJ-1 and its oxidized forms with alpha-synuclein. Native (unoxidized) DJ-1 did not inhibit alpha-synuclein fibrillation, and no evidence for stable interactions between alpha-synuclein and native DJ-1 was observed. However, DJ-1 is very susceptible to oxidation by the addition of two oxygen atoms to form the sulfinic acid of Cys106 (2O DJ-1) (no 1O oxidized state is detectable). 2O DJ-1 was readily prepared by the addition of H(2)O(2) at concentrations up to a 20-fold molar excess. The oxidation of Cys106 to the sulfinic acid had minimal effect on the structural properties of DJ-1. However, 2O DJ-1 was very effective in preventing the fibrillation of alpha-synuclein, and only this form of DJ-1 appears to have significant anti-aggregation properties against alpha-synuclein. Further oxidation of DJ-1 leads to loss of some secondary structure, and to loss of the ability to inhibit alpha-synuclein fibrillation. Our observations confirm the suggestion that DJ-1 may act as an oxidative-stress-induced chaperone to prevent alpha-synuclein fibrillation. Since oxidative stress has been associated with PD, this observation may explain why mutations of DJ-1 could be a contributing factor in PD, and also indicates that excess oxidative stress could also lead to enhanced alpha-synuclein aggregation and hence PD.     

DJ-1 mediates the resistance of cancer cells to dihydroartemisinin through reactive oxygen species removal

Dihydroartemisinin (DHA), one of the main metabolites of artemisinin and its derivatives, presents anti-cancer potential in vitro and in vivo. To explore the mechanisms of resistance toward DHA, a DHA-resistant cell line, HeLa/DHA, was established with a resistance factor of 7.26 in vitro. Upon DHA treatment, apoptotic cells were significantly elicited in parental HeLa cells but minimally induced in HeLa/DHA cells. HeLa/DHA cells also displayed much less sensitivity to DHA-induced tumor suppression in cancer xenograft models than HeLa cells. Intriguingly, DHA-resistant cells did not display a multidrug-resistant phenotype. Based on a proteomic study employing LC–ESI–MS/MS together with pathway analysis, DJ-1 (PARK7) was found to be highly expressed in HeLa/DHA cells. Western blot and immunofluorescence assays confirmed the higher expression of DJ-1 in HeLa/DHA cells than in parental cells in both cell line and xenograft models. DJ-1 is translocated to the mitochondria of HeLa/DHA cells and oxidized, providing DJ-1 with stronger cytoprotection activity. Further study revealed that DJ-1 knockdown in HeLa/DHA cells abolished the observed resistance, whereas overexpression of DJ-1 endowed the parental HeLa cells with resistance toward DHA. Reactive oxygen species (ROS) were also significantly induced by either DHA or hydrogen peroxide in HeLa cells but not in resistant HeLa/DHA cells. When the cells were pretreated with N-acetyl-l-cysteine, the effect of DJ-1 knockdown on sensitizing HeLa/DHA cells to DHA was significantly attenuated. In summary, our study suggests that overexpression and mitochondrial translocation of DJ-1 provides HeLa/DHA cells with resistance to DHA-induced ROS and apoptosis.     

The caspase 6 derived N-terminal fragment of DJ-1 promotes apoptosis via increased ROS production

In pathological conditions, the amount of DJ-1 determines whether a cell can survive or engage a cell death program. This is exemplified in epithelial cancers, in which DJ-1 expression is increased, while autosomal recessive early onset Parkinson''s disease mutations of DJ-1 generally lead to decreased stability and expression of the protein. We have shown previously that DJ-1 is cleaved by caspase-6 during induction of apoptosis. We demonstrate here that the N-terminal cleaved fragment of DJ-1 (DJ-1 Nt) is specifically expressed in the nucleus and promotes apoptosis in SH-SY5Y neuroblastoma cell lines. In addition, overexpression of DJ-1 Nt in different cell lines leads to a loss of clonogenic potential and sensitizes to staurosporin and 1-methyl-4-phenylpyridinium (MPP+)-mediated caspase activation and apoptosis. Importantly, inhibition of endogenous DJ-1 expression with sh-RNA or DJ-1 deficiency mimics the effect of DJ-1 Nt on cell growth and apoptosis. Moreover, overexpression of DJ-1 Nt increases reactive oxygen species (ROS) production, and sensitizes to MPP+-mediated apoptosis and DJ-1 oxidation. Finally, specific exclusion of DJ-1 Nt from the nucleus abrogates its pro-apoptotic effect. Taken together, our findings identify an original pathway by which generation of a nuclear fragment of DJ-1 through caspase 6-mediated cleavage induces ROS-dependent amplification of apoptosis.