Extracellular Signals Induce Glycoprotein M6a Clustering of Lipid Rafts and Associated Signaling Molecules.

Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. To examine how signaling protein complexes are clustered in rafts, we focused on the functions of glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing mouse neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a GPM6a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a, such as Rufy3, Rap2, and Tiam2/STEF, accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation in neuronal development. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of neuronal polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals.SIGNIFICANCE STATEMENT Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. We focused on glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals.

Transcriptional Regulation of DJ-1.

DJ-1 is an oncogene and also a causative gene for familial Parkinson's disease. DJ-1 has various functions, and the oxidative status of a cysteine residue at position 106 (C106) is crucial for determination of the activation level of DJ-1.DJ-1 binds to many proteins, including various transcription factors, and acts as a coactivator or corepressor for regulating their target genes without direct binding to DNA, thereby affecting various cell functions. DJ-1-regulating transcription factors and their modified proteins are the androgen receptor and its regulatory proteins, p53; polypyrimidine tract-binding protein-associated splicing factor (PSF); Keap1, an inhibitor for nuclear factor erythroid2-related factor 2 (Nrf2); sterol regulatory element-binding protein (SREBP); Ras-responsive element-binding protein (RREB1); signal transducer and activator of transcription 1 (STAT1); and Nurr1. Considering oxidative stress response and dopamine synthesis, the regulation of Nrf2, p53, and PSF by DJ-1 is especially important. In addition, SREBP1 and RREB1 functions that are positively regulated by DJ-1 may participate in the onset and pathogenesis of metabolic syndrome.DJ-1 is expressed ubiquitously with high levels in the testis and brain and moderate levels in other tissues. Furthermore, DJ-1 is translocated from the cytoplasm to nucleus during the cell cycle after mitogen stimulation, suggesting that DJ-1 has a growth-related function. In this review, we describe how DJ-1 regulates cell growth/death and dopamine synthesis by targeting various transcription factors.

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.

DJ-1 activates SIRT1 through its direct binding to SIRT1.

The DJ-1 gene is a ras-dependent oncogene and also a causative gene for a familial form of Parkinson's disease park7. DJ-1 is a multi-functional protein and plays roles in regulation of cell growth, cells death, metabolism and mitochondrial homeostasis against oxidative stress. To explore various functions, DJ-1 associates with a number of proteins localized in the nucleus, cytoplasm and mitochondria. The oxidative status of a cysteine residue at an amino acid number 106 (C106) of DJ-1 determines the active level of DJ-1. Precise molecular mechanism of exploration of DJ-1 function is, however, not resolved. In this study, we identified Sirtuin family proteins (SIRT1, 2, and 4-6) as DJ-1-binding proteins, and DJ-1 associated with SIRT1 in cells. Sirtuins like DJ-1 also regulates growth, death and metabolism of cells and mitochondrial homeostasis. We found that DJ-1 stimulated deacetylase activity of SIRT1 and that SIRT1-suppressed transcriptional activity of SIRT1-target p53 was further decreased by DJ-1. Furthermore, SIRT1 activity was reduced in DJ-1-knockout cells, and this reduced activity was restored by re-introduction of wild-type DJ-1 but not of C106-mutant DJ-1 into DJ-1-knockout cells. It is first report showing direct connection of DJ-1 with SIRT1.

DJ-1-dependent protective activity of DJ-1-binding compound no. 23 against neuronal cell death in MPTP-treated mouse model of Parkinson's disease.

Parkinson's disease (PD) is caused by dopaminergic cell death in the substantia nigra, leading to a reduced level of dopamine in the striatum. Oxidative stress is one of the causes of PD. Since symptomatic PD therapies are used, identification of compounds or proteins that inhibit oxidative stress-induced neuronal cell death is necessary. DJ-1 is a causative gene product of familial PD and plays a role in anti-oxidative stress reaction. We have identified various DJ-1-binding compounds, including compound-23, that restored neuronal cell death and locomotion defects observed in neurotoxin-induced PD models. In this study, wild-type and DJ-1-knockout mice were injected intraperitoneally with 1 mg/kg of compound-23 and then with 30 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at 1 h after injection. Five days after administration, the effects of compound-23 on MPTP-induced locomotion deficits, on dopaminergic cell death and on brain dopamine levels were analyzed by rotor rod tests, by staining cells with an anti-TH antibody and by an HPLC, respectively. The results showed that compound-23 inhibited MPTP-induced reduction of retention time on the rotor rod bar, neuronal cell death in the substantia nigra and striatum and dopamine content in wild-type mice but not in DJ-1-knockout mice, indicating a DJ-1-dependent effect of compound-23.

Prefoldin plays a role as a clearance factor in preventing proteasome inhibitor-induced protein aggregation.

Prefoldin is a molecular chaperone composed of six subunits, PFD1-6, and prevents misfolding of newly synthesized nascent polypeptides. Although it is predicted that prefoldin, like other chaperones, modulates protein aggregation, the precise function of prefoldin against protein aggregation under physiological conditions has never been elucidated. In this study, we first established an anti-prefoldin monoclonal antibody that recognizes the prefoldin complex but not its subunits. Using this antibody, it was found that prefoldin was localized in the cytoplasm with dots in co-localization with polyubiquitinated proteins and that the number and strength of dots were increased in cells that had been treated with lactacystin, a proteasome inhibitor, and thapsigargin, an inducer of endoplasmic reticulum stress. Knockdown of prefoldin increased the level of SDS-insoluble ubiquitinated protein and reduced cell viability in lactacystin and thapsigargin-treated cells. Opposite results were obtained in prefoldin-overexpressed cells. It has been reported that mice harboring a missense mutation L110R of MM-1α/PFD5 exhibit neurodegeneration in the cerebellum. Although the prefoldin complex containing L110R MM-1α was properly formed in vitro and in cells derived from L110R MM-1α mice, the levels of ubiquitinated proteins and cytotoxicity were higher in L110R MM-1α cells than in wild-type cells under normal conditions and were increased by lactacystin and thapsigargin treatment, and growth of L110R MM-1α cells was attenuated. Furthermore, the polyubiquitinated protein aggregation level was increased in the brains of L110R MM-1α mice. These results suggest that prefoldin plays a role in quality control against protein aggregation and that dysfunction of prefoldin is one of the causes of neurodegenerative diseases.

Stimulation of vesicular monoamine transporter 2 activity by DJ-1 in SH-SY5Y cells.

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.

Identification of functional marker proteins in the mammalian growth cone.

Identification of proteins in the mammalian growth cone has the potential to advance our understanding of this critical regulator of neuronal growth and formation of neural circuit; however, to date, only one growth cone marker protein, GAP-43, has been reported. Here, we successfully used a proteomic approach to identify 945 proteins present in developing rat forebrain growth cones, including highly abundant, membrane-associated and actin-associated proteins. Almost 100 of the proteins appear to be highly enriched in the growth cone, as determined by quantitative immunostaining, and for 17 proteins, the results of RNAi suggest a role in axon growth. Most of the proteins we identified have not previously been implicated in axon growth and thus their identification presents a significant step forward, providing marker proteins and candidate neuronal growth-associated proteins.

Human DJ-1-specific transcriptional activation of tyrosine hydroxylase gene.

Loss-of-function mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. DJ-1 has multiple functions, including transcriptional regulation, and one of transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene, the product of which is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor polypyrimidine tract-binding protein-associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated TH gene expression and 4-dihydroxy-L-phenylalanine production but that knockdown or knock-out of mouse DJ-1 in mouse cell lines or in mice did not affect such expression and TH activity. In reporter assays using the human TH gene promoter linked to the luciferase gene, stimulation of TH promoter activity was observed in human cells, but not mouse cells, that had been transfected with DJ-1. Although human DJ-1 and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not by mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not the mouse TH gene promoter. These results suggest a novel species-specific transcriptional regulation of the TH promoter by DJ-1 and one of the mechanisms for no reduction of TH in DJ-1-knock-out mice.

Oxidative status of DJ-1-dependent activation of dopamine synthesis through interaction of tyrosine hydroxylase and 4-dihydroxy-L-phenylalanine (L-DOPA) decarboxylase with DJ-1.

Parkinson disease (PD) is caused by loss of dopamine, which is synthesized from tyrosine by two enzymes, tyrosine hydroxylase (TH) and 4-dihydroxy-L-phenylalanine decarboxylase (DDC). DJ-1 is a causative gene for the familial form of PD, but little is known about the roles of DJ-1 in dopamine synthesis. In this study, we found that DJ-1 directly bound to TH and DDC and positively regulated their activities in human dopaminergic cells. Mutants of DJ-1 found in PD patients, including heterozygous mutants, lost their activity and worked as dominant-negative forms toward wild-type DJ-1. When cells were treated with H(2)O(2), 6-hydroxydopamine, or 1-methyl-4-phenylpyridinium, changes in activities of TH and DDC accompanied by oxidation of cysteine 106 of DJ-1 occurred. It was found that DJ-1 possessing Cys-106 with SH and SOH forms was active and that DJ-1 possessing Cys-106 with SO(2)H and SO(3)H forms was inactive in terms of stimulation of TH and DDC activities. These findings indicate an essential role of DJ-1 in dopamine synthesis and contribution of DJ-1 to the sporadic form of PD.

DJ-1-binding compound B enhances Nrf2 activity through the PI3-kinase-Akt pathway by DJ-1-dependent inactivation of PTEN.

DJ-1 was identified as an oncogene and also as a causative gene for a familial form of Parkinson disease (PD). DJ-1 plays various roles in anti-oxidative stress response. Superfluous oxidation of DJ-1 at cysteine residue 106 (C106), an inactive form of DJ-1, was observed in PD patients. DJ-1-binding compound B, which specifically bound to the C106 region of DJ-1, has been isolated and it has been shown to prevent oxidative stress-induced cell death through maintaining active forms of DJ-1 by inhibiting its superfluous oxidation. The molecular mechanism of the action of compound B, however, has not been fully elucidated. In this study, we found that compound B stimulated transcriptional activity of Nrf2 in H2O2-treated SH-SY5Y cells by inhibiting its degradation through the ubiquitin-proteasome system. Although Keap 1 is a major negative regulator of Nrf2, compound B strongly increased Nrf2 activity in Keap1-mutant A549 cells but not in PTEN-null PC3 and PTEN-knockout SH-SY5Y cells. Furthermore, treatment of cells with inhibitors of the PI3-kinase/Akt pathway inhibited the effect of compound B, and compound B increased the binding of PTEN to DJ-1 and decreased lipid phosphatase activity of PTEN concomitantly with increased oxidation of PTEN, an inactive form of PTEN. These results suggest that compound B enhances transcriptional activity of Nrf2 under an oxidative stress condition in a Keap1-independent manner and that its activity is elicited by activation of the PI3Kinase/Akt pathway with DJ-1-dependent inactivation of PTEN, leading to protection of oxidative stress-induced cell death.

DJ-1 binds to mitochondrial complex I and maintains its activity.

Parkinson's disease (PD) is caused by neuronal cell death, and oxidative stress and mitochondrial dysfunction are thought to be responsible for onset of PD. DJ-1, a causative gene product of a familial form of Parkinson's disease, PARK7, plays roles in transcriptional regulation and anti-oxidative stress. The possible mitochondrial function of DJ-1 has been proposed, but its exact function remains unclear. In this study, we found that DJ-1 directly bound to NDUFA4 and ND1, nuclear and mitochondrial DNA-encoding subunits of mitochondrial complex I, respectively, and was colocalized with complex I and that complex I activity was reduced in DJ-1-knockdown NIH3T3 and HEK293 cells. These findings suggest that DJ-1 is an integral mitochondrial protein and that DJ-1 plays a role in maintenance of mitochondrial complex I activity.

DJBP: a novel DJ-1-binding protein, negatively regulates the androgen receptor by recruiting histone deacetylase complex, and DJ-1 antagonizes this inhibition by abrogation of this complex.

DJ-1 was identified by us as a novel oncogene that transforms mouse NIH3T3 cells in cooperation with ras. We later identified PIAS (protein inhibitor of activated STAT)xalpha as a DJ-1-binding protein, and found that DJ-1 restored androgen receptor (AR) transcription activity that was repressed by PIASxalpha. To further characterize the function of DJ-1, we cloned cDNA encoding a novel DJ-1-binding protein, DJBP, by a yeast two-hybrid system. DJBP mRNA was found to be specifically expressed in the testis. In addition to the binding of DJBP to the COOH-terminal region of DJ-1, DJBP was also found to bind in vitro and in vivo to the DNA-binding domain of the AR in a testosterone-dependent manner and to be colocalized with DJ-1 or AR in the nucleus. Furthermore, a co-immunoprecipitation assay showed that the formation of a ternary complex between DJ-1, DJBP, and AR occurred in cells in which DJ-1 bound to the AR via DJBP. It was found that DJBP repressed a testosterone-dependent AR transactivation activity in monkey Cos1 cells by recruiting histone deacetylase (HDAC) complex, including HDAC1 and mSin3, and that DJ-1 partially restored its repressed activity by abrogating DJBP-HDAC complex. These results suggest that AR is positively regulated by DJ-1, which antagonizes the function of negative regulators, including DJBP.

Expression profiles of genes in DJ-1-knockdown and L 166 P DJ-1 mutant cells.

DJ-1 is a novel oncogene and a causative gene for the familial form of Parkinson's disease (PD). DJ-1 has been shown to play roles in anti-oxidative stress by eliminating reactive oxygen species and in transcriptional regulation of genes. Loss of these functions of DJ-1 is thought to trigger the onset of PD. In this study, to identify genes for which expressions are regulated by DJ-1, DNA microarray analyses were carried out using two mouse NIH3T3 cell lines, DJ-1-knockdown cells and cells harboring an exogenously added L 166 P DJ-1 mutant found in PD patients. In both cell lines, drastic changes in expressions of genes, including genes related to stress, apoptosis, oxidative stress and neurotoxicity, were observed and changes in expressions were confirmed by RT-PCR. Of the genes identified, expression level of the extracellular superoxide dismutase (SOD 3) gene was found to decrease in DJ-1-knockdown cells, while expressions of SOD 1 and SOD 2 genes did not change. Furthermore, expression of the tau gene, a gene whose product gives cells neurotoxicity by aggregation, was found to increase at its promoter level in L 166 P DJ-1 cells. These findings suggest that DJ-1 regulates expressions of genes for which functions are thought to be related to cell death or neurodegeneration.

Deficiency of spermatogenesis and reduced expression of spermatogenesis-related genes in prefoldin 5-mutant mice.

MM-1α is a c-Myc-binding protein and acts as a transcriptional co-repressor in the nucleus. MM-1α is also PDF5, a subunit of prefoldin that is chaperon comprised of six subunits and prevents misfolding of newly synthesized nascent polypeptides. Prefoldin also plays a role in quality control against protein aggregation. It has been reported that mice harboring the missense mutation L110R of MM-1α/PFD5 exhibit neurodegeneration in the cerebellum and also male infertility, but the phenotype of infertility has not been fully characterized. In this study, we first analyzed morphology of the testis and epididymis of L110R of MM-1α mice. During differentiation of spermatogenesis, spermatogonia, spermatocytes and round spermatids were formed, but formation of elongated spermatids was compromised in L110R MM-1α mice. Furthermore, reduced number/concentration of sperm in the epididymis was observed. MM-1α was strongly expressed in the round spermatids and sperms with round spermatids, suggesting that MM-1α affects the differentiation and maturation of germ cells. Changes in expression levels of spermatogenesis-related genes in mice testes were then examined. The fatty-acid-binding protein (fabp4) gene was up-regulated and three genes, including sperm-associated glutamate (E)-rich protein 4d (speer-4d), phospholipase A2-Group 3 (pla2g3) and phospholipase A2-Group 10 (pla2g10), were down-regulated in L110R MM-1α mice. L110R MM-1α and wild-type MM-1α bound to regions of up-regulated and down-regulated genes, respectively. Since these gene products are known to play a role in maturation and motility of sperm, a defect of at least MM-1α transcriptional activity is thought to induce expressional changes of these genes, resulting in male infertility.

Immunostaining of oxidized DJ-1 in human and mouse brains.

DJ-1, the product of a causative gene of a familial form of Parkinson disease, undergoes preferential oxidation of Cys106 (cysteine residue at position 106) under oxidative stress. Using specific monoclonal antibodies against Cys106 oxidized DJ-1 (oxDJ-1), we examined oxDJ-1 immunoreactivity in brain sections from DJ-1 knockout and wild-type mice and in human brain sections from cases classified into different Lewy body stages of Parkinson disease and Parkinson disease with dementia. Oxidized DJ-1 immunoreactivity was prominently observed in neuromelanin-containing neurons and neuron processes of the substantia nigra; Lewy bodies also showed oxDJ-1 immunoreactivity. Oxidized DJ-1 was also detected in astrocytes in the striatum, in neurons and glia in the red nucleus, and in the inferior olivary nucleus, all of which are related to regulation of movement. These observations suggest the relevance of DJ-1 oxidation to homeostasis in multiple brain regions, including neuromelanin-containing neurons of the substantia nigra, and raise the possibility that oxDJ-1 levels might change during the progression of Lewy body-associated neurodegenerative diseases.

Identification of the recognition sequence and target proteins for DJ-1 protease.

DJ-1, the product of familial Parkinson's disease gene and an oncogene, is a cysteine protease which plays a role in anti-oxidative stress reaction. In this study, we identified the recognition sequence for DJ-1 protease by using recombinant DJ-1 and a peptide library. Protease activity of DJ-1 lacking C-terminal α-helix (DJ-1ΔH9) was stronger than that of full-sized DJ-1, and the most susceptible sequence digested by DJ-1ΔH9 was valine-lysine-valine-alanine (VKVA) under the optimal conditions of pH 5.5 and 0 mM NaCl. Divalent ions, especially Cu²âº, were inhibitory to DJ-1's protease activity. c-abl oncogene 1 product (ABL1) and kinesin family member 1B (KIF1B) containing VKVA were digested by DJ-1ΔH9.

Transcriptional activation of the cholecystokinin gene by DJ-1 through interaction of DJ-1 with RREB1 and the effect of DJ-1 on the cholecystokinin level in mice.

DJ-1 is an oncogene and also causative gene for familial Parkinson's disease. DJ-1 has multiple functions, including transcriptional regulation. DJ-1 acts as a coactivator that binds to various transcription factors, resulting in stimulation or repression of the expression of their target genes. In this study, we found that the cholecystokinin (CCK) gene is a transcriptional target gene for DJ-1. CCK is a peptide hormone and plays roles in contraction of the gallbladder and in promotion of secretion of pancreatic fluid. CCK is co-localized with dopamine in the substantia nigra to regulate release of dopamine. Reduced expression of CCK mRNA was observed in DJ-1-knockdown cells. The Ras-responsive element (RRE) and Sp1 site were essential for promoter activity, and DJ-1 stimulated promoter activity by binding to RRE-binding protein 1 (RREBP1). The complex of DJ-1 with RREB1 but not with Sp1 bound to the RRE. Furthermore, the reduced CCK level in the serum from DJ-1-knockout mice compared to that from wild-type mice was observed. This is the first report showing that DJ-1 participates in peptide hormone synthesis.

Oxidized DJ-1 inhibits p53 by sequestering p53 from promoters in a DNA-binding affinity-dependent manner.

DJ-1 is an oncogene and the causative gene for familial Parkinson's disease. Although the oxidative status of DJ-1 at cysteine 106 (C106) is thought to affect all of the activities of DJ-1 and excess oxidation leads to the onset of various diseases, the precise molecular mechanisms underlying the effects of oxidation of DJ-1 on protein-protein interactions of DJ-1 remain unclear. In this study, we found that DJ-1 bound to the DNA-binding region of p53 in a manner dependent on the oxidation of C106. Of the p53 target genes, the expression level and promoter activity of the DUSP1 gene, but not those of the p21 gene, were increased in H(2)O(2)-treated DJ-1(-/-) cells and were decreased in wild-type DJ-1- but not C106S DJ-1-transfected H1299 cells through sequestration of p53 from the DUSP1 promoter by DJ-1. DUSP1 downregulated by oxidized DJ-1 activated extracellular signal-regulated kinase (ERK) and decreased apoptosis. The DUSP1 and p21 promoters harbor nonconsensus and consensus p53 recognition sequences, respectively, which have low affinity and high affinity for p53. However, DJ-1 inhibited p21 promoter activity exhibited by p53 mutants harboring low DNA-binding affinity but not by wild-type p53. These results indicate that DJ-1 inhibits the expression of p53 target genes and depend on p53 DNA-binding affinity and oxidation of DJ-1 C106.

Neuroprotective function of DJ-1 in Parkinson's disease.

Parkinson's disease (PD) is caused by dopaminergic neuronal death in the substantia nigra, resulting in a reduced level of dopamine in the striatum. Oxidative stress and mitochondrial dysfunction are thought to be major causes of neurodegeneration in PD. Although genetic and environmental factors are thought to affect the onset of PD, precise mechanisms at the molecular level have not been elucidated. The DJ-1 gene is a causative gene for familial PD (park7) and also an oncogene. DJ-1 has various functions, including transcriptional regulation, antioxidative stress reaction, and chaperone, protease, and mitochondrial regulation, and its activity is regulated by its oxidative status, especially that of cysteine 106 (C106) of DJ-1. Excess oxidation of DJ-1, which renders DJ-1 inactive, has been observed in patients with sporadic PD and Alzheimer's disease, suggesting that DJ-1 also participates in the onset and pathogenesis of sporadic PD as well as familial PD. DJ-1 is also a stress sensor and its expression is increased upon various stresses, including oxidative stress. In this review, we describe functions of DJ-1 against oxidative stress and possible roles of DJ-1 in the pathogenesis of PD.