Takeshi Niki

DJ‐1 has a role in antioxidative stress to prevent cell death

Deletion and point (L166P) mutations of DJ‐1 have recently been shown to be responsible for the onset of familial Parkinsons disease (PD, PARK7). The aim of this study was to determine the role of DJ‐1 in PD. We first found that DJ‐1 eliminated hydrogen peroxide in vitro by oxidizing itself. We then found that DJ‐1 knockdown by short interfering RNA rendered SH‐SY5Y neuroblastoma cells susceptible to hydrogen peroxide‐, MPP+‐ or 6‐hydroxydopamine‐induced cell death and that cells harbouring mutant forms of DJ‐1, including L166P, became susceptible to death in parallel with the loss of oxidized forms of DJ‐1. These results clearly showed that DJ‐1 has a role in the antioxidative stress reaction and that mutations of DJ‐1 lead to cell death, which is observed in PD.

Association of DJ-1 with chaperones and enhanced association and colocalization with mitochondrial Hsp70 by oxidative stress

DJ-1 is a novel oncogene and causative gene for familial form of the Parkinsons disease (PD). DJ-1 has been shown to play a role in anti-oxidative stress by eliminating reactive oxygen species (ROS). The onset of PD is thought to be caused by oxidative stress and mitochondrial injury, which leads to protein aggregation that results in neuronal cell death. However, the mechanism by which DJ-1 triggers the onset of PD is still not clear. In this study, we analyzed association and localization of DJ-1 and its mutants with various chaperones. The results showed that DJ-1 and its mutants were associated with Hsp70, CHIP and mtHsp70/Grp75, a mitochondria-resident Hsp70, and that L166P and M26I mutants found in PD patients were strongly associated with Hsp70 and CHIP compared to wild-type and other DJ-1 mutants. DJ-1 and its mutants were colocalized with Hsp70 and CHIP in cells. Furthermore, association and colocalization of wildtype DJ-1 with mtHsp70 in mitochondria were found to be enhanced by treatment of cells with H2O2. These results suggest that translocation of DJ-1 to mitochondria after oxidative stress is carried out in association with chaperones.

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

Parkinsons 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 Alzheimers 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.

Proper SUMO-1 conjugation is essential to DJ-1 to exert its full activities

DJ-1 is a multifunctional protein that plays roles in transcriptional regulation and antioxidative stress, and loss of its function is thought to result in the onset of Parkinsons disease (PD). Here, we report that DJ-1 was sumoylated on a lysine residue at amino-acid number 130 (K130) by PIASxα or PIASy. The K130 mutation abrogated all of the functions of DJ-1, including ras-dependent transformation, cell growth promotion and anti-UV-induced apoptosis activities. Sumoylation of DJ-1 was increased after UV irradiation concomitant with a pI shift to an acidic point of DJ-1. Furthermore, L166P, a mutant DJ-1 found in PD patients, and K130RX, an artificial mutant containing four mutations in DJ-1, were improperly sumoylated, and they became insoluble, partly localized in the mitochondria and degraded by the proteasome system. Both L166P-expressing cells and DJ-1-knockdown cells were found to be highly susceptible to UV-induced cell apoptosis.

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 H2O2, 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 SO2H and SO3H 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.

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.

DJ-1 interacts with HIPK1 and affects H2O2-induced cell death

DJ-1 is a novel oncogene and causative gene for the familial form of Parkinsons disease (PD). DJ-1 has multiple functions, including anti-oxidative stress by eliminating reactive oxygen species (ROS) and transcriptional regulation as a coactivator, and loss of these functions are thought to trigger the onset of PD. The mechanism underlying the prevention of cell death by DJ-1 is, however, not clear. In this study, we found that DJ-1 directly bound to homeodomaininteracting protein kinase 1 (HIPK1) in vitro and in vivo and that these proteins were colocalized in the nucleus. HIPK1 was then found to be degraded in human H1299 cells transfected with wild-type DJ-1 but not with a C106S DJ-1 mutant, a DJ-1 protein disrupting a catalytic domain of the putative protease, in a dose-dependent manner. Furthermore, although knockdown of either DJ-1 or HIPK1 rendered H1299 cells susceptible to H2O2-induced cell death, double-knockdown of DJ-1 and HIPK1 rendered H1299 cells resistant to H2O2-induced cell death, suggesting that the elevated level of HIPK1 induced by a low level of DJ-1 inhibits oxidative stress-induced cell death.

MSSP promotes ras/myc cooperative cell transforming activity by binding to c‐Myc

MSSPs, mycsingle strand binding proteins, were originally identified as proteins recognizing a putative replication origin/transcriptional enhancer in the human c‐Myc gene. The cDNAs encoding four of the family proteins, MSSP‐1, MSSP‐2, Scr2 and Scr3, were cloned. These proteins carry two copies of the putative RNA binding domains, RNP‐A and RNP‐B, and have been suggested to participate in DNA replication and cell cycle progression from the G1 to the S phase.

Transcriptional Activation of Low-Density Lipoprotein Receptor Gene by DJ-1 and Effect of DJ-1 on Cholesterol Homeostasis

DJ-1 is a novel oncogene and also causative gene for familial Parkinson’s disease park7. DJ-1 has multiple functions that include transcriptional regulation, anti-oxidative reaction and chaperone and mitochondrial regulation. For 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 the low-density lipoprotein receptor (LDLR) gene is a transcriptional target gene for DJ-1. Reduced expression of LDLR mRNA and protein was observed in DJ-1-knockdown cells and DJ-1-knockout mice and this occurred at the transcription level. Reporter gene assays using various deletion and point mutations of the LDLR promoter showed that DJ-1 stimulated promoter activity by binding to the sterol regulatory element (SRE) with sterol regulatory element binding protein (SREBP) and that stimulating activity of DJ-1 toward LDLR promoter activity was enhanced by oxidation of DJ-1. Chromatin immunoprecipitation, gel-mobility shift and co-immunoprecipitation assays showed that DJ-1 made a complex with SREBP on the SRE. Furthermore, it was found that serum LDL cholesterol level was increased in DJ-1-knockout male, but not female, mice and that the increased serum LDL cholesterol level in DJ-1-knockout male mice was cancelled by administration with estrogen, suggesting that estrogen compensates the increased level of serum LDL cholesterol in DJ-1-knockout female mice. This is the first report that DJ-1 participates in metabolism of fatty acid synthesis through transcriptional regulation of the LDLR gene.

DJ-1 cooperates with PYCR1 in cell protection against oxidative stress.

DJ-1, a product of the DJ-1/PARK7 gene, has been suggested to play various functions involved in transcriptional regulation, protease activity, anti-oxidative stress activity, and regulation of mitochondrial complex I. Such a variety of functions of DJ-1 are supposed to be realized through interactions with different partner proteins. Among the candidates for DJ-1-partner proteins detected in TOF-MAS analyses of the cellular proteins co-immunoprecipitated with DJ-1, we focused here pyrroline-5-carboxylate reductase 1, PYCR1, a final key enzyme for proline biosynthesis. DJ-1 directly bound to PYCR1 in vivo and in vitro. DJ-1 and PYCR1 colocalized in mitochondria, and both were suggested to be involved in regulation of mitochondrial membrane potential, but differently. DJ-1 enhanced the enzymatic activity of PYCR1 in vitro. The cells knocked down for DJ-1 and PYCR1 showed lower viability under oxidative stress conditions. No additive nor synergistic results were obtained for the cells that had been knocked down for both DJ-1 and PYCR1, suggesting that DJ-1 and PYCR1 are on the same pathway of anti-oxidative stress protection of the cells.