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 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.

Secretion of DJ-1 into the serum of patients with Parkinson's disease.

DJ-1 was initially identified by us as a novel oncogene and has later been found to be a causative gene for familial Parkinson's disease PARK7. DJ-1 plays role in transcriptional regulation and in oxidative stress function, and loss of its function is thought to be related to onset age, mode of progression and clinical severity of both familial and sporadic forms of Parkinson's disease (PD). DJ-1 is localized both in the cytoplasm and nucleus, and it has been reported to be secreted into the serum or plasma of patients with breast cancer, melanoma, familial amyloidotic polyneuropathy and stroke. In this study, levels of DJ-1 secreted into the serum of healthy controls and patients with sporadic PD were examined by using a DJ-1 ELISA kit, and the level of oxidative stress in the serum was also measured. The results showed that DJ-1 was secreted into the serum of both healthy controls and PD patients. There was no significant difference between the levels of secreted DJ-1 in two groups, and correlations of levels of secreted DJ-1 with age, clinical severity of PD and level of oxidative stress were not found.

Lateral Gene Transfer Between Protozoa-Related Giant Viruses of Family Mimiviridae and Chlamydiae.

Obligate intracellular chlamydiae diverged into pathogenic and environmental chlamydiae 0.7-1.4 billion years ago. While pathogenic chlamydiae have adapted to a wide range of vertebrates, environmental chlamydiae inhabit unicellular amoebae, the free-living Acanthamoeba. However, how and why this divergence occurred remains unclear. Meanwhile, giant viruses consisting of protozoa-related and protozoa-unrelated viruses have been discovered, with the former group being suggested to have more influenced environmental chlamydiae during their evolution while cohabiting host amoebae. Against this background, we attempted to visualize genes of giant viruses in chlamydial genomes by bioinformatic analysis mainly with comparative genome and phylogenic analysis, seeking genes present in chlamydiae that are specifically shared with protozoa-related giant viruses. As a result, in contrast to protozoa-unrelated giant viruses, the genes of protozoa-related giant viruses were significantly shared in both the chlamydia genomes depending on the giant virus type. In particular, the prevalence of Mimiviridae genes among the protozoa-related giant virus genes in chlamydial genomes was significantly high. Meanwhile, the prevalence of protozoa-related giant virus genes in pathogenic chlamydia genomes was consistently higher than those of environmental chlamydiae; the actual number of sequences similar to giant virus was also significantly predominant compared with those in the environmental chlamydial genomes. Among them, the most prevalent of giant virus was in the case of chlamydiae with Megavirus chiliensis; total of 1338 genes of the chlamydiae were found to be shared with the virus (444 genes specific to environmental chlamydiae, 892 genes shared between both chlamydiae, only two genes in the pathogenic chlamydiae). Phylogenic analysis with most prevalent sets (Megavirus chiliensis and Protochlamydia EI2 or Chlamydia trachomatis L2 434Bu) showed the presence of orthologs between these with several clustered. In addition, Pearson's single regression analysis revealed that almost the prevalence of the genes from the giant viruses in chlamydial genomes was negatively and specifically correlated with the number of chlamydial open reading frames (ORFs). Thus, these results indicated the trace of lateral gene transfer between protozoa-related giant viruses of family Mimiviridae and chlamydiae. This is the first demonstration of a putative linkage between chlamydiae and the giant viruses, providing us with a hint to understand chlamydial evolution.

Amoebal endosymbiont Neochlamydia protects host amoebae against Legionella pneumophila infection by preventing Legionella entry.

Acanthamoeba isolated from environmental soil harbors the obligate intracellular symbiont Neochlamydia, which has a critical role in host amoebal defense against Legionella pneumophila infection. Here, by using morphological analysis with confocal laser scanning fluorescence microscopy and transmission electron microscopy, proteome analyses with two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry (LC/MS), and transcriptome analysis with DNA microarray, we explored the mechanism by which the Neochlamydia affected this defense. We observed that when rare uptake did occur, the symbiotic amoebae allowed Legionella to grow normally. However, the symbiotic amoebae had severely reduced uptake of Legionella when compared with the aposymbiotic amoebae. Also, in contrast to amoebae carrying the endosymbiont, the actin cytoskeleton was significantly disrupted by Legionella infection in aposymbiotic amoebae. Furthermore, despite Legionella exposure, there was little change in Neochlamydia gene expression. Taken together, we concluded that the endosymbiont, Neochlamydia prevents Legionella entry to the host amoeba, resulting in the host defense against Legionella infection.

Specific cleavage of DJ-1 under an oxidative condition.

DJ-1 was initially identified by us as a novel oncogene and has recently been found to be a causative gene for familial Parkinson's disease (PD) PARK7. DJ-1 plays roles in transcriptional regulation and in oxidative stress function, and its oxidative state at cysteine residues determines activities of DJ-1. In this study, we found that recombinant DJ-1 expressed in and purified from E. coli was specifically cleaved between glycine and proline at amino acid numbers 157 and 158, respectively, by treatment of DJ-1 with H2O2. A substitution mutant of DJ-1 from cysteine to serine at amino acid number 106, a major oxidation site of DJ-1, was found not to be cleaved under an oxidative condition, suggesting oxidation-dependent cleavage of DJ-1. Cleavage of DJ-1 was also observed in human SH-SY5Y cells that had been treated with H2O2. These results suggest that oxidative stress-induced cleavage of DJ-1 regulates functions of DJ-1.

Protection against nonylphenol-induced cell death by DJ-1 in cultured Japanese medaka (Oryzias latipes) cells.

The Japanese medaka (Oryzias latipes) has been used to investigate diverse aspects of toxicology, genetics and developmental biology and to monitor biological changes caused by endocrine disruptors. In this study, we analyzed a medaka homolog of human DJ-1 (meDJ-1) in cultured medaka cells into which nonylphenol (NP) was added. Like human DJ-1, meDJ-1 was found to be oxidized by treatment with H(2)O(2) and its pI was shifted to acidic points. NP was found to induce cell death with kinetics similar to that of H(2)O(2) in cultured medaka OLHE-13 cells. After OLHE-13 cells had been treated with sub-lethal concentrations of H(2)O(2) and NP, production of reactive oxygen species and oxidation of meDJ-1 were observed. meDJ-1 knockdown by short interfering RNA rendered OLHE-13 cells susceptible to H(2)O(2) and NP-induced cell death, suggesting a protective role of DJ-1 against oxidative stress-induced cell death in medaka cells. These results suggest that meDJ-1 is a suitable biomarker for oxidative stress reactions in medaka.

Draft Genome Sequences of Legionella pneumophila JR32 and Lp01 Laboratory Strains Domesticated in Japan.

We report here the draft genome sequences of two Legionella pneumophila variant strains (JR32 and Lp01_666) originally derived from a Philadelphia-1 clinical isolate, domesticated in Japan, with distinct susceptibility to amoebae. Detailed genomic analysis will allow us to better understand Legionella adaptation and survival mechanisms in host cells.

Monomer DJ-1 and its N-terminal sequence are necessary for mitochondrial localization of DJ-1 mutants.

DJ-1 is a novel oncogene and also a 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. Mitochondrial dysfunction is observed in DJ-1-knockout mice and fry, and mitochondrial DJ-1 is more protective against oxidative stress-induced cell death. Although translocation of DJ-1 into mitochondria is enhanced by oxidative stress that leads to oxidation of cysteine 106 (C106) of DJ-1, the characteristics of mitochondrial DJ-1 and the mechanism by which DJ-1 is translocated into mitochondria are poorly understood. In this study, immunostaining, co-immunoprecipitation, cell fractionation and pull-down experiments showed that mutants of glutamine 18 (E18) DJ-1 are localized in mitochondria and do not make homodimers. Likewise, DJ-1 with mutations of two cysteines located in the dimer interface, C46S and C53A, and pathogenic mutants, M26I and L166P DJ-1, were found to be localized in mitochondria and not to make homodimers. Mutant DJ-1 harboring both E18A and C106S, in which C106 is not oxidized, was also localized in mitochondria, indicating that oxidation of C106 is important but not essential for mitochondrial localization of DJ-1. It should be noted that E18A DJ-1 was translocated from mitochondria to the cytoplasm when mitochondrial membrane potential was reduced by treatment of cells with CCCP, an uncoupler of the oxidative phosphorylation system in mitochondria. Furthermore, deletion or substitution of the N-terminal 12 amino acids in DJ-1 resulted in re-localization of E18A, M26I and L166P DJ-1 from mitochondria into the cytoplasm. These findings suggest that a monomer and the N-terminal 12 amino acids are necessary for mitochondrial localization of DJ-1 mutants and that conformation change induced by C106 oxidation or by E18 mutation leads to translocation of DJ-1 into mitochondria.