Takahiro Taira

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.

Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is primarily characterized by the degeneration of dopaminergic neurons in the nigrostriatal pathway. Previous studies have demonstrated that chronic systemic exposure of Lewis rats to rotenone produced many features of PD, and cerebral tauopathy was also detected in the case of severe weight loss. The present study was designed to assess the neurotoxicity of rotenone after daily oral administration for 28 days at several doses in C57BL/6 mice. In addition, we examined the protective effects of 4‐phenylbutyrate (4‐PBA) on nigral dopamine (DA) neurons in rotenone‐treated mice. 4‐PBA was injected intraperitoneally daily 30 min before each oral administration of rotenone. Chronic oral administration of rotenone at high doses induced specific nigrostriatal DA neurodegeneration, motor deficits and the up‐regulation of α‐synuclein in the surviving DA neurons. In contrast to the Lewis rat model, cerebral tauopathy was not detected in this mouse model. 4‐PBA inhibited rotenone‐induced neuronal death and decreased the protein level of α‐synuclein. These results suggest that this rotenone mouse model may be useful for understanding the mechanism of DA neurodegeneration in PD, and that 4‐PBA has a neuroprotective effect in the treatment of 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.

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.

Identification of heterochromatin protein 1 (HP1) as a phosphorylation target by Pim-1 kinase and the effect of phosphorylation on the transcriptional repression function of HP11

Pim‐1, a protooncogene product, is a serine/threonine kinase and is thought to play a role in signal transduction in blood cells. Few phosphorylated target proteins for Pim‐1, however, have been identified. In the present study, two‐hybrid screening to clone cDNAs encoding proteins binding to Pim‐1 was carried out, and a cDNA for heterochromatin protein 1γ (HP1γ) was obtained. Binding assays both in yeast and in vitro pull‐down using the purified HP1γ and Pim‐1 expressed in Escherichia coli showed that Pim‐1 directly bound to the chromo shadow domain of HP1γ. HP1γ was also associated with Pim‐1 in human HeLa cells and the serine clusters located at the center of HP1γ were phosphorylated by Pim‐1 in vitro. Furthermore, a transcription repression activity of HP1γ was further stimulated by the deletion of the serine clusters targeted by Pim‐1. These results suggest that Pim‐1 affects the structure or silencing of chromatin by phosphorylating HP1.

MM-1, a novel c-Myc-associating protein that represses transcriptional activity of c-Myc.

We have isolated the cDNA encoding a novel c-Myc-binding protein, MM-1, by the yeast two-hybrid screening of a human HeLa cell cDNA library. The protein deduced from the cDNA comprises 167 amino acids and was localized in the nucleus of introduced COS-I cells. The MM-1 mRNA was highly expressed in human pancreas and skeletal muscle and moderately in other tissues. As for the c-Myc binding, glutathione S-transferase MM-1 expressed in Escherichia coli bound in vitro to c-Myc translated in reticulocyte lysate, and almost whole, the MM-1 molecule was necessary for the binding in the yeast two-hybrid system. The mammalian two-hybrid assays in hamster CHO cells revealed that MM-1 interacts in vivo with the N-terminal domain covering themyc box 2, a transcription-activating domain, of c-Myc. Furthermore, MM-1 repressed the activation of E-box-dependent transcription by c-Myc.

Cell Cycle-dependent Switch of Up- and Down-regulation of Human hsp70 Gene Expression by Interaction between c-Myc and CBF/NF-Y

A CCAAT box-binding protein subunit, CBF-C/NF-YC, was cloned as a protein involved in the c-Myc complex formed on the G1-specific enhancer in the human hsp70gene. CBF-C/NF-YC directly bound to c-Myc in vitro andin vivo in cultured cells. The CBF/NF-Y·c-Myc complex required the HSP-MYC-B element as well as CCAAT in the hsp70G1-enhancer, while the purified CBF subunits recognized only CCAAT even in the presence of c-Myc. Both the HSP-MYC-B and CCAAT elements were also required for the enhancer activity. In transient transfection experiments, the CBF/NF-Y·c-Myc complex, as well as transcription due to the G1-enhancer, was increased by the introduction of c-Myc at low doses but decreased at high doses. The repression of both complex formation and transcription by c-Myc at high doses was abrogated by the introduction of CBF/NF-Y in a dose-dependent manner. Furthermore, the CBF/NF-Y·c-Myc complex bound to the G1-enhancer appeared in the early G1 phase of the cell cycle when c-Myc was not higly expressed and gradually disappeared after the c-Myc expression reached its maximum. The results indicate that the cell cycle-dependent expression of the hsp70 gene is regulated by the intracellular amount of c-Myc through the complex formation states between CBF/NF-Y and c-Myc.

Significance of Immunological Detection of Human Telomerase Reverse Transcriptase : Re-Evaluation of Expression and Localization of Human Telomerase Reverse Transcriptase

Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of telomerase and is a potentially useful diagnostic marker for cancers. There have been few studies in which immunological detection of hTERT has been attempted and its subcellular localization has not been precisely defined. In the present study, we re-evaluated expression and localization of hTERT in cancer and normal cells using a newly developed antibody. Immunohistochemistry revealed that hTERT is expressed in approximately 80% of gynecological cancers, but some premalignant lesions exhibited weak expression of hTERT. Interestingly, not only nuclei but also cytoplasm of cancer cells were positive for hTERT staining. This finding was supported by the results of Western blot analysis of cell lines, in which both nuclear and cytoplasmic extracts exhibited significant hTERT bands. Cytoplasmic hTERT in cancer cells may be functional because the telomeric repeat amplification protocol assay of cytoplasmic extracts showed high levels of telomerase activity. Unexpectedly, not all normal primary cells and telomerase-negative cancer cell lines lacked hTERT expression; some exhibited weak TERT signals. In Western analysis, hTERT signals did not always correlate with telomerase activity of the various cell types. These findings suggest that functional hTERT is expressed in both the nucleus and cytoplasm of cancer cells and that hTERT expression does not strictly reflect telomerase activity. Further analysis is needed to clarify the biological significance of cytoplasmic hTERT.

AMY‐1, a novel C‐MYC binding protein that stimulates transcription activity of C‐MYC

The c‐myc proto‐oncogene has been suggested to play key roles in cell proliferation, differentiation, transformation and apoptosis. A variety of functions of C‐MYC, the product of c‐myc, are attributed to protein–protein interactions with various cellular factors including Max, YY1, p107, Bin1 and TBP. Max and YY1 bind to the C‐terminal region of C‐MYC, while p107, Bin1 and TBP bind to the N‐terminal region covering myc boxes. The N‐terminal region is involved in all the biological functions of C‐MYC, and different proteins are therefore thought to interact with the N‐terminal region of C‐MYC to display different functions.

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.