Biomedical subjects

Hiroyoshi Ariga

Publications and source records attributed to Hiroyoshi Ariga.

At least 19 recordsLinked to original sources

PubMed2006

The role of Vpr in the regulation of HIV-1 gene expression.

Expression of the viral protein R, Vpr, of HIV-1 affects many biological events in host cells including cell cycle progression, and modulates HIV-1 gene transcription. Earlier studies implicating the cellular protein p21(WAF1) (p21) in regulation of HIV-1 transcription, led us to investigate the functional and physical interaction of Vpr and p21. Our results show that Vpr modestly activated HIV-LTR in cells lacking p21 gene. Here, we describe the mechanisms by which p21 and Vpr leading to stimulation of HIV-1 transcription. Data from the protein-protein interaction experiments revealed the ability of Vpr, p21 and p300 to form a complex. Further, we show that, Vpr interacts with the N- and the C-terminal domains of p21. Furthermore, in cells expressing Vpr, p21 localizes to both the cytoplasm and the nucleus. Interestingly, expression of Vpr alleviates p21-mediated inhibition of cell departure from G1 phase. Expression of a mutant Vpr, with arginine 73 altered to serine, did not affect the ability of p21 to cause cells arrest or its sub-cellular localization. These observations reveal a new cellular partner for Vpr, and provide a new therapeutic avenue for controlling HIV-1 expression.

Binding Sites↗

PubMed2006

Distribution of DJ-1, Parkinson's disease-related protein PARK7, and its alteration in 6-hydroxydopamine-treated hemiparkinsonian rat brain.

DJ-1 has multiple functions and its dysfunction may be linked to the onset of familial Parkinson's disease PARK7. However, the function and distribution of DJ-1 is unclear. In this study, we determined DJ-1 distribution and change after intranigral injection of 6-hydroxydopamine (6-OHDA). Although distribution of DJ-1 immunoreactivity was not changed in cerebral cortex and striatum, 6-OHDA caused increase of DJ-1 in the particulate fraction and decrease in the cytosolic fraction in substantia nigra. At that time, DJ-1 shifted to acid forms. These results suggest that distributional changes, translocation, and acidic shift of DJ-1 may be compensatory responses to protect against 6-OHDA-induced oxidative stress.

Animals↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2006

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.

Blotting, Western↗

PubMed2006

PARK7 DJ-1 protects against degeneration of nigral dopaminergic neurons in Parkinson's disease rat model.

DJ-1 has recently been shown to be responsible for onset of familial Parkinson's disease (PD), PARK7. DJ-1 has been shown to play roles in transcriptional regulation and anti-oxidative stress, and loss of its function is thought to trigger onset of PD. In this study, a recombinant DJ-1 protein was administrated into the brain of PD model rats that had been injected to 6-hydroxydopamine (6-OHDA) in the left substantia nigra. PD phenotypes, including dopaminergic neuron death in the substantia nigra, decrease in dopamine, and dopamine transporter levels in the striatum, and motor abnormality, were dramatically improved by wild-type DJ-1 but not L166P DJ-1, a mutant form of DJ-1 found in PD patients. Furthermore, production of reactive oxygen species and cell death induced by 6-OHDA in SH-SY5Y cells and mesencephalic neurons were inhibited by addition of the recombinant DJ-1. These findings suggest that DJ-1 is a therapeutic target for PD.

Animals↗

PubMed2006

Establishment of specific antibodies that recognize C106-oxidized DJ-1.

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 PARK7. DJ-1 plays roles in transcriptional regulation and in oxidative stress function, and its oxidative state at the cysteine residue 106 (C106) determines activities of DJ-1. Elevated levels of oxidation of DJ-1 were observed in brain tissues of patients with Parkinson's disease (PD) and patients with Alzheimer's disease (AD). In this study, we established specific antibodies using synthetic peptide containing SO(3)H at C106 of DJ-1 as an immunogen. These antibodies were found by Western blot analysis to recognize DJ-1 specifically oxidized at C106 but not at other cysteines. These antibodies should be useful to study pathophysiologies of PD and AD.

Alzheimer Disease↗

PubMed2006

Distinct localizations and repression activities of MM-1 isoforms toward c-Myc.

MM-1 was identified as a c-Myc-binding protein and has been reported to repress the E-box-dependent transcription activity of c-Myc by recruiting HDAC1 complex via TIF1 beta/KAP1. In this study, originally isolated MM-1 was found to be a fusion protein comprised of the N-terminal 13 amino acids from the sequence of chromosome 14 and of the rest of the amino acids from that of chromosome 12 and was found to be expressed ubiquitously in all human tissues. Four splicing isoforms of MM-1, MM-1alpha, MM-1beta, MM-1gamma, and MM-1delta, which are derived from the sequence of chromosome 12, were then identified. Of these isoforms, MM-1alpha, MM-1gamma, and MM-1delta were found to be expressed in tissue-specific manners and MM-1beta was found to be expressed ubiquitously. Although all of the isoforms potentially possessed c-Myc- and TIF1beta-binding activities, MM-1beta and MM-1delta were found to be mainly localized in the cytoplasm and MM-1alpha and MM-1gamma were found to be localized in the nucleus together with both c-Myc and TIF1beta. Furthermore, when repression activities of MM-1 isoforms toward c-Myc transcription activity were examined by reporter gene assays in HeLa cells, MM-1alpha, MM-1gamma, and MM-1gamma, but not MM-1beta, were found to repress transcription activity of c-Myc, and the degrees of repression by MM-1gamma and MM-1delta were smaller than those by MM-1 and MM-1alpha. These results suggest that each MM-1 isoform distinctly regulates c-Myc transcription activity in respective tissues.

Base Sequence↗

PubMed2006

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 Parkinson's 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.

Apoptosis↗

PubMed2006

Characterization of mouse serum tenascin-X.

The interstitial extracellular matrix tenascin-X (iTNX), which has a molecular mass of roughly 450 kDa, is expressed at high levels in muscular tissues and skin. In this study, we identified the serum form of TNX (sTNX) with a molecular mass of 200 kDa in the mouse. Western blot analysis with specific antibodies against fibronectin type III-like (FNIII) repeats of TNX and N-terminal sequence analysis of 200-kDa sTNX revealed that the N-terminus of sTNX is located in the juncture between the 16th FNIII (M16) and 17th FNIII (M17) repeats of iTNX. The 200-kDa sTNX contains 15 FNIII repeats and a fibrinogen domain identical to the Cterminal portion of the iTNX. TNX-deficient mice lacked not only iTNX but also sTNX. Furthermore, 200-kDa sTNX was generated by cleavage of the spleen iTNX by spleen homogenate, and its generation was inhibited by protease inhibitors. These results suggest that sTNX is generated by proteolytic cleavage of iTNX.

Amino Acid Sequence↗

PubMed2006

AMY-1 (associate of Myc-1) localization to the trans-Golgi network through interacting with BIG2, a guanine-nucleotide exchange factor for ADP-ribosylation factors.

AMY-1 (associate of Myc-1) was originally identified as a c-Myc-binding protein that enhances the c-Myc transcription activity, and subsequently found to interact with A-kinase-anchoring proteins (AKAPs), including AKAP149, S-AKAP84 and AKAP95. We show here that, using anti-AMY-1 antibodies we raised, AMY-1 localizes to the trans-Golgi network (TGN) and the nucleus. To explore the possible function of AMY-1, we have undertaken a search for interacting partners by co-immunoprecipitation experiments using cells stably expressing FLAG-tagged AMY-1. Interestingly, we have found that AMY-1 interacts with BIG2 and BIG1, both of which are high molecular weight guanine-nucleotide exchange factors for ADP-ribosylation factors (ARFs) and mainly localize to the TGN. Furthermore, we have demonstrated that AMY-1 is associated with the TGN through interacting with BIG2 but not with BIG1 using an RNA interference approach, although AMY-1 can interact with both BIG1 and BIG2 in vitro. Taken together with the facts that BIG2 contains domains that bind to regulatory subunits of protein kinase A and that recruitment of ARF1 onto Golgi membranes is mediated, at least in part, by activation of protein kinase A, these results suggest that BIG2 alone or in concert with recruited AMY-1 coordinates ARF-mediated membrane trafficking and signaling pathways.

ADP-Ribosylation Factors↗

PubMed2005

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.

Amino Acid Substitution↗

PubMed2005

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.

Animals↗

PubMed2005

Induction of reactive oxygen species by bisphenol A and abrogation of bisphenol A-induced cell injury by DJ-1.

DJ-1 was first identified as an activated ras-dependent oncogene. DJ-1 is related to male fertility, and its expression in sperm decreases in response to exposure to a number of reproductive toxicants. DJ-1 has been associated with the onset of familial Parkinson's disease (PD) in humans, and has been found to have activity against oxidative damage by eliminating reactive oxygen species (ROS). In this study, we investigated the role of DJ-1 in oxidative stresses by administration of bisphenol A (BPA), which has been reported to induce oxidative stress in rodents, to male mice and cultured cells. In male mice, we found that BPA significantly increased the expression level of DJ-1 in the sperm and brain. In cultured Neuro2a and GC1 cells, we found that BPA induced ROS production and significantly compromised mitochondrial function concomitant with elevated expression and oxidization of DJ-1. DJ-1 was found to maintain the complex I activity against BPA-induced oxidative stress after the localization in mitochondria. The results showed that DJ-1 plays a role in the prevention of mitochondrial injury-induced cell death.

Air Pollutants, Occupational↗

PubMed2005

Positive regulation of Fas gene expression by MSSP and abrogation of Fas-mediated apoptosis induction in MSSP-deficient mice.

MSSP has been identified as a transcription factor that regulates the c-myc gene. MSSP was later found to positively or negatively regulate a variety of genes, including alpha-smooth actin, MHC class I, MHC class 2 and the thyrotropin receptor. The knockout mice for the Mssp gene developed by us revealed that these mice became partially embryonic lethal due to a low concentration of progesterone at E2.5. In this study, we further analyzed Mssp-knockout mice and found that the expression of the Fas gene was repressed, resulting in abrogation of Fas-mediated induction of apoptosis both in Mssp-knockout mice and primary thymocytes. MSSP was then found to stimulate promoter activity of the Fas gene by binding to a region spanning -1035 to -635 in chromatin immunoprecipitation assays. Binding of MSSP in the MSSP-binding sequence, TCTAAT, located in this region was confirmed by mobility shift assays, and deletion of this sequence from the Fas promoter was found to result in loss of MSSP-dependent stimulating activity. The results suggest that MSSP is an important mediator for Fas-induced apoptosis in vivo and in vitro.

Animals↗

PubMed2005

Association of PAP-1 and Prp3p, the products of causative genes of dominant retinitis pigmentosa, in the tri-snRNP complex.

PAP-1 has been identified by us as a Pim-1-binding protein and has recently been implicated as the defective gene in RP9, one type of autosomal dominant retinitis pigmentosa (adRP). We have then shown that PAP-1 plays a role in pre-mRNA splicing. Because four causative genes for adRP, including PAP-1, Prp31, Prp8, and Prp3, encode proteins that function as splicing factors or splicing-modulating factors, we investigated the interaction of PAP-1 with Prp3p and Prp31p in this study. The results showed that PAP-1 interacted with Prp3p but not Prp31p in human cells and yeast, and that the basic region of PAP-1 and the C-terminal region of Prp3p, regions beside spots found in adRP mutations, were needed for binding. Furthermore, both Prp3p and a part of PAP-1 were found to be components of the U4/U6.U5-tri-snRNP complex, one form of the spliceosome, in Ba/F3 and K562 cells by analysis of sucrose density gradients, suggesting that PAP-1 is weakly associated with the spliceosome. These results also suggest that splicing factors implicated in adRP contribute alone or mutually to proper splicing in the retina and that loss of their functions leads to onset of adRP.

Amino Acid Sequence↗

PubMed2005

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 Parkinson's 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.

Animals↗

PubMed2005

Transcription regulatory complex including YB-1 controls expression of mouse matrix metalloproteinase-2 gene in NIH3T3 cells.

Matrix metalloproteinase 2 (MMP-2) is a metalloproteinase belonging to a family of structurally related zinc-dependent endopeptidases capable of degrading extracellular matrix components. To elucidate the functional promoter of the mouse MMP-2 gene, systematic transient expression analysis of the 5'-flanking region of the MMP-2 gene was performed using serially nested deletions. The deletion analysis indicated that the proximal 327-bp sequence from nucleotide positions -313 to +14 relative to the transcription start site is essential for minimal promoter activity and that a 10-bp sequence of the promoter at positions -939 to -930 is required for high expression level of the MMP-2 gene. The 10-bp fragment functioned as a potent stimulator of heterologous SV40 promoter activity. This element is identical to the YB-1 binding motif (Y-box) present within the responsive element-1 (RE-1), which has been shown to act as a potent cis-activator of transcription of the rat MMP-2 gene. The binding of a nuclear factor(s) to the 10-bp fragment was also revealed by electrophoretic mobility shift assays (EMSAs). Antibody-supershift EMSAs of nuclear extracts from NIH 3T3 cells demonstrated YB-1 binding to the RE-1 sequence. It was concluded that the RE-1 is the conserved element for potent expression of MMP-2 gene among rodents.

3T3 Cells↗

PubMed2005

Structure and characterization of AAT-1 isoforms.

A novel protein, AAT-1, was identified as a AMY-1-binding protein and three splicing variants of AAT-1, AAT-1alpha, -beta and -gamma were identified. The function of AAT-1 is thought to be related to spermatogenesis. In this study, we further identified other splicing isoforms of AAT-1, AAT-1L, AAT-1M and AAT-1S, consisting of 767, 603 and 252 amino acids, respectively. These isoforms were found to use a promoter different from that used by AAT-1alpha, -beta and -gamma in the aat-1 gene, which contains 20 exons. Only 60 amino acids in the C-terminal portion of AAT-1 derived from exons 15-17 are common among AAT-1L, AAT-1M, AAT-1S and AAT-1alpha. While AAT-1alpha is specifically expressed in the testis, AAT-1L, AAT-1M, AAT-1S were found to be differentially expressed in human tissues. All of the isoforms of AAT-1 were found to bind to and colocalized with AMY-1 in human cells. While AAT-1L and AAT-1M were found to be localized diffusely in the cytoplasm, AAT-1S, like AAT-1alpha, was found to be localized in the mitochondria-like structure, suggesting different roles of AAT-1 isoforms in cells.

Amino Acid Sequence↗