Updated on 2021/12/16

写真a

 
UEHARA Takashi
 
Organization
Medicine, Dentistry and Pharmaceutical Sciences Professor
Position
Professor
External link

Degree

  • PhD ( Hokkaido University )

Research Areas

  • Life Science / Pharmacology

 

Papers

  • Covalent N-arylation by the pollutant 1,2-naphthoquinone activates the EGF receptor. Reviewed

    Nakahara, K, Hamada, K, Tsuchida, T, Takasugi, N, Abiko, Y, Shien, K, Toyooka, S, Kumagai, Y, Uehara, T

    J. Biol. Chem.   296   100524   2021

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  • Spatiotemporal analysis of the UPR transition induced by methylmercury in the mouse brain. Reviewed

    Hiraoka, H, Nomura, R, Takasugi, N, Akai, R, Iwawaki, T, Kumagai, Y, Fujimura, M, Uehara, T

    Arch. Toxicol.   95 ( 4 )   1241 - 1250   2021

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  • S-Nitrosylation at the active site decreases the ubiquitin-conjugating activity of ubiquitin-conjugating enzyme E2 D1 (UBE2D1), an ERAD-associated protein. International journal

    Kana Fujikawa, Kengo Nakahara, Nobumasa Takasugi, Tadashi Nishiya, Akihiro Ito, Koji Uchida, Takashi Uehara

    Biochemical and biophysical research communications   524 ( 4 )   910 - 915   2020.4

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    S-Nitrosylation of protein cysteine thiol is a post-translational modification mediated by nitric oxide (NO). The overproduction of NO causes nitrosative stress, which is known to induce endoplasmic reticulum (ER) stress. We previously reported that S-nitrosylation of protein disulfide isomerase (PDI) and the ER stress sensor inositol-requiring enzyme 1 (IRE1) decreases their enzymatic activities. However, it remains unclear whether nitrosative stress affects ER-associated degradation (ERAD), a separate ER stress regulatory system responsible for the degradation of substrates via the ubiquitin-proteasomal pathway. In the present study, we found that the ubiquitination of a known ERAD substrate, serine/threonine-protein kinase 1 (SGK1), is attenuated by nitrosative stress. C-terminus of Hsc70-interacting protein (CHIP) together with ubiquitin-conjugating enzyme E2 D1 (UBE2D1) are involved in this modification. We detected that UBE2D1 is S-nitrosylated at its active site, Cys85 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, in vitro and cell-based experiments revealed that S-nitrosylated UBE2D1 has decreased ubiquitin-conjugating activity. Our results suggested that nitrosative stress interferes with ERAD, leading to prolongation of ER stress by co-disruption of various pathways, including the molecular chaperone and ER stress sensor pathways. Given that nitrosative stress and ER stress are upregulated in the brains of patient with Parkinson's disease (PD) and of those with Alzheimer's disease (AD), our findings may provide further insights into the pathogenesis of these neurodegenerative disorders.

    DOI: 10.1016/j.bbrc.2020.02.011

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  • MITOL prevents ER stress-induced apoptosis by IRE1α ubiquitylation at ER-mitochondria contact sites. International journal

    Keisuke Takeda, Shun Nagashima, Isshin Shiiba, Aoi Uda, Takeshi Tokuyama, Naoki Ito, Toshifumi Fukuda, Nobuko Matsushita, Satoshi Ishido, Takao Iwawaki, Takashi Uehara, Ryoko Inatome, Shigeru Yanagi

    The EMBO journal   38 ( 15 )   e100999   2019.8

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    Unresolved endoplasmic reticulum (ER) stress shifts the unfolded protein response signaling from cell survival to cell death, although the switching mechanism remains unclear. Here, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) inhibits ER stress-induced apoptosis through ubiquitylation of IRE1α at the mitochondria-associated ER membrane (MAM). MITOL promotes K63-linked chain ubiquitination of IRE1α at lysine 481 (K481), thereby preventing hyper-oligomerization of IRE1α and regulated IRE1α-dependent decay (RIDD). Therefore, under ER stress, MITOL depletion or the IRE1α mutant (K481R) allows for IRE1α hyper-oligomerization and enhances RIDD activity, resulting in apoptosis. Similarly, in the spinal cord of MITOL-deficient mice, ER stress enhances RIDD activity and subsequent apoptosis. Notably, unresolved ER stress attenuates IRE1α ubiquitylation, suggesting that this directs the apoptotic switch of IRE1α signaling. Our findings suggest that mitochondria regulate cell fate under ER stress through IRE1α ubiquitylation by MITOL at the MAM.

    DOI: 10.15252/embj.2018100999

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  • The Emerging Role of Electrophiles as a Key Regulator for Endoplasmic Reticulum (ER) Stress. International journal

    Nobumasa Takasugi, Hideki Hiraoka, Kengo Nakahara, Shiori Akiyama, Kana Fujikawa, Ryosuke Nomura, Moeka Furuichi, Takashi Uehara

    International journal of molecular sciences   20 ( 7 )   2019.4

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    The unfolded protein response (UPR) is activated by the accumulation of misfolded proteins in the endoplasmic reticulum (ER), which is called ER stress. ER stress sensors PERK, IRE1, and ATF6 play a central role in the initiation and regulation of the UPR; they inhibit novel protein synthesis and upregulate ER chaperones, such as protein disulfide isomerase, to remove unfolded proteins. However, when recovery from ER stress is difficult, the UPR pathway is activated to eliminate unhealthy cells. This signaling transition is the key event of many human diseases. However, the precise mechanisms are largely unknown. Intriguingly, reactive electrophilic species (RES), which exist in the environment or are produced through cellular metabolism, have been identified as a key player of this transition. In this review, we focused on the function of representative RES: nitric oxide (NO) as a gaseous RES, 4-hydroxynonenal (HNE) as a lipid RES, and methylmercury (MeHg) as an environmental organic compound RES, to outline the relationship between ER stress and RES. Modulation by RES might be a target for the development of next-generation therapy for ER stress-associated diseases.

    DOI: 10.3390/ijms20071783

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  • Attenuation of Macrophage Migration Inhibitory Factor-Stimulated Signaling via S-Nitrosylation.

    Kengo Nakahara, Kana Fujikawa, Hideki Hiraoka, Ikuko Miyazaki, Masato Asanuma, Akihiro Ito, Nobumasa Takasugi, Takashi Uehara

    Biological & pharmaceutical bulletin   42 ( 6 )   1044 - 1047   2019

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    Nitric oxide (NO) is a key signaling molecule that has various effects via S-nitrosylation, a reversible post-translational modification that affects the enzymatic activity, localization, and metabolism of target proteins. As chronic nitrosative stress correlates with neurodegeneration, the targets have received focused attention. Macrophage migration inhibitory factor (MIF) plays a pivotal role in the induction of gene expression to control inflammatory responses. MIF acts as a ligand for CD74 receptor and activates the Src-p38 mitogen-activated protein kinase (MAPK) cascade. MIF also elevates the expression of brain-derived neurotrophic factor (BDNF), which contributes to the viability of neurons. Here, we show that MIF is S-nitrosylated by a physiological NO donor. Interestingly, the induction of S-nitrosylation resulted in a loss of MIF activity following stimulation of the Src and p38 MAPK signaling pathways and the induction of BDNF expression. Our results shed light on the pathogenic mechanisms of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

    DOI: 10.1248/bpb.b19-00025

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  • Functional analysis of juxta- and intra-membrane domains of murine APP by genome editing in Neuro2a cells. International journal

    Nanaka Kaneshiro, Ryosuke Imaoka, Masato Komai, Taku Kashiyama, Takashi Sakurai, Takashi Uehara, Nobumasa Takasugi

    Biochemical and biophysical research communications   501 ( 4 )   1023 - 1028   2018.7

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    Amyloid-β precursor protein (APP) correlates with the pathogenesis of certain brain diseases, such as Alzheimer disease (AD). APP is cleaved by several enzymes to produce APP metabolites, including the amyloid beta peptide (Aβ), which accumulates in the brain of AD patients. However, the exact functions of APP metabolites remain elusive. In this study, using genome editing technology, we mutated juxta- and intra-membrane domains of murine APP in the mouse neuroblastoma cell line, Neuro2a. We identified several clones that expressed characteristic patterns of APP metabolites. Mutations in juxta- (deletion 673A), and intra-membrane (deletion 705-6LM) domains of APP, decreased overall levels of APP metabolites or decreased the level of α-secretase-cleaved carboxy-terminal fragment (αCTF), respectively. APP is known to influence neuronal differentiation; therefore, we used theses clones to dissect the function of APP metabolites during neuronal differentiation. One clone (CA), which expressed reduced levels of both FL-APP and αCTF, showed increased expression of the neuronal marker, β3-tubulin, and enhanced retinoic acid (RA)-induced neurite outgrowth. In contrast, a clone that expressed FL-APP, but was devoid of αCTF (CE), showed comparable expression of β3-tubulin and neurite outgrowth compared with normal Neuro2a cells. These data indicate that FL-APP is a suppressor of neurite outgrowth. Our data suggest a novel regulatory function of juxta- and intra-membrane domains on the metabolism and function of APP.

    DOI: 10.1016/j.bbrc.2018.05.102

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  • TMEM30A is a candidate interacting partner for the β-carboxyl-terminal fragment of amyloid-β precursor protein in endosomes. International journal

    Nobumasa Takasugi, Runa Araya, Yuji Kamikubo, Nanaka Kaneshiro, Ryosuke Imaoka, Hao Jin, Taku Kashiyama, Yoshie Hashimoto, Masaru Kurosawa, Takashi Uehara, Nobuyuki Nukina, Takashi Sakurai

    PloS one   13 ( 8 )   e0200988   2018

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    Although the aggregation of amyloid-β peptide (Aβ) clearly plays a central role in the pathogenesis of Alzheimer's disease (AD), endosomal traffic dysfunction is considered to precede Aβ aggregation and trigger AD pathogenesis. A body of evidence suggests that the β-carboxyl-terminal fragment (βCTF) of amyloid-β precursor protein (APP), which is the direct precursor of Aβ, accumulates in endosomes and causes vesicular traffic impairment. However, the mechanism underlying this impairment remains unclear. Here we identified TMEM30A as a candidate partner for βCTF. TMEM30A is a subcomponent of lipid flippase that translocates phospholipids from the outer to the inner leaflet of the lipid bilayer. TMEM30A physically interacts with βCTF in endosomes and may impair vesicular traffic, leading to abnormally enlarged endosomes. APP traffic is also concomitantly impaired, resulting in the accumulation of APP-CTFs, including βCTF. In addition, we found that expressed BACE1 accumulated in enlarged endosomes and increased Aβ production. Our data suggested that TMEM30A is involved in βCTF-dependent endosome abnormalities that are related to Aβ overproduction.

    DOI: 10.1371/journal.pone.0200988

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  • Polysulfide Na2S4 regulates the activation of PTEN/Akt/CREB signaling and cytotoxicity mediated by 1,4-naphthoquinone through formation of sulfur adducts. International journal

    Yumi Abiko, Yasuhiro Shinkai, Takamitsu Unoki, Reiko Hirose, Takashi Uehara, Yoshito Kumagai

    Scientific reports   7 ( 1 )   4814 - 4814   2017.7

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    Electrophiles can activate redox signal transduction pathways, through actions of effector molecules (e.g., kinases and transcription factors) and sensor proteins with low pKa thiols that are covalently modified. In this study, we investigated whether 1,4-naphthoquinone (1,4-NQ) could affect the phosphatase and tensin homolog (PTEN)-Akt signaling pathway and persulfides/polysulfides could modulate this adaptive response. Simultaneous exposure of primary mouse hepatocytes to Na2S4 and 1,4-NQ markedly decreased 1,4-NQ-mediated cell death and S-arylation of cellular proteins. Modification of cellular PTEN during exposure to 1,4-NQ was also blocked in the presence of Na2S4. 1,4-NQ, at up to 10 µM, increased phosphorylation of Akt and cAMP response element binding protein (CREB). However, at higher concentrations, 1,4-NQ inhibited phosphorylation of both proteins. These bell-shaped dose curves for Akt and CREB activation were right-shifted in cells treated with both 1,4-NQ and Na2S4. Incubation of 1,4-NQ with Na2S4 resulted in formation of 1,4-NQ-S-1,4-NQ-OH. Unlike 1,4-NQ, authentic 1,4-NQ-S-1,4-NQ-OH adduct had no cytotoxicity, covalent binding capability nor ability to activate PTEN-Akt signaling in cells. Our results suggested that polysulfides, such as Na2S4, can increase the threshold of 1,4-NQ for activating PTEN-Akt signaling and cytotoxicity by capturing this electrophile to form its sulfur adducts.

    DOI: 10.1038/s41598-017-04590-z

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  • Modulation of Unfolded Protein Response by Methylmercury.

    Hideki Hiraoka, Kengo Nakahara, Yuki Kaneko, Shiori Akiyama, Kosaku Okuda, Takao Iwawaki, Masatake Fujimura, Yoshito Kumagai, Nobumasa Takasugi, Takashi Uehara

    Biological & pharmaceutical bulletin   40 ( 9 )   1595 - 1598   2017

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    Methylmercury (MeHg) results in cell death through endoplasmic reticulum (ER) stress. Previously, we reported that MeHg induces S-mercuration at cysteine 383 or 386 in protein disulfide isomerase (PDI), and this modification induces the loss of enzymatic activity. Because PDI is a key enzyme for the maturation of nascent protein harboring a disulfide bond, the disruption in PDI function by MeHg results in ER stress via the accumulation of misfolded proteins. However, the effects of MeHg on unfolded protein response (UPR) sensors and their signaling remain unclear. In the present study, we show that UPR is regulated by MeHg. We found that MeHg specifically attenuated inositol-requiring enzyme 1α (IRE1α)-x-box binding protein 1 (XBP1) branch, but not the protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcriptional factor 6 (ATF6) branches. Treatment with GSK2606414, a specific PERK inhibitor, significantly inhibited MeHg-induced cell death. These findings suggest that MeHg exquisitely regulates UPR signaling involved in cell death.

    DOI: 10.1248/bpb.b17-00359

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  • A Retrospective Study of the Effects of Oncology Pharmacist Participation in Treatment on Therapeutic Outcomes and Medical Costs.

    Makio Imamura, Daisuke Ogawa, Toshikazu Takatori, Maiko Yamaguchi, Tomoyuki Takata, Tomonori Hada, Yoshiaki Ota, Takashi Uehara

    Biological & pharmaceutical bulletin   40 ( 11 )   1956 - 1962   2017

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    Specialist oncology pharmacists are being trained in Japan to assist cancer treatment teams. These specialized pharmacists address patients' physical and mental problems in pharmacist-managed cancer care clinics, actively participate in formulating treatment policies, and are beneficial in offering qualitative improvements to patient services and team medical care. However, the effect of outpatient treatment by oncology pharmacists on therapeutic outcomes and medical costs is still unknown. A retroactive comparative analysis of the treatment details and clinical course was conducted among three groups of patients: patients who underwent adjuvant chemotherapy managed by a gynecologic oncologist only (S arm), patients managed by a non-oncologist (general practice gynecologist) only (NS arm), and patients managed by both a non-oncologist and a specialist oncology pharmacist (NS+Ph arm). The medical cost per course was significantly lower for patients in the NS+Ph arm than for those in the other two arms. Surprisingly, the outpatient treatment rate in the NS+Ph arm was overwhelmingly high. The involvement of an oncology pharmacist did not make a significant difference in therapeutic outcomes such as recurrence rate and survival. The participation of oncology pharmacists in the management of cancer patients undergoing chemotherapy enables safe outpatient treatment and also reduces medical costs.

    DOI: 10.1248/bpb.b17-00501

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  • Methylmercury, an environmental electrophile capable of activation and disruption of the Akt/CREB/Bcl-2 signal transduction pathway in SH-SY5Y cells. International journal

    Takamitsu Unoki, Yumi Abiko, Takashi Toyama, Takashi Uehara, Koji Tsuboi, Motohiro Nishida, Toshiyuki Kaji, Yoshito Kumagai

    Scientific reports   6   28944 - 28944   2016.6

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    Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as "S-mercuration", potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death.

    DOI: 10.1038/srep28944

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  • Regulation of Unfolded Protein Response via Protein S-nitrosylation.

    Yu Ohkubo, Ryosuke Nakato, Takashi Uehara

    Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan   136 ( 6 )   801 - 4   2016

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    Nitric oxide (NO) plays a pivotal function in neurotransmission, vasodilation, proliferation, and apoptosis in various types of cells via protein S-nitrosylation. Previously we demonstrated that protein disulfide isomerase (PDI) is S-nitrosylated in brains manifesting sporadic neurodegenerative diseases. This modification results in dysfunction of its enzymatic activity and consequently the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER). The aim of this study was to clarify the detailed function of NO on unfolded protein response (UPR) branches. We here found that the ER stress sensor IRE1α is S-nitrosylated. Interestingly, NO specifically abrogates ribonuclease activity, but not oligomerization or autophosphorylation of IRE1α. Site-directed mutagenesis revealed that Cys 931 and Cys951 in IRE1 are targets for S-nitrosylation. These mutants expressing in IRE1α knockout MEF showed a resistant role to the inhibition of nuclease activity by NO. Thus, we elucidated the effects of S-nitrosylation on ER stress sensors that mediate the UPR, and thus contribute to cell death pathways.

    DOI: 10.1248/yakushi.15-00292-1

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  • Regulation of the unfolded protein response via S-nitrosylation of sensors of endoplasmic reticulum stress. International journal

    Ryosuke Nakato, Yu Ohkubo, Akari Konishi, Mari Shibata, Yuki Kaneko, Takao Iwawaki, Tomohiro Nakamura, Stuart A Lipton, Takashi Uehara

    Scientific reports   5   14812 - 14812   2015.10

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    Protein S-nitrosylation modulates important cellular processes, including neurotransmission, vasodilation, proliferation, and apoptosis in various cell types. We have previously reported that protein disulfide isomerase (PDI) is S-nitrosylated in brains of patients with sporadic neurodegenerative diseases. This modification inhibits PDI enzymatic activity and consequently leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen. Here, we describe S-nitrosylation of additional ER pathways that affect the unfolded protein response (UPR) in cell-based models of Parkinson's disease (PD). We demonstrate that nitric oxide (NO) can S-nitrosylate the ER stress sensors IRE1α and PERK. While S-nitrosylation of IRE1α inhibited its ribonuclease activity, S-nitrosylation of PERK activated its kinase activity and downstream phosphorylation/inactivation or eIF2α. Site-directed mutagenesis of IRE1α(Cys931) prevented S-nitrosylation and inhibition of its ribonuclease activity, indicating that Cys931 is the predominant site of S-nitrosylation. Importantly, cells overexpressing mutant IRE1α(C931S) were resistant to NO-induced damage. Our findings show that nitrosative stress leads to dysfunctional ER stress signaling, thus contributing to neuronal cell death.

    DOI: 10.1038/srep14812

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  • Correlation between attenuation of protein disulfide isomerase activity through S-mercuration and neurotoxicity induced by methylmercury. International journal

    Kento Makino, Kosaku Okuda, Eisuke Sugino, Tadashi Nishiya, Takashi Toyama, Takao Iwawaki, Masatake Fujimura, Yoshito Kumagai, Takashi Uehara

    Neurotoxicity research   27 ( 2 )   99 - 105   2015.2

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    Methylmercury (MeHg), an environmental pollutant, causes neuronal death via endoplasmic reticulum (ER) stress; however, the precise mechanism is not fully understood. The aim of this study was to elucidate the possible mechanism of MeHg-induced neurotoxicity. Treatment with MeHg resulted in a loss of cell viability in a concentration-dependent manner accompanying the expression of ER stress marker genes in human neuroblastoma SH-SY5Y cells. We next attempted to identify a target protein for MeHg in the ER. MeHg covalently modified protein disulfide isomerase (PDI), which is important for disulfide bond formation in nascent proteins in the ER lumen. S-Nitrosylation of the catalytic domains of PDI by nitric oxide was attenuated up to 50 % by a MeHg challenge in cells. The MeHg-modified C-terminal catalytic domain in PDI was detected by MALDI-TOF/MS. Furthermore, treatment with MeHg significantly attenuated the enzymatic activity of PDI. Taken together, these observations suggest that MeHg results in ER stress and following the unfolded protein response pathway via ER dysfunction due to S-mercuration of the C-terminus of PDI.

    DOI: 10.1007/s12640-014-9494-8

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  • Endogenous S-sulfhydration of PTEN helps protect against modification by nitric oxide. International journal

    Kazuki Ohno, Kosaku Okuda, Takashi Uehara

    Biochemical and biophysical research communications   456 ( 1 )   245 - 9   2015.1

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    Hydrogen sulfide (H2S) is a gaseous regulatory factor produced by several enzymes, and plays a pivotal role in processes such as proliferation or vasodilation. Recent reports demonstrated the physiological and pathophysiological functions of H2S in neurons. PTEN is a target of nitric oxide (NO) or hydrogen peroxide, and the oxidative modification of cysteine (Cys) residue(s) attenuates its enzymatic activity. In the present study, we assessed the effect of H2S on the direct modification of PTEN and the resulting downstream signaling. A modified biotin switch assay in SH-SY5Y human neuroblastoma cells revealed that PTEN is S-sulfhydrated endogenously. Subsequently, site-directed mutagenesis demonstrated that both Cys71 and Cys124 in PTEN are targets for S-sulfhydration. Further, the knockdown of cystathionine β-synthetase (CBS) using siRNA decreased this modification in a manner that was correlated to amount of H2S. PTEN was more sensitive to NO under these conditions. These results suggest that the endogenous S-sulfhydration of PTEN via CBS/H2S plays a role in preventing the S-nitrosylation that would inhibition its enzymatic activity under physiological conditions.

    DOI: 10.1016/j.bbrc.2014.11.066

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  • Regulation of Histone Deacetylase 6 Activity via S-Nitrosylation.

    Kosaku Okuda, Akihiro Ito, Takashi Uehara

    Biological & pharmaceutical bulletin   38 ( 9 )   1434 - 7   2015

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    Nitric oxide (NO) is a gaseous regulatory factor produced by NO synthases (NOS) and it plays several critical roles via S-nitrosylation of protein cysteine residues. Histone deacetylase (HDAC) functions in the maintenance/balance of chromatin acetylation and contributes to transcriptional supression. It has been reported that S-nitrosylation of HDAC2 is involved in the regulation of deacetylase activity. However, it remains unknown whether other subtypes of the HDAC family are S-nitrosylated. In the present study, we found that HDAC6 is a target of NO. A biotin-switch assay revealed that endogenous HDAC6 is S-nitrosylated by both NO donors and NO derived from the inducible type of NOS in cells treated with cytokines. NO led to suppressed deacetylase activity in vitro and increased acetylated α-tubulin, a major substrate for HDAC6, in A549 cells. These findings suggest that S-nitrosylation of HDAC6 plays a pivotal role in the regulation of protein acetylation.

    DOI: 10.1248/bpb.b15-00364

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  • [Regulation of intracellular signal pathways via sensor proteins by oxidative stress].

    Takashi Uehara

    Clinical calcium   23 ( 11 )   1613 - 9   2013.11

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    It has been known that reactive oxygen species (ROS) control the enzymatic and transcriptional activity of proteins via direct modification of cysteine residues. Hence, oxidation of cysteine thiol could be a vital modulator of signal transduction pathways. These findings indicate that some proteins serve as the sensor proteins highly sensitive to ROS. In this review, I show the relationship between intracellular ROS sensor and the regulation of protein function via oxidation.

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  • [A novel screening system for S-nitrosylated proteins].

    Takashi Uehara

    Seikagaku. The Journal of Japanese Biochemical Society   85 ( 1 )   25 - 30   2013.1

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  • Screening systems for the identification of S-nitrosylated proteins. International journal

    Takashi Uehara, Tadashi Nishiya

    Nitric oxide : biology and chemistry   25 ( 2 )   108 - 11   2011.8

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    S-nitrosylation is a well-characterized reaction involving the covalent binding of nitric oxide (NO) to cysteine residues (Cys) in a protein. Similar to protein phosphorylation, S-nitrosylation is a post-translational modification involved in the regulation of a large number of intracellular functions and signaling events. Moreover, like phosphorylation, S-nitrosylation is precisely regulated in time and space. A procedure known as the biotin-switch method that specifically detects S-nitrosylated proteins (SNO-P) was recently developed by Snyder's group. They found that many proteins are substrates for NO, and several groups have attempted to identify other SNO-P by improving this method. In this review, we describe the SNO-P identified using modified versions of the biotin-switch method.

    DOI: 10.1016/j.niox.2010.11.002

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  • On-off system for PI3-kinase-Akt signaling through S-nitrosylation of phosphatase with sequence homology to tensin (PTEN). International journal

    Naoki Numajiri, Kumi Takasawa, Tadashi Nishiya, Hirotaka Tanaka, Kazuki Ohno, Wataru Hayakawa, Mariko Asada, Hiromi Matsuda, Kaoru Azumi, Hideaki Kamata, Tomohiro Nakamura, Hideaki Hara, Masabumi Minami, Stuart A Lipton, Takashi Uehara

    Proceedings of the National Academy of Sciences of the United States of America   108 ( 25 )   10349 - 54   2011.6

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    Nitric oxide (NO) physiologically regulates numerous cellular responses through S-nitrosylation of protein cysteine residues. We performed antibody-array screening in conjunction with biotin-switch assays to look for S-nitrosylated proteins. Using this combination of techniques, we found that phosphatase with sequence homology to tensin (PTEN) is selectively S-nitrosylated by low concentrations of NO at a specific cysteine residue (Cys-83). S-nitrosylation of PTEN (forming SNO-PTEN) inhibits enzymatic activity and consequently stimulates the downstream Akt cascade, indicating that Cys-83 is a critical site for redox regulation of PTEN function. In ischemic mouse brain, we observed SNO-PTEN in the core and penumbra regions but found SNO-Akt, which is known to inhibit Akt activity, only in the ischemic core. These findings suggest that low concentrations of NO, as found in the penumbra, preferentially S-nitrosylate PTEN, whereas higher concentrations of NO, known to exist in the ischemic core, also S-nitrosylate Akt. In the penumbra, inhibition of PTEN (but not Akt) activity by S-nitrosylation would be expected to contribute to cell survival by means of enhanced Akt signaling. In contrast, in the ischemic core, SNO-Akt formation would inhibit this neuroprotective pathway. In vitro model systems support this notion. Thus, we identify unique sites of PTEN and Akt regulation by means of S-nitrosylation, resulting in an "on-off" pattern of control of Akt signaling.

    DOI: 10.1073/pnas.1103503108

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  • The ECS(SPSB) E3 ubiquitin ligase is the master regulator of the lifetime of inducible nitric-oxide synthase. International journal

    Kazuma Matsumoto, Tadashi Nishiya, Satoshi Maekawa, Takahiro Horinouchi, Kouetsu Ogasawara, Takashi Uehara, Soichi Miwa

    Biochemical and biophysical research communications   409 ( 1 )   46 - 51   2011.5

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    The ubiquitin-proteasome pathway is an important regulatory system for the lifetime of inducible nitric-oxide synthase (iNOS), a high-output isoform compared to neuronal NOS (nNOS) and endothelial NOS (eNOS), to prevent overproduction of NO that could trigger detrimental effects such as cytotoxicity. Two E3 ubiquitin ligases, Elongin B/C-Cullin-5-SPRY domain- and SOCS box-containing protein [ECS(SPSB)] and the C-terminus of Hsp70-interacting protein (CHIP), recently have been reported to target iNOS for proteasomal degradation. However, the significance of each E3 ubiquitin ligase for the proteasomal degradation of iNOS remains to be determined. Here, we show that ECS(SPSB) specifically interacted with iNOS, but not nNOS and eNOS, and induced the subcellular redistribution of iNOS from dense regions to diffused expression as well as the ubiquitination and proteasomal degradation of iNOS, whereas CHIP neither interacted with iNOS nor had any effects on the subcellular localization, ubiquitination, and proteasomal degradation of iNOS. These results differ from previous reports. Furthermore, the lifetime of the iNOS(N27A) mutant, a form of iNOS that does not bind to ECS(SPSB), was substantially extended in macrophages. These results demonstrate that ECS(SPSB), but not CHIP, is the master regulator of the iNOS lifetime.

    DOI: 10.1016/j.bbrc.2011.04.103

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  • Regulation of inducible nitric-oxide synthase by the SPRY domain- and SOCS box-containing proteins. International journal

    Tadashi Nishiya, Kazuma Matsumoto, Satoshi Maekawa, Emi Kajita, Takahiro Horinouchi, Masahiro Fujimuro, Kouetsu Ogasawara, Takashi Uehara, Soichi Miwa

    The Journal of biological chemistry   286 ( 11 )   9009 - 19   2011.3

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    Inducible nitric-oxide synthase (iNOS, NOS2) plays a prominent role in macrophage bactericidal and tumoricidal activities. A relatively large amount of NO produced via iNOS, however, also targets the macrophage itself for apoptotic cell death. To uncover the intrinsic mechanisms of iNOS regulation, we have characterized the SPRY domain- and SOCS box-containing protein 1 (SPSB1), SPSB2, and SPSB4 that interact with the N-terminal region of iNOS in a D-I-N-N-N sequence-dependent manner. Fluorescence microscopy revealed that these SPSB proteins can induce the subcellular redistribution of iNOS from dense regions to diffused expression in a SOCS box-dependent manner. In immunoprecipitation studies, both Elongin C and Cullin-5, components of the multi-subunit E3 ubiquitin ligase, were found to bind to iNOS via SPSB1, SPSB2, or SPSB4. Consistently, iNOS was polyubiquitinated and degraded in a proteasome-dependent manner when SPSB1, SPSB2, or SPSB4 was expressed. SPSB1 and SPSB4 had a greater effect on iNOS regulation than SPSB2. The iNOS N-terminal fragment (residues 1-124 of human iNOS) could disrupt iNOS-SPSB interactions and inhibit iNOS degradation. In lipopolysaccharide-treated macrophages, this fragment attenuated iNOS ubiquitination and substantially prolonged iNOS lifetime, resulting in a corresponding increase in NO production and enhanced NO-dependent cell death. These results not only demonstrate the mechanism of SPSB-mediated iNOS degradation and the relative contributions of different SPSB proteins to iNOS regulation, but also show that iNOS levels are sophisticatedly regulated by SPSB proteins in activated macrophages to prevent overproduction of NO that could trigger detrimental effects, such as cytotoxicity.

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  • Participation of metabotropic glutamate receptors in pentetrazol-induced kindled seizure. International journal

    Yusuke Watanabe, Yuko Kaida, Satoko Fukuhara, Kenshi Takechi, Takashi Uehara, Chiaki Kamei

    Epilepsia   52 ( 1 )   140 - 50   2011.1

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    PURPOSE: The present study was undertaken to clarify the effects of (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), a metabotropic glutamate receptor (mGluR) 1 antagonist, (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate ((2R,4R)-APDC), a mGluR2/3 agonist, and L-(+)-2-amino-4-phosphonobutyric acid (L-AP4), a mGluR4/8 agonist, on pentetrazol-induced kindled seizures. METHODS: Mice were anesthetized with pentobarbital; the electrodes and guide cannula were chronically implanted into the cortex and lateral ventricle. To induce kindling, pentetrazol at a dose of 40 mg/kg was injected once every 48 h. Behavioral and electroencephalographic seizures were monitored for 20 min following pentetrazol administration. Fully kindled mice were used for pharmacologic studies. RESULTS: Intracerebroventricular injection of AIDA and L-AP4 showed significant inhibitory effects on pentetrazol-induced kindled seizures. In addition, simultaneous use of AIDA and (2R,4R)-APDC or L-AP4 caused more potent inhibition of seizure activities. The inhibitory effect of AIDA on pentetrazol-induced kindled seizures was antagonized by (RS)-3,5-dihydroxyphenylglycine ((RS)-3,5-DHPG), a group I mGluR agonist; (2S)-a-ethylglutamic acid (EGLU), a group II mGluR antagonist; and (RS)-α-methyl-4-phosphonophenylglycine (MPPG), a group III mGluR antagonist. On the other hand, the inhibitory effect of L-AP4 was antagonized only by MPPG. DISCUSSION: It is proposed that mGluR1 antagonists and mGluR4/8 agonists show anticonvulsive effects on pentetrazol-induced kindled seizures. Furthermore, it is also proposed that the simultaneous use of an mGluR1 antagonist and an mGluR2/3 or mGluR4/8 agonist is a potential novel therapeutic strategy in epileptic disorders.

    DOI: 10.1111/j.1528-1167.2010.02764.x

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  • Sustained activation of ERK signaling in astrocytes is critical for neuronal injury-induced monocyte chemoattractant protein-1 production in rat corticostriatal slice cultures. International journal

    Takahiro Katayama, Emi Sakaguchi, Yosuke Komatsu, Takahiro Oguma, Takashi Uehara, Masabumi Minami

    The European journal of neuroscience   31 ( 8 )   1359 - 67   2010.4

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    We previously demonstrated that N-methyl-D-aspartate (NMDA) treatment (50 microM, 3 h) induced astrocytic production of monocyte chemoattractant protein-1 (MCP-1, CCL2), a CC chemokine implicated in ischemic and excitotoxic brain injury, in rat corticostriatal slice cultures. In this study, we investigated the signaling mechanisms for NMDA-induced MCP-1 production in slice cultures. The results showed a close correlation between NMDA-induced neuronal injury and MCP-1 production, and an abrogation of NMDA-induced MCP-1 production in NMDA-pretreated slices where neuronal cells had been eliminated. These results collectively indicate that NMDA-induced neuronal injury led to astrocytic MCP-1 production. NMDA-induced MCP-1 production was significantly inhibited by U0126, an inhibitor of extracellular signal-regulated kinase (ERK). Immunostaining for phosphorylated ERK revealed that transient neuronal ERK activation was initially induced and subsided within 30 min, followed by sustained ERK activation in astrocytes. Treatment with U0126 during only the early phase (U0126 was washed out at 15 or 30 min after NMDA administration) suppressed early activation of ERK in neuronal cells, but not later activation of ERK in astrocytes. In this case, MCP-1 production was not suppressed, suggesting that activation of neuronal ERK is not necessary for MCP-1 production. In contrast, delayed application of U0126 at 3 h after the beginning of NMDA treatment inhibited MCP-1 production to the same degree as that observed when U0126 was applied from 3 h before NMDA administration. These findings suggest that sustained activation of the ERK signaling pathway in astrocytes plays a key role in neuronal injury-induced MCP-1 production.

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  • Reciprocal regulation of ATPgammaS-induced monocyte chemoattractant protein-1 production by ERK and p38 MAP kinases in rat corticostriatal slice cultures. International journal

    Takahiro Katayama, Misato Ito, Shuji Kaneko, Masamichi Satoh, Takashi Uehara, Masabumi Minami

    Journal of neuroscience research   87 ( 7 )   1573 - 81   2009.5

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    Monocyte chemoattractant protein-1 (MCP-1, CCL2) is a well-defined chemokine implicated in the pathology of various neurodegenerative diseases and brain injuries, such as Alzheimer's disease, multiple sclerosis, stroke, and traumatic injury. We investigated the effect of the activation of P2 purinoceptors on MCP-1 production in rat corticostriatal slice cultures. Treatment with adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), a hydrolysis-resistant adenosine triphosphate (ATP) analog, induced MCP-1 production in astrocytes. The induction was in a concentration-dependent manner and was antagonized by a P2 purinoceptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid. The inhibition of an extracellular signal-regulated kinase (ERK) pathway by PD98059 and U0126 significantly suppressed ATPgammaS-induced MCP-1 mRNA expression and protein production, while inhibition of c-Jun N-terminal kinase by SP600125 resulted in the partial suppression. Conversely, SB203580, a p38 mitogen-activated protein (MAP) kinase inhibitor, significantly enhanced ATPgammaS-induced MCP-1 production. Similar effects of ERK and p38 MAP kinase inhibitors on MCP-1 production were observed in the slices stimulated by ATP and BzATP. These results demonstrate that astrocytic MCP-1 production induced by P2 purinoceptor stimulation is reciprocally regulated by ERK and p38 MAP kinases in the organotypic slice cultures.

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  • Neuronal injury induces cytokine-induced neutrophil chemoattractant-1 (CINC-1) production in astrocytes.

    Takahiro Katayama, Hiroki Tanaka, Tadashi Yoshida, Takashi Uehara, Masabumi Minami

    Journal of pharmacological sciences   109 ( 1 )   88 - 93   2009.1

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    Accumulating evidence indicates a pivotal role for neuroinflammation in ischemic and excitotoxic brain injury. Cytokine-induced neutrophil chemoattractant-1 (CINC-1) is a CXC chemokine implicated in the infiltration of inflammatory cells into the brain parenchyma. In this study, we investigated the effect of N-methyl-D-aspartate (NMDA)-induced neuronal injury on CINC-1 production in the organotypic cortico-striatal slice cultures. Treatment with 50 microM NMDA for 3 - 4 h caused devastating neuronal damage and increased CINC-1 production. Immunohistochemical analysis revealed that the CINC-1 immunoreactivity was predominantly detected in astrocytes. NMDA failed to induce CINC-1 production in enriched astrocyte cultures or neuron-depleted slice cultures, suggesting that NMDA acted on neuronal cells to induce astrocytic CINC-1 production. NMDA-induced CINC-1 mRNA expression was significantly inhibited by U0126, a mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor. These results suggest that NMDA-evoked neuronal injury induced astrocytic CINC-1 production via a MEK/ERK signaling pathway. Manipulation of this signaling pathway may serve as a target for suppressing neuroinflammation and, thereby, treating ischemic brain injury.

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  • GMEB1, a novel endogenous caspase inhibitor, prevents hypoxia- and oxidative stress-induced neuronal apoptosis. International journal

    Tadashi Nakagawa, Kazuhiro Tsuruma, Takashi Uehara, Yasuyuki Nomura

    Neuroscience letters   438 ( 1 )   34 - 7   2008.6

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    The interaction of glucocorticoid modulatory element-binding protein 1 (GMEB1) with procaspase-2, -8, or -9 prevents caspase oligomerization and maturation. In the present study, we examined the effect of GMEB1 on neuronal apoptosis induced by hypoxia and oxidative stress. GMEB1 effectively attenuated caspase activation and apoptosis caused by these stresses in human neuroblastoma SK-N-MC cells, indicating that it functions as a potent inhibitor of caspase activation and apoptosis in response to oxidative stress. We propose that GMEB1 blocks pro-apoptosis signals induced by a variety of stresses.

    DOI: 10.1016/j.neulet.2008.04.023

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  • Accumulation of misfolded protein through nitrosative stress linked to neurodegenerative disorders. International journal

    Takashi Uehara

    Antioxidants & redox signaling   9 ( 5 )   597 - 601   2007.5

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    Protein quality control is a critical feature of intracellular homeostasis. In particular, unfolded or misfolded proteins resulting from environmental stresses or free radicals are rapidly degraded via the ubiquitin-proteasome pathway. Nitric oxide (NO), a free radical gas, has been reported to be involved in such processes as vasorelaxation and neurotransmission. Conversely, NO also is implicated in neuronal cell death or neurodegeneration. Recent reports suggest that S-nitrosylation of proteins is a significant cause of neural dysfunction leading to neurodegenerative disorders. Specifically, S-nitrosylation of parkin eventually leads to the accumulation of unfolded proteins and subsequent neuronal death. The focus of this review is the identity of the target of NO. Nitrosative stress prevents normal functioning of the endoplasmic reticulum (ER) via S-nitrosylation of protein-disulfide isomerase (PDI), which is located in the ER lumen. This may contribute to the accumulation of misfolded proteins, as well as sustained activation of the unfolded protein response (UPR) pathway. These phenomena may be linked to the development of sporadic neurodegenerative diseases.

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  • Involvement of the bed nucleus of the stria terminalis in the negative affective component of visceral and somatic pain in rats. International journal

    Satoshi Deyama, Takayuki Nakagawa, Shuji Kaneko, Takashi Uehara, Masabumi Minami

    Behavioural brain research   176 ( 2 )   367 - 71   2007.1

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    Using a conditioned place paradigm, we examined the involvement of the bed nucleus of the stria terminalis (BST) in the negative affective component of visceral and somatic pain induced by intraperitoneal acetic acid and intraplantar formalin injections, respectively, in rats. Bilateral BST lesions suppressed both the acetic acid- and formalin-induced conditioned place aversion, suggesting the crucial role of the BST in the negative affective component of visceral and somatic pain.

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  • A ubiquitin ligase HRD1 promotes the degradation of Pael receptor, a substrate of Parkin. International journal

    Tomohiro Omura, Masayuki Kaneko, Yasunobu Okuma, Yasuko Orba, Kazuo Nagashima, Ryosuke Takahashi, Noboru Fujitani, Satoshi Matsumura, Akihisa Hata, Kyoko Kubota, Karin Murahashi, Takashi Uehara, Yasuyuki Nomura

    Journal of neurochemistry   99 ( 6 )   1456 - 69   2006.12

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    It has been proposed that in autosomal recessive juvenile parkinsonism (AR-JP), a ubiquitin ligase (E3) Parkin, which is involved in endoplasmic reticulum-associated degradation (ERAD), lacks E3 activity. The resulting accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R), a substrate of Parkin, leads to endoplasmic reticulum stress, causing neuronal death. We previously reported that human E3 HRD1 in the endoplasmic reticulum protects against endoplasmic reticulum stress-induced apoptosis. This study shows that HRD1 was expressed in substantia nigra pars compacta (SNC) dopaminergic neurons and interacted with Pael-R through the HRD1 proline-rich region, promoting the ubiquitylation and degradation of Pael-R. Furthermore, the disruption of endogenous HRD1 by small interfering RNA (siRNA) induced Pael-R accumulation and caspase-3 activation. We also found that ATF6 overexpression, which induced HRD1, accelerated and caused Pael-R degradation; the suppression of HRD1 expression by siRNA partially prevents this degradation. These results suggest that in addition to Parkin, HRD1 is also involved in the degradation of Pael-R.

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  • Suppressive effects of 4-phenylbutyrate on the aggregation of Pael receptors and endoplasmic reticulum stress. International journal

    Kyoko Kubota, Yoshifumi Niinuma, Masayuki Kaneko, Yasunobu Okuma, Mami Sugai, Tomohiro Omura, Mai Uesugi, Takashi Uehara, Toru Hosoi, Yasuyuki Nomura

    Journal of neurochemistry   97 ( 5 )   1259 - 68   2006.6

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    Endoplasmic reticulum (ER) stress is defined as an accumulation of unfolded proteins in the endoplasmic reticulum. 4-phenylbutyrate (4-PBA) has been demonstrated to promote the normal trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) mutant from the ER to the plasma membrane and to restore activity. We have reported that 4-PBA protected against cerebral ischemic injury and ER stress-induced neuronal cell death. In this study, we revealed that 4-PBA possesses chemical chaperone activity in vitro, which prevents the aggregation of denatured alpha-lactalbumin and bovine serum albumin (BSA). Furthermore, we investigated the effects of 4-PBA on the accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R) pathologically relevant to the loss of dopaminergic neurons in autosomal recessive juvenile parkinsonism (AR-JP). Interestingly, 4-PBA restored the normal expression of Pael-R protein and suppressed ER stress induced by the overexpression of Pael-R. In addition, we showed that 4-PBA attenuated the activation of ER stress-induced signal transduction pathways and subsequent neuronal cell death. Moreover, 4-PBA restored the viability of yeasts that fail to induce an ER stress response under ER stress conditions. These results suggest that 4-PBA suppresses ER stress by directly reducing the amount of misfolded protein, including Pael-R accumulated in the ER.

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  • S-nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration. International journal

    Takashi Uehara, Tomohiro Nakamura, Dongdong Yao, Zhong-Qing Shi, Zezong Gu, Yuliang Ma, Eliezer Masliah, Yasuyuki Nomura, Stuart A Lipton

    Nature   441 ( 7092 )   513 - 7   2006.5

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    Stress proteins located in the cytosol or endoplasmic reticulum (ER) maintain cell homeostasis and afford tolerance to severe insults. In neurodegenerative diseases, several chaperones ameliorate the accumulation of misfolded proteins triggered by oxidative or nitrosative stress, or of mutated gene products. Although severe ER stress can induce apoptosis, the ER withstands relatively mild insults through the expression of stress proteins or chaperones such as glucose-regulated protein (GRP) and protein-disulphide isomerase (PDI), which assist in the maturation and transport of unfolded secretory proteins. PDI catalyses thiol-disulphide exchange, thus facilitating disulphide bond formation and rearrangement reactions. PDI has two domains that function as independent active sites with homology to the small, redox-active protein thioredoxin. During neurodegenerative disorders and cerebral ischaemia, the accumulation of immature and denatured proteins results in ER dysfunction, but the upregulation of PDI represents an adaptive response to protect neuronal cells. Here we show, in brains manifesting sporadic Parkinson's or Alzheimer's disease, that PDI is S-nitrosylated, a reaction transferring a nitric oxide (NO) group to a critical cysteine thiol to affect protein function. NO-induced S-nitrosylation of PDI inhibits its enzymatic activity, leads to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response. S-nitrosylation also abrogates PDI-mediated attenuation of neuronal cell death triggered by ER stress, misfolded proteins or proteasome inhibition. Thus, PDI prevents neurotoxicity associated with ER stress and protein misfolding, but NO blocks this protective effect in neurodegenerative disorders through the S-nitrosylation of PDI.

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  • Glucocorticoid modulatory element-binding protein 1 binds to initiator procaspases and inhibits ischemia-induced apoptosis and neuronal injury. International journal

    Kazuhiro Tsuruma, Tadashi Nakagawa, Nobutaka Morimoto, Masabumi Minami, Hideaki Hara, Takashi Uehara, Yasuyuki Nomura

    The Journal of biological chemistry   281 ( 16 )   11397 - 404   2006.4

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    Caspases are divided into two classes: initiator caspases, which include caspase-8 and -9 and possess long prodomains, and effector caspases, which include caspase-3 and -7 and possess short prodomains. Recently, we demonstrated that glucocorticoid modulatory element-binding protein 1 (GMEB1) interacts with the prodomain of procaspase-2, thereby disrupting its autoactivation and the induction of apoptosis. Here we show that GMEB1 is also capable of binding to procaspase-8 and -9. GMEB1 attenuated the Fas-mediated activation of these caspases and the subsequent apoptosis. The knockdown of endogenous GMEB1 using RNA interference revealed that cells with decreased GMEB1 expression are more sensitive to stress and undergo accelerated apoptosis. Transgenic mice expressing a neurospecific GMEB1 had smaller cerebral infarcts and less brain swelling than wild-type mice in response to transient focal ischemia. These results suggest that GMEB1 is an endogenous regulator that selectively binds to initiator procaspases and inhibits caspase-induced apoptosis.

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  • Astrapterocarpan isolated from Astragalus membranaceus inhibits proliferation of vascular smooth muscle cells. International journal

    Susumu Ohkawara, Yasunobu Okuma, Takashi Uehara, Takashi Yamagishi, Yasuyuki Nomura

    European journal of pharmacology   525 ( 1-3 )   41 - 7   2005.11

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    The inhibitory effects of astrapterocarpan, formononetin, and calycosin isolated from Astragalus membraneceus on platelet-derived growth factor (PDGF)-BB-induced proliferative response in rat vascular smooth muscle cells (A10 cells) were investigated. Astrapterocarpan significantly inhibited PDGF-BB-induced cell proliferation and DNA synthesis in a concentration-dependent manner. This inhibition was not attributed to toxicity. In contrast, formononetin and calycosin had no effect. We next examined the effect of astrapterocarpan on PDGF-BB signal transduction. Astrapterocarpan inhibited PDGF-BB-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERIC1/2) mitogen-activated protein (MAP) kinase. However, this compound had no effect on phosphorylation of PDGF-beta-receptor, Akt kinase and p38 MAP kinase. These results indicated that astrapterocarpan inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and that this effect may be mediated, at least in part, by inhibition of the ERK1/2 MAP kinase cascade.

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  • Comment on "S-nitrosylation of parkin regulates ubiquitination and compromises parkin's protective function". International journal

    Stuart A Lipton, Tomohiro Nakamura, Dongdong Yao, Zhong-Qing Shi, Takashi Uehara, Zezong Gu

    Science (New York, N.Y.)   308 ( 5730 )   1870; author reply 1870   2005.6

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  • Caspase recruitment domain of procaspase-2 could be a target for SUMO-1 modification through Ubc9. International journal

    Hiromi Shirakura, Naoko Hayashi, Shin-ichi Ogino, Kazuhiro Tsuruma, Takashi Uehara, Yasuyuki Nomura

    Biochemical and biophysical research communications   331 ( 4 )   1007 - 15   2005.6

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    To identify the binding proteins that regulate the function of procaspase-2, we screened for proteins using the yeast two-hybrid method and isolated human Ubc9 and SUMO-1 as the candidates. Ubc9 and SUMO-1 interacted with the caspase recruitment domain of procaspase-2 in its N-terminal. We elucidated the covalent modification of procaspase-2 by SUMO-1 in mammalian cells by immunoprecipitation followed by Western blot analysis. Procaspase-2 and SUMO-1 were co-localized by dot-like structures in the nucleus that are related to promyelocytic leukemia bodies. Interestingly, a conjugation-deficient mutant (K60R) procaspase-2 resulted in a delay of its enzyme maturation (appearance of p12 subunit) compared to that of wild-type. Thus, the modification with SUMO-1 may play a critical role in the nuclear localization and the activation (maturation) of procaspase-2.

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  • Regulation of procaspase-2 by glucocorticoid modulatory element-binding protein 1 through the interaction with caspase recruitment domain. International journal

    Kazuhiro Tsuruma, Tadashi Nakagawa, Hiromi Shirakura, Naoko Hayashi, Takashi Uehara, Yasuyuki Nomura

    Biochemical and biophysical research communications   325 ( 4 )   1246 - 51   2004.12

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    Caspases are the primary executioners of apoptosis. Although procaspases believe to exist as inactive forms in cells, the detailed regulatory system remains unclear. Here we show that glucocorticoid modulatory element-binding protein 1 (GMEB1) is capable of binding to the prodomain of caspase-2. We found that this molecule inhibits the autoproteolytic activation of procaspase-2 by oligomerization on a chemical compound-dependent system. These findings indicated that GMEB1 might be an endogenous inhibitory protein that selectively interacts with prodomain of caspase-2 to disrupt the autoactivation.

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  • Possible involvement of pyruvate kinase in acquisition of tolerance to hypoxic stress in glial cells. International journal

    Takahiro Shimizu, Takashi Uehara, Yasuyuki Nomura

    Journal of neurochemistry   91 ( 1 )   167 - 75   2004.10

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    Neurons are highly vulnerable to ischemic/hypoxic stress, while glial cells show tolerance to such stress. However, the mechanisms for tolerance acquisition in glial cells have yet to be established. We attempted to isolate and identify a stress protein that is upregulated in response to hypoxia in human astrocytoma CCF-STTG1 cells. In particular, pyruvate kinase (PK) was upregulated by hypoxia in CCF-STTG1 cells. Hypoxia-inducible factor 1 (HIF-1), the primary transcription factor that is responsible for multiple gene activation under hypoxia, plays a critical role in PK expression during hypoxic challenge. To determine whether newly synthesized PK is involved in tolerance to hypoxic stress, we established the PK-overexpressing neuronal cells. Overexpression of the wild-type, but not the kinase-negative mutant, resulted in attenuation of the loss of cell viability and the typical apoptotic features by hypoxia or oxidative stress in SK-N-MC cells. These findings suggest that upregulation of PK may result in acquisition of tolerance against hypoxic stress, and that the antioxidant effect may be involved in the protective effect of PK.

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  • Nitrosative stress linked to sporadic Parkinson's disease: S-nitrosylation of parkin regulates its E3 ubiquitin ligase activity. International journal

    Dongdong Yao, Zezong Gu, Tomohiro Nakamura, Zhong-Qing Shi, Yuliang Ma, Benjamin Gaston, Lisa A Palmer, Edward M Rockenstein, Zhuohua Zhang, Eliezer Masliah, Takashi Uehara, Stuart A Lipton

    Proceedings of the National Academy of Sciences of the United States of America   101 ( 29 )   10810 - 4   2004.7

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    Many hereditary and sporadic neurodegenerative disorders are characterized by the accumulation of aberrant proteins. In sporadic Parkinson's disease, representing the most prevalent movement disorder, oxidative and nitrosative stress are believed to contribute to disease pathogenesis, but the exact molecular basis for protein aggregation remains unclear. In the case of autosomal recessive-juvenile Parkinsonism, mutation in the E3 ubiquitin ligase protein parkin is linked to death of dopaminergic neurons. Here we show both in vitro and in vivo that nitrosative stress leads to S-nitrosylation of wild-type parkin and, initially, to a dramatic increase followed by a decrease in the E3 ligase-ubiquitin-proteasome degradative pathway. The initial increase in parkin's E3 ubiquitin ligase activity leads to autoubiquitination of parkin and subsequent inhibition of its activity, which would impair ubiquitination and clearance of parkin substrates. These findings may thus provide a molecular link between free radical toxicity and protein accumulation in sporadic Parkinson's disease.

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  • Herbimycin A abrogates nuclear factor-kappaB activation by interacting preferentially with the IkappaB kinase beta subunit. International journal

    Shinichi Ogino, Kazuhiro Tsuruma, Takashi Uehara, Yasuyuki Nomura

    Molecular pharmacology   65 ( 6 )   1344 - 51   2004.6

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    NF (nuclear factor)-kappaB is known to be a critical transcription factor in inflammatory responses. We have reported that herbimycin A, a potent Src tyrosine kinase inhibitor, attenuates the NF-kappaB activation triggered by cytokines, bacterial endotoxin, and hydrogen peroxide. Accompanying the suppression by this agent, NF-kappaB-dependent gene expressions, such as cytokine, chemokine, and inducible-type nitric oxide, are specifically inhibited in glial cells. In the present study, we attempted to elucidate the possible target protein for herbimycin A on this pathway. We demonstrate here that herbimycin A preferentially inhibits IKK (IkappaB kinase)beta. Furthermore, substituting alanine for the cysteine at 59 (Cys59) in IKKbeta resulted in the insensitivity to herbimycin A, suggesting that this compound may interact with the Cys59 residue located near the catalytic ATP binding site. Taken together, these results indicate that herbimycin A can be considered a novel candidate for an anti-inflammatory drug agent through its specific inhibition of IKKbeta, which results in prevention of the expression of NF-kappaB-dependent genes implicated in the pathogenesis of inflammatory responses.

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  • Ubiquilin interacts with ubiquitylated proteins and proteasome through its ubiquitin-associated and ubiquitin-like domains. International journal

    Han Seok Ko, Takashi Uehara, Kazuhiro Tsuruma, Yasuyuki Nomura

    FEBS letters   566 ( 1-3 )   110 - 4   2004.5

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    Mammalian cells acquire tolerance against multiple stressors through the high-level expression of stress-responsible genes. We have previously demonstrated that protein-disulfide isomerase (PDI) together with ubiquilin are up-regulated in response to hypoxia/brain ischemia, and play critical roles in resistance to these damages. We show here that ubiquilin interacts preferentially with poly-ubiquitin chains and 19S proteasome subunits. Taken together, these results suggest that ubiquitin could serve as an adaptor protein that both interacts with PDI and mediates the delivery of poly-ubiquitylated proteins to the proteasome in the cytosol in the vicinity of the endoplasmic reticulum membrane.

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  • Correlation between delayed neuronal cell death and selective decrease in phosphatidylinositol 4-kinase expression in the CA1 subfield of the hippocampus after transient forebrain ischemia. International journal

    Yudai Furuta, Takashi Uehara, Yasuyuki Nomura

    Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism   23 ( 8 )   962 - 71   2003.8

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    Transient forebrain ischemia induces a delayed neuronal death in the CA1 area of the hippocampus. However, the mechanism leading to this phenomenon has yet to be established. The authors used an mRNA differential-display method to isolate genes for which mRNA levels change only in the hippocampus during ischemia/reperfusion. They succeeded in identifying the product of one down-regulated gene as phosphatidylinositol 4-kinase (PI 4-K). Compared with control levels, PI 4-K mRNA expression in the hippocampus, but not the cerebral cortex, was significantly decreased by 30% and about 80% 1 and 7 days after ischemia/reperfusion, respectively. Interestingly, PI 4-K and PI bisphosphate levels were selectively decreased in the CA1 region, but not other regions, whereas TUNEL-positive cells could be detected 3 days after ischemia. Consistent with these results, PI 4-K expression was suppressed by hypoxia in SK-N-MC neuroblastoma cells before loss of cell viability. Overexpression of wild-type PI 4-K, but not the kinase-negative mutant of PI 4-K (K1789A), recovered the loss of viability induced by hypoxia. These findings strongly suggest that a prior decrease in PI 4-K and PI bisphosphate levels caused by brain ischemia/hypoxia is partly involved in delayed neuronal cell death.

    PubMed

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  • Role of ubiquilin associated with protein-disulfide isomerase in the endoplasmic reticulum in stress-induced apoptotic cell death. International journal

    Han Seok Ko, Takashi Uehara, Yasuyuki Nomura

    The Journal of biological chemistry   277 ( 38 )   35386 - 92   2002.9

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    Language:English   Publishing type:Research paper (scientific journal)  

    Up-regulation of several stress proteins such as heat-shock proteins and glucose-regulated proteins participate in tolerance against environmental stress. Previously, we found that protein-disulfide isomerase (PDI) is specifically up-regulated in response to hypoxia/brain ischemia in astrocytes. In addition, the overexpression of this gene into neurons protects against apoptotic cell death induced by hypoxia/brain ischemia. To address the detailed function of PDI, we screened for proteins that interact with PDI using the yeast two-hybrid system. We report here that PDI interacts with ubiquilin, which has a ubiquitin-like domain and a ubiquitin-associated domain. Interestingly, ubiquilin is also up-regulated in response to hypoxia in glial cells with a time course similar to that of PDI induction. In hypoxia-treated glial cells, the endogenous ubiquilin and PDI were almost completely co-localized, suggesting that ubiquilin is an endoplasmic reticulum-associated protein. Overexpression of this gene in neuronal cells resulted in significant inhibition of the DNA fragmentation triggered by hypoxia, but not that induced by nitric oxide or staurosporine. Moreover, ubiquilin has the ability to attenuate CHOP induction by hypoxia. These observations suggested that ubiquilin together with PDI have critical functions as regulatory proteins for CHOP-mediated cell death, and therefore up-regulation of these proteins may result in acquisition of tolerance against ischemic stress in glial cells.

    PubMed

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MISC

  • Downregulation of BNDF expression by extracellular HMGB1 in rat cortico-striatal slice cultures

    Hiroki Tanaka, Yosuke Komatsu, Takahiro Katayama, Soichiro Ide, Takashi Uehara, Masabumi Minami

    NEUROSCIENCE RESEARCH   65   S162 - S162   2009

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    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:ELSEVIER IRELAND LTD  

    DOI: 10.1016/j.neures.2009.09.839

    Web of Science

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Awards

  • 特別研究員等審査会専門委員表彰

    2016.7   日本学術振興会  

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  • 科研費助成事業第一段審査委員表彰

    2011.9   日本学術振興会  

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  • 奨励賞

    2005.9   日本神経化学会  

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  • 奨励賞

    2003.3   日本薬学会  

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Research Projects

  • 小胞体ストレスセンサー分子特異的な酸化修飾阻止薬の開発

    Grant number:19K22498  2019.06 - 2021.03

    日本学術振興会  科学研究費助成事業 挑戦的研究(萌芽)  挑戦的研究(萌芽)

    上原 孝

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    Grant amount:\6500000 ( Direct expense: \5000000 、 Indirect expense:\1500000 )

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  • Environmental electrophiles exposome and reactive sulfur species as its regulator molecule

    Grant number:18H05293  2018.06 - 2023.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (S)  Grant-in-Aid for Scientific Research (S)

    熊谷 嘉人, 上原 孝, 西田 基宏

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    Grant amount:\195260000 ( Direct expense: \150200000 、 Indirect expense:\45060000 )

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  • 一酸化窒素誘発性エピゲノム変化の病態生理的意義の解明と特異的阻害薬の薬効評価

    Grant number:18H02579  2018.04 - 2021.03

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    上原 孝, 竹内 靖雄, 伊藤 昭博

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    Grant amount:\17290000 ( Direct expense: \13300000 、 Indirect expense:\3990000 )

    平成30年度は,2つのテーマを推進し,以下に示す結果を得ることに成功した.
    1.NO依存的誘導遺伝子の特定とそのエピジェネティックな制御の有無を検討した.まず,マウス初代培養神経細胞ならびに株化ヒト胃がん細胞を用いて,NO 刺激に伴って発現が誘導される遺伝子をDNAマイクロアレイ解析ならびに次世代シークエンサーを用いたSAGE法で単離した.その結果,これまでに報告のないNO刺激依存的な遺伝子を特定することに成功した.これらについて,ゲノムにおけるCpGサイトの有無を確認すると同時に,私たちが開発したDNMT3ニトロシル化阻害薬であるDBICの効果について解析を進めている.
    2.DBICの効果ならびにその誘導体の薬理学的特性を明らかにすることを目指した.まず,DNMTの誘導体を9種合成することに成功した.DNMT3のSーニトロシル化抑制能を細胞レベルで検討したところ,DBICよりも数十倍効力が高いものがあった.これらの結果はin silicoシミュレーションのそれと一致しており,さらに活性の高い誘導体の作出を進めることとした.次に,in vivoでの作用を検討するために最適化を計った.DBICは水にほとんど不溶であることから,動物への長期間投与が出来ない.そこで,DBIC塩酸塩の作出を試みたところ,0.8 mg/ml程度まで溶解することが判明した.なお,DBICと同様に大量合成も可能であることを確認した.一方,DBICはその一部が結晶中で代謝されることも,初期的に明らかにしている.将来的には,脳内での効果も期待していることから,プロドラッグ化を進めることにした.DBICの水酸基をサクシニル化した誘導体の合成に着手し,期待通りの化合物の作出に成功した.本化合物も水やbufferに約0.8 mg/ml (2 mM)まで溶解することを確認した.

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  • Biological effect and preventive method for human serum albumin binding to transboundary air borne PM2.5.

    Grant number:18H03039  2018.04 - 2021.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    荻野 景規, 尾長谷 靖, 菅沼 成文, 栄徳 勝光, 荻野 学芳, 荻野 志穂奈, 市村 宏, 長岡 憲次郎, 高柴 正悟, 伊藤 達男, 早川 和一, 中村 裕之, 上原 孝, 浜田 博喜, 岡野 光博, 江口 依里, 竹本 圭

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    Grant amount:\12870000 ( Direct expense: \9900000 、 Indirect expense:\2970000 )

    チロシンをニトロ化したヒトアルブミン(NO2-hAlb)とPM2.5をマウスに投与した結果、NO2-hAlb単独投与では、気道の好産球の増加を認め、PM2.5を加えることにより好産球数がさらに増加した。さらに、肺組織のPCRによりIL-5、エオタキシン2、muc5ac、muc5bがNO2-hAlb単独で上昇し、PM2.5が加わると相乗的に増加した。NO2-hAlbにはアレルギー性があり、PM2.5の存在でその作用が増強されることが判明した。大気中から粒径0.1mm以下のnanoPMを純粋に大気中から収集することに世界で初めて成功し、PM2.5とnanoPMの電子顕微鏡による形状を比較すると、PM2.5は丸みを帯びた構造物で、nanoPMは、角張った構造物であった。気管支上皮細胞への毒性を透過型電子顕微鏡で検討すると、PM2.5に比しnanoPMは、ミトコンドリア障害が顕著であった。さらに細胞影響を、PCRで検討すると、PM2.5及びnanoPM曝露は、クラスリン関連因子、ミトコンドリアに局在する抗酸化酵素SOD2が有意に発現上昇し、ヒトアルブミン添加でその発現上昇がさらに増強した。PM2.5及びnanoPMは、大気中のヒトアルブミンの存在下、クラスリン受容体を介して細胞内へ侵入し、ミトコンドリア障害を引き起こすことを証明した。このことはPM2.5及びPM2.5に含まれるnanoPMが、大気中の存在するヒトアルブミンと結合し、細胞に侵入しやすくなっていることを世界で初めて証明したことになる。また一方、企業健診受診者の炎症関連バイオマーカーの研究として、チロシンニトロ化ヒトアルブミンと無修飾ヒトアルブミンに対する自己抗体(IgG)を測定した結果、血清中抗体化が高い程、血圧が低い結果となった。ニトロチロシンに対する抗体が、ニトロ化蛋白質を排除している可能性が示唆された。

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  • Novel regulatory mechanism of Abeta degradation via ER stress

    Grant number:17K19490  2017.06 - 2019.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Challenging Research (Exploratory)  Challenging Research (Exploratory)

    Uehara Takashi

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    Grant amount:\6500000 ( Direct expense: \5000000 、 Indirect expense:\1500000 )

    To clarify the role of E3 ligase that is involved in ER stress signaling, we attempted to find its substrate. In this study, we succeeded in identifying an Aβ degrading enzymes (proteases). The substrate protein was polyubiquitinated but not degraded by proteasome. Next, we examined if ubiquitination results in the change in the enzymatic activity. Hydrolysis of Aβ peptide was significantly suppressed by this modification. Therefore, it is suggested that under the endoplasmic reticulum stress environment, an E3 ligases is highly expressed, and ubiquitination of the substrate may alter intracellular localization by regulating the enzyme activity.

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  • Life science basis of short-lived reactive species originated from foods

    Grant number:17H06170  2017.05 - 2022.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (S)  Grant-in-Aid for Scientific Research (S)

    内田 浩二, 赤池 孝章, 上原 孝, 安達 貴弘

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    Grant amount:\204230000 ( Direct expense: \157100000 、 Indirect expense:\47130000 )

    1. 抗酸化剤に由来する短寿命分子種の同定・検出:ワインなどに含まれるポリフェノールとして有名なレスベラトロールの代謝物(ピセアタンノール)がタンパク質に作用して生成される短寿命分子として酸化リジン (α-aminoadipic semialdehyde) を同定し、さらにLC-ESI-MS/MSによる高感度検出にも成功した。ピセアタンノールとの反応により生成するタンハク質の構造変化をLC-ESI-MS/MSを用いて機器分析により詳細に解析した結果、リジン2分子が架橋したdehydrolysinonorleucineを検出した。
    2. 短寿命抗酸化剤代謝物によるタンパク質の新機能獲得:酸化型ビタミンCと反応させたタンパク質が自然抗体IgM産生を伴う自然免疫機構を活性化することを見出した。また、カテキンとして知られるポリフェノールEGCGと反応させたタンパク質が、自然抗体IgMだけでなくアポリポタンパク質Eとも相互作用することを発見した。
    3. 過硫黄分子によるタンパク質パースルフィド化:イオウ関連分子の生理機能解明のために、質量分析による生体内イオウ代謝物質のメタボローム解析を行い、新規ポリスルフィド合成系としてシステイン-tRNA合成酵素(CARS)を同定した。また、CARSが産生するシステインパースルフィドがミトコンドリアにおけるエネルギー産生の制御に関わることを明らかにした。
    4. 短寿命分子種による細胞内タンパク質機能制御:活性硫黄種がPDI のポリサルファー化を介して酵素活性を亢進させ、小胞体での異常タンパク質の蓄積を抑制することでストレス抵抗性を獲得する可能性を明らかにした。また、一酸化窒素の標的として同定したDNAメチル基転移酵素の修飾部位に関して、LC-MS/MS 解析を進めた。

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  • Regulation of epigenetic enzymes via nitrosative stress and development of its specific modulator

    Grant number:15H04649  2015.04 - 2018.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    Uehara Takashi

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    Grant amount:\16770000 ( Direct expense: \12900000 、 Indirect expense:\3870000 )

    We could demonstrate that nitric oxide (NO) regulates epigenetic enzymes. A cysteine residue was a target of NO and its covalent modification contributed to the inhibition of enzymatic activity in cells. From these findings, we speculated that NO may be involved in the up-regulation of several genes via this mechanism. Next, we conducted DNA micro array for isolating genes that is sensitive to NO. We could isolate several genes regulated by NO. Thus, we could propose a novel system of gene expression via nitrosative stress in this study.

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  • Regulation of unfolded protein response by oxidative stress

    Grant number:15K14952  2015.04 - 2017.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research  Grant-in-Aid for Challenging Exploratory Research

    Uehara Takashi

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    Grant amount:\3770000 ( Direct expense: \2900000 、 Indirect expense:\870000 )

    S-nitrosylation modulates important cellular processes in many cell types. We attempted to elucidate the effects of S-nitrosylation on unfolded protein response (UPR) pathway. We found that nitric oxide (NO) can S-nitrosylate the ER stress sensors IRE1α and PERK. However S-nitrosylation of IRE1α inhibited its ribonuclease activity, S-nitrosylation of PERK activated its kinase activity and downstream phosphorylation/inactivation or eIF2α. Overexpression of IRE1α(Cys931) prevented S-nitrosylation and inhibition of its enzymatic activity, indicating Cys931 is the predominant site of S-nitrosylation. These results indicated that nitrosative stress leads to dysfunctional ER stress signaling, thus contributing to neuronal cell death.

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  • Polysulfur metabolome and regulation of anti-oxidative stress responses

    Grant number:26111008  2014.07 - 2019.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)  Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

    Akaike Takaaki

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    Grant amount:\105430000 ( Direct expense: \81100000 、 Indirect expense:\24330000 )

    Polysulfides is reactive persulfide species which have extra sulfur atoms in cysteine thiol and cellular proteins contain polysulfides abundantly (protein polysulfuration). In this study, we investigated the translation-coupled mechanism of protein polysulfidation mediated by cysteinyl-tRNA synthetase (CARS) and its biological functions. CARSs from various biological species including bacteria and mammals are found to have cysteine persulfide-producing activity and to be critically involved in protein polysulfuration. Protein polysulfuration is suggested to play important roles in the regulation of cellular function such as mitochondrial morphogenesis.

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  • Identification of novel target proteins of NO with new screening system

    Grant number:25670029  2013.04 - 2015.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research  Grant-in-Aid for Challenging Exploratory Research

    UEHARA Takashi

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    Grant amount:\3900000 ( Direct expense: \3000000 、 Indirect expense:\900000 )

    In this study, we attempted to isolate the novel target proteins of NO using new screening system. In addition, we tried to identify the substrates of NO with several specific antibodies. We could isolate several enzymes involved in epigenetics as the targets of NO. We found that those candidates are S-nitrosylated in vitro and in vivo. These results indicated that those proteins are certainly the targets of NO. We are now investigated the role of S-nitrosylation on its enzymatic activity.

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  • Detection of oxidized ERprotein by nitrosative/oxidative stress with a specific antibody

    Grant number:23659041  2011 - 2012

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research  Grant-in-Aid for Challenging Exploratory Research

    UEHARA Takashi

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    Grant amount:\3770000 ( Direct expense: \2900000 、 Indirect expense:\870000 )

    The aim of this study is to establish the assay system to detect oxidized PDI (PDI-SO3H) with a specific antibody. We succeeded to isolate several specific antibodies against PDI-SO3H. An antibody could detect the oxidized form of PDI treated with H2O2, NO, or rotenone. This antibody might be useful for diagnosis of sporadic neurodegenerative disprders such as Alzheimer’s and Parkinson’s diseases.

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  • Molecular mechanism of neuronal cell death induced by methyl mercury via endoplasmic reticulum stress

    Grant number:22310039  2010 - 2012

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    UEHARA Takashi

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    Grant amount:\18070000 ( Direct expense: \13900000 、 Indirect expense:\4170000 )

    We found that methyl mercury results in neuronal cell death and the reduction of S-nitrosylated PDI formation in a concentration-dependent manner in SH-SY5Y cells. These observations suggested methyl mercury affects same cysteine residue that is oxidized by nitric oxide. Treatment with methyl mercury stimulated the specific splicing of XBP1 mRNA and phosphorylation of IRE1α. Thus, methyl mercury might induce apoptosis via endoplasmic reticulum stress caused by dysfunction of protein maturation system.

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  • 新規小胞体膜ユビキチンリガーゼの基質探索と神経生理機能特定

    Grant number:21659013  2009 - 2010

    日本学術振興会  科学研究費助成事業 挑戦的萌芽研究  挑戦的萌芽研究

    上原 孝

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    Grant amount:\3000000 ( Direct expense: \3000000 )

    私たちはこれまでにヒトゲノム情報から推定上の新規な小胞体膜に存在するユビキチンリガーゼの特定を行なってきた.その生理あるいは病態生理的特徴を解明するために,基質の同定を試みた,まず,動物細胞内に当該ユビキチンリガーゼを強制発現させ,特異的抗体により免疫沈降反応を行った.免疫沈降物をSDS-PAGEで展開し,ゲルを染色した後,得られたバンドを切り出して酵素処理した.これらをMALDI TOF/MASS解析し,蛋白質を特定した.数種の候補蛋白質が単離されたが,その一つはインスリン分解酵素(IDE)であった.IDEは末梢ばかりでなく中枢神経系にも高発現している,さらには脳内ではアルツハイマー病発症に関わるAβの分解に寄与していることが明らかとなっている.そこでこのユビキチンリガーゼとIDEの関係の詳細を調べた.両者を神経細胞で強制発現させた際に結合が観察されたが,ユビキチンリガーゼ単独で発現した際にも内在性のIDEと強く結合することから,両者の結合は非常に強いものであることがわかった.さらには,このときIDEはポリユビキチン化されることを見いだした.また,このユビキチン鎖はLys48結合であることを特異的抗体から明らかにした,さらに,内在性IDEはHRD1を過剰発現させることで急速に分解されることを明らかにした,逆に,HRD 1をノックダウンさせるとIDEの半減期は有意に延長されることがわかった.今後は,さらに本研究を発展させ,ヒト病態におけるタンパク質量の変化を明らかにし,神経変性疾患発症との関わりについて明らかにする予定である.

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  • Signaling Functions of Reactive Oxygen Species

    Grant number:20117001  2008.11 - 2014.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)  Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

    AKAIKE Takaaki, SUMIMOTO Hideki, UCHIDA Koji, MATSUMOTO Akio, URANO Yasuteru, ARIMOTO Hirokazu, ITOH Ken, SHIMOKAWA Hiroaki, TSUTSUI Hiroyuki, UEHARA Takashi, KAMATA Hideaki, NISHIDA Motohiro

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    Grant amount:\58500000 ( Direct expense: \45000000 、 Indirect expense:\13500000 )

    活性酸素は生体分子に非特異的な化学損傷をもたらす単なる毒性因子ではなく、シグナル分子として生体内で様々な役割を担っていることが明らかになってきた。本領域研究は、化学と生物系が融合したケミカルバイオロジーの新たな視点から、活性酸素によるシグナル伝達研究を展開することにより、多彩な生命現象と疾患病態に関与している活性酸素の生理機能の解明と生物種に普遍的に発現されている活性酸素シグナルの統合的理解を目指して発足した。本年度は、領域研究にて得られた成果を国内外に情報発信することを目的とした。具体的には、下記の学会での共催として、領域メンバーおよび招待演者における講演をおこなった。(1)ジョイントシンポジウム「生体ストレス応答:酸素毒から環境汚染物質まで」(領域メンバー5名、海外招待演者1名)(平成25年7月12日、熊本、日本生体防御学会との共催)、(2)東北医学会(海外招待演者1名、平成26年3月20日、仙台)。本領域研究で取扱う「活性酸素シグナル」は、基礎生物学、農学(植物学)、医学生物学を含めた生命科学の幅広い分野に関係している。また、活性酸素の生理機能の解明なしには、活性酸素の制御異常がもたらす疾病である、メタボリックシンドローム、感染・炎症、老化、発がんなどの病態解明と予防対策、治療戦略は確立できない。したがって、本領域研究で得られた成果は、今後益々重要となる「活性酸素シグナル」をキーワードとした異分野融合型プロジェクトの形成推進に大きく資するものと期待される。

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  • Molecular mechanism of nitric oxide/nitrosative stress implicating in onset of neurodegenerative disorders

    Grant number:20117011  2008 - 2012

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)  Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

    UEHARA Takashi, NISHIYA Tadashi

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    Grant amount:\71630000 ( Direct expense: \55100000 、 Indirect expense:\16530000 )

    We performed antibody-array screening in conjunction with biotin-switch assays to identify the novel S-nitrosylated proteins. Using this assay system, we found that phosphatase with sequence homology to tensin (PTEN) is selectively S-nitrosylated by low concentrations of NO at a specific cysteine residue. In addition, we demonstrated the mechanism of SPSB-mediated iNOS degradation and the relative contributions of different SPSB proteins to iNOS regulation.

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  • The inhibitory mechanism of novel proteins induced in the endoplasmic reticulum and of drugs on neurodegenerative diseases

    Grant number:19300135  2007 - 2008

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    NOMURA Yasuyuki, OKUMA Yasunobu, TAKAHASHI Ryosuke, UEHARA Takashi, KANEKO Masayuki, IZUMO Nobuo, ISUZUGAWA Kazuto

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    Grant amount:\19370000 ( Direct expense: \14900000 、 Indirect expense:\4470000 )

    本研究では,抗脳神経変性疾患薬創製に向けて, 細胞内小器官小胞体への変性タンパク質蓄積抑制機構に関する研究を行った. 小胞体のタンパク質分解系(ERAD)分子の生体での生理的役割, とくに脳変性疾患への関与を明らかにするとともに, ERAD 分子をターゲットとした脳変性疾患抑制薬の基盤研究を行った. また, タンパク質凝集抑制作用を有する化合物を作成し, 神経細胞死抑制作用について検討した.

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  • The role of nitrosylation of neurotransmitter transporterin pain-induced aversion

    Grant number:19390149  2007 - 2008

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    MINAMI Masabumi, UEHARA Takashi, KATAYAMA Takahiro

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    Grant amount:\19500000 ( Direct expense: \15000000 、 Indirect expense:\4500000 )

    本研究では、痛みによる不安や抑うつ、嫌悪などの不快情動の生成・制御メカニズムを明らかにするため、分界条床核の役割に関して研究を進め、腹側分界条床核におけるノルアドレナリン遊離亢進とβアドレナリン受容体-アデニル酸シクラーゼ-proteinkinase A(PKA)系活性化が痛みによる不快情動生成に重要な役割を果たしていることを明らかにした。一方、ノルアドレナリントランスポーターの酸化的修飾は、その機能に影響を与えなかった。

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  • 神経変性疾患発症における一酸化窒素による小胞体膜存在ユビキチンリガーゼの機能変化

    Grant number:19044002  2007 - 2008

    日本学術振興会  科学研究費助成事業 特定領域研究  特定領域研究

    上原 孝

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    Grant amount:\7000000 ( Direct expense: \7000000 )

    私たちはこれまでにバイオインフォーマティックスによってヒトゲノムライブラリーから新規小胞体膜存在ユビキチンリガーゼを網羅的に単離することに成功してきた. また, 小胞体における蛋白質品質管理系にユビキチンプロテアソームが関わっていること, とくにE3リガーゼであるパーキンが小胞体膜蛋白質を基質としていることが報告されてきた. このパーキンが一酸化窒素によって酸化されることで機能が変化し, 変性基質蛋白質の蓄積をもたらすことでドパミン神経細胞死/パーキンソン病を発症する可能性を明らかにしてきた. そこで本年度は, 神経細胞内で比較的発現量の高い小胞体膜存在E3リガーゼであるHRD1の新たな基質の探索を試みた.
    HRD1は酵母から哺乳類まで存在が確認されている分子であり, 小胞体膜に存在していることから小胞体関連蛋白質分解に関わることが指摘されてきた. この生理的基質に関してはAPPなどが示されてきたものの, その結合様式については不明である. そこで, より直接的な結合蛋白質を同定するためにHRD1と共免疫沈降してくる蛋白質をSDS-PAGEで分離し, MALDI-TOFMSを利用して同定を試みた. その結果, 神経変性疾患発症に関わっていることが示唆されている数種の細胞質蛋白質が単離同定された. これまでにこれらの分解機構についての報告は無く, 生理的あるいは病態生理的役割を推察する上で興味がもたれた. 現在, 細胞内局在ならびに分解機構の詳細について解析しているところである.

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  • Pathophysiological function of nitric oxide on protein quality control system in endoplasmic reticulum.

    Grant number:19590051  2006 - 2008

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    UEHARA Takashi

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    Grant amount:\4680000 ( Direct expense: \3600000 、 Indirect expense:\1080000 )

    脳虚血時による細胞内Ca^(2+)の上昇は, 一酸化窒素(NO)合成酵素の活性化を介してNOを過剰産生し, 小胞体シャペロンであるカルレチキュリン(CRT)をS-ニトロシル(SNO)化することが示唆された.また, CRT のSNO 化によるシャペロン活性の低下は, 小胞体ストレスに対する細胞死に関連することが示唆された. 以上より, NO と小胞体ストレスの関連性に新たな見解を提示することができた.

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  • Possible relationship between S-nitrosylated proteins and neurodegenerative disorders

    Grant number:17590050  2005 - 2006

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    UEHARA Takashi

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    Grant amount:\3500000 ( Direct expense: \3500000 )

    Stress proteins located in the cytosol or endoplasmic reticulum (ER) participate in maintenance of cell homeostasis and development of tolerance against severe insults. In neurodegenerative diseases, a number of chaperones can ameliorate accumulation of misfolded proteins triggered by oxidative/nitrosative stress or mutated gene products. Here we show that one such ER chaperone, protein-disulfide isomerase (PDI), is S-nitrosylated in brains manifesting sporadic Parkinson's or Alzheimer's disease. Nitric oxide-induced S-nitrosylation of PDI inhibits its enzymatic activity, leads to accumulation of polyubiquitinated proteins, and activates the unfolded protein response (UPR). S-Nitrosylation also abrogates PDI-mediated attenuation of neuronal cell death triggered by ER stress or proteasome inhibition. Thus, PDI prevents neurotoxicity associated with ER stress and protein misfolding, but nitric oxide blocks this protective effect in neurodegenerative disorders via PDI S-nitrosylation.

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  • 小胞体に蓄積する変性蛋白質の修復/分解系を用いた神経変性疾患防御機構

    Grant number:16659016  2004 - 2005

    日本学術振興会  科学研究費助成事業 萌芽研究  萌芽研究

    野村 靖幸, 大熊 康修, 上原 孝, 金子 雅幸

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    Grant amount:\3500000 ( Direct expense: \3500000 )

    1.ケミカルシャペロンとして知られている4-フェニル酪酸(4-PBA)の小胞体ストレス抑制作用について明らかにするため,小胞体ストレスに対する4-PBAの作用を検討した。その結果,4-PBAは小胞体ストレスを抑制し,それによる細胞死を抑制、した。さらに4-フェニル酪酸の作用機序を明らかにするため,in vitroにおけるシャペロン活性を検討したところ,4-PBAはin vitroにおいてタンパク質の凝集を抑制することを明らかにした。家族性パーキンソン病(AR-JP)の原因遺伝子Parkin(ユビキチンリガーゼ:E3)の基質タンパク質Pael-Rは,AR-JPにおいて小胞体に蓄積し,小胞体ストレスを引き起こす。Pael受容体に対する4-PBAの作用を検討したところ,4-PBAはPael受容体の正常な発現を促進し,Pael受容体蓄積による小胞体ストレスとそれによる細胞死を抑制した。
    2.小胞体のタンパク質の分解系である小胞体関連分解(ER associated degradation:ERAD)に関与し,小胞体ストレス抑制作用を有する遺伝子の同定と機能解析を行った。RING-fingerドメインを有すること,膜蛋白質であること,小胞体に局在することを条件として,バイオインフォマティクス的解析によりERAD関連新規E3の候補を検索したところ,51の遺伝子を選出した。そのうちの8遺伝子が小胞体ストレスによって誘導された。つぎに,絞り込まれたERADに関与すると考えられる遺伝子7個をクローニングした。そのうち3遺伝子が,小胞体に局在してE3活性を有し,小胞体ストレスによる細胞死に対して保護作用を示したことから,ERADに関与する新規のE3であることが示唆された。

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  • Studies on the Regulatory Mechanism of Neuronal Death: Isolation of Novel Factors and Title of Preparation of Model of Neurodegenerative Disease.

    Grant number:15109002  2003 - 2006

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (S)  Grant-in-Aid for Scientific Research (S)

    NOMURA Yasuyuki, TAKAHASHI Ryosuke, OKUMA Yasunobu, UEHARA Takashi, KANEKO Masayuki

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    Grant amount:\112190000 ( Direct expense: \86300000 、 Indirect expense:\25890000 )

    Protein-disulphide isomerase (PDI), which exists in endoplasmic reticulum (ER), and assists in the maturation and transport of unfolded secretory proteins. PDI has two domains that function as independent active sites with homology to the small, redox-active protein thioredoxin. We demonstrated that PDI was S-nitrosylated, a reaction transferring a nitric oxide (NO) group to a critical cysteine thiol to affect protein function. NO-induced S-nitrosylation of PDI inhibited its enzymatic activity, leads to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response. Furthermore, we showed in brains manifesting sporadic Parkinson's or Alzheimer's disease, that PDI is S-nitrosylated. Thus, PDI prevents neurotoxicity associated with ER stress and protein misfolding, but NO blocks this protective effect in neurodegenerative disorders through the S-nitrosylation of PDI.
    Upregulation of Parkin associated endothelin-receptor like receptor (Pael-R) in the ubiquitin-protein ligase Parkin deficient mice leads to death of dopaminergic neurons. The cell death in these animals was protected or aggaravated in upregulated or deficient mice of the ER chaperone ORP150 (150 kDa oxygen-regulated protein), respectively. We interbreed Pael-R-upregulated and Parkin knockout mice. In the mice, we observed ER-stress in the brain, selective cell death of catecholaminergic neurons in the substantia nigra and the locus ceruleus, and deficiency of mitochondria complex I. These data suggest a model in which ER-and dopamine-related stress are major contributors to decreased viability of dopaminergic neurons in a setting relevant to Parkinson's disease.

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  • 小胞体での変性蛋白質分解に関与する分子の単離・同定と神経変性疾患防御機構

    Grant number:14657574  2002 - 2003

    日本学術振興会  科学研究費助成事業 萌芽研究  萌芽研究

    野村 靖幸, 金子 雅幸, 上原 孝

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    Grant amount:\3200000 ( Direct expense: \3200000 )

    ラットに四血管閉塞/再灌流を行い,一過性前脳虚血モデルを作成して,脳虚血に伴って発現量の変化する遺伝子をディファレンシャル・ディスプレイ法を用いて単離することを試み,減少する遺伝子としてphosphatidylinositol 4-kinase (PI 4-K)の同定に成功した.PI 4-K蛋白質は海馬ニューロンに多く発現していること,また,虚血に伴って細胞死が起こるCA1領域においては,細胞死が観察される前に先んじて減少することがわかった.さらに,PI 4-Kの過剰発現は有意に低酸素による細胞死を抑制すること,PI 4-KはAktの活性化を増強・維持することがわかった.
    脳虚血モデルマウスにおいて,HRD1 mRNA発現を検討したところ,HRD1 mRNAが大脳皮質および線条体において増加することが明らかとなった.さらにin vitroの系で低酸素/再酸素化を負荷するとHRD1 mRNAはグリア細胞においては発現が上昇したが,一方神経芽細胞腫Neuro2aにおいては発現に変化は認められなかった.したがって,脳虚血は小胞体機能を障害すること,HRD1は虚血による細胞死に保護的に関与することが示唆された.
    小胞体に蓄積した変性タンパク質の分解系である小胞体関連分解(ER associated degradation : ERAD)に関与する遺伝子KF-1をバイオインフォマティクス的解析により,単離・同定した.KF-1は実際に小胞体に局在し,ユビキチンリガーゼ活性を有していることを示した.また,小胞体ストレス誘導されることが判明した.さらに,KF-1を哺乳類細胞に過剰発現させることによって小胞体ストレスによる神経細胞死が抑制されるか解析したところ,小胞体ストレス特異的に細胞死を抑制した.

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  • 脳ニューロン・グリア細胞相関系のストレス応答機構に関する細胞分子生物学的研究

    Grant number:13307064  2001 - 2003

    日本学術振興会  科学研究費助成事業 基盤研究(A)  基盤研究(A)

    野村 靖幸, 金子 雅幸, 上原 孝, 大熊 康修

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    Grant amount:\41340000 ( Direct expense: \31800000 、 Indirect expense:\9540000 )

    ラットに四血管閉塞/再灌流を行い,一過性前脳虚血モデルを作成して,脳虚血に伴って発現量の変化する遺伝子をディファレンシャル・ディスプレイ法を用いて単離することを試み,減少する遺伝子としてphosphatidylinositol 4-kinase(PI 4-K)の同定に成功した.PI 4-K蛋白質は海馬ニューロンに多く発現していること,また,虚血に伴って細胞死が起こるCA1領域においては,細胞死が観察される前に先んじて減少することがわかった.さらに,PI 4-Kの過剰発現は有意に低酸素による細胞死を抑制すること,PI 4-KはAktの活性化を増強・維持することがわかった.
    低酸素ストレスや脳虚血によって誘導されるPDI結合蛋白質として小胞体膜蛋白質 ubiquilinを単離し,それがPDIの低酸素ストレスによるニューロン死抑制作用を増強することを明らかにした.さらに,ubiquilinが低酸素ストレスによる小胞体ストレスによって誘導されるアポトーシス促進転写因子CHOPの誘導を抑制したことから,PDIやubiquilin の低酸素ストレス防御作用は小胞体ストレス抑制によるもにであることが,示唆される結果を得た.
    哺乳類における小胞体ストレス誘導遺伝子HRD1をバイオインフォマティクス的解析により,単離・同定した.これらの遺伝子の特徴として,小胞体に局在し,小胞体に蓄積した変性タンパク質の分解系である小胞体関連分解(ER associated degradation : ERAD)に関与する遺伝子であることが判明した.また,HRD1を哺乳類細胞に過剰発現させることによって小胞体ストレスによる細胞死が抑制されるか解析したところ,小胞体ストレス特異的に細胞死を抑制することを示した.

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  • 脳虚血によるニューロン死惹起機構に関わる新規因子の探索・同定

    Grant number:13771364  2001 - 2002

    日本学術振興会  科学研究費助成事業 若手研究(B)  若手研究(B)

    上原 孝

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    Grant amount:\2100000 ( Direct expense: \2100000 )

    ラットに四血管閉塞/再灌流を行い,一過性前脳虚血モデルを作成して,脳虚血に伴って発現量の変化する遺伝子をディファレンシャル・ディスプレイ法を用いて単離することを試み,減少する遺伝子としてphosphatidylinositol 4-kinase (PI 4-K)の同定に成功した.PI 4-Kの発現の変化を詳細に検討したところ,PI 4-K蛋白質は海馬ニューロンに多く発現していること,また,虚血に伴って細胞死が起こるCA1領域においては,細胞死が観察される前に先んじて減少することがわかった.さらに,ノーザンブロット解析から,PI 4-K mRNAは脳虚血1日後から有意に減少することが明かとなった.また,細胞死が起こらない海馬CA3ならびに歯状回において,PI 4-Kの変化は認められなかった.また,培養神経芽細胞腫に低酸素ストレスを負荷するとアポトーシス様の細胞死が観察されるが,その際にもPI 4-Kの著明な減少が認められた.さらに,PI 4-Kの過剰発現は有意に低酸素による細胞死を抑制したこと,キナーゼ活性を有しない優性抑制変異型PI 4-Kには細胞死抑制効果はないことから,PI 4-Kの酵素活性が重要であることが示唆された.低酸素ストレスによって細胞は死から免れるために,細胞死抑制シグナルであるAktを活性化させる.PI 4KはAktの活性化を増強・維持することがわかった.したがって,PI 4Kの脳虚血/低酸素による減少は細胞死抑制シグナルを減弱させ,その結果,ニューロン死が惹起する可能性が推定された.

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  • Preparation of animal model with higher brain dysfunction : studies on evaluation methods of active chemicals

    Grant number:12357015  2000 - 2002

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)  Grant-in-Aid for Scientific Research (A)

    NOMURA Yasuyuki, SYUTO Satoshi, MURAYAMA Toshihiko, OKUMA Yasunobu, SAITO Hiroshi, UEHARA Takashi

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    Grant amount:\41570000 ( Direct expense: \35600000 、 Indirect expense:\5970000 )

    Senescence-accelerated mouse prone 8 (SAMP8) shows marked impairment of learning and memory, whereas SAMP10 shows brain atrophy and aging-associated depressive behavior. Hippocampal GDNF mRNA expression in 2-month-old SAMP8 and SAMP10 strains was less than in SAMR1 specimens of the same age. The number of surviving neurons in the CA1 region decreased with age in SAMP8 and SAMP10. These findings suggest that low GDNF expression in young SAMP8 and SAMP10 may be involved in hippocampal dysfunctions, such as age-related learning impairment and neuronal death. We investigated genetic characteristic of learning and memory impairment in SAMP8 by cross-mating between SAMP8 and normal mice, JF1. Results of the incidence of learning deficit in backcross generation and quantitative trait Loci analysis (QTL) suggest that at least one major gene may involves in learning impairment of SAMP8. CV-159, dihydropyridine derivative, 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pryridinedicarboxylic acid methyl 6-(5-phenyl-3- pyrazolyloxyl ester that blocks the L-type calcium channel and inhibits the calmodulin-dependent pathway. We found that CV-159 protects against ischemic brain injury. This might be mediated by both blocking the L-type calcium channel and inhibiting calmodulin-dependent function. We have attempted to isolate the genes whose levels were changed in response to transient cerebral ischemia. We found that hippocampal expression of phosphatididylinositol 4-kinase (PI4-K) was decreaed after the brain ischemia, and demonstrated the protective role of PI4-K on ischemia-induced neuronal death. Application of a brief period of ischemia has been known to produce ischemic tolerance. We found that the phosphorylation of CREB in the penumbra region was more rapidly enhanced in the preconditioned rats. The result suggests that the immediate enhancement in the phosphorylation of CREB in penumbra region prevented the spread of infarction in the preconditioned animal.

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  • ニューロン・グリアにおける脳虚血ストレス応答の細胞内機構

    Grant number:11771418  1999 - 2000

    日本学術振興会  科学研究費助成事業 奨励研究(A)  奨励研究(A)

    上原 孝

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    Grant amount:\2100000 ( Direct expense: \2100000 )

    ヒト由来株化ニューロンモデル細胞SH-SY5Yに一酸化窒素(NO)を負荷すると,クロマチンの凝縮,DNA断片化,低DNA含量細胞や細胞膜上へのフォスファチジルセリンの露出を伴うアポトーシス様の死が起こることをこれまでに報告している.そこで,今年度はその詳細なアポトーシス実行過程を明らかにする目的で研究を行い,以下の知見を得た.
    NOはミトコンドリアからのチトクロームcの漏出を惹起し,それに続いてカスパーゼ-2,-3,-6,-7,および-9の活性化を引き起こした.また,カスパーゼ-3の基質蛋白質であり,DNA断片化に関与するDNaseであるCADに結合して活性を制御しているinhibitor of CAD(ICAD)の分解もNOによって起こることが分かった.カスパーゼ-3阻害ペプチドを処理すると,カスパーゼ-2,および-7の切断が抑制されたことから,カスパーゼ-3はカスパーゼ-2および-7の上流で働いていることが示唆された.一方,NOは時間依存的にミトコンドリア内膜の膜電位を低下させることが明かとなった.内膜において,透過性を調節する複合体に作用するシクロスポリンAおよびbongkrekic acidはNOによるミトコンドリア膜電位の低下を有意に抑制し,さらにチトクロムCの細胞質への漏出とそれに続くDNAの断片化も抑制した.
    以上のことから,NOはミトコンドリアに作用し,その膜電位を低下させることでアポトーシス実行因子であるチトクロムCを細胞質に放出させ,カスパーゼおよびDNaseを活性化して,アポトーシスを惹起している可能性が示唆された.

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  • Mechanisms of cellular and intracellular functions in brain-immune qnetwork.

    Grant number:10307055  1998 - 2000

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A).  Grant-in-Aid for Scientific Research (A).

    NOMURA Yasuyuki, MURAYAMA Toshihiko, UEHARA Takashi, OKUMA Yasunobu

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    Grant amount:\37200000 ( Direct expense: \37200000 )

    We investigated the mechanisms of 1) ischemia-induced neuronal cell death and 2) acquisition of tolerance against ischemic stress in glial cells. Hypoxia, an ischemic stress, induced neuronal apoptosis via cytochrome c release from mitochondria and following caspase activation. On the other hand, this stress up-regulated some stress proteins such as a 70 kDa heat-shock protein (HSP70) and a 78 kDa glucose-regulated protein (GRP78). In addition, we found that HSP70 induction was sensitive to SB203580, suggesting that p38 MAP kinase was positively involved in this expression. We further attempted to identify a stress protein that was enhanced or induced by hypoxia or brain ischemia in glial cells. We isolated protein-disulfide isomerase (PDI) as a candidate protein and this protein certainly induced in glial cells of ischemic brain. Overexpression of PDI in vitro and in vivo resulted in attenuation of the loss of cell viability in neuroblastoma SK-N-MC cells and a reduction in the number of DNA-fragmented cells in the CA1 subfield of the hippocampus in brain ischemic rats, respectively. Thus, up-regulated PDI may play a critical role in resistance to ichemic damage.

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  • Development of model animals and cells for neuronal disease

    Grant number:09357019  1997 - 1999

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)  Grant-in-Aid for Scientific Research (A)

    NOMURA Yasuyuki, SHUTO Satoshi, MURAYAMA Toshihiko, OKUMA Yasunobu, HATTORI Masao, UEHARA Takashi

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    Grant amount:\35300000 ( Direct expense: \35300000 )

    The senescence accelerated mouse (SAM) is known as a murine model of aging. SAM consists of senescence accelerated-prone mouse (SAMP) and senescence accelerated-resistant mouse (SAMR). Previously, we reported that SAMP8 and SAMP10 exhibit age-related learning impairments . In this study, we investigated the changes in emotional behavior and in neurotansmitter receptor in SAMP10, and the effect of onion extract on learning ability in Morris's water maze test.
    1) SAMP10 at 8 months showed an increases of immobility in a forced swimming test compared with SAMR1. Treatment with desipramine (25 mg/kg, I.p., 3 days) in SAMP10 caused a decrease in immobility.
    2) In the cortex from SAMP1O, [ィイD13ィエD1H]quinpirol binding to D2/D3 dopamine receptors increased significantly. In the hippocampus from SAMP10, [ィイD13ィエD1H]hydroxy DPAT binding to 5-HTィイD11AィエD1 receptors increased.
    3) In Morris's water maze task, in a control strain of SAMR1 at 8 months, the escape latency and path length decreased with increasing trial days, in contrast, the escape latency and path length did not change in SAMP8. Treatment with onion extract (5 ml/kg, p.o., 2 months) in SAMP8 improved the learning and memory in the task.
    4) In the control rats pretreated with the vehicle, transient forebrain ischemia-induced delayed neuronal death in the hippocampal CA 1 region was observed 7 days after reperfusion. Pretreatment with glial cell line-derived neurotrophic factor (1 μg), which was directly microinjected into the right hippocampal CA1 region, gave significant protection against the neuronal death. CV-159 (a dihydropyridine derivative) that blocks the L-type CaィイD12+ィエD1 channel and inhibits the calmodulin-dependent pathway. CV-159 gave protection against delayed neuronal death in the CA1 region after 15-min transient forebrain ischemia.

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  • 脂肪細胞分化を誘導するインスリンの細胞内情報伝達系の解析

    Grant number:09771961  1997 - 1998

    日本学術振興会  科学研究費助成事業 奨励研究(A)  奨励研究(A)

    上原 孝

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    Grant amount:\1900000 ( Direct expense: \1900000 )

    3T3-L1線維芽細胞はインスリン・デキサメサゾン・IBMXの同時添加によって白色脂肪細胞へと分化することが報告されているものの,細胞分化を誘導する情報伝達系については不明な点が多く残されている.前年度までに私たちはSwissマウス由来3T3-L1細胞の脂肪細胞への分化が,3量体GTP結合蛋白質の機能を消失させる百日咳毒素(PTX)の前処理によって抑制されることを見い出した.さらには,phosphatidylinositol 3-kinase(PI3-キナーゼ)の特異的阻害薬であるwortmanninの前処理によっても本細胞の分化が抑制されることを明らかにした.この事実から,PTXとwortmanninは細胞分化を誘導する情報伝達系を解析する上で,有用なプローブである可能性が示唆された,そこで,脂肪細胞分化へのkey factorであるインスリンの情報伝達系を解析したところ,PTX、wortmanninは共にインスリン刺激によるRasの活性化とそれに続くMAPキナーゼ系のそれを抑制した.この時,インスリンと同様に受容体チロシンキナーゼを活性化させるepidermal growth factor(EGF)刺激に伴うRasおよびMAPキナーゼ系の活性化に対しては無効であった.また,インスリン(あるいはIGF-1)受容体自己チロシンリン酸化反応、基質蛋白質(IRS-1)のチロシンリン酸化とPI3-キナーゼとの結合に対しては影響を及ぼさなかったものの,PTXはインスリン刺激に伴うPI3-キナーゼ活性を50%抑制した.このことから,インスリンのPI3-キナーゼ活性化機構には異なる2つの経路が存在し,1つは受容体キナーゼ→IRS-1→PI3-キナーゼ,他方は受容体→PTX感受性蛋白質,おそらく3量体G蛋白質→PI3-キナーゼを介するものであることが示唆された。したがって、PTXはインスリンによるPI3-キナーゼ活性を抑制することで,その下流に存在するカスケードを抑制する結果,脂肪細胞への分化を抑制していることが推定された.

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  • マウス3T3-L1細胞の分化におけるインスリンの情報伝達系の解析

    Grant number:08772073  1996

    日本学術振興会  科学研究費助成事業 奨励研究(A)  奨励研究(A)

    上原 孝

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    Grant amount:\1000000 ( Direct expense: \1000000 )

    3T3-L1線維芽細胞はインスリン・デキサメサゾン・IBMXの同時添加によって白色脂肪細胞へと分化することが報告されているものの、細胞分化を誘導する情報伝達系については不明な点が多く残されている。私たちは3T3-L1細胞の脂肪細胞への分化が3量体GTP結合蛋白質の機能を消失させる百日咳毒素(PTX)の前処理によって抑制されることを見い出した。さらには、インスリンによって活性化されるphosphatidylinositol 3-kinase (PI 3-キナーゼ)の特異的阻害薬であるwortmanninの前処理によっても脂肪細胞への分化が抑制されることを明らかにしてきた。このことから、PTXとwortmaninは細胞分化を誘導する情報伝達系を解析する上で、有用なプローブであることが予想された。そこで、脂肪細胞分化へのkey factorであるインスリンの情報伝達系を調べた。PTX、wortmanminは共にインスリン刺激に伴うRasの活性化とMAPキナーゼ活性やMAPキナーゼの活性化に必須なチロシン残基のリン酸化を抑制した。この時、インスリンと同様に受容体チロシンキナーゼを活性化させるepidermal growth factor(EGF)刺激に伴うRasおよびMAPキナーゼの活性化に対しては抑制作用を示さなかった。受容体自己チロシンリン酸化反応、基質蛋白質(IRS-1)のチロシンリン酸化とPI 3-キナーゼとの結合に対しては影響を及ぼさなかった。しかしながら、PTXはインスリン刺激に伴うPI 3-キナーゼ活性を50%抑制した。このことから、インスリンのPI 3-キナーゼ活性化機構には異なる2つの経路が存在し、1つは受容体キナーゼ→IRS-1→PI 3-キナーゼ、他方は受容体→PTX感受性蛋白質、おそらく3量体G蛋白質→PI 3-キナーゼを介するものであることが示唆された。したがって、PTXはインスリンによるPI 3-キナーゼ活性を抑制することで、その下流に存在するカスケードを抑制する結果、脂肪細胞への分化を抑制していることが推定された。

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  • Establishment and estimation of pharmacological bioassay system for therapeutic drugs of aging in central nervous system.

    Grant number:07557148  1995 - 1996

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)  Grant-in-Aid for Scientific Research (A)

    NOMURA Yasuyuki, NAMBA Tsuneo, UEHARA Takashi, MURAYAMA Toshihiko, TOKUMITSU Yukiko, MATSUDA Akira

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    Grant amount:\14700000 ( Direct expense: \14700000 )

    To clarify the physiological roles of muscarinic acetylcholine (mACh) receptor subtypes, M1 and M2, on learning and memory, we examined the effects of three antagonists, atropine (non-selective), pirenzepine (M1 selective) and 11-[[2[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one, AF-DX 116 (M2 selective), on stpe-through passive avoidance tasks in mice. Pre-training (5min before) administration of atropine (1-40 nmol) and pirenzepine (10 and 40 nmol) shortened the response latency in retention tests at 14 d after acquisition training. Pre-test (5min befor) and post-training (immediately after the acquisition) administration of atropine slightly but not significantly impaired retention scores. The administration of AF-DX 116 did not apparently affect the scores in any of tests. Thus, the M1 receptor subtype coupling systemes seem to be more important in the acquisition-consolidation process rather than in the retrieval process.

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  • マウス3T3-L1細胞の分化におけるインスリンの情報伝達系の解析

    Grant number:07772147  1995

    日本学術振興会  科学研究費助成事業 奨励研究(A)  奨励研究(A)

    上原 孝

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    Grant amount:\1000000 ( Direct expense: \1000000 )

    3T3-L1線維芽細胞はインスリン・デキサメサゾン・IBMXの同時添加によって白色脂肪細胞へと分化する。しかしながら、細胞分化を誘導する情報伝達系については不明である、そこで、3T3-L1細胞の分化にはインスリンがkey roleを担っていることから、インスリンの情報伝達系を解析することで、脂肪細胞分化の誘導機構を調べた。インスリンをコンフルエントの3T3-L1線維芽細胞に添加すると、インスリン受容体チロシンキナーゼの自己リン酸化が速やかに起こった。さらに、細胞の増殖や分化に関与していると考えられている低分子量G蛋白質のRasとRasの下流に存在するmitogen-activated protein kinase(MAPキナーゼ)の活性化が起こった。また、インスリンによる糖取り込み作用に関与していることが示唆されているphosphatidylinositol 3-kinase(PI3-キナーゼ)も活性化することが分かった。一方、PI3-キナーゼの特異的阻害薬であるwortmanninは3T3-L1線維芽細胞の脂肪細胞への分化を濃度依存的に抑制することが分かった。さらには、wortmanninはインスリン刺激に伴うRasの活性化とMAPキナーゼ活性やMAPキナーゼの活性化に必須なチロシン残基のリン酸化を抑制した。この時、インスリンと同様に受容体チロシンキナーゼを活性化させるepidermal growth factor(EGF)刺激に伴うRasおよびMAPキナーゼの活性化に対してwortmanninは抑制作用を示さなかった。このことから、3T3-L1細胞においてインスリンとEGFの情報伝達系は異なっており、脂肪細胞分化を引き起こすインスリンの情報伝達系はPI3-キナーゼ→Ras→MAPキナーゼというカスケードを経ていることが推定された。

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