2024/10/12 更新

写真a

オシカワ キヨタカ
押川 清孝
OSHIKAWA Kiyotaka
所属
教育研究院 医歯学系 医学系列 准教授
医歯学総合研究科 分子細胞医学専攻 遺伝子制御 准教授
職名
准教授
外部リンク

学位

  • 博士(理学) ( 2000年10月   福岡大学 )

  • 修士(理学) ( 1997年3月 )

研究キーワード

  • プロテオミクス

  • 機能生物化学

研究分野

  • ライフサイエンス / 機能生物化学

経歴(researchmap)

  • 新潟大学   大学院医歯学総合研究科 オミクス生物学分野   准教授

    2019年11月 - 現在

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  • 九州大学   分子医科学分野   学術研究員

    2009年4月 - 2019年10月

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  • 九州大学   Medical Institute of Bioregulation   非常勤研究員

    2008年4月 - 2009年3月

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  • 株式会社ロコモジェン   研究員

    2006年8月 - 2008年3月

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  • 徳島大学分子酵素学研究センター   情報細胞学部門   COE研究員

    2004年4月 - 2006年7月

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  • 九州大学   分子発現制御学分野   学振特別研究員

    2002年4月 - 2004年3月

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  • 九州大学   発生工学実験施設   非常勤研究員

    2000年4月 - 2002年3月

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▶ 全件表示

経歴

  • 新潟大学   医歯学総合研究科 分子細胞医学専攻 遺伝子制御   准教授

    2019年11月 - 現在

学歴

  • 福岡大学   Graduate School, Division of Natural Science

    - 2000年

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  • 福岡大学   理学研究科   化学専攻

    - 2000年

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    国名: 日本国

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  • 福岡大学   Faculty of Science

    - 1995年

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  • 福岡大学   理学部   化学科

    - 1995年

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    国名: 日本国

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所属学協会

 

論文

  • Cell cycle-dependent localization of the proteasome to chromatin. 査読 国際誌

    Yuki Kito, Masaki Matsumoto, Atsushi Hatano, Tomoyo Takami, Kiyotaka Oshikawa, Akinobu Matsumoto, Keiichi I Nakayama

    Scientific reports   10 ( 1 )   5801 - 5801   2020年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    An integrative understanding of nuclear events including transcription in normal and cancer cells requires comprehensive and quantitative measurement of protein dynamics that underlie such events. However, the low abundance of most nuclear proteins hampers their detailed functional characterization. We have now comprehensively quantified the abundance of nuclear proteins with the use of proteomics approaches in both normal and transformed human diploid fibroblasts. We found that subunits of the 26S proteasome complex were markedly down-regulated in the nuclear fraction of the transformed cells compared with that of the wild-type cells. The intranuclear proteasome abundance appeared to be inversely related to the rate of cell cycle progression, with restraint of the cell cycle being associated with an increase in the amount of proteasome subunits in the nucleus, suggesting that the nuclear proteasome content is dependent on the cell cycle. Furthermore, chromatin enrichment for proteomics (ChEP) analysis revealed enrichment of the proteasome in the chromatin fraction of quiescent cells and its apparent dissociation from chromatin in transformed cells. Our results thus suggest that translocation of the nuclear proteasome to chromatin may play an important role in control of the cell cycle and oncogenesis through regulation of chromatin-associated transcription factors.

    DOI: 10.1038/s41598-020-62697-2

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  • A shift in glutamine nitrogen metabolism contributes to the malignant progression of cancer. 査読 国際誌

    Manabu Kodama, Kiyotaka Oshikawa, Hideyuki Shimizu, Susumu Yoshioka, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Chisa Tateishi, Takeshi Tomonaga, Masaki Matsumoto, Keiichi I Nakayama

    Nature communications   11 ( 1 )   1320 - 1320   2020年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Glucose metabolism is remodeled in cancer, but the global pattern of cancer-specific metabolic changes remains unclear. Here we show, using the comprehensive measurement of metabolic enzymes by large-scale targeted proteomics, that the metabolism both carbon and nitrogen is altered during the malignant progression of cancer. The fate of glutamine nitrogen is shifted from the anaplerotic pathway into the TCA cycle to nucleotide biosynthesis, with this shift being controlled by glutaminase (GLS1) and phosphoribosyl pyrophosphate amidotransferase (PPAT). Interventions to reduce the PPAT/GLS1 ratio suppresses tumor growth of many types of cancer. A meta-analysis reveals that PPAT shows the strongest correlation with malignancy among all metabolic enzymes, in particular in neuroendocrine cancer including small cell lung cancer (SCLC). PPAT depletion suppresses the growth of SCLC lines. A shift in glutamine fate may thus be required for malignant progression of cancer, with modulation of nitrogen metabolism being a potential approach to SCLC treatment.

    DOI: 10.1038/s41467-020-15136-9

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  • A fail-safe system to prevent oncogenesis by senescence is targeted by SV40 small T antigen 査読

    Oshikawa K, Matsumoto M, Kodama M, Shimizu H, Nakayama KI

    Oncogene   39 ( 10 )   2170 - 2186   2020年3月

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    掲載種別:研究論文(学術雑誌)  

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  • A large-scale targeted proteomics assay resource based on an in vitro human proteome 査読

    Masaki Matsumoto, Fumiko Matsuzaki, Kiyotaka Oshikawa, Naoki Goshima, Masatoshi Mori, Yoshifumi Kawamura, Koji Ogawa, Eriko Fukuda, Hirokazu Nakatsumi, Tohru Natsume, Kazuhiko Fukui, Katsuhisa Horimoto, Takeshi Nagashima, Ryo Funayama, Keiko Nakayama, Keiichi I. Nakayama

    NATURE METHODS   14 ( 3 )   251 - +   2017年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Targeted proteomics approaches are of value for deep and accurate quantification of protein abundance. Extending such methods to quantify large numbers of proteins requires the construction of predefined targeted assays. We developed a targeted proteomics platform-in vitro proteome-assisted multiple reaction monitoring (MRM) for protein absolute quantification (iMPAQT)-by using > 18,000 human recombinant proteins, thus enabling protein absolute quantification on a genome-wide scale. Our platform comprises experimentally confirmed MRM assays of mass tag (mTRAQ)-labeled peptides to allow for rapid and straightforward measurement of the absolute abundance of predefined sets of proteins by mass spectrometry. We applied iMPAQT to delineate the quantitative metabolic landscape of normal and transformed human fibroblasts. Oncogenic transformation gave rise to relatively small but global changes in metabolic pathways resulting in aerobic glycolysis (Warburg effect) and increased rates of macromolecule synthesis. iMPAQT should facilitate quantitative biology studies based on protein abundance measurements.

    DOI: 10.1038/NMETH.4116

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  • Proteome-wide Identification of Ubiquitylation Sites by Conjugation of Engineered Lysine-less Ubiquitin 査読

    Kiyotaka Oshikawa, Masaki Matsumoto, Koji Oyamada, Keiichi I. Nakayama

    JOURNAL OF PROTEOME RESEARCH   11 ( 2 )   796 - 807   2012年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    Ubiquitin conjugation (ubiquitylation) plays important roles not only in protein degradation but also in many other cellular functions. However, the sites of proteins that are targeted for such modification have remained poorly characterized at the proteomic level. We have now developed a method for the efficient identification of ubiquitylation sites in target proteins with the use of an engineered form of ubiquitin (K0-Ub), in which all seven lysine residues are replaced with arginine. K0-Ub is covalently attached to lysine residues of target proteins via an isopeptide bond, but further formation of a polyubiquitin chain does not occur on K0-Ub. We identified a total of 1392 ubiquitylation sites of 794 proteins from HEK293T cells. Profiling of ubiquitylation sites indicated that the sequences surrounding lysine residues targeted for ubiquitin conjugation do not share a common motif or structural feature Furthermore, we identified a critical ubiquitylation site of the cyclin-dependent kinase inhibitor p27(KiP1). Mutation of this site thus inhibited ubiquitration of and stabilized p27(KiP1), suggesting that this lysine residue is the target site of p27(Kip1) for ubiquitin conjugation in vivo. In conclusion, our method based on K0-Ub is a powerful tool for proteome-wide identification of ubiquitylation sites of target proteins.

    DOI: 10.1021/pr200668y

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  • Comprehensive study of protein ubiquitylation sites by conjugation of engineered lysine-less ubiquitin

    Kiyotaka Oshikawa, Masaki Matsumoto, Keiichi I. Nakayama

    Seikagaku   84 ( 6 )   479 - 487   2012年

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

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  • ヒトプロテオーム絶対定量プロジェクト:網羅的ターゲットプロテオミクスの開発と応用

    松本 雅記, 押川 清孝, 松崎 芙美子, 中山 敬一

    日本プロテオーム学会大会要旨集   2012   56 - 56   2012年

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    記述言語:日本語   出版者・発行元:日本プロテオーム学会(日本ヒトプロテオーム機構)  

    DOI: 10.14889/jhupo.2012.0.56.0

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  • CHD8 suppresses p53-mediated apoptosis through histone H1 recruitment during early embryogenesis 査読

    Masaaki Nishiyama, Kiyotaka Oshikawa, Yu-ichi Tsukada, Tadashi Nakagawa, Shun-ichiro Iemura, Tohru Natsume, Yuhong Fan, Akira Kikuchi, Arthur I. Skoultchi, Keiichi I. Nakayama

    NATURE CELL BIOLOGY   11 ( 2 )   172 - U139   2009年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    The chromodomain helicase DNA-binding (CHD) family of enzymes is thought to regulate gene expression, but their role in the regulation of specific genes has been unclear. Here we show that CHD8 is expressed at a high level during early embryogenesis and prevents apoptosis mediated by the tumour suppressor protein p53. CHD8 was found to bind to p53 and to suppress its transactivation activity. CHD8 promoted the association of p53 and histone H1, forming a trimeric complex on chromatin that was required for inhibition of p53-dependent transactivation and apoptosis. Depletion of CHD8 or histone H1 resulted in p53 activation and apoptosis. Furthermore, Chd8(-/-) mice died early during embryogenesis, manifesting widespread apoptosis, whereas deletion of p53 ameliorated this developmental arrest. These observations reveal a mode of p53 regulation mediated by CHD8, which may set a threshold for induction of apoptosis during early embryogenesis by counteracting p53 function through recruitment of histone H1.

    DOI: 10.1038/ncb1831

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  • ST2 gene induced by type 2 helper T cell (Th2) and proinflammatory cytokine stimuli may modulate lung injury and fibrosis 査読

    Shunji Tajima, Masashi Bando, Shoji Ohno, Yukihiko Sugiyama, Katsuhisa Oshikawa, Shin-ichi Tominaga, Kouichi Itoh, Toshinori Takada, Eiichi Suzuki, Fumitake Gejyo

    EXPERIMENTAL LUNG RESEARCH   33 ( 2 )   81 - 97   2007年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:TAYLOR & FRANCIS INC  

    The authors have investigated gene expression of ST2 in the lung tissue of a bleomycin (BLM)-induced lung fibrosis model in vivo and in a human lung fibroblast cell line, WI38, and a human type II alveolar epithelial cell line, A549, reacting to proinflammatory and type 2 helper T cell (Th2)-type cytokine stimuli in vitro. The lung mRNA expression of interleukin (IL)-4, IL-5, IL-1 beta, and tumor necrosis factor (TNT)-alpha increased significantly at day 7 after instillation of BLM, whereas interferon (IFN)-gamma mRNA expression did not increase. ST2 and transforming growth factor (TGF)-beta 1 mRNA expression of the lung increased significantly between days 7 and 21, and increased to maximal levels at day 14 post-BLM challenge. ST2 mRNA expression statistically correlated with TGF-beta 1 mPLNA expression. In addition, the combination of IL-1 beta, TAT-alpha, and IL-4 had an additive effect on ST2 mRNA expression from A549 cells and WI38 cells. These findings suggest that soluble ST2 gene may increase, possibly reflecting the development of the inflammatory process and the Th2-type immune response in the fibrotic lung tissue, and may modulate a process of pulmonary fibrosis.

    DOI: 10.1080/01902140701198583

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  • Alteration of intra-pancreatic target-organ specificity by abrogation of Aire in NOD mice 査読

    S Niki, K Oshikawa, Y Mouri, F Hirota, A Matsushima, M Yano, H Han, Y Bando, K Izumi, M Matsumoto, KI Nakayama, N Kuroda, M Matsumoto

    JOURNAL OF CLINICAL INVESTIGATION   116 ( 5 )   1292 - 1301   2006年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC CLINICAL INVESTIGATION INC  

    Factors that determine the spectrum of target organs involved in autoimmune destruction are poorly understood. Although loss of function of autoimmune regulator (AIRE) in thymic epithelial cells is responsible for autoimmunity, the pathogenic roles of AIRE in regulating target-organ specificity remain elusive. In order to gain insight into this issue, we have established NOD mice, an animal model of type 1 diabetes caused by autoimmune attack against P cell islets, in which Aire has been abrogated. Remarkably, acinar cells rather than P cell islets were the major targets of autoimmune destruction in Aire-deficient NOD mice, and this alteration of intra-pancreatic target-organ specificity was associated with production of autoantibody against pancreas-specific protein disulfide isomerase (PDIp), an antigen expressed predominantly by acinar cells. Consistent with this pathological change, the animals were resistant to the development of diabetes. The results suggest that Aire not only is critical for the control of self-tolerance but is also a strong modifier of target-organ specificity through regulation of T cell repertoire diversification. We also demonstrated that transcriptional expression of PDIp was retained in the Aire-deficient NOD thymus, further supporting the concept that Aire may regulate the survival of autoreactive T cells beyond transcriptional control of self-protein expression in the thymus.

    DOI: 10.1172/JCI26971

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  • Essential role of I kappa B kinase alpha in thymic organogenesis required for the establishment of self-tolerance 査読

    Dan Kinoshita, Fumiko Hirota, Tsuneyasu Kaisho, Michiyuki Kasai, Keisuke Izumi, Yoshimi Bando, Yasuhiro Mouri, Akemi Matsushima, Shino Niki, Hongwei Han, Kiyotaka Oshikawa, Noriyuki Kuroda, Masahiko Maegawa, Minoru Irahara, Kiyoshi Takeda, Shizuo Akira, Mitsuru Matsumoto

    JOURNAL OF IMMUNOLOGY   176 ( 7 )   3995 - 4002   2006年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER ASSOC IMMUNOLOGISTS  

    I kappa B kinase (IKK) alpha exhibits diverse biological activities through protein kinase-dependent and -independent functions, the former mediated predominantly through a noncanonical NF-kappa B activation pathway. The in vivo function of IKK alpha, however, still remains elusive. Because a natural strain of mice with mutant NF-kappa B-inducing kinase (NIK) manifests autoimmunity as a result of disorganized thymic structure with abnormal expression of Rel proteins in the thymic stroma, we speculated that the NIK-IKK alpha axis might constitute an essential step in the thymic organogenesis that is required for the establishment of self-tolerance. An autoimmune disease phenotype was induced in athymic nude mice by grafting embryonic thymus from IKK alpha-deficient mice. The thymic microenvironment that caused autoimmunity in an IKK alpha-dependent manner was associated with defective processing of NF-kappa B2, resulting in the impaired development of thymic epithelial cells. Thus, our results demonstrate a novel function for IKK alpha in thymic organogenesis for the establishment of central tolerance that depends on its protein kinase activity in cooperation with NIK.

    DOI: 10.4049/jimmunol.176.7.3995

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  • Subcellular expression of autoimmune regulator is organized in a spatiotemporal manner 査読

    H Akiyoshi, S Hatakeyama, J Pitkanen, Y Mouri, Doucas, V, J Kudoh, K Tsurugaya, D Uchida, A Matsushima, K Oshikawa, KI Nakayama, N Shimizu, P Peterson, M Matsumoto

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 32 )   33984 - 33991   2004年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Autoimmune regulator ( AIRE) is responsible for the development of organ-specific autoimmune disease in a monogenic fashion. Rare and low levels of tissue expression together with the lack of AIRE-expressing cell lines have hampered a detailed analysis of the molecular dynamics of AIRE. Here we have established cell lines stably transfected with AIRE and studied the regulatory mechanisms for its subcellular expression. We found that nuclear body ( NB) formation by AIRE was dependent on the cell cycle. Biochemical fractionation revealed that a significant proportion of AIRE is associated with the nuclear matrix, which directs the functional domains of chromatin to provide sites for gene regulation. Upon proteasome inhibition, AIRE NBs were increased with concomitant reduced expression in the cytoplasm, suggesting that subcellular targeting of AIRE is regulated by a ubiquitin-proteasome pathway. We also found that AIRE NBs compete for cAMP-response element-binding protein-binding protein/p300, a common coactivator of transcription, with the promyelocytic leukemia gene product. These results suggest that the transcriptional regulating activities of AIRE within a cell are controlled and organized in a spatiotemporal manner.

    DOI: 10.1074/jbc.M400702200

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  • Preferential interaction of TIP120A with Cul1 that is not modified by NEDD8 and not associated with Skp1 査読

    K Oshikawa, M Matsumoto, M Yada, T Kamura, S Hatakeyama, KI Nakayama

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   303 ( 4 )   1209 - 1216   2003年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    The SCF complex, which consists of the invariable components Skp1, Cul1, and Rbx1 as well as a variable F-box protein, functions as an E3 ubiquitin ligase. The mechanism by which the activity of this complex is regulated, however, has been unclear. The application of tandem affinity purification has now resulted in the identification of a novel Cul1-binding protein: TATA-binding protein-interacting protein 120A (TIP120A, also called CAND1). Immunoprecipitation, immunoblot, and immunofluorescence analyses with mammalian cells revealed that TIP120A physically associates with Cull in the nucleus and that this interaction is mediated by a central region of Cull distinct from its binding sites for Skp1 and Rbx1. Furthermore, TIP120A was shown to interact selectively with Cull that is not modified by NEDD8. The Cul1-TIP120A complex does not include Skp1, raising the possibility that TIP120A competes with Skp1 for binding to Cul1. These observations thus suggest that TIP120A may function as a negative regulator of the SCF complex by binding to nonneddylated Cul1 and thereby preventing assembly of this ubiquitin ligase. (C) 2003 Elsevier Science (USA). All rights reserved.

    DOI: 10.1016/S0006-291X(03)00501-1

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  • Degradation of p27(Kip1) at the G(0)-G(1) transition mediated by a Skp2-independent ubiquitination pathway 査読

    T Hara, T Kamura, K Nakayama, K Oshikawa, S Hatakeyama, KI Nakayama

    JOURNAL OF BIOLOGICAL CHEMISTRY   276 ( 52 )   48937 - 48943   2001年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Targeting of the cyclin-dependent kinase inhibitor p27(Kip1) for proteolysis has been thought to be mediated by Skp2, the F-box protein component of an SCF ubiquitin ligase complex. Degradation of p27(Kip1) at the Go-G, transition of the cell cycle has now been shown to proceed normally in Shp2(-/-) lymphocytes, whereas p27(Kip1) proteolysis during S-G(2) phases is impaired in these Skp2-deficient cells. Degradation of p27(Kip1) at the G,-G(1), transition was blocked by lactacystin, a specific proteasome inhibitor, suggesting that it is mediated by the ubiquitin-proteasome pathway. The first cell cycle of stimulated Shp2(-/-) lymphocytes appeared normal, but the second cycle was markedly inhibited, presumably as a result of P27(Kip1) accumulation during S-G, phases of the first cell cycle. Polyubiquitination of p27(Kip1) in the nucleus is dependent on Skp2 and phosphorylation of P27(Kip1) on threonine 187. However, polyubiquitination activity was also detected in the cytoplasm of Shp2(-/-) cells, even with a threonine 187 --> alanine mutant of P27(Kip1) as substrate. These results suggest that a polyubiquitination activity in the cytoplasm contributes to the early phase of p27(Kip1) degradation in a Skp2-independent manner, thereby promoting cell cycle progression from Go to G,.

    DOI: 10.1074/jbc.M107274200

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  • Purification and characterization of a basic amino acid-specific peptidase from seeds of jack bean (Canavalia ensiformis) 査読

    K Oshikawa, K Aoki, Y Yoshino, S Terada

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   64 ( 10 )   2186 - 2192   2000年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:TAYLOR & FRANCIS LTD  

    A peptidase was purified from seeds of Canavalia ensiformis by extraction with water, ammonium sulfate precipitation, and successive chromatographies on DEAE-Toyopearl 650M, butyl-Toyopearl 650M, and G-3000 SW columns. The enzyme has an apparent molecular weight of 41,000. Activity is maximal at pH 9 and 60 degreesC. The enzyme hydrolyzed synthetic substrates at Arg-X and Lys-X bonds more rapidly than bovine trypsin did, and did not cleave protein or ester substrates. The enzyme was inhibited by alkylamines and several serine protease inhibitors such as diisopropylfluorophosphate, chymostatin, leupeptin, and benzamidine. Cysteine protease-, metalloprotease-, and proteinous trypsin inhibitors were ineffective. Inhibition by alkylamines was dependent on length of the alkyl chains. From the substrate specificity and susceptibility to chemicals, the enzyme is a unique peptidase with trypsin-like specificity.

    DOI: 10.1271/bbb.64.2186

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  • Primary structure and autoproteolysis of brevilysin H6 from the venom of Gloydius halys brevicaudus 査読

    S Fujimura, K Oshikawa, S Terada, E Kimoto

    JOURNAL OF BIOCHEMISTRY   128 ( 2 )   167 - 173   2000年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPANESE BIOCHEMICAL SOC  

    The complete amino acid sequence of brevilysin H6 (H6), a zinc-protease isolated from Gloydius halys brevicaudus venom, was determined by a manual Edman degradation method. HG has an amino-terminal pyroglutamic acid and consists of a total of 419 residues, An N-linked sugar chain is attached at Asn-181, The molecule is composed of three domains (metalloprotease, disintegrin-like and cysteine-rich domains), as commonly found in other high molecular mass: metalloproteases from snake venoms. In the absence of calcium ions, H6 is autocatalytically degraded with a half-life of 47 min to give 29 and 45 kDa fragments, which correspond to residues 208-419 and 99-419 of H6, respectively. Thus, the autoproteolysis seemed to start from the cleavage of either the Leu(98)-Leu(99) or Asp(207)-Ile(208) bond. Calcium ions suppressed both the formation of the 45 kDa fragment and the rate of autoproteolysis, Calcium ions also contributed to the stability of BG against pH, heating, urea and cysteine. More than twenty-five peptide bonds adjacent to hydrophobic residues in the metalloprotease domain were progressively cleaved during the autoproteolysis.

    DOI: 10.1093/oxfordjournals.jbchem.a022737

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  • Ussuristatin 2, a novel KGD-bearing disintegrin from Agkistrodon ussuriensis venom 査読

    K Oshikawa, S Terada

    JOURNAL OF BIOCHEMISTRY   125 ( 1 )   31 - 35   1999年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPANESE BIOCHEMICAL SOC  

    Two platelet aggregation inhibitors, ussuristatin 1 (US-l) and 2 (US-2), were newly isolated from the venom of Chinese viper (Agkistrodon ussuriensis) by SP-Toyopearl 650M column chromatography and reverse-phase HPLC. The Ms of these polypeptides were estimated to be about 8,000 by SDS-PAGE. Analytical gel filtration revealed that US-2 exists as a dimer, Both polypeptides comprised 71 amino acids, whose sequences showed high similarities to those of other disintegrins. US-1 had a typical Arg-Gly-Asp (RGD) sequence, which is responsible for blocking the binding of fibrinogen to the receptor. In US-2, the corresponding sequence was Lys-G;ly-Asp (KGD). US-1 strongly suppressed platelet aggregation induced by ADP, collagen, thrombin, and epinephrine with IC50=17-33 nM. US-2 also inhibited the platelet aggregation, but the IC(50)s were about ten times higher. US-1 also dose-dependently inhibited the adhesion of human melanoma cells to fibrinogen and fibronectin, while US-2 did not inhibit the cell adhesion to fibronectin. This indicates that the KGD-bearing disintegrin is a specific inhibitor for the fibrinogen receptor.

    DOI: 10.1093/oxfordjournals.jbchem.a022264

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MISC

共同研究・競争的資金等の研究

  • 次世代プロテオミクスを用いた細胞老化代謝ネットワークの全体像の解明

    研究課題/領域番号:19K06543

    2019年4月 - 2023年3月

    制度名:科学研究費助成事業

    研究種目:基盤研究(C)

    提供機関:日本学術振興会

    押川 清孝

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    配分額:4290000円 ( 直接経費:3300000円 、 間接経費:990000円 )

    老化に伴い細胞は特殊な代謝状態を取ることが知られているが、これまで老化代謝を対象にした解析はあまり行われていない。そこで本研究課題では、われわれが独自に開発した次世代プロテオミクス技術(iMPAQTシステム)を駆使して、老化過程で起こるタンパク質発現量変化の網羅的な解析を実施している。本研究の実施・遂行の結果として代謝経路の人為的操作により老化状態を制御することが可能となれば、多くの細胞老化と関わると推定されている種々の疾患の診断や治療あるいは予防に役立つだけでなく、逆に老化を促進することによってがん細胞の撲滅を図る戦略も可能になることが期待できる。
    前年度までに3種類の老化誘導モデル(複製老化、酸化ストレス誘導老化、がん遺伝子誘導老化)の全代謝酵素関連タンパク質の情報基盤多重モニタリング法と、転写産物量の計測を実施した。その結果、3種類の老化細胞に共通する代謝変動パターンと各種老化細胞特異的な代謝ネットワークのリモデリングを見出すことに成功している。
    令和三年度は、3種類の老化細胞で発現が減少していたDNA合成関連酵素群の過剰発現およびノックダウン実験を実施し、代謝酵素量の介入実験による細胞老化の制御が可能かを検証した。このうち、Ribonucleotide reductase subunits (RRM)遺伝子の発現を正常細胞で低下させたところ、細胞の増殖が停止した。このノックダウン細胞を詳細に解析したところ、老化細胞の表現型を示すことを見出した。RRM遺伝子の低下に伴い、p16, p21の発現量の上昇およびLamin B1の発現量の低下していた。また老化マーカーであるSA-β-Gal染色も陽性であった。以上から、DNA合成酵素の発現を低下させることで細胞老化が生じることを明らかにした。

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  • トランスオミクス解析によるがん悪性進展機構の解明

    2018年4月 - 2020年3月

    制度名:新学術領域研究(研究領域提案型)

    提供機関:科研費

    押川 清孝

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    担当区分:研究代表者  資金種別:競争的資金

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  • 次世代プロテオミクスを用いた低酸素代謝ネットワークの全体像の解明

    2016年4月 - 2017年3月

    制度名:挑戦的萌芽研究

    提供機関:科研費

    押川 清孝

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    担当区分:研究代表者  資金種別:競争的資金

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  • 情報基盤定量法によるがんのワールブルグ効果の分子機構解明

    2013年4月 - 2016年3月

    制度名:基盤研究(C)

    提供機関:科研費

    押川 清孝

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    担当区分:研究代表者  資金種別:競争的資金

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  • 大規模リン酸化プロテオーム解析による細胞周期制御機構の解明

    研究課題/領域番号:20058028

    2008年 - 2009年

    制度名:科学研究費助成事業

    研究種目:特定領域研究

    提供機関:日本学術振興会

    松本 雅記, 押川 清孝

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    配分額:6600000円 ( 直接経費:6600000円 )

    前年度までにリン酸化ペプチドの精製技術を確立した。21年度は細胞内リン酸化細胞周期の全過程の解析には少なくとも4点以上の定量が必要となるため、多検体間比較定量法を構築した。当初、一般的に質量分析計による定量法として安定同位体標識アミノ酸代謝標識法(SILAC)が主流であり、これを応用した多点定量法の構築を試みたが、使用できる培養細胞が限られるなどの欠点があるため、新規リン酸化定量プロテオミクス法であるphospho-iTRAQ法の構築を試みた。
    iTRAQ法は4種類,あるいは8種類の試料間の同時比較定量が可能であるため、細胞周期など時系列解析に適している。しかしながら、リン酸化の解析に適用する場合、試料の消化・リン酸ペプチドの精製後に標識を行うため、消化や精製過程での誤差が生じやすい。そこで、カゼインなどのリン酸化タンパク質を内部標準として各々の試料に少量添加することで、消化や精製の誤差を補正することを考案した。実際、実験過程で生じる誤差を内部標準由来のリン酸化ペプチドの定量値を用いて補正することが可能であった。本方法を用いて増殖因子刺激や細胞周期進行過程でリン酸化の変動を大規模に解析したところ、約10,000種類以上のリン酸化部位を同定・定量が可能であった。
    得られた新規細リン酸化の細胞周期制御上の意味を検証するため当該タンパク質の機能撹乱やリン酸化部位変異体の導入による細胞周期への影響の検討、さらに、細胞内局在や他のタンパク質との相互作用の細胞周期依存性等を横断的に解析している。

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  • プロテオミクスによる遺伝性自己免疫疾患の原因タンパク質の機能解析

    2006年4月 - 2008年3月

    制度名:若手研究(B)

    提供機関:科研費

    押川 清孝

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    担当区分:研究代表者  資金種別:競争的資金

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  • 単一遺伝子の異常によってもたらされる自己免疫疾患の病態解析

    研究課題/領域番号:17390291

    2005年 - 2006年

    制度名:科学研究費助成事業

    研究種目:基盤研究(B)

    提供機関:日本学術振興会

    松本 満, 黒田 範行, 押川 清孝

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    配分額:14400000円 ( 直接経費:14400000円 )

    AIRE欠損NODマウスは、既に樹立しているAIRE欠損マウスを10代以上にわたりNODマウスに戻し交配して樹立した。AIRE欠損NODマウスではNODマウスにおける本来の標的組織である膵ラ氏島の破壊を認めなくなり、かわって外分泌組織である膵腺房細胞の著明な破壊像を認めた。この病理学的な所見に一致して、AIRE欠損NODマウスは糖尿病の発症に対して抵抗性を獲得していた。AIREを欠損させたNODマウスにおける臓器特異性の変化は、AIREが単に自己免疫病態の発症を規定するのみならず、どの臓器を標的として免疫破壊を行うかという、いわゆる臓器特異性にも関与していることを明確に示した結果である。したがって、AIRE欠損にともなう臓器特異性の変化がどのような分子および細胞学的メカニズムによって起こるかを明らかにすることで、多彩な病状を示す自己免疫疾患の病態理解に貴重な情報が得られるものと思われる。
    他方、AIRE欠損NODマウスの血中に存在する自己抗体の対応抗原の一つを同定したところ、膵臓腺房細胞に発現するpancreas-specific protein disulfide isomerase (PDIp)であることが判明した。AIRE依存的自己免疫病態の発症機構として、AIREが胸腺上皮細胞内で種々の末梢自己抗原(peripheral tissue-restricted antigen)の発現を転写レベルで制御している可能性が示唆されている。しかしながら、AIRE欠損NODマウス胸腺におけるPDIpの発現は低下しておらず、このことからAIRE依存的自己免疫病態の発症にはAIREによるPDIp転写制御以外のメカニズムの存在が想定された。すなわち、AIREは胸腺上皮細胞内で種々の自己抗原の転写レベルを制御するのみならず、抗原提示過程を制御している可能性がある。

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  • G1-S期を制御するタンパク質分解システムの解明

    2002年4月 - 2004年3月

    制度名:特別研究員奨励費

    提供機関:科研費

    押川 清孝

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    担当区分:研究代表者  資金種別:競争的資金

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担当経験のある授業科目

  • 分子生物学

    2024年
    -
    現在
    機関名:新潟大学

  • 生体内物質と代謝

    2024年
    -
    現在
    機関名:新潟大学