Updated on 2025/06/06

写真a

 
JUGE Narinobu
 
Organization
Scheduled update Assistant Professor
Position
Assistant Professor
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Research Interests

  • Transporter

  • PfCRT

Research Areas

  • Life Science / Functional biochemistry  / Biochemistry

  • Life Science / Pharmaceutical hygiene and biochemistry  / Biochemistry

Research History

  • Japan Science and Technology Agency

    2014.10 - 2018.3

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  • 海外特別研究員

    2010.4 - 2012.3

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Professional Memberships

 

Papers

  • Neurotransmitter recognition by human vesicular monoamine transporter 2. International journal

    Dohyun Im, Mika Jormakka, Narinobu Juge, Jun-Ichi Kishikawa, Takayuki Kato, Yukihiko Sugita, Takeshi Noda, Tomoko Uemura, Yuki Shiimura, Takaaki Miyaji, Hidetsugu Asada, So Iwata

    Nature communications   15 ( 1 )   7661 - 7661   2024.9

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

    Human vesicular monoamine transporter 2 (VMAT2), a member of the SLC18 family, plays a crucial role in regulating neurotransmitters in the brain by facilitating their uptake and storage within vesicles, preparing them for exocytotic release. Because of its central role in neurotransmitter signalling and neuroprotection, VMAT2 is a target for neurodegenerative diseases and movement disorders, with its inhibitor being used as therapeutics. Despite the importance of VMAT2 in pharmacophysiology, the molecular basis of VMAT2-mediated neurotransmitter transport and its inhibition remains unclear. Here we show the cryo-electron microscopy structure of VMAT2 in the substrate-free state, in complex with the neurotransmitter dopamine, and in complex with the inhibitor tetrabenazine. In addition to these structural determinations, monoamine uptake assays, mutational studies, and pKa value predictions were performed to characterize the dynamic changes in VMAT2 structure. These results provide a structural basis for understanding VMAT2-mediated vesicular transport of neurotransmitters and a platform for modulation of current inhibitor design.

    DOI: 10.1038/s41467-024-51960-z

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  • Structures suggest a mechanism for energy coupling by a family of organic anion transporters. International journal

    Jonathan B Leano, Samir Batarni, Jacob Eriksen, Narinobu Juge, John E Pak, Tomomi Kimura-Someya, Yaneth Robles-Colmenares, Yoshinori Moriyama, Robert M Stroud, Robert H Edwards

    PLoS biology   17 ( 5 )   e3000260   2019.5

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

    Members of the solute carrier 17 (SLC17) family use divergent mechanisms to concentrate organic anions. Membrane potential drives uptake of the principal excitatory neurotransmitter glutamate into synaptic vesicles, whereas closely related proteins use proton cotransport to drive efflux from the lysosome. To delineate the divergent features of ionic coupling by the SLC17 family, we determined the structure of Escherichia coli D-galactonate/H+ symporter D-galactonate transporter (DgoT) in 2 states: one open to the cytoplasmic side and the other open to the periplasmic side with substrate bound. The structures suggest a mechanism that couples H+ flux to substrate recognition. A transition in the role of H+ from flux coupling to allostery may confer regulation by trafficking to and from the plasma membrane.

    DOI: 10.1371/journal.pbio.3000260

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  • Purification and reconstitution of polyspecific H+/organic cation antiporter human MATE1. International journal

    Tatsuya Kawasaki, Takuya Matsumoto, Yuma Iwai, Mamiyo Kawakami, Narinobu Juge, Hiroshi Omote, Tomohiro Nabekura, Yoshinori Moriyama

    Biochimica et biophysica acta. Biomembranes   1860 ( 11 )   2456 - 2464   2018.11

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

    Human MATE1 (multidrug and toxin extrusion 1, hMATE1) is a H+/organic cation (OC) exchanger responsible for the final step of toxic organic cation excretion in the kidney and liver. To investigate the mechanism of transport, we have established an in vitro assay procedure that includes its expression in insect cells, solubilization with octyl glucoside, purification, and reconstitution into liposomes. The resultant proteoliposomes containing hMATE1 as the sole protein component took up radiolabeled tetraethylammonium (TEA) in a ∆pH-dependent and electroneutral fashion. Furthermore, lipid-detergent micelle containing hMATE1 showed ∆pH-dependent TEA binding similar to transport. Mutated hMATE1 with replacement E273Q completely lacked these TEA binding and transport. In the case of divalent substrates, transport was electrogenic. These observations indicate that the stoichiometry of OC/H+ exchange is independent of substrate charge. Purification and reconstitution of hMATE1 is considered to be suitable for understanding the detailed molecular mechanisms of hMATE1. The results suggest that Glu273 of hMATE1 plays essential roles in substrate binding and transport.

    DOI: 10.1016/j.bbamem.2018.07.005

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  • Outward open conformation of a Major Facilitator Superfamily multidrug/H+ antiporter provides insights into switching mechanism. International journal

    Kumar Nagarathinam, Yoshiko Nakada-Nakura, Christoph Parthier, Tohru Terada, Narinobu Juge, Frank Jaenecke, Kehong Liu, Yunhon Hotta, Takaaki Miyaji, Hiroshi Omote, So Iwata, Norimichi Nomura, Milton T Stubbs, Mikio Tanabe

    Nature communications   9 ( 1 )   4005 - 4005   2018.10

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    Multidrug resistance (MDR) poses a major challenge to medicine. A principle cause of MDR is through active efflux by MDR transporters situated in the bacterial membrane. Here we present the crystal structure of the major facilitator superfamily (MFS) drug/H+ antiporter MdfA from Escherichia coli in an outward open conformation. Comparison with the inward facing (drug binding) state shows that, in addition to the expected change in relative orientations of the N- and C-terminal lobes of the antiporter, the conformation of TM5 is kinked and twisted. In vitro reconstitution experiments demonstrate the importance of selected residues for transport and molecular dynamics simulations are used to gain insights into antiporter switching. With the availability of structures of alternative conformational states, we anticipate that MdfA will serve as a model system for understanding drug efflux in MFS MDR antiporters.

    DOI: 10.1038/s41467-018-06306-x

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  • Efficient Mass Spectral Analysis of Active Transporters Overexpressed in Escherichia coli. International journal

    Mamiyo Kawakami, Narinobu Juge, Yuri Kato, Hiroshi Omote, Yoshinori Moriyama, Takaaki Miyaji

    Journal of proteome research   17 ( 3 )   1108 - 1119   2018.3

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

    Structural analysis of purified active membrane proteins can be performed by mass spectrometry (MS). However, no large-scale expression systems for active eukaryotic membrane proteins are available. Moreover, because membrane proteins cannot easily be digested by trypsin and ionized, they are difficult to analyze by MS. We developed a method for mass spectral analysis of eukaryotic membrane proteins combined with an overexpression system in Escherichia coli. Vesicular glutamate transporter 2 (VGLUT2/SLC17A6) with a soluble α-helical protein and histidine tag on the N- and C-terminus, respectively, was overexpressed in E. coli, solubilized with detergent, and purified by Ni-NTA affinity chromatography. Proteoliposomes containing VGLUT2 retained glutamate transport activity. For MS analysis, the detergent was removed from purified VGLUT2 by trichloroacetic acid precipitation, and VGLUT2 was then subjected to reductive alkylation and tryptic digestion. The resulting peptides were detected with 88% coverage by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS with or without liquid chromatography. Vesicular excitatory amino acid transporter and vesicular acetylcholine transporter were also detected with similar coverage by the same method. Thus this methodology could be used to analyze purified eukaryotic active transporters. Structural analysis with chemical modifiers by MS could have applications in functional binding analysis for drug discovery.

    DOI: 10.1021/acs.jproteome.7b00777

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MISC

  • Identification and characterization of a vesicular nucleotide transporter

    Keisuke Sawada, Miki Hiasa, Noriko Echigo, Narinobu Juge, Takaaki Miyaji, Hiroshi Omote, Yoshinori Moriyama

    NEUROSCIENCE RESEARCH   65   S77 - S77   2009

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    Language:English   Publishing type:Research paper, summary (international conference)  

    DOI: 10.1016/j.neures.2009.09.280

    Web of Science

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  • Characterization of transport properties of purified vesicular inhibitory amino acid transporter (VIAAT)

    Narinobu Juge, Akiko Muroyama, Miki Hiasa, Hiroshi Omote, Yoshinori Moriyama

    NEUROSCIENCE RESEARCH   65   S77 - S77   2009

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    Language:English   Publishing type:Research paper, summary (international conference)  

    DOI: 10.1016/j.neures.2009.09.281

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  • Identification and characterization of a vesicular nucleotide transporter

    Keisuke Sawada, Noriko Echigo, Narinobu Juge, Takaaki Miyaji, Miki Hiasa, Masato Otsuka, Hiroshi Omote, Yoshinori Moriyama

    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN   128   65 - 66   2008

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    Language:English   Publishing type:Research paper, summary (international conference)  

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  • L-glutamate signaling regulates transcytotic vesicle function in osteoclasts.

    S. Uehara, R. Morimoto, S. Yatsushiro, N. Juge, M. Hayashi, S. Senoh, T. Mizoguchi, T. Ninomiya, N. Udagawa, Z. Hua, H. Omote, A. Yamamoto, R. H. Edwards, Y. Moriyama

    JOURNAL OF BONE AND MINERAL RESEARCH   21   S401 - S401   2006.9

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    Language:English   Publishing type:Research paper, summary (international conference)  

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Presentations

  • トランスポーターの生化学的・構造生物学的展開 Invited

    樹下成信

    GEヘルスケア・ジャパン「初めてのライフサイエンス基礎講座」  2015.5.15 

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    Language:Japanese   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

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Research Projects

  • Neuronal signal transmission under high fat diet condition..

    Grant number:24K09796  2024.04 - 2027.03

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

    樹下 成信

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

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  • MFS型多剤排出輸送体の多剤認識と輸送機構の解明

    Grant number:19H03186  2019.04 - 2023.03

    日本学術振興会  科学研究費助成事業  基盤研究(B)

    田辺 幹雄, 野村 紀通, 樹下 成信

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

    細菌が薬剤耐性を持つ原因の一つに多剤排出トランスポーターによる投与薬剤の排出が挙げられる。本課題ではグラム陰性菌の細胞質からペリプラズムに薬剤を排出するMajor facilitator superfamily(MFS)型の多剤排出トランスポーターの薬剤認識とその輸送機構の解明を目指しており、その目標達成のために特に2つの研究テーマを掲げている。
    1)MFS型-多剤排出トランスポーターのモデルタンパク質であるMdfAを使い生化学、構造解析、シミュレーションを用いて、薬剤分子の認識やその排出の輸送サイクルを明らかにする。
    2) 新規の病原菌由来の輸送体の構造解析を行い、薬剤輸送機構の解明を目指す。
    本年度の研究実績として、
    1)については、MdfAを認識する4つの抗体を用いてそれらがどのような構造状態を認識しているかをクライオ電顕を用いて確認することを目指した。前年度は分解能が10オングストローム程度で留まっていたデータについて、様々な条件で精製し直し、グリッドを作成したものの、分解能は10オングストロームから僅かに更新されたのみであった。MDシミュレーションを中心とした解析では現在も進行中であり、内開きから中間体を経て外開きの構造変化を順次追っている最中であるがその検証には時間を有する。MdfAと抗体のクロスリンク実験は進行中で、より安定な複合体の取得に取り組んでいる。
    2)前年度にタンパク精製に成功したS.aureusのトランスポーターにおいては阻害剤存在下での精製に成功し、結晶を得ることができた。回折能は10オングストローム程度であったため、こちらも阻害剤の存在下でのタンパク質の構造を認識する抗体の取得について実験が進んでいる。

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  • 小胞型神経伝達物質トランスポーターの脂質制御機構の解明

    2018.04 - 2022.03

    KAKEN  JSPS 

    Narinobu Juge

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    Authorship:Principal investigator  Grant type:Competitive

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  • 小胞容量測定系の確立

    2016.04 - 2018.03

    KKEN  JSPS 

    Narinobu Juge

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    Authorship:Principal investigator  Grant type:Competitive

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  • グルタミン酸のシナプス小胞充填機構の構造生物学的展開

    2014.10 - 2018.03

    JST  PREST 

    Narinobu Juge

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    Authorship:Principal investigator  Grant type:Competitive

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Class subject in charge

  • Life science for drug discovery (2024academic year) Third semester  - 木5~6

  • Drug/biomembrane interactions I (2024academic year) special  - その他

  • Drug/biomembrane interactions II (2024academic year) special  - その他

  • Experimental Molecular Biology (2024academic year) Second semester  - その他5~9

  • Experimental Molecular Biology (2024academic year) Second semester  - その他5~9

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