Updated on 2024/03/29

写真a

 
ITO Kosuke
 
Organization
Academic Assembly Institute of Science and Technology CHIKYU SEIBUTSU KAGAKU KEIRETU Associate Professor
Faculty of Science Associate Professor
Graduate School of Science and Technology Life and Food Sciences Associate Professor
College of Creative Studies Creative Studies Course Associate Professor
Title
Associate Professor
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Degree

  • 博士(農学) ( 2003.3   東京大学 )

  • 修士(農学) ( 1999.3   東京大学 )

  • 学士(理学) ( 1997.3   学習院大学 )

Research Interests

  • 翻訳

  • リボソーム

  • 遺伝子発現

  • DNA

  • X線結晶構造解析

  • タンパク質

  • RNA

Research Areas

  • Life Science / Structural biochemistry

  • Life Science / Molecular biology

Research History (researchmap)

  • Niigata University   Faculty of Science/Graduate School of Science and Technology Life and Food Sciences   Associate Professor

    2019.4

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  • Niigata University   Faculty of Science/Graduate School of Science and Technology Life and Food Sciences   Assistant Professor

    2009.4 - 2019.3

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  • Niigata University   Center for Transdisciplinary Research   Assistant Professor

    2008.1 - 2009.3

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  • The University of Tokyo   Graduate School of Frontier Sciences

    2007.4 - 2007.12

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  • The University of Tokyo   Graduate School of Agricultural and Life Sciences

    2003.4 - 2007.3

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Research History

  • Niigata University   Creative Studies Course, College of Creative Studies   Associate Professor

    2021.4

  • Niigata University   Faculty of Science   Associate Professor

    2019.4

  • Niigata University   Graduate School of Science and Technology Life and Food Sciences   Associate Professor

    2019.4

  • Niigata University   Faculty of Science   Assistant Professor

    2009.4 - 2019.3

  • Niigata University   Graduate School of Science and Technology Life and Food Sciences   Assistant Professor

    2009.4 - 2019.3

  • Niigata University   Center for Transdisciplinary Research   Assistant Professor

    2008.1 - 2009.3

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Education

  • The University of Tokyo   Graduate School of Agricultural and Life Sciences   Applied Biological Chemistry

    1999.4 - 2003.3

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  • The University of Tokyo   Graduate School of Agricultural and Life Sciences   Biotechnology

    1997.4 - 1999.3

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  • Gakushuin University   Faculty of Science   Department of Chemistry

    1993.4 - 1997.3

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Professional Memberships

 

Papers

  • The flexible N-terminal motif of uL11 unique to eukaryotic ribosomes interacts with P-complex and facilitates protein translation. Reviewed International journal

    Lei Yang, Ka-Ming Lee, Conny Wing-Heng Yu, Hirotatsu Imai, Andrew Kwok-Ho Choi, David K Banfield, Kosuke Ito, Toshio Uchiumi, Kam-Bo Wong

    Nucleic acids research   50 ( 9 )   5335 - 5348   2022.5

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    Eukaryotic uL11 contains a conserved MPPKFDP motif at the N-terminus that is not found in archaeal and bacterial homologs. Here, we determined the solution structure of human uL11 by NMR spectroscopy and characterized its backbone dynamics by 15N-1H relaxation experiments. We showed that these N-terminal residues are unstructured and flexible. Structural comparison with ribosome-bound uL11 suggests that the linker region between the N-terminal domain and C-terminal domain of human uL11 is intrinsically disordered and only becomes structured when bound to the ribosomes. Mutagenesis studies show that the N-terminal conserved MPPKFDP motif is involved in interacting with the P-complex and its extended protuberant domain of uL10 in vitro. Truncation of the MPPKFDP motif also reduced the poly-phenylalanine synthesis in both hybrid ribosome and yeast mutagenesis studies. In addition, G→A/P substitutions to the conserved GPLG motif of helix-1 reduced poly-phenylalanine synthesis to 9-32% in yeast ribosomes. We propose that the flexible N-terminal residues of uL11, which could extend up to ∼25 Å from the N-terminal domain of uL11, can form transient interactions with the uL10 that help to fetch and fix it into a position ready for recruiting the incoming translation factors and facilitate protein synthesis.

    DOI: 10.1093/nar/gkac292

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  • A novel ribosome-dimerization protein found in the hyperthermophilic archaeon Pyrococcus furiosus using ribosome-associated proteomics. Reviewed International journal

    Chiaki Yaeshima, Natsumi Murata, Sonoko Ishino, Ikuko Sagawa, Kosuke Ito, Toshio Uchiumi

    Biochemical and biophysical research communications   593   116 - 121   2022.2

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    Ribosome dimerization is one of the bacterial events that suppresses protein synthesis in the stationary phase. Protein factors responsible for ribosome dimerization in bacteria are well characterized, whereas no information is available for the corresponding factors in archaeal and eukaryotic cells. Here we describe a protein found among the ribosome-associated proteins which dimerizes the 30S ribosomal subunit of the archaeon Pyrococcus furiosus. The ribosome-associated proteins were prepared by high-salt wash of crude ribosomes, and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). Of the detected proteins we focused on a protein (PF0560) whose Protein Score was the highest of all of the function-unknown proteins. PF0560 protein had a pronounced effect on the sedimentation pattern of the 30S ribosomal subunit; addition of this protein to isolated 30S subunit reduced the 30S fraction and increased the amount of the 50S fraction. This increase presumably corresponds to the dimer of the 30S subunit. The PF0560-dependent 30S-dimerization, was also observed by gel electrophoretic analysis. This effect was not observed in EDTA-treated 30S subunit, with protein-free 16S rRNA or with bacterial/eukaryotic ribosomal small subunits. Furthermore, PF0560 protein suppressed the formation of functional 70S ribosomes. These results suggest that PF0560 is a novel 30S dimerization factor, which might participate in regulation of archaeal translation.

    DOI: 10.1016/j.bbrc.2022.01.043

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  • Structural insights into the Switching Off of the Interaction between the Archaeal Ribosomal Stalk and aEF1A by Nucleotide Exchange Factor aEF1B. Reviewed International journal

    Suzuki T, Ito K, Miyoshi T, Murakami R, Uchiumi T

    Journal of molecular biology   433 ( 15 )   167046 - 167046   2021.7

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    The ribosomal stalk protein plays a crucial role in functional interactions with translational GTPase factors. It has been shown that the archaeal stalk aP1 binds to both GDP- and GTP-bound conformations of aEF1A through its C-terminal region in two different modes. To obtain an insight into how the aP1•aEF1A binding mode changes during the process of nucleotide exchange from GDP to GTP on aEF1A, we have analyzed structural changes in aEF1A upon binding of the nucleotide exchange factor aEF1B. The isolated archaeal aEF1B has nucleotide exchange ability in the presence of aa-tRNA but not deacylated tRNA, and increases activity of polyphenylalanine synthesis 4-fold. The aEF1B mutation, R90A, results in loss of its original nucleotide exchange activity but retains a remarkable ability to enhance polyphenylalanine synthesis. These results suggest an additional functional role for aEF1B other than in nucleotide exchange. The crystal structure of the aEF1A•aEF1B complex, resolved at 2.0 Å resolution, shows marked rotational movement of domain 1 of aEF1A compared to the structure of aEF1A•GDP•aP1, and this conformational change results in disruption of the original aP1 binding site between domains 1 and 3 of aEF1A. The loss of aP1 binding to the aEF1A•aEF1B complex was confirmed by native gel analysis. The results suggest that aEF1B plays a role in switching off the interaction between aP1 and aEF1A•GDP, as well as in nucleotide exchange, and promote translation elongation.

    DOI: 10.1016/j.jmb.2021.167046

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  • Crystal structure of rice defensin OsAFP1 and molecular insight into lipid-binding. Reviewed

    Ochiai A, Ogawa K, Fukuda M, Suzuki M, Ito K, Tanaka T, Sagehashi Y, Taniguchi M

    Journal of bioscience and bioengineering   130 ( 1 )   6 - 13   2020.7

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    Defensins are antibacterial peptides that function in the innate immune system. OsAFP1, a defensin identified from Oryza sativa (rice), exhibits antimicrobial activity against rice pathogens. Intriguingly, OsAFP1 was also shown to demonstrate potent antifungal activity against the human pathogenic fungus Candida albicans by inducing apoptosis in target cells, suggesting that OsAFP1 represents a potential new antibiotic candidate; however, further analyses, particularly at the structural level, are required to elucidate the mechanistic underpinnings of OsAFP1 antifungal activity. Here, we determined the three-dimensional structure of OsAFP1 using X-ray crystallography. OsAFP1 features the cysteine-stabilized αβ structure highly conserved in plant defensins and presents a dimeric structure that appears necessary for antifungal activity. Superimposition of the OsAFP1 structure with that of Nicotiana alata NaD1 complexed with phosphatidic acid indicated that the target molecule is likely trapped between the S2-S3 loops of each OsAFP1 dimer. In lipid-binding analyses performed using nitrocellulose membranes immobilized with various membrane lipid components, OsAFP1 was found to bind to phosphatidylinositols (PIPs) harboring phosphate groups, particularly PI(3)P. These results indicate that OsAFP1 exerts antifungal activity by binding to PI(3)P contained in the C. albicans cell membrane, thereby applying cellular stress and inducing apoptosis. Furthermore, the OsAFP1 structure and site-specific-mutation analyses revealed that Arg1, His2, Leu4, Arg9, and Phe10 play critical roles in OsAFP1 dimer formation. Thus, our study provides novel insights into the antifungal mechanism of OsAFP1.

    DOI: 10.1016/j.jbiosc.2020.02.011

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  • Arabidopsis ROOT PHOTOTROPISM2 Is a Light-Dependent Dynamic Modulator of Phototropin1. Reviewed International journal

    Kimura T, Tsuchida-Mayama T, Imai H, Okajima K, Ito K, Sakai T

    The Plant Cell   10.1101/862649   2020.3

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    The Arabidopsis thaliana blue-light photoreceptor phototropin1 (phot1) is a blue light-activated Ser/Thr protein kinase that mediates various light responses including phototropism. The function of phot1 in hypocotyl phototropism is dependent on the light induction of ROOT PHOTOTROPISM2 (RPT2) proteins within a broad range of blue light intensities. It is not yet known however how RPT2 contributes to the photosensory adaptation of phot1 to high intensity blue light and the phototropic responses under bright light conditions. We show that RPT2 suppresses the activity of phot1 and demonstrate that RPT2 binds to the PHOT1 LOV1 (light, oxygen or voltage sensing 1) domain which is required for its high photosensitivity. Our biochemical analyses revealed that RPT2 inhibits autophosphorylation of phot1, suggesting that it suppresses the photosensitivity and/or kinase activity of phot1 through the inhibition of LOV1 function. We found that RPT2 proteins are degraded via a ubiquitin-proteasome pathway when phot1 is inactive and are stabilized under blue light in a phot1-dependent manner. We propose that RPT2 is a molecular rheostat that maintains a moderate activation level of phot1 under any light intensity conditions.

    DOI: 10.1105/tpc.19.00926

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  • Switch of the interactions between the ribosomal stalk and EF1A in the GTP- and GDP-bound conformations. Reviewed

    Maruyama K, Imai H, Kawamura M, Ishino S, Ishino Y, Ito K, Uchiumi T

    Scientific reports   9 ( 1 )   14761   2019.10

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    DOI: 10.1038/s41598-019-51266-x

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  • Structural and Mutagenesis Studies Evince the Role of the Extended Protuberant Domain of Ribosomal Protein uL10 in Protein Translation. Reviewed

    Choi KA, Yang L, Lee KM, Yu CW, Banfield DK, Ito K, Uchiumi T, Wong KB

    Biochemistry   58 ( 36 )   3744 - 3754   2019.9

  • High-resolution crystal structure of peptidyl-tRNA hydrolase from Thermus thermophilus. Reviewed International journal

    Matsumoto A, Uehara Y, Shimizu Y, Ueda T, Uchiumi T, Ito K

    Proteins   87 ( 3 )   226 - 235   2019.3

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    Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are the products of defective translation, to recycle the tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its activity is essential for bacterial viability. Here, we have determined the crystal structure of Pth from Thermus thermophilus (TtPth) at 1.00 Å resolution. This is the first structure of a Pth from a thermophilic bacterium and the highest resolution Pth structure reported so far. The present atomic resolution data enabled the calculation of anisotropic displacement parameters for all atoms, which revealed the directionality of the fluctuations of key regions for the substrate recognition. Comparisons between TtPth and mesophilic bacterial Pths revealed that their structures are similar overall. However, the structures of the N- and C-terminal, loop-helix α4, and helix α6 regions are different. In addition, the helix α1 to strand β4 region of TtPth is remarkably different from those of the mesophilic bacterial Pths, because this region is 9 or 10 amino acid residues shorter than those of the mesophilic bacterial Pths. This shortening seems to contribute to the thermostability of TtPth. To further understand the determinants for the thermostability of TtPth, we compared various structural factors of TtPth with those of mesophilic bacterial Pths. The data suggest that the decreases in accessible surface area and thermolabile amino acid residues, and the increases in ion pairs, hydrogen bonds, and proline residues cooperatively contribute to the thermostability of TtPth.

    DOI: 10.1002/prot.25643

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  • The ribosomal stalk protein is crucial for the action of the conserved ATPase ABCE1. Reviewed International journal

    Imai H, Abe T, Miyoshi T, Nishikawa SI, Ito K, Uchiumi T

    Nucleic acids research   46 ( 15 )   7820 - 7830   2018.9

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    The ATP-binding cassette (ABC) protein ABCE1 is an essential factor in ribosome recycling during translation. However, the detailed mechanochemistry of its recruitment to the ribosome, ATPase activation and subunit dissociation remain to be elucidated. Here, we show that the ribosomal stalk protein, which is known to participate in the actions of translational GTPase factors, plays an important role in these events. Biochemical and crystal structural data indicate that the conserved hydrophobic amino acid residues at the C-terminus of the archaeal stalk protein aP1 binds to the nucleotide-binding domain 1 (NBD1) of aABCE1, and that this binding is crucial for ATPase activation of aABCE1 on the ribosome. The functional role of the stalk•ABCE1 interaction in ATPase activation and the subunit dissociation is also investigated using mutagenesis in a yeast system. The data demonstrate that the ribosomal stalk protein likely participates in efficient actions of both archaeal and eukaryotic ABCE1 in ribosome recycling. The results also show that the stalk protein has a role in the function of ATPase as well as GTPase factors in translation.

    DOI: 10.1093/nar/gky619

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  • The Interaction between the Ribosomal Stalk Proteins and Translation Initiation Factor 5B Promotes Translation Initiation. Reviewed International journal

    Murakami R, Singh CR, Morris J, Tang L, Harmon I, Takasu A, Miyoshi T, Ito K, Asano K, Uchiumi T, ibute, Corresponding authors

    Molecular and cellular biology   38 ( 16 )   e00067-18   2018.8

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    Ribosomal stalk proteins recruit translation elongation GTPases to the factor-binding center of the ribosome. Initiation factor 5B (eIF5B in eukaryotes and aIF5B in archaea) is a universally conserved GTPase that promotes the joining of the large and small ribosomal subunits during translation initiation. Here we show that aIF5B binds to the C-terminal tail of the stalk protein. In the cocrystal structure, the interaction occurs between the hydrophobic amino acids of the stalk C-terminal tail and a small hydrophobic pocket on the surface of the GTP-binding domain (domain I) of aIF5B. A substitution mutation altering the hydrophobic pocket of yeast eIF5B resulted in a marked reduction in ribosome-dependent eIF5B GTPase activity in vitro In yeast cells, the eIF5B mutation affected growth and impaired GCN4 expression during amino acid starvation via a defect in start site selection for the first upstream open reading frame in GCN4 mRNA, as observed with the eIF5B deletion mutant. The deletion of two of the four stalk proteins diminished polyribosome levels (indicating defective translation initiation) and starvation-induced GCN4 expression, both of which were suppressible by eIF5B overexpression. Thus, the mutual interaction between a/eIF5B and the ribosomal stalk plays an important role in subunit joining during translation initiation in vivo.

    DOI: 10.1128/MCB.00067-18

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  • Binding of translation elongation factors to individual copies of the archaeal ribosomal stalk protein aP1 assembled onto aP0 Reviewed

    Takayoshi Honda, Hirotatsu Imai, Takahiro Suzuki, Tomohiro Miyoshi, Kosuke Ito, Toshio Uchiumi

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   483 ( 1 )   153 - 158   2017.1

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    Ribosomes in all organisms contain oligomeric and flexible proteins called stalks, which are responsible for the recruitment of translational GTPase factors to the ribosome. Archaeal ribosomes have three stalk homodimers (aP1)(2) that constitute a heptameric complex with the anchor protein aP0. We investigated the factor binding ability of aP1 proteins assembled onto aP0, by gel-retardation assays. The isolated aP0(aP1)(2)(aP1)(2)(aP1)(2) complex, as well as the form bound to the Escherichia coli 50S core, as a hybrid 50S particle, interacted strongly with elongation factor aEF2, but weakly with aEF1A. These interactions were disrupted by a point mutation, F107S, at the C-terminus of aP1. To examine the ability of each copy of aP0-associated aP1 to bind to elongation factors, we constructed aP0.aP1 variant trimers, composed of an aP0 mutant and a single (aP1) 2 dimer. Biochemical and quantitative analyses revealed that the resultant three trimers, aP0(aP1)(2)(I), aP0(aP1)(2)(II), and aP0(aP1)(2)(III), individually bound two molecules of aEF2, suggesting that each copy of the aP1 C-terminal region in the aP0.aP1 trimers can bind tightly to aEF2. Interestingly, the unstable binding of aEF1A to each of the three aP0.aP1 trimers was remarkably stabilized in the presence of aEF2. The stability of the aEF1A binding to the stalk complex may be affected by the presence of aEF2 bound to the complex, by an unknown mechanism. (C) 2016 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2016.12.175

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  • Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute Reviewed

    *Miyoshi T, *Ito K, Murakami R, Uchiumi T

    NATURE COMMUNICATIONS   7   11846   2016.6

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    Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 angstrom crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson-Crick base-pairing even in the 30-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 50 base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region.

    DOI: 10.1038/ncomms11846

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  • Crystal structure of translation initiation factor 5B from the crenarchaeon Aeropyrum pernix Reviewed

    Ryo Murakami, Tomohiro Miyoshi, Toshio Uchiumi, Kosuke Ito

    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS   84 ( 5 )   712 - 717   2016.5

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    Initiation factor 5B (IF5B) is a universally conserved translational GTPase that catalyzes ribosomal subunit joining. In eukaryotes, IF5B directly interacts via a groove in its domain IV with initiation factor 1A (IF1A), another universally conserved initiation factor, to accomplish efficient subunit joining. Here, we have determined the first structure of a crenarchaeal IF5B, which revealed that the archaea-specific region of IF5B (helix 15) binds and occludes the groove of domain IV. Therefore, archaeal IF5B cannot access IF1A in the same manner as eukaryotic IF5B. This fact suggests that different relationships between IF5B and IF1A exist in archaea and eukaryotes. Proteins 2016; 84:712-717. (c) 2016 Wiley Periodicals, Inc.

    DOI: 10.1002/prot.25009

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  • Functional role of the C-terminal tail of the archaeal ribosomal stalk in recruitment of two elongation factors to the sarcin/ricin loop of 23S rRNA Reviewed

    Imai H, Miyoshi T, Murakami R, Ito K, Ishino Y, Uchiumi T

    GENES TO CELLS   20 ( 7 )   613 - 624   2015.7

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    Two types of elongation factors alternate in their binding to the factor-binding center of the ribosome. Both binding events are accompanied by GTP hydrolysis and drive the translation elongation cycle. The multicopy ribosomal protein family, termed the stalk, contributes actively to the elongation process. Recent evidence indicates that the mobile C-terminal tail of archaeal stalk aP1 directly interacts with both the elongation factors aEF1A and aEF2. To investigate the functional significance of these interactions in recruitment of elongation factors to the factor-binding center of the ribosome, we substituted the archaeal stalk complex aL10 center dot aP1 for the bL10 center dot bL12 stalk complex in the Escherichia coli 50S subunit. The resultant hybrid ribosome accessed archaeal aEF1A and aEF2 in a manner dependent on the C-terminal tail containing the hydrophobic residues Leu103, Leu106 and Phe107. Bases G2659 and A2660 in the sarcin/ricin loop (SRL) of 23S rRNA were protected against DMS modification by both factors as was A1067 by aEF2. Mutagenesis indicated that this protection was dependent on the intact C-terminal tail of aP1. The results suggest a crucial role for the interactions between the stalk C-terminal tail and elongation factors in their recruitment to the SRL of 23S rRNA within the ribosome.

    DOI: 10.1111/gtc.12256

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  • Molecular insights into the interaction of the ribosomal stalk protein with elongation factor 1 alpha Reviewed

    Kosuke Ito, Takayoshi Honda, Takahiro Suzuki, Tomohiro Miyoshi, Ryo Murakami, Min Yao, Toshio Uchiumi

    NUCLEIC ACIDS RESEARCH   42 ( 22 )   14042 - 14052   2014.12

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    In all organisms, the large ribosomal subunit contains multiple copies of a flexible protein, the so-called 'stalk'. The C-terminal domain (CTD) of the stalk interacts directly with the translational GTPase factors, and this interaction is required for factor-dependent activity on the ribosome. Here we have determined the structure of a complex of the CTD of the archaeal stalk protein aP1 and the GDP-bound archaeal elongation factor aEF1 alpha at 2.3 angstrom resolution. The structure showed that the CTD of aP1 formed a long extended alpha-helix, which bound to a cleft between domains 1 and 3 of aEF1 alpha, and bridged these domains. This binding between the CTD of aP1 and the aEF1 alpha center dot GDP complex was formed mainly by hydrophobic interactions. The docking analysis showed that the CTD of aP1 can bind to aEF1 alpha center dot GDP located on the ribosome. An additional biochemical assay demonstrated that the CTD of aP1 also bound to the aEF1 alpha center dot GTP center dot aminoacyl-tRNA complex. These results suggest that the CTD of aP1 interacts with aEF1 alpha at various stages in translation. Furthermore, phylogenetic perspectives and functional analyses suggested that the eukaryotic stalk protein also interacts directly with domains 1 and 3 of eEF1 alpha, in a manner similar to the interaction of archaeal aP1 with aEF1 alpha.

    DOI: 10.1093/nar/gku1248

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  • Crystal structure of alpha-amylase from Oryza sativa: molecular insights into enzyme activity and thermostability Reviewed

    Akihito Ochiai, Hiroshi Sugai, Kazuki Harada, Seiya Tanaka, Yohei Ishiyama, Kosuke Ito, Takaaki Tanaka, Toshio Uchiumi, Masayuki Taniguchi, Toshiaki Mitsui

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   78 ( 6 )   989 - 997   2014.6

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    AmyI-1 is an alpha-amylase from Oryza sativa (rice) and plays a crucial role in degrading starch in various tissues and at various growth stages. This enzyme is a glycoprotein with an N-glycosylated carbohydrate chain, a unique characteristic among plant alpha-amylases. In this study, we report the first crystal structure of AmyI-1 at 2.2-angstrom resolution. The structure consists of a typical (beta/alpha)(8)-barrel, which is well-conserved among most alpha-amylases in the glycoside hydrolase family-13. Structural superimposition indicated small variations in the catalytic domain and carbohydrate-binding sites between AmyI-1 and barley alpha-amylases. By contrast, regions around the N-linked glycosylation sites displayed lower conservation of amino acid residues, including Asn-263, Asn-265, Thr-307, Asn-342, Pro-373, and Ala-374 in AmyI-1, which are not conserved in barley alpha-amylases, suggesting that these residues may contribute to the construction of the structure of glycosylated AmyI-1. These results increase the depths of our understanding of the biological functions of AmyI-1.

    DOI: 10.1080/09168451.2014.917261

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  • Solution structure of human P1 center dot P2 heterodimer provides insights into the role of eukaryotic stalk in recruiting the ribosome-inactivating protein trichosanthin to the ribosome Reviewed

    Ka-Ming Lee, Kazuyuki Yusa, Lai-On Chu, Conny Wing-Heng Yu, Moe Oono, Tomohiro Miyoshi, Kosuke Ito, Pang-Chui Shaw, Kam-Bo Wong, Toshio Uchiumi

    NUCLEIC ACIDS RESEARCH   41 ( 18 )   8776 - 8787   2013.10

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    Lateral ribosomal stalk is responsible for binding and recruiting translation factors during protein synthesis. The eukaryotic stalk consists of one P0 protein with two copies of P1 center dot P2 heterodimers to form a P0(P1 center dot P2)(2) pentameric P-complex. Here, we have solved the structure of full-length P1 center dot P2 by nuclear magnetic resonance spectroscopy. P1 and P2 dimerize via their helical N-terminal domains, whereas the C-terminal tails of P1 center dot P2 are unstructured and can extend up to similar to 125 A away from the dimerization domains. N-15 relaxation study reveals that the C-terminal tails are flexible, having a much faster internal mobility than the N-terminal domains. Replacement of prokaryotic L10(L7/L12)(4)/L11 by eukaryotic P0(P1 center dot P2)(2)/eL12 rendered Escherichia coli ribosome, which is insensitive to trichosanthin (TCS), susceptible to depurination by TCS and the C-terminal tail was found to be responsible for this depurination. Truncation and insertion studies showed that depurination of hybrid ribosome is dependent on the length of the proline-alanine rich hinge region within the C-terminal tail. All together, we propose a model that recruitment of TCS to the sarcin-ricin loop required the flexible C-terminal tail, and the proline-alanine rich hinge region lengthens this C-terminal tail, allowing the tail to sweep around the ribosome to recruit TCS.

    DOI: 10.1093/nar/gkt636

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  • Crystallization and preliminary X-ray analysis of peptidyl-tRNA hydrolase from Thermus thermophilus HB8 Reviewed

    Matsumoto A, Shimizu Y, Takemoto C, Ueda T, Uchiumi T, *Ito K (*責任著者)

    Acta Crystallographica Section F: Structural Biology and Crystallization Communications   69 ( 3 )   332 - 335   2013.3

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    Peptidyl-tRNA is produced from the ribosome as a result of aborted translation. Peptidyl-tRNA hydrolase cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, to recycle tRNA for further rounds of protein synthesis. In this study, peptidyl-tRNA hydrolase from Thermus thermophilus HB8 (TthPth) was crystallized using 2-methyl-2,4-pentanediol as a precipitant. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 47.45, b = 53.92, c = 58.67Å, and diffracted X-rays to atomic resolution (beyond 1.0Å resolution). The asymmetric unit is expected to contain one TthPth molecule, with a solvent content of 27.13% (V M = 1.69Å3Da-1). The structure is being solved by molecular replacement. © 2013 International Union of Crystallography All rights reserved.

    DOI: 10.1107/S1744309113003424

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  • Structural basis for the substrate recognition and catalysis of peptidyl-tRNA hydrolase Reviewed

    Kosuke Ito, Ryo Murakami, Masahiro Mochizuki, Hao Qi, Yoshihiro Shimizu, Kin-ichiro Miura, Takuya Ueda, Toshio Uchiumi

    NUCLEIC ACIDS RESEARCH   40 ( 20 )   10521 - 10531   2012.11

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    Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are produced by aborted translation, to recycle tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its enzymatic activity is essential for bacterial viability. We have determined the crystal structure of Escherichia coli Pth in complex with the tRNA CCA-acceptor-T Psi C domain, the enzyme-binding region of the tRNA moiety of the substrate, at 2.4 A resolution. In combination with site-directed mutagenesis studies, the structure identified the amino acid residues involved in tRNA recognition. The structure also revealed that Pth interacts with the tRNA moiety through the backbone phosphates and riboses, and no base-specific interactions were observed, except for the interaction with the highly conserved base G53. This feature enables Pth to accept the diverse sequences of the elongator-tRNAs as substrate components. Furthermore, we propose an authentic Pth:peptidyl-tRNA complex model and a detailed mechanism for the hydrolysis reaction, based on the present crystal structure and the previous studies' results.

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  • Analysis of chimeric ribosomal stalk complexes from eukaryotic and bacterial sources: structural features responsible for specificity of translation factors Reviewed

    Masahiro Mochizuki, Masumi Kitamyo, Tomohiro Miyoshi, Kosuke Ito, Toshio Uchiumi

    GENES TO CELLS   17 ( 4 )   273 - 284   2012.4

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    Ribosomal protein P0 forms a pentameric complex with two heterodimers of the flexible stalk proteins P1 center dot P2 and plays a role in the functional interaction of eukaryotic ribosomes with translational factors. To investigate the functionality of domains of P0 characteristic to eukaryotes, we constructed various chimeras between silkworm P0 and Escherichia coli counterpart L10. Replacement of the C-terminal region of L10 with that of P0 allowed the binding of two P1 center dot P2 heterodimers, which supported ribosomal activity dependent on eukaryotic elongation factors eEF-2/eEF-1a, but not activity dependent on bacterial factors EF-G/EF-Tu. Conversely, replacement of the C-terminal region of P0 with that of L10 allowed binding of two bacterial L12 homodimers, which resulted in a low level of activity dependent on bacterial factors. Insertion of the extended region of P0 that is absent in the bacterial counterpart into L10 did not affect L12 binding or bacterial factor-dependent activity, but deletion of this region from P0 resulted in a 40% reduction in eukaryotic factor-dependent activity. The results indicate that the C-terminal regions of P0 and L10 are responsible for binding of the cognate stalk dimers and ribosomal specificity for translation factors and suggest that the extended region participates in accessibility only for eukaryotic factors.

    DOI: 10.1111/j.1365-2443.2012.01586.x

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  • Crystallization and preliminary X-ray analysis of peptidyl-tRNA hydrolase from Escherichia coli in complex with the acceptor-T?C domain of tRNA Reviewed

    Kosuke Ito, Hao Qi, Yoshihiro Shimizu, Ryo Murakami, Kin-ichiro Miura, Takuya Ueda, Toshio Uchiumi

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS   67 ( Pt 12 )   1566 - 1569   2011.12

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    Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are the product of aborted translation. In the present work, Pth from Escherichia coli was crystallized with the acceptor-TC domain of tRNA using 1,4-butanediol as a precipitant. The crystals belonged to the hexagonal space group P61, with unit-cell parameters a=b = 55.1, c = 413.1 angstrom, and diffracted X-rays beyond 2.4 angstrom resolution. The asymmetric unit is expected to contain two complexes of Pth and the acceptor-TC domain of tRNA (VM = 2.8 angstrom 3 Da-1), with a solvent content of 60.8%. The structure is being solved by molecular replacement.

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  • Expansion of substrate specificity and catalytic mechanism of azoreductase by X-ray crystallography and site-directed mutagenesis Reviewed

    Ito K, Nakanishi M, Lee WC, Zhi Y, Sasaki H, Zenno S, Saigo K, Kitade Y, Tanokura M

    JOURNAL OF BIOLOGICAL CHEMISTRY   283 ( 20 )   13889 - 13896   2008.5

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    AzoR is an FMN-dependent NADH-azoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. We previously reported the crystal structure of the enzyme in the oxidized form. In the present study, different structures of AzoR were determined under several conditions to obtain clues to the reaction mechanism of the enzyme. AzoR in its reduced form revealed a twisted butterfly bend of the isoalloxazine ring of the FMN cofactor and a rearrangement of solvent molecules. The crystal structure of oxidized AzoR in a different space group and the structure of the enzyme in complex with the inhibitor dicoumarol were also determined. These structures indicate that the formation of a hydrophobic part around the isoalloxazine ring is important for substrate binding and an electrostatic interaction between Arg-59 and the carboxyl group of the azo compound causes a substrate preference for methyl red over p-methyl red. The substitution of Arg-59 with Ala enhanced the V-max value for p-methyl red 27-fold with a 3.8-fold increase of the K-m value. This result indicates that Arg-59 decides the substrate specificity of AzoR. The V-max value for the p-methyl red reduction of the R59A mutant is comparable with that for the methyl red reduction of the wild-type enzyme, whereas the activity toward methyl red was retained. These findings indicate the expansion of AzoR substrate specificity by a single amino acid substitution. Furthermore, we built an authentic model of the AzoR-methyl red complex based on the results of the study.

    DOI: 10.1074/jbc.M710070200

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  • The structure and reaction mechanism of azoreductase Invited

    Ito K, Tanokura M

    Seikagaku   80 ( 6 )   550 - 559   2008

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  • Crystal structure of thioredoxin domain of ST2123 from thermophilic archaea Sulfalabus tokodaii strain7 Reviewed

    Hua Ming, Yusuke Kato, Ken-ichi Miyazono, Kosuke Ito, Masayuki Kamo, Koji Nagata, Masaru Tanokura

    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS   69 ( 1 )   204 - 208   2007.10

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    DOI: 10.1002/prot.21467

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  • Three-dimensional structure of AzoR from Escherichia coli - An oxidereductase conserved in microorganisms Reviewed

    Ito K, Nakanishi M, Lee WC, Sasaki H, Zenno S, Saigo K, Kitade Y, Tanokura M

    JOURNAL OF BIOLOGICAL CHEMISTRY   281 ( 29 )   20567 - 20576   2006.7

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    The crystal structure of AzoR (azoreductase) has been determined in complex with FMN for two different crystal forms at 1.8 and 2.2 angstrom resolution. AzoR is an oxidoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. This enzyme is an FMN-dependent NADH-azoreductase and catalyzes the reductive cleavage of azo groups by a ping-pong mechanism. The structure suggests that AzoR acts in a homodimeric state forming the two identical catalytic sites to which both monomers contribute. The structure revealed that each monomer of AzoR has a flavodoxin-like structure, without the explicit overall amino acid sequence homology. Superposition of the structures from the two different crystal forms revealed the conformational change and suggested a mechanism for accommodating substrates of different size. Furthermore, comparison of the active site structure with that of NQO1 complexed with substrates provides clues to the possible substrate-binding mechanism of AzoR.

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  • Crystal structure of monomeric sarcosine oxidase from Bacillus sp. NS-129 reveals multiple conformations at the active-site loop Reviewed

    Nagata K, Sasaki H, Ohtsuka J, Hua M, Okai M, Kubota K, Kamo M, Ito K, Ichikawa T, Koyama Y, Tanokura M

    Proceedings of the Japan Academy Series B: Physical and Biological Sciences   81 ( 6 )   220 - 224   2005.6

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    Monomeric sarcosine oxidase (MSOX) is a flavoprotein that catalyzes the oxidation of sarcosine to generate formaldehyde, glycine, and hydrogen peroxide, and is utilized in quantification of creatinine in serum. Crystal structure of MSOX from Bacillus sp. NS-129 (without ligand) has been determined at 1.86 Å. Unlike the published structures of MSOX from Bacillus sp. B-0618 (without or with carboxylate-containing ligand), the two molecules in the asymmetric unit adopt distinct conformations at the active site loop (Gly56 to Glu60) with a maximal root-mean-square (RMS) displacement of 3.3 Å for Cα atom of Arg59. The multiple conformations seen at the active-site loop suggest that high flexibility of the loop would be important for the activity of MSOX.

    DOI: 10.2183/pjab.81.220

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  • Crystal structure of azoreductase AzoR from Escherichia coli Reviewed

    Ito K, Nakanishi M, Lee WC, Sasaki H, Zenno S, Saigo K, Kitade Y, Tanokura M

    Proceedings of the Japan Academy Series B: Physical and Biological Sciences   81 ( 6 )   225 - 228   2005.6

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    Azoreductase AzoR is an oxidoreductase isolated from Escherichia coli as an enzyme responsible for the reduction of azo compounds. As the first step toward the elucidation of the molecular mechanism of function, we determined the three-dimensional structure of the enzyme by X-ray crystallography at 1.8 Å resolution. The crystal structure has revealed that AzoR is an FMN-containing and homodimeric enzyme. Each monomer consists of a twisted central parallel β-sheet surrounded on both sides by helices. The overall folding of the protein resembles NQO1, originally called DT-diaphorase [NAD(P)H: quinone reductase, EC 1.6.99.2], a mammalian FAD containing protein without significant sequence identity.

    DOI: 10.2183/pjab.81.225

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  • Recombinant porcine zona pellucida glycoproteins expressed in Sf9 cells bind to bovine sperm but not to porcine sperm Reviewed

    N Yonezawa, K Kudo, H Terauchi, S Kanai, N Yoda, M Tanokura, K Ito, K Miura, T Katsumata, M Nakano

    JOURNAL OF BIOLOGICAL CHEMISTRY   280 ( 21 )   20189 - 20196   2005.5

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    The zona pellucida, which surrounds the mammalian oocyte, consists of the ZPA, ZPB, and ZPC glycoproteins and plays roles in species-selective sperm-egg interactions via its carbohydrate moieties. In the pig, this activity is conferred by tri- and tetraantennary complex type chains; in cattle, it is conferred by a chain of 5 mannose residues. In this study, porcine zona glycoproteins were expressed as secreted forms, using the baculovirus-Sf9 insect cell system. The sperm binding activities of the recombinant proteins were examined in three different assays. The assays clearly demonstrated that recombinant ZPB bound bovine sperm weakly but did not bind porcine sperm; when recombinant ZPC was also present, bovine sperm binding activity was greatly increased, but porcine sperm still was not bound. The major sugar chains of ZPB were pauci and high mannose type chains that were similar in structure to the major neutral N-linked chain of the bovine zona. In fact, the nonreducing terminal alpha-mannose residues were necessary for the sperm binding activity. These results show that the carbohydrate moieties of zona glycoproteins, but not the polypeptide moieties, play an essential role in species-selective recognition of porcine and bovine sperm. Moreover, Asn to Asp mutations at either of two of the N-glycosylation sites of ZPB, residue 203 or 220, significantly reduced the sperm binding activity of the ZPB/ZPC mixture, whereas a similar mutation at the third N-glycosylation site, Asn-333, had no effect on binding. These results suggest that the N-glycans located in the N-terminal half of the ZP domain of porcine ZPB are involved in sperm-zona binding.

    DOI: 10.1074/jbc.M414242200

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  • Crystallization and preliminary X-ray analysis of AzoR (azoreductase) from Escherichia coli Reviewed

    K Ito, M Nakanishi, WC Lee, H Sasaki, S Zenno, K Saigo, Y Kitade, M Tanokura

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS   61 ( Pt 4 )   399 - 402   2005.4

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    DOI: 10.1107/S1744309105007918

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  • Crystallization and preliminary X-ray analysis of carboxypeptidase 1 from Thermus thermophilus Reviewed

    K Nagata, S Tsutsui, WC Lee, K Ito, M Kamo, Y Inoue, M Tanokura

    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY   60 ( 8 )   1445 - 1446   2004.8

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    Carboxypeptidase 1 from the thermophilic eubacterium Thermus thermophilus (TthCP1, 58 kDa), a member of the M32 family of metallocarboxypeptidases, was crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals diffracted X-rays to beyond 2.6 Angstrom resolution using a synchrotron-radiation source. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 171.0, b = 231.6, c = 124.9 Angstrom. The crystal contains three molecules in an asymmetric unit (V-M = 2.11 Angstrom(3) Da(-1)) and has a solvent content of 61.5%.

    DOI: 10.1107/S0907444904012557

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Books

  • Flavins and Flavoproteins

    Nishino T, Miura R, Tanokura M, Fukui K. ed( Role: Joint author)

    ARchiTect inc. Tokyo  2005.11 

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  • 「食品の科学」

    上野川修一, 田之倉優( Role: Joint author)

    東京化学同人  2005.3 

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    Language:Japanese Book type:Textbook, survey, introduction

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  • 「ナノバイオテクノロジーの最前線」

    植田充美( Role: Joint author)

    シーエムシー出版  2003.10 

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    Language:Japanese Book type:Scholarly book

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  • 「動物細胞工学ハンドブック」

    動物細胞工学会編( Role: Joint author)

    朝倉書店  2000.10 

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MISC

  • ペプチジルtRNA加水分解酵素・プロテアソーム輸送タンパク質複合体のX線結晶構造解析

    市邨晃久, 笠原杏子, 今井大達, 上原祐二, 西川周一, 内海利男, 伊東孝祐

    日本分子生物学会年会プログラム・要旨集(Web)   41st   2018

  • イネ由来α-アミラーゼの立体構造とその熱安定性に関与する構造要因の解析

    落合秋人, 菅井寛, 伊東孝祐, 内海利男, 田中孝明, 谷口正之, 三ツ井敏明

    日本生化学会大会(Web)   87th   2014

  • 2P-042 Crystal Structure of α-Amylase AmyI-1 from Oryza sativa

    Ochiai Akihito, Sugai Hiroshi, Harada Kazuki, Itoh Kousuke, Uchiumi Toshio, Tanaka Takaaki, Taniguchi Masayuki, Mitsui Toshiaki

    66   117 - 117   2014

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  • 高度好熱菌T.thermophilusペプチジルtRNA加水分解酵素のX線結晶構造解析

    松本愛弥, 伊東孝祐, 竹本千重, 内海利男

    日本分子生物学会年会プログラム・要旨集(Web)   34th   2011

  • アゾ還元酵素の構造と反応機構

    伊東孝祐, 田之倉優

    生化学   80 ( 6 )   550 - 559   2008.6

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  • Expression of Xenopus laevis translation initiation factor 4E (eIF-4E) by baculovirus-insect cell system.

    Miyoshi H, Ito K, Sakai N, Mizushima J, Okamoto K, Hori H, Nishino T, Wakiyama M, Miura K

    Nucleic Acids Symposium Series   37   191 - 192   1997.12

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Industrial property rights

  • 抗原結合用キャリア及びその使用

    内海利男, 須田真広, 藤間真紀, 伊東孝祐

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Awards

  • B.B.B.論文賞

    2015.3   日本農芸化学会  

    Ochiai A, Sugai H, Harada K, Tanaka S, Ishiyama Y, Ito K, Tanaka T, Uchiumi T, Taniguchi M, Mitsui T

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Research Projects

  • 結核菌の休眠誘導の構造基盤解明

    Grant number:23H02417

    2023.4 - 2026.3

    System name:科学研究費助成事業 基盤研究(B)

    Research category:基盤研究(B)

    Awarding organization:日本学術振興会

    伊東 孝祐, 松本 壮吉, 真柳 浩太

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    Grant amount:\18720000 ( Direct Cost: \14400000 、 Indirect Cost:\4320000 )

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  • 天然変性蛋白質(IDP)を標的とする、中分子化合物による、新しい創薬フィールドの開拓

    2022.10 - 2028.3

    System name:革新的先端研究開発支援事業 感染症創薬基盤研究開発領域

    Research category:感染症創薬基盤研究開発領域

    Awarding organization:日本医療研究開発機構

    松本 壮吉, 古寺 哲幸, 伊東 孝祐, 西山 晃史, 真柳 浩太, 廣明 秀一, 岩本 啓, 白井 剛

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    Authorship:Coinvestigator(s) 

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  • 生態系保全のための新規抗結核薬の開発研究

    Grant number:14861

    2022.7 - 2024.3

    System name:試験研究費助成

    Awarding organization:佐々木環境技術振興財団

    伊東孝祐

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  • 次世代型環境浄化酵素生産システムの開発 (リボソームのエネルギー代謝中心部位の改良研究)

    Grant number:R04-1-019

    2022.7 - 2023.3

    System name:試験研究費助成

    Awarding organization:財団法人内田エネルギー科学振興財団

    伊東孝祐

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  • Elucidation of the relationship between protein synthesis system and protein degradation system

    Grant number:22K19267

    2022.6 - 2025.3

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)

    Research category:Grant-in-Aid for Challenging Research (Exploratory)

    Awarding organization:Japan Society for the Promotion of Science

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    Grant amount:\6370000 ( Direct Cost: \4900000 、 Indirect Cost:\1470000 )

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  • 休眠結核菌を殺傷する薬剤の開発

    2022.6 - 2023.3

    System name:科学技術研究費助成金制度

    Awarding organization:公益財団法人ユニオンツール育英奨学会

    伊東孝祐

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  • Development of Next-Generation Platform for Light-induced Drugs

    Grant number:20K21541

    2020.7 - 2023.3

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)

    Research category:Grant-in-Aid for Challenging Research (Exploratory)

    Awarding organization:Japan Society for the Promotion of Science

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  • Development of CRISPR-Cas-type antibacterial agents specifically targeting MRSA

    Grant number:20K21671

    2020.7 - 2022.3

    System name:Grants-in-Aid for Scientific Research

    Research category:Grant-in-Aid for Challenging Research (Exploratory)

    Awarding organization:Japan Society for the Promotion of Science

    Terao Yutaka

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

    The estimated death by MRSA infections in Japan is more than 10,000 per year. Furthermore, in the latest infection control surveillance, the MRSA detection rate at domestic medical institutions was approximately 100%. Nevertheless, research and development of new drugs against drug-resistant bacteria is facing the reality of inadequacy. Therefore, in this present study, we applied CRISPR-Cas genome editing technology to develop a DNA antibacterial drug that specifically deletes the resistance factor PBP2' in MRSA. By using DNA (CRISPR-Cas expression plasmid) as a material, the possibility of side reactions will be low, and the drug target will be able to exchange simply by changing the guide RNA sequence according to the evolution of resistance. This was because it could be an antibacterial drug.

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  • 天然変性ヒストン様蛋白質による、結核菌の個性の創出と多様性獲得の分子機構

    Grant number:20H03483

    2020.4 - 2024.3

    System name:科学研究費助成事業 基盤研究(B)

    Research category:基盤研究(B)

    Awarding organization:日本学術振興会

    松本 壮吉, 白井 剛, 伊東 孝祐, 真柳 浩太

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    Grant amount:\17680000 ( Direct Cost: \13600000 、 Indirect Cost:\4080000 )

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  • Versatile molecular functions of the ribosomal stalk protein in translation cycle

    Grant number:19H03155

    2019.4 - 2022.3

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

    Research category:Grant-in-Aid for Scientific Research (B)

    Awarding organization:Japan Society for the Promotion of Science

    Uchiumi Toshio

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    Grant amount:\17550000 ( Direct Cost: \13500000 、 Indirect Cost:\4050000 )

    Translation of genetic information proceeds on the ribosome with amino acids being brought efficiently to the ribosome by the translation factor EF1A one after another. There are many unclear points about the mechanism for achieving the high efficiency. In this project, we have demonstrated that the ribosomal stalk protein contributes to translation efficiency by binding, via its C-terminal region, to two different conformations of EF1A, that is, the active EF1A-GTP form that carries the aminoacyl-tRNA to the ribosome and the inactive EF1A-GDP form that is dissociated from the ribosome, and that the interaction between the stalk and EF1A is disrupted by anothor factor EF1B which promote nucleotide exchange from EF1A-GDP to EF1A-GTP. Furthermore, we also detected ability of the stalk to bind to a stress-response factor, YchF. These results represent the versatile functions of the ribosomal stalk in translation efficiency and control.

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  • Elucidation of the mechanism of peptidyl-tRNA hydrolysis in the stalled eukaryotic ribosome

    Grant number:18K06080

    2018.4 - 2021.3

    System name:Grants-in-Aid for Scientific Research

    Research category:Grant-in-Aid for Scientific Research (C)

    Awarding organization:Japan Society for the Promotion of Science

    Ito Kosuke

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\4420000 ( Direct Cost: \3400000 、 Indirect Cost:\1020000 )

    During the course of protein synthesis, ribosomes occasionally stall due to various reasons and thus produce peptidyl-tRNAs, which are immature translation products. This unfavorable situation is solved by hydrolyzing the peptidyl-tRNA into the peptide and tRNA components. However, the mechanism of the peptidyl-tRNA hydrolysis in the stalled ribosome still remains unclear in eukaryotes. Our study revealed that Vms1 is not sufficient to hydrolyze the peptidyl-tRNA in the stalled ribosome. We also clarified the structural basis of the substrate recognition and the hydrolysis reaction of Pth, which acts in the cytosol to hydrolyze the peptidyl-tRNAs.

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  • 水環境汚染問題解決を目指した新規抗生物質開発研究

    2017 - 2019

    System name:試験研究費助成

    Awarding organization:佐々木環境技術振興財団

    伊東孝祐

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  • Functional role of the ribosomal stalk protein in translation recycling

    Grant number:16H04741

    2016.4 - 2019.3

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

    Research category:Grant-in-Aid for Scientific Research (B)

    Awarding organization:Japan Society for the Promotion of Science

    Uchiumi Toshio, IMAI Hirotatsu

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    Grant type:Competitive

    Grant amount:\17420000 ( Direct Cost: \13400000 、 Indirect Cost:\4020000 )

    Translation of genetic information, i.e., protein synthesis, on a macromolecular complex called the ribosome occurs in four stages: initiation, elongation, termination, and recycling. So far in research, much less is known about mechanism of the recycling. In this project, we have focused on a ribosome recycling factor, ABCE1 (ATPase), and investigated molecular mechanism of the action of ABCE1 by using techniques of biochemistry and structural biology. As results, we have clarified that a ribosomal protein component, termed the “stalk”, directly binds to a specific site of ABCE1, recruits it to the functional site within the ribosome, and stimulates ATP hydrolysis. Thus, our results demonstrate that interaction between ABCE1 and the stalk protein leads to dissociation of ribosomes to subunits, or ribosome recycling.

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  • Research for the mechanism of DNA silencing using small RNA as a guide strand

    Grant number:16K07246

    2016.4 - 2019.3

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

    Research category:Grant-in-Aid for Scientific Research (C)

    Awarding organization:Japan Society for the Promotion of Science

    Miyoshi Tomohiro

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    Grant type:Competitive

    Grant amount:\4940000 ( Direct Cost: \3800000 、 Indirect Cost:\1140000 )

    Argonaute (RsAgo) of Rhodobacter sphaeroides binds in a sequence-specific manner to a target DNA using small RNA as a guide strand. RsAgo is an important protein involved in the development of new gene analysis technology. In this study, the new results were obtained regarding (1) guide strand selection mechanisms of RsAgo, (2) the influence of each domain structure on nucleic acid recognition and function, and (3) the functional role of DNA silencing.

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  • Elucidation of the structural basis for the interaction of the ribosomal stalk protein with aminoacyl-tRNA synthetase

    Grant number:15K06964

    2015.4 - 2018.3

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

    Research category:Grant-in-Aid for Scientific Research (C)

    Awarding organization:Japan Society for the Promotion of Science

    Kosuke Ito, UCHIUMI Toshio

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\4940000 ( Direct Cost: \3800000 、 Indirect Cost:\1140000 )

    The ribosomal stalk protein moves in a broad range on the ribosome. The ribosomal stalk protein directly interacts with and recruits the translation factors to the ribosome. On the other hand, recently it was found that the ribosomal stalk protein also binds to aminoacyl-tRNA synthetase and contributes to the efficient supply of aminoacyl-tRNA to the translation elongation factor. In our present research, we first conducted the analysis of the interaction between the ribosomal stalk protein and aminoacyl-tRNA synthetase. The results suggested that the N-terminal region of the C-terminal domain of the stalk protein is important for the binding. In addition, we obtained the microcrystals of the stalk protein-aminoacyl-tRNA complex.

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  • 環境浄化酵素生産システムの開発

    2013 - 2014

    System name:試験研究費助成

    Awarding organization:佐々木環境技術振興財団

    伊東孝祐

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    Authorship:Principal investigator  Grant type:Competitive

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  • Ribosomal stalk complex driving translation cycle: molecular basis for high-speed and high-efficiency

    Grant number:24370073

    2012.4 - 2015.3

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

    Research category:Grant-in-Aid for Scientific Research (B)

    Awarding organization:Japan Society for the Promotion of Science

    UCHIUMI Toshio, ITO Kosuke, YAO Minn, MIYOSHI Tomohiro, UCHIYAMA Susumu

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    Grant type:Competitive

    Grant amount:\18330000 ( Direct Cost: \14100000 、 Indirect Cost:\4230000 )

    We have studied on the ribosomal stalk protein complex, which determines the rate and efficiency of protein synthesis (translation), by using biochemical and crystallographic approaches, as follows: 1) multiple copies of the stalk proteins all bind to the ribosomal core via their N-terminal domains, and the C-terminal region of each stalk is flexible and moving widely around the ribosome; 2) the crystal structural data indicate that the C-terminal region of the stalk protein binds to individual translation factors through hydrophobic interactions; 3) the interactions between the C-terminal region of the stalk protein and translation factors is important to recruit the factors to the functional center within the ribosomal core. These lines of evidence demonstrate the crucial contribution of the stalk protein to efficient translation.

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  • tRNA再生酵素-基質複合体のX線結晶構造解析

    2012 - 2015

    System name:若手研究(B)

    Research category:若手研究(B)

    Awarding organization:日本学術振興会

    伊東孝祐

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    Authorship:Principal investigator  Grant type:Competitive

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  • Creation of the tailor-made system for antibody production by using an autoantigenic complex

    Grant number:23657087

    2011 - 2013

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

    Research category:Grant-in-Aid for Challenging Exploratory Research

    Awarding organization:Japan Society for the Promotion of Science

    UCHIUMI Toshio, ITO Koshuke, AOYAGI Yutaka

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    Grant type:Competitive

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

    The aim of this study is to clarify the relationship between the structure and antigenicity of ribosomal autoantigen (P0-P1-P2 complex), and to develop a useful antibody-production system. Epitope analysis of the ribosomal autoantigen identified the 3 amino acids at the C-terminus, which is shared by P0/P1/P2 and responsible for anti-P recognition. It was also found that phosphorylation at Ser residues adjacent to the 3 amino acids enhanced the anti-P binding, suggesting that phosphorylation of the autoantigen is related to the antigenicity. We also found that aP1, the archaeal homologue of human P1/P2, forms a stable tetramer and that immunization of this tetramer resulted in production of antibodies to the conserved C-terminal part. When the C-terminal amino acid sequence was replaced with a sequence of another ribosomal protein, the antibody specific to the introduced sequence was produced. We thus developed a novel method to produce efficiently the antibody for a desired sequence.

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  • タンパク質生合成系の保守管理を担うペプチジルtRNA分解酵素の分子機構解明

    2011 - 2012

    System name:科学技術研究費助成金制度

    Awarding organization:ユニオンツール育英奨学会

    伊東孝祐

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    Authorship:Principal investigator  Grant type:Competitive

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  • Molecular Dissection of the Ribosomal Protein Complex that Plays a as Drive Part of Translation apparatus

    Grant number:21370078

    2009 - 2011

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

    Research category:Grant-in-Aid for Scientific Research (B)

    Awarding organization:Japan Society for the Promotion of Science

    UCHIUMI Toshio, ITO Kosuke, YAO Minn

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    Grant amount:\18590000 ( Direct Cost: \14300000 、 Indirect Cost:\4290000 )

    Ribosomes in all organisms have a unique flexible structure composed of ribosomal protein complex, termed the stalk, which plays a crucial role in ribosome function. In this research subject, we have investigated the structure and function of the ribosomal stalk in a hyperthermophilic archaeon by biochemical and crystallographic analyses. The results showed that the archaeal stalk is composed of aP0 and aP1, which form the heptameric complex aP0(aP1)_2(aP1)_2(aP1)_2, and that the C-terminal sequence, which is shared by aP1 and aP0, directly binds to translation factors. We concluded that the 7 copies of C-terminal tails of aP1/aP0 in the complex participate in efficient recruitment of translation factors onto the ribosome.

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  • リボソーム結合性GTPaseファミリーの構造生物学

    2009 - 2011

    System name:若手研究(B)

    Research category:若手研究(B)

    Awarding organization:日本学術振興会

    伊東孝祐

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    Authorship:Principal investigator  Grant type:Competitive

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  • タンパク質合成システムにおけるエネルギー代謝中心の機能制御研究

    2009 - 2010

    System name:研究助成

    Awarding organization:財団法人内田エネルギー科学振興財団

    伊東孝祐

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    Authorship:Principal investigator  Grant type:Competitive

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  • 超高熱古細菌リボソームストークタンパク質とGTP結合翻訳因子の構造・機能研究

    2008 - 2009

    System name:若手研究 (スタートアップ)

    Research category:若手研究(スタートアップ)

    Awarding organization:日本学術振興会

    伊東孝祐

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    Authorship:Principal investigator  Grant type:Competitive

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  • タンパク質低速合成大腸菌株AM68を利用した有用タンパク質生産システムの開発

    2008 - 2009

    System name:シーズ発掘試験A(発掘型)

    Awarding organization:科学技術振興機構

    伊東孝祐

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    Authorship:Principal investigator  Grant type:Competitive

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Teaching Experience (researchmap)

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Teaching Experience

  • 生物学実習IV

    2023
    Institution name:新潟大学

  • 生体情報学

    2022
    Institution name:新潟大学

  • 生物学実習I

    2022
    Institution name:新潟大学

  • 生物学基礎B

    2022
    Institution name:新潟大学

  • 基礎生物科学実習II

    2022
    Institution name:新潟大学

  • 構造生物学特論

    2022
    Institution name:新潟大学

  • 基礎生物科学実習I

    2022
    Institution name:新潟大学

  • 自然科学総論IV

    2022
    Institution name:新潟大学

  • 領域概説 B (理学)

    2021
    Institution name:新潟大学

  • リフレクションデザインIII

    2021
    Institution name:新潟大学

  • リフレクションデザインII

    2021
    Institution name:新潟大学

  • 生物学実験

    2021
    -
    2022
    Institution name:新潟大学

  • タンパク質・核酸化学特論

    2021
    Institution name:新潟大学

  • 構造生物学特論

    2020
    -
    2022
    Institution name:新潟大学

  • 先端科学技術総論

    2020
    Institution name:新潟大学

  • 生物学特論 V

    2020
    Institution name:新潟大学

  • 細胞生物学I

    2019
    Institution name:新潟大学

  • タンパク質・核酸化学特論

    2019
    -
    2021
    Institution name:新潟大学

  • 日本事情自然系A

    2019
    Institution name:新潟大学

  • 自然科学基礎実験

    2018
    Institution name:新潟大学

  • 課題研究II(生物学)

    2018
    Institution name:新潟大学

  • 理学スタディ・スキルズ

    2018
    -
    2020
    Institution name:新潟大学

  • 課題研究I(生物学)

    2017
    Institution name:新潟大学

  • 生物学総合演習

    2017
    Institution name:新潟大学

  • 生物学基礎実習a

    2017
    Institution name:新潟大学

  • 総合力アクティブ・ラーニング

    2017
    -
    2018
    Institution name:新潟大学

  • 生物学特論I

    2017
    Institution name:新潟大学

  • 生物学基礎演習

    2017
    Institution name:新潟大学

  • 生体分子機能学実習

    2016
    Institution name:新潟大学

  • 基礎生物科学実習II

    2015
    -
    2022
    Institution name:新潟大学

  • 生物学実習

    2015
    Institution name:新潟大学

  • 生命科学への招待(生物学学習法)

    2014
    Institution name:新潟大学

  • 生物英語I

    2013
    Institution name:新潟大学

  • 細胞生物学演習

    2013
    Institution name:新潟大学

  • 基礎生物科学実習I

    2013
    -
    2022
    Institution name:新潟大学

  • 生命・食料科学セミナーBⅡ

    2013
    -
    2015
    Institution name:新潟大学

  • 文献詳読Ⅱ

    2013
    -
    2015
    Institution name:新潟大学

  • 生物化学実習

    2013
    -
    2015
    Institution name:新潟大学

  • 生命・食料科学特定研究BⅡ

    2013
    -
    2015
    Institution name:新潟大学

  • 研究発表演習(中間発表)

    2013
    -
    2014
    Institution name:新潟大学

  • 課題研究I(生物学科)

    2013
    Institution name:新潟大学

  • 課題研究II

    2013
    Institution name:新潟大学

  • 生命・食料科学特定研究BⅠ

    2012
    -
    2014
    Institution name:新潟大学

  • 生命・食料科学セミナーBⅠ

    2012
    -
    2014
    Institution name:新潟大学

  • 文献詳読Ⅰ

    2012
    -
    2014
    Institution name:新潟大学

  • 原書講読

    2012
    Institution name:新潟大学

  • 生物学実験 I

    2010
    -
    2019
    Institution name:新潟大学

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Social Activities

  • 新潟大学公開講座「目指せ!未来の科学者」

    Role(s): Lecturer

    2018.9 - 2018.10

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  • 独立行政法人科学技術振興機構「未来の科学者養成講座」

    Role(s): Lecturer

    2011.9 - 2012.3

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  • 新潟県内外高等学校 出前授業

    Role(s): Lecturer

    2011

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Media Coverage

  • 新聞報道:「遺伝子解析へ新手法」

    新潟日報新聞  2016.8

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  • 新聞報道:「イネの主要α-アミラーゼ立体構造を解明」

    科学新聞  2014.8

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  • 新聞報道:「アゾ還元酵素解明」

    科学新聞  2006.8

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  • 新聞報道:「東大、環境を汚染する合成色素を浄化する酵素の立体構造を解明」

    日刊工業新聞  2006.7

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