Doi, Nobuhide

写真a

Affiliation

Faculty of Science and Technology, Department of Biosciences and Informatics (Yagami)

Position

Professor

Related Websites
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Profile Summary 【 Display / hide 】

  • '''''''This laboratory focuses on the biophysics/biotechnology of macromolecules, especially proteins, used to develop new methodologies for solving various biological problems that are resistant to conventional analytical approaches. Also studied is the development of new methods for in vitro selection and directed evolution of proteins, protein folding and design, origin and evolution of life, and high-throughput screening of protein interactions.'''''''

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Career 【 Display / hide 】

  • 1997.04
    -
    2000.03

    三菱化学生命科学研究所

  • 2000.04
    -
    2002.03

    慶應義塾大学理工学部応用化学科 助手

  • 2002.04
    -
    2003.03

    慶應義塾大学理工学部生命情報学科 助手

  • 2003.04
    -
    2008.03

    慶應義塾大学理工学部生命情報学科 専任講師

  • 2005.10
    -
    2007.09

    兼慶應義塾大学理工学部生命情報学科 広報委員

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Academic Background 【 Display / hide 】

  • 1997.03

    Hokkaido University, Graduate School of Environmental Earth Sciences, 生態環境科学専攻

    Graduate School, Completed, Doctoral course

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Academic Degrees 【 Display / hide 】

  • Environmental Science, Hokkaido University, 1997.03

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Research Areas 【 Display / hide 】

  • Manufacturing Technology (Mechanical Engineering, Electrical and Electronic Engineering, Chemical Engineering) / Biofunction and bioprocess engineering (生物機能工学)

  • Life Science / Applied molecular and cellular biology (バイオテクノロジー)

  • Life Science / Molecular biology

  • Life Science / Biophysics

  • Life Science / System genome science (蛋白質ネットワーク)

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Research Keywords 【 Display / hide 】

  • Drug delivery system

  • Proteomics

  • Synthetic Biology

  • Antibody Engineering

  • Evolutionary Molecular Engineering

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Books 【 Display / hide 】

  • ドラッグデリバリーシステム - バイオ医薬品創成に向けた組織、細胞内、核内送達技術の開発-

    土居信英, シーエムシー出版, 2018.06

    Scope: pp.131-137

  • 医療・診断をささえるペプチド科学 - 再生医療・DDS・診断への応用 -

    土居信英, シーエムシー出版, 2017.10

    Scope: pp.232-238

  • ペプチド医薬品のスクリーニング・安定化・製剤化技術

    須藤慧，藤原慶，土居信英, 技術情報協会, 2017

  • 進化分子工学　～高速分子進化によるタンパク質・核酸の開発～

    土居 信英、柳川弘志, NTS出版, 2013.10

    Scope: pp.275-286

  • 次世代医薬開発に向けた抗体工学の最前線

    土居信英, シーエムシー出版, 2012.12

    Scope: pp.66-73

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Papers 【 Display / hide 】

  • STALL-seq: mRNA-display selection of bacterial and eukaryotic translational arrest sequences from large random-sequence libraries

    Hamano T., Nagumo Y., Umehara T., Hirono K., Fujiwara K., Taguchi H., Chadani Y., Doi N.

    Journal of Biological Chemistry 300 ( 12 ) 107978 2024.12

    ISSN  00219258

    　View Summary

    The translational arrest is a phenomenon wherein a temporary pause or slowing of the translation elongation reaction occurs due to the interaction between ribosome and nascent peptide. Recent studies have revealed that translational arrest peptides are involved in intracellular protein homeostasis regulatory functions, such as gene expression regulation at the translational level and regulation of cotranslational protein folding. Herein, we established a method for the large-scale in vitro selection of translational arrest peptides from DNA libraries by combining a modified mRNA display method and deep sequencing. We performed in vitro selection of translational arrest sequences from random-sequence libraries via mRNA display based on the Escherichia coli PURE system or wheat germ extract. Following several rounds of affinity selection, we obtained various candidate sequences that were not similar to known arrest peptides and subsequently confirmed their ribosome stalling activity by peptidyl-tRNA detection and toeprinting assay. Following the site-directed mutagenesis of the selected sequences, these clones were found to contain novel arrest peptide motifs. This method, termed STALL-seq (Selection of Translational Arrest sequences from Large Library sequencing), could be useful for the large-scale investigation of translational arrest sequences acting on both bacterial and eukaryotic ribosomes and could help discover novel intracellular regulatory mechanisms.

  • Lipid nanoparticle-encapsulated DOCK11-siRNA efficiently reduces hepatitis B virus cccDNA level in infected mice

    Okada H., Sakamoto T., Nio K., Li Y., Kuroki K., Sugimoto S., Shimakami T., Doi N., Honda M., Seiki M., Kaneko S., Yamashita T.

    Molecular Therapy Methods and Clinical Development 32 ( 3 ) 101289 2024.09

    ISSN  2329-0501

    　View Summary

    The hepatitis B virus (HBV) infects many people worldwide. As HBV infection frequently leads to liver fibrosis and carcinogenesis, developing anti-HBV therapeutic drugs is urgent. Therapeutic drugs for preventing covalently closed circular DNA (cccDNA) production, which can eliminate HBV infection, are unavailable. The host factor dedicator of cytokinesis 11 (DOCK11) is involved in the synthesis and maintenance of HBV cccDNA in vitro. However, the effectiveness of DOCK11 as a target for the in vivo elimination of HBV cccDNA remains unclear. In this study, we assess whether DOCK11 inhibitors suppress HBV cccDNA production in mouse models of HBV infection. The tocopherol-conjugate hetero- gapmer, a DNA/RNA duplex of gapmer/complementary RNA targeting the DOCK11 sequence, partially reduces the expression of DOCK11, but not that of HBV cccDNA, in the livers of HBV-infected human hepatocyte chimeric mice, along with weight loss and decreased serum human albumin levels. Lipid nanoparticle-encapsulated chemically modified siRNAs specific for DOCK11 suppress DOCK11 expression and decrease HBV cccDNA levels without adverse effects in the mice. Therefore, nucleic acid-based drugs targeting DOCK11 in hepatocytes are potentially effective anti-HBV therapeutics that can reduce HBV cccDNA levels in vivo.

  • Cell-Free Protein Expression by a Reconstituted Transcription–Translation System Energized by Sugar Catabolism

    Sato G., Miyazawa S., Doi N., Fujiwara K.

    Molecules 29 ( 13 )  2024.07

    　View Summary

    Cooperation between catabolism and anabolism is crucial for maintaining homeostasis in living cells. The most fundamental systems for catabolism and anabolism are the glycolysis of sugars and the transcription–translation (TX-TL) of DNA, respectively. Despite their importance in living cells, the in vitro reconstitution of their cooperation through purified factors has not been achieved, which hinders the elucidation of the design principle in living cells. Here, we reconstituted glycolysis using sugars and integrated it with the PURE system, a commercial in vitro TX-TL kit composed of purified factors. By optimizing key parameters, such as glucokinase and initial phosphate concentrations, we determined suitable conditions for their cooperation. The optimized system showed protein synthesis at up to 33% of that of the original PURE system. We observed that ATP consumption in upstream glycolysis inhibits TX-TL and that this inhibition can be alleviated by the co-addition of glycolytic intermediates, such as glyceraldehyde 3-phosphate, with glucose. Moreover, the system developed here simultaneously synthesizes a subset of its own enzymes, that is, glycolytic enzymes, in a single test tube, which is a necessary step toward self-replication. As glycolysis and TX-TL provide building blocks for constructing cells, the integrated system can be a fundamental material for reconstituting living cells from purified factors.

  • Metabolic Tug-of-War between Glycolysis and Translation Revealed by Biochemical Reconstitution

    Sato G., Kinoshita S., Yamada T.G., Arai S., Kitaguchi T., Funahashi A., Doi N., Fujiwara K.

    ACS Synthetic Biology 13 ( 5 ) 1572 - 1581 2024.05

    　View Summary

    Inside cells, various biological systems work cooperatively for homeostasis and self-replication. These systems do not work independently as they compete for shared elements like ATP and NADH. However, it has been believed that such competition is not a problem in codependent biological systems such as the energy-supplying glycolysis and the energy-consuming translation system. In this study, we biochemically reconstituted the coupling system of glycolysis and translation using purified elements and found that the competition for ATP between glycolysis and protein synthesis interferes with their coupling. Both experiments and simulations revealed that this interference is derived from a metabolic tug-of-war between glycolysis and translation based on their reaction rates, which changes the threshold of the initial substrate concentration for the success coupling. By the metabolic tug-of-war, translation energized by strong glycolysis is facilitated by an exogenous ATPase, which normally inhibits translation. These findings provide chemical insights into the mechanism of competition among biological systems in living cells and provide a framework for the construction of synthetic metabolism in vitro.

  • Multimolecular Competition Effect as a Modulator of Protein Localization and Biochemical Networks in Cell-Size Space

    Nishikawa S., Sato G., Takada S., Kohyama S., Honda G., Yanagisawa M., Hori Y., Doi N., Yoshinaga N., Fujiwara K.

    Advanced Science 11 ( 6 ) e2308030 2024.02

    　View Summary

    Cells are small, closed spaces filled with various types of macromolecules. Although it is shown that the characteristics of biochemical reactions in vitro are quite different from those in living cells, the role of the co-existence of various macromolecules in cell-size space remains still elusive. Here, using a constructive approach, it is demonstrated that the co-existence of various macromolecules themselves has the ability to tune protein localization for spatiotemporal regulation and a biochemical reaction system in a cell-size space. Both experimental and theoretical analyses reveal that enhancement of interfacial effects by a large surface-area-to-volume ratio facilitates membrane localization of molecules in the cell-size space, and the interfacial effects are alleviated by competitive binding to lipid membranes among multiple proteins even if their membrane affinities are weak. These results indicate that competition for membrane binding among various macromolecules in the cell-size space plays a role in regulating the spatiotemporal molecular organization and biochemical reaction networks. These findings shed light on the importance of surrounding molecules for biochemical reactions using purified elements in small spaces.

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Papers, etc., Registered in KOARA 【 Display / hide 】

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Reviews, Commentaries, etc. 【 Display / hide 】

  • mRNAディスプレイによる翻訳アレスト配列の大規模探索

    濱野理，土居信英

    生物工学 (日本生物工学会)  97 ( 8 ) 10 - 12 2019.08

    Article, review, commentary, editorial, etc. (scientific journal), Joint Work

  • ヒト由来膜融合ペプチドによるバイオ医薬のDDS

    土居 信英

    細胞 49 ( 12 ) 602 - 605 2017.10

    Article, review, commentary, editorial, etc. (scientific journal), Single Work

  • マイクロカプセルを利用した制限酵素および受容体リガンドの試験管内選択法

    土居 信英, 柳川 弘志

    バイオテクノロジージャーナル 1 ( 1-2 ) 84 - 86 2005.01

    Article, review, commentary, editorial, etc. (scientific journal), Joint Work

  • 生命の理解と制御に向けた遺伝子ネットワーク解析

    土居 信英, 柳川 弘志

    Bioベンチャー 4 ( 4 ) 54 - 56 2004.07

    Article, review, commentary, editorial, etc. (scientific journal), Joint Work

  • タンパク質相互作用のハイスループット解析に向けて―新しい蛍光標識法とマイクロアレイ技術の融合

    土居 信英, 柳川 弘志

    Bioベンチャー 2   102 - 105 2002.06

    Article, review, commentary, editorial, etc. (scientific journal), Joint Work

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Presentations 【 Display / hide 】

  • Second extracellular loop of human CD9 is a cell-penetrating peptide

    Kumeno, Y., Sudo, K., Fujiwara, K., Doi, N.

    The 2021 International Chemical Congress of Pacific Basin Societies (Honolulu, Hawaii, online) , 

    2021.12
    , 

    Poster presentation

  • Application of human syncytin1-derived fusogenic peptide to direct intracellular delivery of Cas9 protein-gRNA complex

    Furuya, T., Fujiwara, K., Doi, N.

    The 2021 International Chemical Congress of Pacific Basin Societies (Honolulu, Hawaii, online) , 

    2021.12
    , 

    Poster presentation

  • がん細胞選択的DDSに向けたEGFRに対する環境応答性単一ドメイン抗体の創出

    明石尚之，清野真梨子，藤原慶，土居信英

    第44回日本分子生物学会年会 (横浜) , 

    2021.12
    , 

    Poster presentation

  • 膜透過性ペプチドを用いたCas9タンパク質-gRNA複合体の直接的な細胞内送達

    古屋智規，藤原慶，土居信英

    第43回日本分子生物学会年会 (オンライン) , 

    2020.12
    , 

    Poster presentation

  • 細胞選択的なタンパク質デリバリーのためのヒト・シンシチン1由来膜透過促進ペプチドの最適化

    前村帆南，藤原慶，土居信英

    第43回日本分子生物学会年会 (オンライン) , 

    2020.12
    , 

    Poster presentation

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Research Projects of Competitive Funds, etc. 【 Display / hide 】

  • 感染症の標的となる細胞内因子に対するバイオ医薬の創出・送達技術の開発

    2023.04
    -
    2027.03

    Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (A), Principal investigator

    　View Summary

    本研究では、申請者らが発見したヒト由来の膜透過促進ペプチドを利用したタンパク質・核酸の細胞質送達技術と、独自のmRNAディスプレイ法による小型抗体の試験管内選択技術を利用して、感染症の標的となる細胞内の因子に対するバイオ医薬の創出・送達手法を確立することを目的とする。これまで感染症に対するバイオ医薬の標的はウイルス・細菌の表面タンパク質や受容体など細胞外の分子に限られていたが、本研究を契機として、病原体の耐性獲得に左右されにくい細胞内の分子を標的とすることが可能になれば、我が国の感染症に対するバイオ創薬技術の基盤強化、ひいては国民の医療・福祉の向上に大いに貢献することが期待できる。
    （1）HBV感染細胞のcccDNAを完全駆除する核酸医薬の開発（土居・岡田）
    肝細胞特異的に発現しているアシアロ糖タンパク質受容体(ASGR)に結合する小型抗体やASGRのリガンドであるtri-GalNAcと、ヒトSyncytin1やIZUMO1由来の様々な膜透過促進ペプチドを組み合わせて融合したAgo2タンパク質をキャリアとして、siRNA医薬を肝細胞選択的に効率よく細胞質送達できる全く新しいDDSを開発するために、蛍光タンパク質をモデルとして共焦点顕微鏡観察することで、コンストラクトの最適化を進めた。また、DOCK11以外の新たな標的を見い出すために、野生型とAAV8-HBV1.3mer慢性感染モデルマウスの肝組織を用いたシングルセルトランスクリプトーム解析を行い、HBV関連遺伝子と宿主側の遺伝子を区別して空間的な情報を得ることができた。
    （2）クレブシエラ菌感染を阻害する小型抗体医薬の開発（土居・津川）
    Axlの細胞内ドメイン(ICD)の様々な部位に結合する多様な小型抗体を１次セレクションし、その中から、所望の経路のみを活性化する小型抗体の２次スクリーニングを行うために、１年目は、Axl(ICD)の部分断片にビーズ固定用のタグを融合した遺伝子の発現用プラスミドを構築し、大量発現・精製した。Axl(ICD)断片が非天然変性領域を含むため苦労したが、ビーズへの固定を確認することができた。また、クレブシエラ菌感染下におけるAxl/Gas6シグナルの亢進がTight-junctionの増強と同時に腸管上皮細胞における細胞増殖も増強することをin vitro感染モデル試験にてki67染色を指標に明らかにした。このin vitroモデルを小型抗体の2次スクリーニングに利用することで、細胞増殖の誘導を引き起こさない医薬品の開発が可能になると考えられる。
    ASGRに結合する小型抗体やASGRのリガンドである tri-GalNAc と、ヒト Syncytin1 や IZUMO1 由来の様々な膜透過促進ペプチドを組み合わせて融合したタンパク質の肝細胞選択的な取り込みを確認できたこと、また、Axl(ICD) の各部位に結合する多様な小型抗体のセレクションに必要となるAxl(ICD) の部分断片を調製できたことから、おおむね順調に進展していると判断した。
    (1) HBV感染細胞のcccDNAを完全駆除する核酸医薬の開発（土居・岡田）
    前年度に引き続き、Ago2融合タンパク質とsiRNAの複合体をヒト培養細胞に添加し、肝細胞選択的に細胞内に送達されることを共焦点顕微鏡観察により確認することで、コンストラクトの最適化を進める。また、複合体が細胞質で標的mRNAを分解していることを確認するために、ヒト培養細胞の内在遺伝子やDOCK11に対するsiRNAとの複合体を肝細胞由来の培養細胞に送達し、培養細胞から抽出した標的遺伝子に対するmRNA量をRT-qPCRで定量する。さらに、前年度のHBV複製に関わる遺伝子情報の包括的解析から、核酸創薬に適した標的細胞から数種類の標的遺伝子まで絞り込み、先行したDOCK11の研究基盤データと比較して、新たな核酸創薬に最適な標的遺伝子を同定する。
    (2) クレブシエラ菌感染を阻害する小型抗体医薬の開発（土居・津川）
    mRNAディスプレイ法により、すでに構築済みのヒト単一ドメイン抗体ライブラリーから、前年度に調製したAxl(ICD)に結合する小型抗体を試験管内選択する。その後、１次セレクションで得られた小型抗体を腸管上皮細胞由来 Caco-2細胞で発現させることでTight-junctionタンパク質の発現が変化するクローンを２次スクリーニングする。このときTight-junctionタンパク質のプロモーター細胞の構築と合わせて、クレブシエラ菌感染下におけるAxl/Gas6シグナルを確認し、クレブシエラ感染症予防に最適な２次スクリーニング技術を確立し利用する。

  • 肝微小環境の構造理解に基づく新たな代謝性肝疾患治療の確立

    2021.04
    -
    2025.03

    Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (A), Research grant, Coinvestigator(s)

    　View Summary

    NASHは脂肪化・炎症・線維化を伴って進行し、肝不全や肝発癌発生の要因となる。本研究では肝微小環境の構造理解に基づく新たな代謝性肝疾患治療の確立を行う。肝類洞内皮で高発現するSema6AはSema6A-PlxnA2シグナルを介して肝星細胞の活性化を抑制し抗線維化治療分子として有用な可能性が示唆される。Sema6Aの肝類洞内皮における本来の生理機能を解き明かすことにより、肝再生機構、肝類洞の細動脈化機構、線維化、炎症の機転を解明する。類洞という肝微小構造の解剖学的特徴を利用し、効果的なSema6Aの投与方法を確立する。さらには、PlxnA2をターゲットとした新たな代謝性肝疾患治療を確立する。

  • 独自の膜透過促進ペプチドを利用した簡便かつ汎用的なゲノム編集技術の開発

    2020.10
    -
    2022.03

    科学技術振興機構　A-STEPトライアウト, Research grant, Principal investigator

  • Development of cancer cell-selective membrane-permeable antibodies against intracellular disease targets

    2020.04
    -
    2024.03

    MEXT,JSPS, Grant-in-Aid for Scientific Research, Doi Nobuhide, Grant-in-Aid for Scientific Research (B), Research grant, Principal investigator

    　View Summary

    In this study, by using human-derived membrane permeation-enhancing peptides that we discovered and our mRNA display technology for in vitro selection of fragment antibodies and ligand peptides, we established elemental technologies for the development of revolutionary anticancer drugs that target intracellular targets that have not been targeted for biopharmaceuticals.
    First, using the mRNA display method, we created human VH single-domain antibodies that bind to marker proteins on the surface of cancer cells or intracellular disease targets. By combining these antibodies with the human-derived membrane permeation-enhancing peptides, we succeeded in cell-selective delivery of proteins, peptides and nucleic acids as biopharmaceutical models.

  • Development of a two-step molecular targeted therapy to eliminate gastric cancer stem cells and progenitor cells

    2020.04
    -
    2023.03

    Grants-in-Aid for Scientific Research, Suzuki Hidekazu, Grant-in-Aid for Scientific Research (B), Research grant, Coinvestigator(s)

    　View Summary

    Analysis of the mucosoid model, which is a two-dimensional development of gastric organoids, showed that butyric acid, a short-chain fatty acid with HDAC inhibitory activity, markedly increased CAPZA1 expression, and under H. pylori infection, CagA accumulated in a CAPZA1 expression-dependent manner. Furthermore, we confirmed the existence of commensal bacteria other than H. pylori that confer intragastric butyrate environment in human gastric juice. On the other hand, we obtained human VH single-domain antibodies that specifically bind to CAPZA1 and developed a technology that can deliver them into the nucleus while maintaining the binding activity of the antibodies. In addition, by using a ternary complex of luciferase gene/chitosan/hyaluronic acid, we obtained intracellular transduction of genes specific to CD44v9-positive cells, microtubule-dependent transport to the nucleus, and gene expression activity.

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Intellectual Property Rights, etc. 【 Display / hide 】

  • 融合タンパク質又は複合タンパク質，細胞内送達用担体，部分ペプチド，細胞膜透過促進剤，DNA，及びベクター

    Date applied： PCT/JP2016/066455  2015.06 

    Date issued： 6864364 

    Date registered： 2021.04

    Patent, Joint

  • 物質と蛋白質との間の相互作用の検出方法

    Date applied： 2001-239175  2001.08 

    Date issued： 3706942  2004.11

    Patent, Joint

  • C末端修飾タンパク質の製造とその利用方法

    Date applied： 2000-373105  2000.11 

    Date issued： 3750020  2005.12

    Patent, Joint

  • タンパク質-ＤＮＡ連結分子及びその利用

    Date applied： 2000-244061  2000.08 

    Date announced： 2001-128690  2001.05 

    Date issued： 04122694 

    Date registered： 2008.05

    Patent, Joint

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Courses Taught 【 Display / hide 】

  • TOPICS IN BIOSCIENCES AND INFORMATICS A

    2025

  • TOPICS IN BIOSCIENCES AND INFORMATICS 1

    2025

  • SEMINAR IN BIOSCIENCES AND INFORMATICS

    2025

  • MOLECULAR BIOLOGY 2

    2025

  • MOLECULAR BIOLOGY 1

    2025

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Courses Previously Taught 【 Display / hide 】

  • 微生物学

    Keio University

    2015.04
    -
    2016.03
    , 

    Spring Semester, Lecture

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Memberships in Academic Societies 【 Display / hide 】

  • 日本生物工学会, 

    2014
    -
    Present

  • 日本DDS学会, 

    2014
    -
    Present

  • 細胞を創る研究会, 

    2013
    -
    Present

  • 日本癌学会, 

    2010
    -
    Present

  • 日本分子生物学会, 

    1994
    -
    Present

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Committee Experiences 【 Display / hide 】

  • 2018.01
    -
    2021.12

    JB編集委員会 編集委員 (Associate Editor), 日本生化学会

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