Structural analysis of NOD2, a protein involved in inflammatory bowel disease pathogenesis

• Project/Area Number: 20K15730
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 43020:Structural biochemistry-related
• Research Institution: The University of Tokyo
• Principal Investigator: ZHANG ZHIKUAN 東京大学, 大学院薬学系研究科(薬学部), 助教 (60866937)
• Project Period (FY): 2020-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2021: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2020: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
• Keywords: 炎症性腸疾患 / 自然免疫 / NOD2 / クライオ電顕 / NLR / NOD-like receptor / クライオ電子顕微鏡

Outline of Research at the Start

炎症性腸疾患 (IBD) は小腸や大腸の組織細胞が患者自身の免疫細胞に攻撃され、腸の粘膜に炎症反応が引き起こされる自己免疫疾患であり、世界的に年々増加する傾向を示している。日本においては、多くの炎症性腸疾患は指定難病とされている。
多数のIBD患者においてNOD2遺伝子に変異を有することが見つかり、NOD2の機能不全はIBDの原因として同定されている。しかし、NOD2の活性化機構およびNOD2の機能不全によるIBDの発症機構の分子基盤は解明されていない。本課題では、脂質膜に制御されるNOD2の構造生物学的研究を実施し、NOD2の活性化機構およびその機能破綻によるIBDの発症機構の解明を目指す。

Outline of Final Research Achievements

The molecular mechanisms of the activation and regulation of NOD2, a pathogenic protein involved in inflammatory bowel disease, have not been elucidated. This making it difficult to understand the detailed molecular mechanisms of NOD2-related inflammatory bowel disease pathogenesis and to develop therapeutic methods targeting NOD2. This project aimed to elucidate the activation mechanism of NOD2. We established an original PEGylation method for modification of proteins in order to obtain more suitable samples for cryo-EM analysis. As a result, the application of PEGylation method to NOD2 enabled us to analyze the full-length structure of NOD2 at high resolution by cryo-EM (Structure, 2021). Besides, we also identified the interactions of NOD2 with acidic lipid membranes and a novel ligand, heparin, suggesting that they may be required for NOD2 activation. We will now analyze the complex structure of NOD2 with these interaction partners.

Academic Significance and Societal Importance of the Research Achievements

炎症性腸疾患 (IBD) は指定難病の一種であり、世界中の患者数が年々増加傾向である。IBDの病因タンパク質NOD2の活性化機能の解明はIBDの分子機構の解明に繋がり、有効な治療方法の開発に重要である。本研究で開発されたPEGylation分子修飾法のクライオ電子顕微鏡への適応はNOD2を始め、様々なタンパク質の高分解能クライオ電顕解析を可能とさせる新たなサンプル調製法を提供している。また、本研究で同定したNOD2の新規相互作用分子 (酸性脂質およびヘパリン) は、NOD2の活性制御機構の解明に向ける重要な知見を提供した。

Research Products

• [Journal Article] Structural basis for the oligomerization-mediated regulation of NLRP3 inflammasome activation (2022)
  • Author(s): Ohto Umeharu、Kamitsukasa Yukie、Ishida Hanako、Zhang Zhikuan、Murakami Karin、Hirama Chie、Maekawa Sakiko、Shimizu Toshiyuki
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 119 Issue: 11
  • DOI: 10.1073/pnas.2121353119
  • Peer Reviewed
• [Journal Article] Structural Basis for the Inhibition Mechanism of TLR7 (2021)
  • Author(s): ZHANG Zhikuan
  • Journal Title: Nihon Kessho Gakkaishi Volume: 63 Issue: 3 Pages: 181-182
  • DOI: 10.5940/jcrsj.63.181
  • NAID: 130008083573
  • ISSN: 0369-4585, 1884-5576
  • Year and Date: 2021-08-31
  • Peer Reviewed / Open Access
• [Journal Article] Improving particle quality in cryo-EM analysis using a PEGylation method (2021)
  • Author(s): Zhang Zhikuan、Shigematsu Hideki、Shimizu Toshiyuki、Ohto Umeharu
  • Journal Title: Structure Volume: 29 Issue: 10 Pages: 1192-1199.e4
  • DOI: 10.1016/j.str.2021.05.004
  • Peer Reviewed
• [Journal Article] Cryo-EM structures of Toll-like receptors in complex with UNC93B1 (2021)
  • Author(s): Ishida Hanako、Asami Jinta、Zhang Zhikuan、Nishizawa Tomohiro、Shigematsu Hideki、Ohto Umeharu、Shimizu Toshiyuki
  • Journal Title: Nature Structural & Molecular Biology Volume: 28 Issue: 2 Pages: 173-180
  • DOI: 10.1038/s41594-020-00542-w
  • Peer Reviewed / Open Access
• [Journal Article] Structural analysis reveals TLR7 dynamics underlying antagonism (2020)
  • Author(s): Tojo Shingo、Zhang Zhikuan、Matsui Hiroyuki、Tahara Masahiro、Ikeguchi Mitsunori、Kochi Mami、Kamada Mami、Shigematsu Hideki、Tsutsumi Akihisa、Adachi Naruhiko、Shibata Takuma、Yamamoto Masaki、Kikkawa Masahide、Senda Toshiya、Isobe Yoshiaki、Ohto Umeharu、Shimizu Toshiyuki
  • Journal Title: Nature Communications Volume: 11 Issue: 1 Pages: 1-11
  • DOI: 10.1038/s41467-020-19025-z
  • Peer Reviewed / Open Access
• [Remarks]
  • URL: https://www.f.u-tokyo.ac.jp/topics.html?key=1646880551
• [Remarks]
  • URL: http://www.f.u-tokyo.ac.jp/topics.html?page=2&key=1603192010