ユビキチン修飾による腸管恒常性維持機構の解明

• Project/Area Number: 17H04024
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Environmental physiology(including physical medicine and nutritional physiology)
• Research Institution: Fukushima Medical University (2019); Tokyo Medical and Dental University (2017-2018)
• Principal Investigator: 鬼澤 道夫 福島県立医科大学, 医学部, 助教 (30783352)
• Co-Investigator(Kenkyū-buntansha): 渡辺 守 東京医科歯科大学, 高等研究院, 特別栄誉教授 (10175127)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Project Status: Granted (Fiscal Year 2019)
• Budget Amount: ¥17,810,000 (Direct Cost: ¥13,700,000、Indirect Cost: ¥4,110,000); Fiscal Year 2019: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2018: ¥5,980,000 (Direct Cost: ¥4,600,000、Indirect Cost: ¥1,380,000); Fiscal Year 2017: ¥6,630,000 (Direct Cost: ¥5,100,000、Indirect Cost: ¥1,530,000)
• Keywords: 腸内環境 / 炎症性腸疾患 / 微生物 / 下部消化管 / 食品 / 修飾後翻訳

Outline of Annual Research Achievements

本邦において炎症性腸疾患(IBD)の罹患者数は増加を続け、その有効な予知・予防法は存在せず、内科的治療に抵抗する難治症例の克服は世界的な課題である。腸管は腸管上皮を介して外界と接し、微生物や食餌成分と共生している。IBDは、その共生関係の破綻により過剰な免疫反応が惹起された結果、発症すると考えられている。本研究では宿主のユビキチン化修飾反応に着目することにより、腸管恒常性の維持機構を解明し、IBDの病因・病態を明らかにすることを目的とする。本年度は、病原体関連分子パターン（PAMPs）に反応して発現するタンパク及びその制御タンパクに着目し解析を継続した。ユビキチンに対する免疫沈降法では、あるPAMPs反応タンパク及びその制御タンパクそれぞれがユビキチン化修飾を受けることを認めた。CRISPR-Cas9法を用いてそれぞれの遺伝子欠損細胞を作成し機能解析を行った。制御タンパクについて、ユビキチン化推定部位の変異タンパク発現ベクターを作成し、遺伝子欠損細胞に発現させ機能を解析した。また、PAMPs反応タンパクについて、質量分析を用いてそのユビキチン化修飾部位を推定し、今後はユビキチン化修飾部位の変異タンパク強制発現系を用いて機能解析を行う予定である。さらにはPAMPs反応タンパクに対する免疫沈降を行い質量分析で解析した結果、PAMPs反応タンパクの発現を制御する新規のタンパクも複数同定した。次年度はPAMPs反応タンパクの新規制御機構の詳細を解明する予定である。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

PAMPsに反応するタンパクの発現制御機構について、ユビキチン化修飾に着目した結果、新たなメカニズムを見出したため。この新規発現制御機構は、腸管恒常性維持の一翼を担う新たなメカニズムであると考えられる。現在まで、遺伝子欠損細胞や変異タンパク強制発現系による機能解析、タンパク質複合体の相互作用解析などを行い、概ね順調に進展していると考える。

Strategy for Future Research Activity

前年度に同定された新規の制御タンパクの遺伝子ノックダウンを行い、それら制御タンパクの機能を詳細に解析する。また、PAMPs反応タンパクにおける、ユビキチン化修飾部位の変異タンパク強制発現系により、そのユビキチン化修飾の機能を明らかにする。最終的に実験結果を取りまとめ、新たな腸管恒常性維持機構を明らかとし、IBDの新規治療戦略を提案する。

Research Products

• [Journal Article] Genetic and environmental factors drive personalized medicine for Crohn’s disease. (2019)
  • Author(s): Oshima Shigeru、Watanabe Mamoru
  • Journal Title: J Clin Invest. Volume: 128 Pages: 4758-4760
  • DOI: 10.1172/jci124303
  • Peer Reviewed
• [Journal Article] Ubiquitin D is up-regulated by synergy of Notch signaling and TNF-α in the inflamed intestinal epithelia of IBD patients. (2019)
  • Author(s): Kawamoto Ami、Nagata Sayaka、Anzai Sho、Takahashi Junichi、Kawai Mao、Hama Minami、Nogawa Daichi、Yamamoto Kouhei、Kuno Reiko、Suzuki Kohei、Shimizu Hiromichi、Hiraguri Yui、Yui Shiro、Oshima Shigeru、Tsuchiya Kiichiro、Nakamura Tetsuya、Ohtsuka Kazuo、Kitagawa Masanobu、Okamoto Ryuichi、Watanabe Mamoru
  • Journal Title: J Crohns Colitis Volume: 13 Pages: 495-509
  • DOI: 10.1093/ecco-jcc/jjy180
  • Peer Reviewed
• [Journal Article] Novel polyubiquitin imaging system, PolyUb-FC, reveals that K33-linked polyubiquitin is recruited by SQSTM1/p62. (2018)
  • Author(s): Yoichi Nibe, Shigeru Oshima, Masanori Kobayashi, Chiaki Maeyashiki, Yu Matsuzawa, Kana Otsubo, Hiroki Matsuda, Emi Aonuma, Yasuhiro Nemoto, Takashi Nagaishi, Ryuichi Okamoto, Kiichiro Tsuchiya, Tetsuya Nakamura, Shinichiro Nakada, Mamoru Watanabe
  • Journal Title: Autophagy Volume: 22 Pages: 347-358
  • DOI: 10.1080/15548627.2017.1407889
  • Peer Reviewed / Open Access
• [Journal Article] Contribution of ATOH1+ cells to the homeostasis, repair and tumorigenesis of the colonic epithelium. (2018)
  • Author(s): Ishibashi Fumiaki、Shimizu Hiromichi、Nakata Toru、Fujii Satoru、Suzuki Kohei、Kawamoto Ami、Anzai Sho、Kuno Reiko、Nagata Sayaka、Ito Go、Murano Tatsuro、Mizutani Tomohiro、Oshima Shigeru、Tsuchiya Kiichiro、Nakamura Tetsuya、Watanabe Mamoru、Okamoto Ryuichi
  • Journal Title: Stem Cell Rep. Volume: 10 Pages: 27-42
  • DOI: 10.1016/j.stemcr.2017.11.006
  • Peer Reviewed / Open Access
• [Journal Article] YAP/TAZ-dependent reprogramming of colonic epithelium links ECM remodeling to tissue regeneration. (2018)
  • Author(s): Yui S、Azzolin L、Maimets M、Pedersen MT、Fordham RP、Hansen SL、Larsen HL、Guiu Jordi、Alves MRP、Rundsten CF、Johansen JV、Li Y、Madsen CD、Nakamura T、Watanabe M、Nielsen OH、Schweiger PJ、Piccolo S、Jensen KB
  • Journal Title: Cell Stem Cell Volume: 22 Pages: 35-49
  • DOI: 10.1016/j.stem.2017.11.001
  • Peer Reviewed / Open Access
• [Journal Article] Single cell analysis of Crohn’s disease patient-derived small intestinal organoids reveals disease activity-dependent modification of stem cell properties. (2018)
  • Author(s): Suzuki Kohei、Murano Tatsuro、Shimizu Hiromichi、Ito Go、Nakata Toru、Fujii Satoru、Ishibashi Fumiaki、Kawamoto Ami、Anzai Sho、Kuno Reiko、Kuwabara Konomi、Takahashi Junichi、Tsuchiya Kiichiro、Nakamura Tetsuya、Ohtsuka Kazuo、Watanabe Mamoru、Okamoto Ryuichi
  • Journal Title: J Gastroenterol. Volume: 印刷中 Pages: 1035-1047
  • DOI: 10.1007/s00535-018-1437-3
  • Peer Reviewed / Open Access
• [Journal Article] B cell activation in the cecal patches during the development of an experimental colitis model (2018)
  • Author(s): Taro Watabe, Takashi Nagaishi, Naoya Tsugawa, Yudai Kojima, Nisha Jose, Akinori Hosoya, Michio Onizawa, Yasuhiro Nemoto, Shigeru Oshima, Tetsuya Nakamura, Hajime Karasuyama, Takahiro Adachi, Mamoru Watanabe
  • Journal Title: Biochem Biophys res Commun. Volume: 496 Pages: 367-373
  • DOI: 10.1016/j.bbrc.2018.01.053
  • Peer Reviewed / Open Access
• [Presentation] UC-related and segment-specific properties of patientderived colonic organoids. (2019)
  • Author(s): Suzuki K, Shimizu H, Kawai M, Takahashi J, Anzai S, Kawamoto A, Nagata S, Hiraguri Y, Yui S, Tsuchiya K, Nakamura T, Ohtsuka K, Okamoto R, Watanabe M
  • Organizer: ECCO 2019
  • Int'l Joint Research
• [Presentation] Synergy of Notch signalling and TNF-α in the inflamed intestinal epithelia of IBD patients leads to up-regulation of UBD, a ubiquitin-like protein. (2019)
  • Author(s): Kawamoto A, Nagata S, Anzai S, Takahashi J, Kawai M, Hama M, Nogawa D, Yamamoto K, Kuno R, Suzuki K, Shimizu H, Hiraguri Y, Yui S, Oshima S, Tsuchiya K, Nakamura T, Ohtsuka K, Kitagawa M, Okamoto R, Watanabe M
  • Organizer: ECCO 2019
  • Int'l Joint Research
• [Presentation] TP53 mutation in human colonic organoids acquires resistance to in vitro long-term inflammation. (2019)
  • Author(s): Tsuchiya K, Watanabe S, Shirasaki T, Nishimura R, Katsukura N, Hibiya S, Okamoto R, Nakamura T, Watanabe M
  • Organizer: ECCO 2019
  • Int'l Joint Research
• [Presentation] Fetalization of colonic epithelium medicated by YAP/TAZ links ECM remodeling to tissue regeneration. (2018)
  • Author(s): Yui S, Okamoto R, Tsuchiya K, Nakamura T, Watanabe M
  • Organizer: GI Research Academy
  • Int'l Joint Research
• [Presentation] Single-cell level analysis of organoids derived from CD patients reveals disease-status related modifications of small intestinal stem cells. (2018)
  • Author(s): Suzuki K, Kuwabara K, Takahashi J, Anzai S, Kuno R, Kawamoto A, Ishibashi F, Nagata S, Hiraguri Y, Yui S, Tsuchiya K, Nakamura T, Ohtsuka K, Okamoto R, Watanabe M
  • Organizer: DDW 2018
  • Int'l Joint Research
• [Presentation] Analysis of ileocecal immune response in an animal model of colitis using intra-vital imaging. (2018)
  • Author(s): Watabe T, Nagaishi T, Tsugawa N, Kojima Y, Jose N, Hosoya A, Adachi T, Watanabe M
  • Organizer: DDW 2018
  • Int'l Joint Research
• [Presentation] Identification of lesion-specific epithelial function of ulcerative colitis by comparing colonic organoids from lesion and non-lesion parts of same patients. (2018)
  • Author(s): Shirasaki T, Tsuchiya K, Nishimura R, Watanabe S, Hibiya S, Katsukura N, Okamoto R, Nakamura T, Watanabe M
  • Organizer: DDW 2018
  • Int'l Joint Research
• [Presentation] Human colonic organoid treated with inflammatory factors might mimic the pathophysiology of epithelial cells in ulcerative colitis. (2018)
  • Author(s): Hibiya S, Tsuchiya K, Watanabe S, Nishimura R, Shirasaki T, Oshima S, Okamoto R, Nakamura T, Watanabe M
  • Organizer: DDW 2018
  • Int'l Joint Research
• [Presentation] Mice overexpressing MKL1 in macrophages are susceptible to the development of colitis. (2018)
  • Author(s): An J, Nagaishi T, Watabe T, Naruse TK., Watanabe M, Kimura A
  • Organizer: DDW 2018
  • Int'l Joint Research
• [Presentation] Mouse colonic secretory cells de-differentiate into intestinal stem cells and promote mucosal repair through activation of NF-KB signaling. (2018)
  • Author(s): Ishibashi F, Shimizu H, Kawamoto A, Suzuki K, Anzai S, Kuwabara K, Takahashi J, Nagata S, Oshima S, Tsuchiya K, Nakamura T, Watanabe M, Okamoto R
  • Organizer: ISSCR 2018
  • Int'l Joint Research
• [Presentation] Analysis of cecal immune response in a murine model of colitis using intravital imaging. (2018)
  • Author(s): Nagaishi T, Watabe T, Tsugawa N, Kojima Y, Jose N, Yamada D, Adachi T, Watanabe M
  • Organizer: FOCIS 2018
  • Int'l Joint Research
• [Presentation] Mice overexpressing MKL1 in macrophages are susceptible to DSS-induced colitis. (2018)
  • Author(s): An J, Nagaishi T, Watabe T, Naruse TK., Watanabe M, Kimura
  • Organizer: FOCIS 2018
  • Int'l Joint Research
• [Presentation] Overexpression of MKL1 in Macrophages leads to the development of murine colitis. (2018)
  • Author(s): An J, Nagaishi T, Watabe T, Naruse TK., Watanabe M, Kimura A
  • Organizer: MICS 2018
  • Int'l Joint Research
• [Presentation] Development of a new mouse model of short bowel syndrome that may allow for the assessment of therapeutic efficacy of heterotopic transplantation of small intestinal organoids. (2018)
  • Author(s): Matsumoto Y, Watanabe M, Nakamura T
  • Organizer: 5th TERMIS World Congress 2018
  • Int'l Joint Research
• [Presentation] Crohn’s disease patient-derived small intestinal organoids reveal disease-status related modification of stem cell properties. (2018)
  • Author(s): Suzuki K, Murano T, Hiraguri Y, Takahashi J, Shimizu H,Anzai S, Kuwabara K, Kawamoto A, Ishibashi F, Yui S, Tsuchiya K, Nakamura T, Ohtsuka K, Watanabe M, Okamoto R
  • Organizer: 5th TERMIS World Congress 2018
  • Int'l Joint Research
• [Presentation] Regenerative medicine for the treatment of inflammatory bowel disease. (2018)
  • Author(s): Okamoto R, Watanabe M
  • Organizer: 5th TERMIS World Congress 2018
  • Int'l Joint Research
• [Presentation] Ectopic expression of reg3a in the mice distal Colon is mediated by interactions between notch and Il-22 signaling pathways, and promotes tissue repair By the augmentation of EGFR signaling. (2018)
  • Author(s): Ishibashi F, Kuwabara K, Kawamoto A, Anzai S, Takahashi J, Nagata S, Shimizu H, Yui S, Oshima S, Tsuchiya K, Nakamura T, Watanabe M, Okamoto R
  • Organizer: UEGW 2018
  • Int'l Joint Research
• [Presentation] Long-lived secretory cells residing in the mouse proximal colon serve as reserve stem cells under DNA damage-induced mucosal injury. (2018)
  • Author(s): Kuwabara K, Ishibashi F, Kawamoto A, Anzai S, Takahashi J, Nagata S, Shimizu H, Yui S, Oshima S, Tsuchiya K, Nakamura T, Watanabe M, Okamoto R
  • Organizer: UEGW 2018
  • Int'l Joint Research
• [Presentation] Tp53 mutation enhances cell proliferation and stemness in human colon epithelial organoids and promotes a resistance against long-term inflammation. (2018)
  • Author(s): Watanabe S, Tsuchiya K, Shirasaki T, Hibiya S, Nishimura S, Katsukura N, Oshima S, Okamoto R, Tetsuya N, Watanabe M
  • Organizer: UEGW 2018
  • Int'l Joint Research
• [Presentation] Lesion-specific gene expression in the Epithelial cells of Crohn’s disease by comparing Small intestinal organoids from active and inactive Lesion in the same patient. (2018)
  • Author(s): Hibiya S, Tsuchiya K, Nishimura R, Shirasaki T, Watanabe S, Katsukura N, Okamoto R, Nakamura T, Watanabe M
  • Organizer: UEGW 2018
  • Int'l Joint Research
• [Presentation] B cell-mediated ileocecal immune response is activated in the early phase of colitis development. (2018)
  • Author(s): Kojima Y, Nagaishi T, Watabe T, Tsugawa N, Jose N, Yamada D, Hosoya A, Adachi T, Watanabe M
  • Organizer: UEGW 2018
  • Int'l Joint Research
• [Presentation] Mice overexpressing MKL1 in macrophages experience fulminant colitis. (2018)
  • Author(s): An J, Nagaishi T, Watabe T, Naruse TK., Watanabe M, Kimura A
  • Organizer: UEGW 2018
  • Int'l Joint Research
• [Presentation] Downregulated APL expression in T cells in the setting of chronic colitis. (2018)
  • Author(s): Kojima Y, Nagaishi T, Watabe T, Yamada D, Tsugawa N, Jose N, Hosoya A, Shimizu M, Onizawa M, Watanabe M
  • Organizer: 第47回日本免疫学会学術集会
  • Int'l Joint Research
• [Presentation] Introduction to IBD Centre at Tokyo Medical and Dental University. (2018)
  • Author(s): Watanabe M
  • Organizer: IBD Center of Excellence
  • Int'l Joint Research / Invited
• [Presentation] Intestinal Epithelial Stem Cell Organoid and IBD. (2018)
  • Author(s): Watanabe M
  • Organizer: 第55回日本消化器免疫学会
  • Int'l Joint Research / Invited
• [Presentation] Regeneration of intestinal epithelial cell and IBD (2018)
  • Author(s): Watanabe M
  • Organizer: Falk symposium 2018
  • Int'l Joint Research / Invited