Study on molecular mechanism of p62-Keap1-Nrf2 system

• Project/Area Number: 16H05137
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: General medical chemistry
• Research Institution: Niigata University
• Principal Investigator: Ichimura Yoshinobu 新潟大学, 医歯学系, 准教授 (80400993)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥17,680,000 (Direct Cost: ¥13,600,000、Indirect Cost: ¥4,080,000); Fiscal Year 2018: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2017: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000); Fiscal Year 2016: ¥7,280,000 (Direct Cost: ¥5,600,000、Indirect Cost: ¥1,680,000)
• Keywords: 選択的オートファジー / p62-Keap1-Nrf2経路 / p62 / Keap1-Nrf2経路 / リン酸化 / Keap1-Nrf2システム / p62-Keap1-Nrf2システム / Keap1-Nrf2 / p62-Keap1-Nrf2 / キナーゼ / p62/SQSTM1 / Keap1

Outline of Final Research Achievements

We have previously reported that phosphorylation of p62 at Ser349 markedly increases p62’s binding affinity for Keap1, an adaptor of the Cul3-ubiquitin E3 ligase complex responsible for degrading Nrf2, and induces expression of cytoprotective Nrf2 targets (p62-Keap1-Nrf2 axis). Further, we have demonstrated that the S349-phosphorylated p62 causes rearrangement of glucose and glutamine metabolism through persistent activation of Nrf2, which provides HCC cells with both tolerance to anti-cancer drugs and proliferation potency. However, the molecular mechanisms regulating the p62-Keap1-Nrf2 axis are not yet fully understood. In this study, we performed high-content screening with human whole siRNA library. Further, we established derivatives of K67, inhibitor for the Keap1-phosphorylated p62 interaction, and developed fluorescent K67-derivertive.

Academic Significance and Societal Importance of the Research Achievements

p62-Keap1-Nrf2経路は、p62のリン酸化を介して転写因子Nrf2が活性化される経路である。重要なことに、本経路は肝がん細胞株や肝がん患者組織では恒常的に活性化されており、がん細胞の増殖に有利な代謝リプログラミングや薬剤耐性を介してがんの悪性化に寄与している。すなわち、p62-Keap1-Nrf2経路を制御することが、がんの有効な治療手段となる。しかしながら、p62-Keap1-Nrf2経路の制御機構については十分に理解されていない。本研究課題ではp62-Keap1-Nrf2経路の制御に関わる新規な低分子化合物を同定し、そのメカニズムを明らかにした。さらに制御因子の探索を進めた。

Research Products

• [Journal Article] Biallelic UFM1 and UFC1 mutations expand the essential role of ufmylation in brain development. (2018)
  • Author(s): Nahorski MS, Maddirevula S, Ishimura R, Alsahli S, Brady AF, Begemann A, Mizushima T, Guzman-Vega FJ, Obata M, Ichimura Y, Alsaif HS, Anazi S, Ibrahim N, Abdulwahab F, Hashem M, Monies D, Abouelhoda M, Meyer BF, Alfadhel M, Eyaid W, Zweier M, Steindl K, Rauch A, Arold ST, Woods CG, Komatsu M, Alkuraya FS.
  • Journal Title: Brain Volume: 141 Issue: 7 Pages: 1934-1945
  • DOI: 10.1093/brain/awy135
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Negative regulation of the Keap1-Nrf2 pathway by a p62/Sqstm1 splicing variant. (2018)
  • Author(s): Kageyama Shun、Saito Tetsuya、Obata Miki、Koide Ryouhei、 Ichimura Yoshinobu、Komatsu Masaaki.
  • Journal Title: Mol Cell Biol. Volume: 38 Issue: 7
  • DOI: 10.1128/mcb.00642-17
  • Peer Reviewed / Open Access
• [Journal Article] Activation of p62/SQSTM1-Keap1-Nuclear Factor Erythroid 2-Related Factor 2 Pathway in Cancer. (2018)
  • Author(s): Ichimura Y, Komatsu M.
  • Journal Title: Frontiers in Oncology Volume: 8 Pages: 210-210
  • DOI: 10.3389/fonc.2018.00210
  • Peer Reviewed / Open Access
• [Journal Article] p62を介した選択的オートファジー (2018)
  • Author(s): 一村義信, 小松雅明
  • Journal Title: 医学のあゆみ Volume: 267 Pages: 1023-1028
• [Journal Article] Discovery of benzo[ g ]indoles as a novel class of non-covalent Keap1-Nrf2 protein-protein interaction inhibitor (2017)
  • Author(s): Yasuda Daisuke、Yuasa Akihiro、Obata Rika、Nakajima Mao、Takahashi Kyoko、Ohe Tomoyuki、Ichimura Yoshinobu、Komatsu Masaaki、Yamamoto Masayuki、Imamura Riyo、Kojima Hirotatsu、Okabe Takayoshi、Nagano Tetsuo、Mashino Tadahiko
  • Journal Title: Bioorg Med Chem Lett Volume: 27 Issue: 22 Pages: 5006-5009
  • DOI: 10.1016/j.bmcl.2017.10.008
  • Peer Reviewed / Open Access
• [Journal Article] Structural and functional analysis of a novel interaction motif within UFM1-activating enzyme 5 (UBA5) required for binding to ubiquitin-like proteins and ufmylation. (2016)
  • Author(s): Habisov S, Huber J, Ichimura Y, Akutsu M, Rogova N, Loehr F, McEwan DG, Johansen T, Dikic I, Doetsch V, Komatsu M, Rogov VV, Kirkin V.
  • Journal Title: J Biol Chem. Volume: - Issue: 17 Pages: 9025-9041
  • DOI: 10.1074/jbc.m116.715474
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] p62/Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming (2016)
  • Author(s): Saito T, Ichimura Y, Taguchi K, Suzuki T, Mizushima T, Takagi K, Hirose Y, Nagahashi M, Iso T, Fukutomi T, Ohishi M, Endo K, Uemura T, Nishito Y, Okuda S, Obata M, Kouno T, Imamura R, Tada Y, Obata R, Yasuda D, Takahashi K, Fujimura T, Pi J, Lee MS, Ueno T, Ohe T, Mashino T, Wakai T, Kojima H, Okabe T, Nagano T et al.
  • Journal Title: Nature Commun Volume: 7 Issue: 1 Pages: 12030-12030
  • DOI: 10.1038/ncomms12030
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Biallelic Variants in UBA5 Link Dysfunctional UFM1 Ubiquitin-like Modifier Pathway to Severe Infantile-Onset Encephalopathy. (2016)
  • Author(s): Muona M, Ishimura R, Laari A,...... , Komatsu M.
  • Journal Title: Am J Hum Genet. Volume: 99 Issue: 3 Pages: 683-94
  • DOI: 10.1016/j.ajhg.2016.06.020
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Autophagy linked FYVE (Alfy/WDFY3) is required for establishing neuronal connectivity in the mammalian brain. (2016)
  • Author(s): Dragich JM, Kuwajima T, Hirose-Ikeda M, Yoon MS, Eenjes E, Bosco JR, Fox LM, Lystad AH, Oo TF, Yarygina O, Mita T, Waguri S, Ichimura Y, Komatsu M, Simonsen A, Burke RE, Mason CA, Yamamoto A.
  • Journal Title: Elife Volume: 20
  • DOI: 10.7554/elife.14810
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Synthesis of Keap1-phosphorylated p62 and Keap1-Nrf2 protein-protein interaction inhibitors and their inhibitory activity. (2016)
  • Author(s): Yasuda D, Nakajima M, Yuasa A, Obata R, Takahashi K, Ohe T, Ichimura Y, Komatsu M, Yamamoto M, Imamura R, Kojima H, Okabe T, Nagano T, Mashino T.
  • Journal Title: Bioorg Med Chem Lett Volume: 26 Issue: 24 Pages: 5956-5959
  • DOI: 10.1016/j.bmcl.2016.10.083
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Regulation of the Keap1-Nrf2 pathway by p62/SQSTM1 (2016)
  • Author(s): Katsuragi Y, Ichimura Y, Komatsu M.
  • Journal Title: Curr Opin Toxicol. Volume: 1 Pages: 54-61
  • DOI: 10.1016/j.cotox.2016.09.005
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Presentation] リン酸化p62抗体を利用したオートファジーフラックスアッセイ (2018)
  • Author(s): 一村義信、Omar Faruk、小幡美貴、小松雅明
  • Organizer: 第11回オートファジー研究会
• [Presentation] リン酸化p62を指標とするオートファジーFlux assay (2018)
  • Author(s): 一村義信、Omar Faruk、小幡美貴、小松雅明
  • Organizer: 第41回日本分子生物学会年会
• [Presentation] High-throughput screening for inhibitors of p62-Keap1 protein-protein interaction (2017)
  • Author(s): Yoshinobu Ichimura, Tetsuya Saito, Masaaki Komatsu
  • Organizer: The 8th International Symposium on Autophagy (ISA)
  • Int'l Joint Research
• [Presentation] リン酸化p62とKEAP1の結合を特異的に阻害する化合物K67の抗がん作用 (2016)
  • Author(s): 一村義信、斉藤哲也、小松雅明
  • Organizer: 第10回オートファジー研究会・第4回新学術「オートファジー」班会議
  • Place of Presentation: NASPA ニューオータニ (新潟県南魚沼郡湯沢町)
  • Year and Date: 2016-11-13
• [Presentation] p62/Sqstm1のKeap1相互作用領域をリン酸化するキナーゼのスクリーニング (2016)
  • Author(s): 一村義信、小松雅明
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 仙台国際センター、東北大学川内北キャンパス（宮城県仙台市）
  • Year and Date: 2016-09-25
• [Presentation] 肝臓がんを抑制する新規化合物K67の同定とその作用機序 (2016)
  • Author(s): 一村義信
  • Organizer: 第４回創薬等支援技術基盤プラットフォーム公開シンポジウム
  • Place of Presentation: 有楽町朝日ホール（東京都千代田区）
  • Invited