肝細胞における遊離コレステロールの毒性発現機構

• Project/Area Number: 17K08295
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Biological pharmacy
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: 牧野 麻美 国立研究開発法人理化学研究所, 開拓研究本部, 協力研究員 (20373368)
• Project Period (FY): 2017-04-01 – 2019-03-31
• Project Status: Discontinued (Fiscal Year 2018)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2018: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2017: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: コレステロール / 脂肪滴 / 肝細胞 / 中性脂質

Outline of Annual Research Achievements

私たちは培養肝細胞Huh7を用い、アシルコレステロール・アシルトランスフェラーゼ（ACAT）阻害剤、(1s,2s)-2-[3-(2,2-dimethylpropyl)-3-
nonylureido]aminocyclohexane-1-yl 3-[N-(2,2,5,5-tetramethyl-1,3-dioxane-4-carbonyl)amino]propionate (F1394)を加えることにより細胞内の遊離コレステロールを上昇させると、低分子量Gタンパク質、Rab18活性に依存して脂肪滴と小胞体の融合が起こることを見出した（Makino et al. Mol Biol Cell 27, 3293(2016)）。遊離コレステロールの導入に利用されるメチル-b;-シクロデキストリンとコレステロールとの複合体（Mb;CD/Chol）をF1394と併用すると細胞死が観察される。この実験系を用い、siRNAライブラリーを用いて不活性化すると細胞死を起こさないタンパク質の検索を行い、Golgi associated, gamma adaptin earcontaining, ARF binding protein 2 (GGA2)、sphingosine-1-phosphate phosphatase 1 (SGPP1)、Rab18、ADP ribosylation factor 1 (ARF1)、inositol polyphosphate-5-phosphatase K (INPP5K)の５つのタンパク質を同定した。本研究では脂肪滴のサイズを正確に測定することが求められる。これまで私たちは精製した脂肪滴のサイズ測定には光散乱を用いていたが、光散乱は粒子サイズが不均一の場合誤差が大きいため、個々の脂肪滴のサイズを測定できるナノ粒子マルチアナライザーを導入した。

Research Products

• [Journal Article] Photoswitchable phospholipid FRET acceptor: Detergent free intermembrane transfer assay of fluorescent lipid analogs (2017)
  • Author(s): Sumi Mariko、Makino Asami、Inaba Takehiko、Sako Yasushi、Fujimori Fumihiro、Greimel Peter、Kobayashi Toshihide
  • Journal Title: Scientific Reports Volume: 7 Issue: 1 Pages: 1-10
  • DOI: 10.1038/s41598-017-02980-x
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Raft-based sphingomyelin interactions revealed by new fluorescent sphingomyelin analogs (2017)
  • Author(s): Masanao Kinoshita, Kenichi GN Suzuki, Nobuaki Matsumori, Misa Takada, Hikaru Ano, Kenichi Morigaki, Mitsuhiro Abe, Asami Makino, Toshihide Kobayashi, Koichiro M Hirosawa, Takahiro K Fujiwara, Akihiro Kusumi, Michio Murata
  • Journal Title: J Cell Biol Volume: 未定 Issue: 4 Pages: 1183-1204
  • DOI: 10.1083/jcb.201607086
  • NAID: 120005998045
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] A novel spingomyeling/cholesterol specific probe reveals the dynamics of the membrane domain during virus and in Nieman-Pick type C (2016)
  • Author(s): Makino, A., Sako, Y. et al.
  • Journal Title: FASEB J. Volume: 31 Issue: 4 Pages: 1301-1322
  • DOI: 10.1096/fj.201500075r
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Phospholipase C beta1 induces membrane tubulation and is involved in caveola e formation (2017)
  • Author(s): Toshihide Kobayashi, Takehiko Inaba, Takuma Kishimoto, Motohide Murate, Takuya Tajima, Mitsuhiro Abe, Asami Makino, Nario Tomishige, Shinji Takeoka
  • Organizer: 第９１回日本生化学会大会「
  • Invited
• [Presentation] A NOVEL SPHINGOMYELIN/CHOLESTEROL DOMAIN-SPECIFIC PROBE REVEALS THE DYNAMICS OF THE MEMBRANE DOMAINS DURING VIRUS RELEASE AND IN NIEMANN-PICK TYPE C (2017)
  • Author(s): Makino A, Abe M, Ishitsuka R, Murate M, Kishimoto T, Sakai S, Hullin-Matsuda F, Inaba T, Greimel P and Kobayashi T
  • Organizer: XII Sphingolipid Club Meeting
  • Int'l Joint Research
• [Presentation] Acute accumulation of free cholesterol induces the degradation of perilipin 2 and Rab18‐ dependent fusion of ER and lipid droplets in cultured human hepatocytes (2017)
  • Author(s): A.Makino, F. Hullin‐Matsuda, M. Murate, M. Abe, N. Tomishige, H. Vidal, M. Lagarde, I. Delton and T. Kobayashi
  • Organizer: 17th journee scientifique de l’IMBL Glycosciences et Lipidomique
  • Int'l Joint Research
• [Presentation] Phospholipase Cb1 induces membrane tubulation and is involved in caveolae formation (2017)
  • Author(s): Takehiko Inaba, Takuma Kishimoto, Motohide Murate, Takuya Tajima, Shota Sakai, Mitsuhiro Abe, Asami Makino, Nario Tomishige, Reiko Ishitsuka, Yasuo Ikeda, Shinji Takeoka, and Toshihide Kobayashi
  • Organizer: EMBO Symposium Molecular and Cell Biology of Membranes
  • Int'l Joint Research