Identification of phosphatidylcholine-specifc phospholipase C

2016 Fiscal Year Annual Research Report

• Project/Area Number: 15K14470
• Research Institution: Chiba University
• Principal Investigator: 坂根 郁夫 千葉大学, 大学院理学研究科, 教授 (10183815)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Keywords: ホスファチジルコリン特異的ホスホリパーゼC / Coiled-coil structure / 相互作用 / 液体クロマトグラフィー/質量分析 / PH domain / sterile α motif domain

Outline of Annual Research Achievements

「研究の目的」，「研究実施計画」に沿って幾つかの興味ある知見が得られた．具体的には，「Ⅰ．哺乳類ホスファチジルコリン特異的ホスホリパーゼC（PC-PLC）の分子実体の解明」について以下の結果が得られた．
ジアシルグリセロールキナーゼ（DGK）δをpleckstrin-homology domain (PH), C1 domain (C1), 介在領域を含めたcatalytic domain (CD), 2つのcoiled-coil structureを含む領域 (CC), sterile α motif domain (SAM) の5つに分け，マウス脳破砕物を用いてPC-PLC活性沈降能の強さを解析した.PC-PLC活性はCCによる沈降物が最も高く, 対照と比較して有意な差があり, 約7倍の活性を示した. 一方, 他の変異体（PH, C1, CD, SAM）では沈降したPC-PLC活性が低く, 対照と有意な差は認められなかった.そして今後，DGKδ-CC との相互作用を利用してPC-PLCの分子実体の解明を目指す．
また，従来のPC-PLC活性測定法では，バックグラウンド活性が高く，弱いPC-PLC活性を定量性良く測定することが困難であった．そこで，液体クロマトグラフィー/質量分析を用いた，感度が高く定量性の良い測定法を確立した（Anal. Biochem. 2017）．すなわちち，本法は従来法に比べ，低いバックグラウンド活性 (0.02% 対 91%), 高いシグナル/ノイズ比 (7294 対 4.4), 高い感度（32倍以上），低い定量限界 (0.04 pmol 対 0.69 pmol of PC-PLC)を持っていた．

Research Products

• [Int'l Joint Research] Cambridge University(英国)
  • Country Name: UNITED KINGDOM
  • Counterpart Institution: Cambridge University
• [Int'l Joint Research] IGBMC(フランス)
  • Country Name: FRANCE
  • Counterpart Institution: IGBMC
• [Journal Article] Dioleoyl-phosphatidic acid selectively binds to α-synuclein and strongly induces its aggregation. (2017)
  • Author(s): Mizuno, S., Sasai, H., Kume, A., Takahashi, D., Satoh, M., Kado, S. and Sakane, F.
  • Journal Title: FEBS Lett. Volume: 591 Pages: 784-791
  • DOI: 10.1002/1873-3468.12592
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Development of a liquid chromatography-mass spectrometry based enzyme activity assay for phosphatidylcholine-specific phospholipase C. (2017)
  • Author(s): Murakami, C., Mizuno, S., Kado, S. and Sakane, F.
  • Journal Title: Anal. Biochem. Volume: 526 Pages: 43-49
  • DOI: 10.1016/j.ab.2017.03.010
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Deficiency of diacylglycerol kinase η induces lithium-sensitive mania-like behaviors. (2016)
  • Author(s): Isozaki, T., Komenoi, S., Lu Q., Usuki, T., Tomokata, S., Matsutomo, D., Sakai, H. Bando, K., Kiyonari, H. and Sakane, F.
  • Journal Title: J. Neurochem. Volume: 138 Pages: 448-456
  • DOI: 10.1111/jnc.13661
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Myristic acid enhances diacylglycerol kinase δ-dependent glucose uptake in myotubes. (2016)
  • Author(s): Wada, Y., Sakiyama, S., Sakai, H. and Sakane, F.
  • Journal Title: Lipids Volume: 51 Pages: 897-903
  • DOI: 10.1007/s11745-016-4162-9
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Distinct 1- and 2-monoacylglycerol kinase activities of diacylglycerol kinase isozymes. (2016)
  • Author(s): Sato, Y., Murakami, C., Yamaki, A., Mizuno, S., Sakai, H. and Sakane, F.
  • Journal Title: Biochim. Biophys. Acta - Proteins Proteomics Volume: 1864 Pages: 1170-1176
  • DOI: 10.1016/j.bbapap.2016.06.012
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Behavioral and pharmacological phenotypes of brain-specific diacylglycerol kinase δ-knockout mice. (2016)
  • Author(s): Usuki, T., Takato, T., Lu Q., Sakai, H., Bando, K., Kiyonari, H. and Sakane, F.
  • Journal Title: Brain Res. Volume: 1648 Pages: 193-201
  • DOI: 10.1016/j.brainres.2016.07.017
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Activation of conventional and novel protein kinase C isozymes by different diacylglycerol molecular species. (2016)
  • Author(s): Kamiya, Y., Mizuno, S., Komenoi, S., Sakai, H., and Sakane, F.
  • Journal Title: Biochem. Biophys. Rep. Volume: 7 Pages: 361-366
  • DOI: 10.1016/j.bbrep.2016.07.017
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Cloning and characterization of novel testis-specific diacylglycerol kinase η splice variant 3 and 4. (2016)
  • Author(s): Murakami, E., Shionoya, T. Komenoi, S., Suzuki, Y. and Sakane, F.
  • Journal Title: PLOS ONE Volume: 11 Pages: e0162997
  • DOI: 10.1371/journal.pone.0162997
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Diacylglycerol kinase ζ generates dipalmitoyl-phosphatidic acid species during neuroblastoma cell differentiation. (2016)
  • Author(s): Mizuno, S., Kado, S., Goto, K., Takahashi, D. and Sakane, F.
  • Journal Title: Biochem. Biophys. Rep. Volume: 8 Pages: 352-359
  • DOI: 10.1016/j.bbrep.2016.10.004
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Diacylglycerol kinases as emerging potential drug targets for a variety of diseases: An update. (2016)
  • Author(s): Sakane, F., Mizuno, S. and Komenoi, S.
  • Journal Title: Front. Cell Dev. Biol. Volume: 4 Pages: Article 82
  • DOI: 10.3389/fcell.2016.00082
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] Roles of diacylglycerol kinase isozymes and phosphatidic acid species in neurological functions and disorders. (2017)
  • Author(s): Sakane, F.
  • Organizer: The 7th International Symposium on Diacylglycerol Kinase
  • Place of Presentation: Kobe, , Hyogo, Japan
  • Year and Date: 2017-03-13 – 2017-03-13
  • Int'l Joint Research / Invited
• [Presentation] Expression and purification of diacylglycerol kinase α catalytic domain for crystallographic studies. (2017)
  • Author(s): Takahashi, D., Satoh, E. and Sakane, F.
  • Organizer: The 7th International Symposium on Diacylglycerol Kinase
  • Place of Presentation: Kobe, , Hyogo, Japan
  • Year and Date: 2017-03-13 – 2017-03-13
  • Int'l Joint Research
• [Presentation] New evaluation method of diacylglycerol-generating activity of phosphatidylcholine-specific phospholipase C using mass spectrometry. (2017)
  • Author(s): Murakami, C. and Sakane, F.
  • Organizer: The 7th International Symposium on Diacylglycerol Kinase
  • Place of Presentation: Kobe, , Hyogo, Japan
  • Year and Date: 2017-03-13 – 2017-03-13
  • Int'l Joint Research
• [Presentation] 神経分化誘導時に産生されるジアシルグリセロール及びホスファチジン酸分子種の解析 (2016)
  • Author(s): 水野悟，坂根郁夫
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 宮城・仙台
  • Year and Date: 2016-09-25 – 2016-09-27
• [Presentation] ジアシルグリセロールキナーゼηノックアウトマウスのそう様行動惹起の分子メカニズムアシルグリセロールキナーゼηノックアウトマウスのそう様行動惹起の分子メカニズム (2016)
  • Author(s): 米野井優，坂根郁夫
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 宮城・仙台
  • Year and Date: 2016-09-25 – 2016-09-27
• [Presentation] 質量分析法を用いた新規ホスファチジルコリン特異的ホスホリパーゼC活性測定法の開発 (2016)
  • Author(s): 村上千明，水野悟，坂根郁夫上千明，水野悟，坂根郁夫
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 宮城・仙台
  • Year and Date: 2016-09-25 – 2016-09-27
• [Presentation] ジアシルグリセロールキナーゼη欠損によるリチウム感受性躁様行動の誘導 (2016)
  • Author(s): 陸強，磯崎丈志，米野井優，坂根郁夫
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 仙台
  • Year and Date: 2016-09-25 – 2016-09-27
• [Presentation] ジアシルグリセロールキナーゼηのプレクストリン相同ドメインの精製とその性質アシルグリセロールキナーゼηのプレクストリン相同ドメインの精製とその性質 (2016)
  • Author(s): 川瀬功暉，坂根郁夫
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 宮城・仙台
  • Year and Date: 2016-09-25 – 2016-09-27
• [Presentation] メラノーマ細胞に対するジアシルグリセロールキナーゼα選択的活性制御化合物の細胞死誘導効果 (2016)
  • Author(s): 八巻篤実，劉可，国井奈央子，坂根郁夫
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 宮城・仙台
  • Year and Date: 2016-09-25 – 2016-09-27
• [Presentation] C2C12筋芽細胞の筋分化におけるジアシルグリセロールキナーゼδの機能 (2016)
  • Author(s): 堺弘道，松本健一，坂根郁夫
  • Organizer: 第58回日本脂質生化学会
  • Place of Presentation: 秋田・秋田
  • Year and Date: 2016-06-09 – 2016-06-10
• [Presentation] 神経分化誘導時に産生されるジアシルグリセロール及びホスファチジン酸分子種の解析 (2016)
  • Author(s): 水野悟，坂根郁夫
  • Organizer: 第58回日本脂質生化学会
  • Place of Presentation: 秋田・秋田
  • Year and Date: 2016-06-09 – 2016-06-10
• [Presentation] ジアシルグリセロールキナーゼη欠損によるリチウム感受性躁様行動の誘導 (2016)
  • Author(s): 陸強，磯崎丈志，米野井優，坂根郁夫
  • Organizer: 第58回日本脂質生化学会
  • Place of Presentation: 秋田・秋田
  • Year and Date: 2016-06-09 – 2016-06-10
• [Presentation] Diacylglycerol kinase ζ produces 16:0-containing phosphatidic acid molecular species during neuroblastoma cell differentiation. (2016)
  • Author(s): Mizuno, S. and Sakane, F.
  • Organizer: 2016 Annual Meeting of the American Society for Biochemistry and Molecular Biology at Experimental Biology 2016
  • Place of Presentation: San Diego, California, USA
  • Year and Date: 2016-04-02 – 2016-04-06
  • Int'l Joint Research
• [Remarks] 千葉大学大学院理学研究科 基盤理学専攻 生体機能化学研究室
  • URL: http://sakane32.wixsite.com/biofunctionchemistry
• [Patent(Industrial Property Rights)] ＤＧＫδノックアウトマウス及びこれを用いた方法 (2017)
  • Inventor(s): 坂根郁夫
  • Industrial Property Rights Holder: 坂根郁夫
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 特願2017-004729
  • Filing Date: 2017-01-13
• [Patent(Industrial Property Rights)] ＤＧＫηノックアウトマウス及びこれを用いた方法 (2016)
  • Inventor(s): 坂根郁夫
  • Industrial Property Rights Holder: 坂根郁夫
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 特願2016-220080
  • Filing Date: 2016-11-10