細胞内スフィンゴ脂質選別輸送の分子機構と生体膜ドメイン形成にはたす役割

• Project/Area Number: 12140206
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: National Institute of Infectious Diseases
• Principal Investigator: 花田 賢太郎 国立感染症研究所, 細胞化学部, 室長 (30192701)
• Co-Investigator(Kenkyū-buntansha): 深澤 征義 国立感染症研究所, 細胞化学部, 主任研究官 (20291130)
• Project Period (FY): 2000 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥68,400,000 (Direct Cost: ¥68,400,000); Fiscal Year 2004: ¥17,200,000 (Direct Cost: ¥17,200,000); Fiscal Year 2003: ¥17,200,000 (Direct Cost: ¥17,200,000); Fiscal Year 2002: ¥17,000,000 (Direct Cost: ¥17,000,000); Fiscal Year 2001: ¥17,000,000 (Direct Cost: ¥17,000,000)
• Keywords: スフィンゴミエリン / セラミド / スフィンゴ脂質 / 細胞内輸送 / 脂質 / CERT / ホスファチジルイノシトール4リン酸 / レトロウイルスベクター / 遺伝性感覚神経障害 / セリンパルミトイル転移酵素 / 突然変異株 / コレステロール / 膜輸送 / ラフト / 膜ドメイン

Research Abstract

膜脂質の細胞内選別輸送のメカニズムはほとんど未解明のままである.主要膜リン脂質の一つであるスフィンゴミエリン(SM)の生合成では,小胞体で合成されたセラミドがゴルジ体に移行してSMへと変換される.我々は、細胞内セラミド選別輸送の欠損CHO細胞変異株を分離・解析し、その欠損を回復する遺伝子をクローニングする手法を通じて、セラミド選別輸送を担う特異的因子CERTの同定に成功した。CERTは、小胞体に会合するドメイン、ゴルジ体に会合するドメイン、およびセラミドを脂質膜から特異的に引き抜くドメインを持つことを昨年度までに明らかにした。
本年度は、人工膜を用いた膜間脂質転移アッセイによってCERTの基質認識選択性を詳細に解析した。その結果、CERTは、(1)セラミドの4つの立体化学異性体のうち、天然型のD-erythoro-セラミドのみを認識し、(2)スフィンゴシン、SM、コレステロールなどの他の脂質は認識できない。ただし、ジアシルグリセロールはわずかに認識する一方、(3)ジヒドロ型およびファイト型セラミドは認識し、また、様々なアミド鎖長のセラミドも認識することが明らかとなった。また、CERTとセラミド基質の結合比は、1対1であることも明らかにした。さらに、我々が以前開発したセラミド輸送阻害剤・HPA12は、CERTのアンタゴニストであることも示した。
一方、海外の研グループとの共同研究によって、酸化ストレスによる細胞死において新合成のセラミド蓄積が重要であることや、ジフテリア毒素の侵入に宿主細胞スフィンゴ脂質が必要であることなどを示唆した。

Research Products

• [Journal Article] Differential effects of modification of membrane cholesterol and sphingolipids on the conformation, function, and trafficking of the G protein-coupled cholecystokinin receptor (2005)
  • Author(s): Kaleeckal G.Harikumar
  • Journal Title: Journal of Biological Chemistry 280 Pages: 2176-2185
• [Journal Article] CERT mediates intermembrane transfer of various molecular species of ceramides (2005)
  • Author(s): Keigo Kumagai
  • Journal Title: Journal of Biological Chemistry 280 Pages: 6488-6495
• [Journal Article] Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids (2005)
  • Author(s): Bjorn Spilsberg
  • Journal Title: Biochemical and Biopysical Research Communications 329 Pages: 465-473
• [Journal Article] De novo ceramide accumulation due to inhibition of its conversion to complex sphingolipids in apoptotic photosensitized cells (2004)
  • Author(s): Vladislav Dolgachev
  • Journal Title: Journal of Biological Chemistry 279 Pages: 23238-23249
• [Journal Article] Ceramide sensitizes astrocytes to oxidative stress : protective role of cannabinoids (2004)
  • Author(s): Arkaitz Carracedo
  • Journal Title: Biochemical Journal 380 Pages: 435-440
• [Publications] Kentaro Hanada: "Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism"Biochimica et Biophysics Acta. 1632. 16-30 (2003)
• [Publications] Vladislav Dalgachev: "The reactive oxygen species generation is independent of de novo sphingolipids in apoptotic photosensitized cells"Experimental Cell Research. 288. 425-436 (2003)
• [Publications] Yoshitaka Nakamura: "Stereoselective synthesis and structure-activity relationship of novel ceramide trafficking inhibitors, (1R,3R)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12) and its analogues"Journal of Medicinal Chemistry. 46. 3688-3695 (2003)
• [Publications] Masayuki Nakamura: "Chemistry and biology of khafrefungin. Large-scale synthesis, design, and structure-activity relationship of khafrefungin, an antifungal agent"Organic & Biomolecular Chemistry. 1. 3362-3376 (2003)
• [Publications] Kentaro Hanada: "Molecular machinery for non-vesicular trafficking of ceramide"Nature. 426. 803-809 (2003)
• [Publications] Yukiko Miura: "Hydrolysis of sphingosylphosphocholine by neutral sphingomyelinases"FEBS Letters. 557. 288-292 (2004)
• [Publications] Khemissa Bejaoui: "Hereditary sensory neuropathy type 1 mutations confer dominant-negative effects on serine palmitoyltransferase, critical for sphingolipid synthesis"The Journal of Clinical Investigation. 110・9. 1301-1308 (2002)
• [Publications] Frank Lafont: "Initial steps of Shigella infection depend on the cholesterol-sphingolipids raft-mediated CD44-IpaB interaction"The EMBO Journal. 21・17. 4449-4457 (2002)
• [Publications] Bambang Wispriyono: "A role of the de novo sphingolipids in apoptosis of photosensitized cells"Experimental Cell Research. 279. 153-165 (2002)
• [Publications] Satoshi Yasuda: "Localization, topology, and function of the LCB1 subunit of serine palmitoyltransferase in mammalian cells"The Journal of Biological Chemistry. 278・6. 4176-4183 (2003)
• [Publications] Kentaro Hanada: "Purification of mammalian serine palmitoyltransferase, a hetero-subunit enzyme for sphingolipid biosynthesis, by affinity-peptide chromatography"Membrane Protein Protocols. (印刷中).
• [Publications] Satoshi Yasuda: "A novel inhibitor of ceramids Tisfficking from the endoplasmic reticulum to the site of sphingompelin synthesis"The Journal of Biological Chemistry. 276巻47号. 43994-44002 (2001)
• [Publications] Masaharu Ueno: "Catalytic enantio selective synthesis of a novel inhibitor of ceramide trafficking,(1R,3R)-N-13-hydroxy-1-hydrixymethyl-3-plerylpropyl)-dodecomida(HPA-12)"Tetrahedron Letters. 42巻. 7863-7865 (2001)
• [Publications] 花田_賢太郎: "高等動物細胞におけるスフィンゴ脂質の生合成と細胞内輸送"生化学. 73巻・12号. 1397-1409 (2001)
• [Publications] Samit Chatterjee: "GPI anchoring leads to sphingolipid-dependent retention of endocytosed proteins in the recycling endasomal compartment"The EMBO Journal. 20巻・7号. 1583-1592 (2001)
• [Publications] Shu Kobayashi: "Convergent total synthesis of Khafrefungin and its inhibitory activity of fungal sphingolipid syntheses"The Journal of Organic Chemistry. 66巻・16号. 5580-5584 (2001)
• [Publications] Yukiko Miura: "Gs signaling is intact after disruption of lipidrafts"Biochemistry. 40巻・50号. 15418-15423 (2001)
• [Publications] Kentaro Hanada: "D-Serine inhibits serine palmitoyltrans ferose, the enzyme Catalyzing the first step of sphingolipid biosynthesis"FEBS Letters. 474巻. 63-65 (2000)
• [Publications] Tomoko Funakoshi: "Reconstitution of ATP-and Cytosol-dependent transport of de novo synthesized ceramide to the site of sphingomyelin synthesis in semi-intact cells"The Journal of Biologicol Chemistry. 275巻. 29938-29945 (2000)
• [Publications] Kentaro Hanada: "Selection of mammalian cell mutants in sphingolipid biosynthesis"Methods in Enzymology. 312巻. 304-317 (2000)
• [Publications] Masayoshi Fukasawa: "Reduction of sphingomyelin level without accumulation of cevamide in eninese hamster ovary cells aflects detergent-resistant membrane domains"The Journal of Biological Chemistry. 275巻. 34028-34034 (2000)
• [Publications] Christiaan van Ooji: "Host cell-derived sphingolipids are reguired for the intracellular growth of Chlamydia trachomatis"Cellular Microbiology. 2巻. 627-637 (2000)