Analysis of membrane lipid sorting by using an in vitro reconstitution system

使用体外重构系统分析膜脂分选

• 批准号: 12680621
• 负责人: HANADA Kentaro
• 金额: $ 2.3万
• 依托单位: National Institute of Infectious Diseases
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680621/
• 关键词: Sphingomyelin; Ceramide; Sphingolipids; Intracellular trafficking; Lipids; Hereditary sensory neuropathy; Serine palmitoyltransferase; mutant; セリンパルミトイル転移酵素; 知覚神経障害; 阻害剤; 膜脂質; 膜輸送; HPA-12

Transport of ceramide synthesized at the endoplasmic reticulum to the Golgi compartment where sphingomyelin(SM) synthase exists was reconstituted within semi-intact Chinese hamster ovary (CHO) cells. When [^3H]ceramide that had been produced from [^3H]sphingosine at 15℃ in perforated cells was chased at 37℃ [^3H]ceramide-to-[^3H]SM conversion occurred in a cytosol-dependent manner. In various aspects (i. e., kinetics, ATP-dependence, and temperature-dependence), [^3H]ceramide-to-[^3H]SM conversion in perforated cells was consistent with that in intact cells. The cytosol from LY-A strain, a CHO cell mutant defective in endoplasmic reticulum-to-Golgi transport of ceramide, did not support [^3H]ceramide-to-^3H]SM conversion in perforated wild-type cells, while the wild-type cytosol rescued the conversion in perforated LY-A cells. Brefeldin A-treated cells, in which the endoplasmic reticulum and the Golgi apparatus were merged, no longer required cytosol for conversion of [^3H]ceramide to … More [^3H]SM. These results indicated that the assay of [^3H]ceramide-to-[^3H]SM conversion in semi-intact cells is a faithful in vitro assay for the activity of cytosol-dependent transport of ceramide, and that LY-A cells are defective in a cytosolic factor involved in ceramide transport. Moreover, we developed a novel inhibitor of ceramide transport. (1R,3R)-HPA-12, an analog of ceramide, inhibits ATP-dependent transport of ceramide from the ER to the site of SM synthesis without appreciable inhibition of ER-to-Golgi trafficking of glycoproteins.During the period of this research, we also made progress in the study on serine palmitoyltransfease (SPT), the enzyme catalyzing the initial step of sphingolpid biosynthesis. SPT consists of two subunits, LCB1 and LCB2. We showed that the LCB1 subunit is indispensable for the maintenance of the LCB2 subunit in mammalian cells. Hereditary sensory neuropathy type 1 (HSN1), a dominantly inherited degenerative disorder of the peripheral nerves, is accompanied by specific misssense mutations in the LCB1 subunit. We indicated that the HSN1-associated mutations in LCB1 confer dominant-negative effects on the SPT enzyme. Less

在半独立性的中国仓鼠卵巢（CHO）细胞中重构，在内质网中合成的神经酰胺在内质网中合成的转运。当[^3H]在穿孔细胞中从[^3H]在15°处产生的[^3H]鞘氨酸产生的神经酰胺以37℃[^3H]的神经酰胺至[^3H]的sm转化以细胞溶胶依赖性方式发生。在各个方面（即，动力学，ATP依赖性和温度依赖性），[^3H] ceramide-to- [^3H]在穿孔细胞中的SM转化与完整的细胞中的SM转化与该sm转化。 Ly-A菌株的胞质溶液是一种CHO细胞突变体在内质性网状到高尔基体的转运，不支持[^3H] ceramide-to-^3H] SM在穿孔的野生型细胞中转化，而野生型细胞质则在野生型胞质溶液中拯救了培养的ly-a ly-a细胞的转化。 Brefeldin A处理的细胞合并了内质网和高尔基体，不再需要细胞溶胶将[^3H]神经酰胺转化为…更多[^3H] SM。这些结果表明，半独立性细胞中[^3H]神经酰胺至 - [^3H] SM转化的测定是一种忠实的体外测定法，用于依赖神经酰胺的细胞质依赖性转运的活性，而LY-A细胞在陶瓷酰胺转运中所涉及的细胞醇中有缺陷。此外，我们开发了一种新型的神经酰胺运输抑制剂。 （1R，3R）-HPA-12（一种类似神经酰胺）抑制了ATP依赖于神经酰胺从ER到SM合成部位的转运，而无需欣赏糖蛋白的ER到高尔基糖蛋白的良好抑制。在这项研究时期，我们还在塞氨酸palmitoyltransfease（SPT）的研究中取得了进展，该研究在SPTEN上（SPT）（SPT）（SPT）（SPT）（SPT）（SPT）（SPT）（SPT）（SPT）（鞘磷脂生物合成。 SPT由LCB1和LCB2的两个亚基组成。我们表明，LCB1亚基对于哺乳动物细胞中LCB2亚基的维持是必不可少的。遗传性感觉神经病1型（HSN1）是外围神经的主要遗传退化性疾病，是通过LCB1亚基中的特定误差突变来完成的。我们指出，LCB1会议会议中与SPT酶的显性阴性作用的相关突变。较少的

项目成果

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. 1397-1409 (2001)

Kentaro Hanada：“高等动物细胞中鞘脂的生物合成和细胞内运输”生物化学 73. 1397-1409 (2001)。

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)

安田聪：“哺乳动物细胞中丝氨酸棕榈酰转移酶的LCB1亚基的定位、拓扑结构和功能”生物化学杂志278・6（2003）。

Satoshi Yasuda, Hidetoshi Kitagawa, Masaharu Ueno, Haruo Ishitani, Masayoshi Fukasawa, Masahiro Nishijima, Shu Kobayashi and Kentaro Hanada: "A novel inhibitor of ceramide trafficking from endoplasmic reticulum to the site of sphingomyelin synthesis"J. Bi

Satoshi Yasuda、Hidetoshi Kitakawa、Masaharu Ueno、Haruo Ishitani、Masayoshi Fukasawa、Masahiro Nishijima、Shu Kobayashi 和 Kentaro Hanada：“神经酰胺从内质网运输到鞘磷脂合成位点的新型抑制剂”J.

Khemissa Bejaoui: "Hereditary sensory neuropathy type 1 mutations confer dominant-negative effects on serine palmitoyltransferase, critical sphingolipid synthesis"The Journal of Clinical Investigation. 110. 1301-1308 (2002)

Khemissa Bejaoui：“遗传性感觉神经病 1 型突变对丝氨酸棕榈酰转移酶、关键鞘脂合成产生显性负效应”《临床研究杂志》。

Kentaro Hanada: "Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism"Biochimica et Biophysica Acta. 1632. 16-30 (2003)

Kentaro Hanada：“丝氨酸棕榈酰转移酶，鞘脂代谢的关键酶”Biochimica et Biophysicala Acta。