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New approaches to functional elucidation of unknown genes related to lipid metabolism : combination of mutational approaches using C. elegans and metabolite analyses using mass spectrometry

与脂质代谢相关的未知基因功能阐明的新方法：使用秀丽隐杆线虫的突变方法和使用质谱的代谢物分析相结合

基本信息

批准号：
17207008
负责人：
ARAI Hiroyuki
金额：
$ 32.86万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17207008/
关键词：
C. elegans phosnholinid fatty acid acvltransferase

项目摘要

Various kinds of fatty acids are distributed in membrane phospholipids in mammalian cells and tissues. The fatty acyl residues of individual phospholipids appear to be under strict metabolic regulation. In general, saturated fatty acids are esterified at the sn-1 position, whereas polyunsaturated fatty acids (PUFAs), such as arachidonic acid, are commonly found at the sn-2 position. The rapid turnover of the sn-2 acyl chains of glycerophospholipids was originally described by Lands, who proposed that acyl moieties of membrane phospholipids are rapidly metabolized by the action of phospholipase A_2s and subsequently by lysophospholipid acyltransferases (Lands' cycle); however the genes involved in this turnover have not been reported.In this project, we established a screening system to identify genes required for utilization of exogenous PUFAs in C. elegans and found that an uncharacterized gene, which we named mboa-7, is required for incorporation of PUFAs into phosphatidylinositol (P … More I). Incorporation of exogenous PUFA into PI of the living worms and lysoPI acyltransferase (LPIAT) activity in the microsomes were greatly reduced in mboa-7 mutants. Furthermore, the membrane fractions of transgenic worms expressing recombinant mboa-7 and its human homologue exhibited remarkably increased LPIAT activity. mboa-7 encodes a member of the membrane-bound O-acyltransferase family, suggesting that mboa-7 is LPIAT. We also identified an acyltransferase which catalyzes acyltransferase activity toward lysoPC, lysoPS, and lysoPE.In regards to phospholipase A_2s, we generated knockout mice of PAF-AH (II), which hydrolyzes oxidatively fragmented fatty acyl chains attached to phospholipids and was predominantly expressed in epithelial cells such as kidney proximal and distal tubules, intestinal column epithelium and hepatocytes. Pafah2^<-/-> mice developed normally and were phenotypically indistinguishable from wild-type mice. However, mouse embryonic fibroblasts derived from Pafah2% mice were more sensitive to tert-butylhydroperoxide treatment than those derived from wild-type mice. When carbon tetrachloride (CCl_4) was injected into mice, Pafah2^<-/-> mice showed a delay in hepatic injury recovery. Moreover, following CCl_4 administration, liver levels of the esterified form of 8-iso-PGF_2a, a known in vitro substrate of PAF-AH (II), were higher in Pafah2^<-/-> mice than in wild-type mice. These results indicate that PAF-AH (II) is involved in the metabolism of esterified 8-iso-PGF_2a and protects tissue from oxidative stress-induced injury. Less

哺乳动物细胞和组织的细胞膜磷脂中分布着多种脂肪酸。个别磷脂的脂肪酰基残基似乎受到严格的代谢调节。一般来说，饱和脂肪酸在sn-1位酯化，而多不饱和脂肪酸(PUFA)，如花生四烯酸，通常在sn-2位发现。甘油磷脂sn-2酰基链的快速周转最初是由Lands描述的，他提出膜磷脂的酰基部分在磷脂酶A_2s的作用下迅速代谢，然后被溶血磷脂酰基转移酶(Lands‘s Cycle)代谢，但参与这种代谢的基因还没有报道。在这个项目中，我们建立了一个筛选系统来鉴定线虫利用外源多不饱和脂肪酸所需的基因，并发现了一个未被鉴定的基因，我们命名为mboA-7，以将多不饱和脂肪酸结合到磷脂酰肌醇(P…更多I)。在MboA-7突变体中，活虫PI中掺入外源PUFA的量和微粒体中的溶菌素PI酰基转移酶(LPIAT)活性显著降低。此外，表达重组MboA-7及其人类同源物的转基因蠕虫的膜部分显示出显著的LPIAT活性。MboA-7编码膜结合型O-酰基转移酶家族的一个成员，提示它是LPIAT。在磷脂酶A_2S方面，我们获得了PAF-AH(II)基因敲除小鼠，它能降解附着在磷脂上的氧化断裂的脂肪酰链，主要在肾近端和远端小管、肠柱上皮和肝细胞等上皮细胞中表达。Pafah2^&lt；-/-&gt；小鼠发育正常，与野生型小鼠没有明显区别。然而，来自Pafah2%小鼠的小鼠胚胎成纤维细胞比来自野生型小鼠的胚胎成纤维细胞对叔丁基氢过氧化氢处理更敏感。小鼠体内注射四氯化碳(CCl_4)后，Pafah2^&lt；-/-&gt；小鼠肝损伤恢复明显延迟。此外，给予CCl_4后，PAF-AH(II)的体外底物8-iso-PGF2a的酯化形式在Pafah2^&lt；-/-&gt；小鼠肝脏中的水平高于野生型小鼠。这些结果表明，PAF-AH(II)参与了酯化的8-iso-PGF2a的代谢，对氧化应激诱导的组织损伤具有保护作用。较少