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

在哺乳动物细胞和组织中，各种脂肪酸分布于膜磷脂中。个别磷脂的脂肪酰基残基似乎受到严格的代谢调节。通常，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。甘油磷脂的sn-2酰基链的快速转换最初由Lands描述，他提出膜磷脂的酰基部分通过磷脂酶A_2s的作用和随后通过溶血磷脂酰基转移酶的作用而快速代谢（土地周期）;然而，参与这种转换的基因还没有报道。在这个项目中，我们建立了一个筛选系统，以鉴定C.发现一个未知的基因，我们命名为mboa-7，是将PUFAs整合到磷脂酰肌醇中所必需的（P 关于我们 （一）。在mboa-7突变体中，将外源性PUFA掺入活蠕虫的PI和微粒体中的lysoPI酰基转移酶（LPIAT）活性大大降低。此外，表达重组mboa-7及其人类同源物的转基因蠕虫的膜组分表现出显著增加的LPIAT活性。mboa-7编码膜结合O-酰基转移酶家族的成员，表明mboa-7是LPIAT。磷脂酶A_2s（PAF-AH（II））基因敲除小鼠，其主要表达于上皮细胞，如肾近端小管、远端小管、肠柱状上皮细胞和肝细胞。Pafah 2 ^<-/->小鼠发育正常，在表型上与野生型小鼠无区别。然而，来自Pafah2%小鼠的小鼠胚胎成纤维细胞对叔丁基过氧化氢处理比来自野生型小鼠的那些更敏感。四氯化碳（CCl_4）注射后，Pafah 2 ^<-/->小鼠肝损伤恢复延迟。此外，CCl_4处理后，PAF-AH（II）的体外底物8-iso-PGF_2 α的酯化形式在Pafah 2 ^<-/->小鼠肝脏中的含量高于野生型小鼠。这些结果表明PAF-AH（II）参与了酯化的8-iso-PGF_2 α的代谢，对氧化应激引起的组织损伤具有保护作用。少

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