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Biochemical and pathological research of acyl-CoA : lysophosphatidic acid acyltransferases.

酰基辅酶A的生化和病理学研究：溶血磷脂酸酰基转移酶。

基本信息

批准号：
16590062
负责人：
YAMASHITA Atsushi
金额：
$ 1.98万
依托单位：
Teikyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590062/
关键词：
Lysophosphatidic acid (LPA)Acyl-CoA Acyltransferase LPAAT Phospholipid Triacylglycerol

项目摘要

Lysophosphatidic acid (LPA) acyltransferase (LPAAT) is one of the key enzymes involved in de novo biosynthesis of glycerolipids, including phospholipids and triacylglycerol. Recent reports have suggested that LPAAT may be involved in signal transduction pathways and various diseases including the progression of cancer, an inflammation, and obesity through the synthesis of phosphatidic acid (PA), bioactive lipid second messenger. LPAATα is uniformly expressed throughout most tissues ; whereas LPAATβ is differentially expressed, with the highest level found in adipose tissues. Mutations in LPAATβ are reported to cause congenital generalized lipodystrophy, suggesting that β-isoform is involved in triacylglycerol synthesis and storage in adipocytes. To understand the physiological roles and properties of LPAAT isoforms, we investigated detailed enzymology of human LPAATα and β expressed in Sf9 and mammalian cells. Alignment of amino acid sequences from various acyltransferases (LPAATs, sn- … More glycerol-3-phosphate (G3P) acyltransferase) reveals four regions with strong homology (acyltransferase motifs). We examined the role of the highly conserved amino acid residues in these four motifs in human LPAATα activity by site-directed mutagenesis. We found that the H104, D109, F146, R149, E178, and R181, all play a role in LPAAT catalysis. R149 but not R181 could be exchangeable to basic amino acid K for the activity. T180 could be exchangeable to S. These results suggest that acyltransferase motifs seem to form a catalytically important site in this family of acyltransferases. In contrast, the roles of N- or C-terminal domains have not established. The program-based prediction and accessibility of trypsin to N- or C-terminal tag suggested that C-terminus of LPAATα or β isoforms faces to cytosolic or lumen side, respectively. In contrast, N-terminus of LPAATα and β was predicted that both faced to cytosolic side. N-terminal tag of LPAATα resisted tryptic digestion, however, addition of PA, the product of the enzyme, rendered them labile to trypsin. These results suggest that the N-terminus faces to cytoslic side but hided behind the membranes, and PA induced the conformational changes to expose of the N-terminus. Deletion of 25 amino acids at N-terminal domains of LPAATα (up to predicted first transmembrane domain) reduced the LPAAT activity, suggesting that these regions are not essential but affect for the activity. Deletion of 20 amino acids at C-terminal domains of LPAATα increased the activity, suggesting that these regions are not essential and intrinsically inhibited the activity. Taken together, these results suggested the regulatory roles of N-terminal and C-terminal domains of the enzyme. Less

溶血磷脂酸（LPA）酰基转移酶（LPAAT）是参与甘油（包括磷脂和三酰甘油）从头生物合成的关键酶之一。近年来的研究表明，LPAAT可能通过合成磷脂酸（phosphatidic acid，PA）这一具有生物活性的脂质第二信使，参与信号转导途径和多种疾病的发生发展，包括癌症、炎症和肥胖症。LPAATα在大多数组织中均匀表达;而LPAATβ则差异表达，在脂肪组织中水平最高。据报道，LPAATβ的突变可引起先天性全身性脂肪营养不良，表明β-同种型参与脂肪细胞中三酰甘油的合成和储存。为了了解LPAAT亚型的生理作用和特性，我们研究了Sf 9和哺乳动物细胞中表达的人LPAATα和β的详细酶学。来自各种酰基转移酶（LPAAT，sn-125）的氨基酸序列的比对 ...更多信息甘油-3-磷酸（G3 P）酰基转移酶）揭示了具有强同源性的四个区域（酰基转移酶基序）。我们通过定点突变研究了这四个基序中高度保守的氨基酸残基在人LPAATα活性中的作用。我们发现H104、D109、F146、R149、E178和R181都在LPAAT催化中起作用。R149可与碱性氨基酸K交换，而R181不能。T180可转化为S.这些结果表明，酰基转移酶基序似乎形成一个催化的重要网站在这个家庭的酰基转移酶。相反，N-或C-末端结构域的作用尚未确立。基于程序的预测和胰蛋白酶对N-或C-末端标签的可接近性表明，LPAATα或β异构体的C-末端分别朝向胞质或管腔侧。而LPAATα和β的N端均朝向胞浆侧。LPAATα的N-末端标签能抵抗胰蛋白酶的消化，但加入酶的产物PA后，它们对胰蛋白酶不稳定。这些结果表明，N-端朝向胞浆侧，但隐藏在膜后，PA引起构象变化，暴露的N-端。LPAATα N端25个氨基酸的缺失（直至预测的第一个跨膜结构域）降低了LPAAT活性，表明这些区域不是必需的，但对活性有影响。LPAATα C端20个氨基酸的缺失增加了活性，表明这些区域不是必需的，并且内在地抑制了活性。综上所述，这些结果表明，N-末端和C-末端结构域的酶的调节作用。少