ENZYMES IN PHOSPHATIDIC ACID BIOSYNTHESIS

磷脂酸生物合成中的酶

• 批准号: 2906047
• 负责人: Hei Sook Sul
• 金额: $ 19.03万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-10 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2906047
• 关键词: Escherichia coli; active sites; acyl coA; acyltransferase; antisense nucleic acid; crosslink; endoplasmic reticulum; enzyme activity; enzyme structure; esterification; fatty acylation; lipid biosynthesis; lipid metabolism; messenger RNA; mitochondria; mutant; oxidation; phosphatidate; phosphoglycerides; site directed mutagenesis; tissue /cell culture

DESCRIPTION (Adapted from applicant's abstract): Phosphatidic acid, which serves as the backbone of glycerophospholipid biosynthesis, is synthesized de novo by the sequential acylation of glycerol-3-phosphate. Glycerol-3-phosphate acyltransferase (GPAT) acylates the sn-1 position to form 1-acylglycerol-3-phosphate (lysophosphatidate) and this is the first committed and presumed to be rate-limiting, step in glycerophospholipid synthesis. 1-Acylglycerol-3-phosphate acyltransferase (AGPAT) acylates the sn-2 position to generate 1,2-diacylglycerol-3-phosphate (phosphatidate). Both GPAT and AGPA activities are found in the endoplasmic reticulum. In addition, GPAT that prefers saturated fatty acyl-CoA is found in mitochondria. Fatty acyl-CoA preferences of the mitochondrial GPAT and microsomal AGPAT may play key roles in the asymmetric distribution of fatty acids, ie, saturated at the sn-1 position and unsaturated at the sn-2 position, which is considered to be of major importance in maintaining the functional and structural function of glycerophospholipids. The applicant cloned the murine mitochondrial GPAT, expressed the protein in insect cells, and purified and reconstituted the GPAT activity. Recently, the applicant also cloned a full-length mammalian cDNA sequence homologous to known AGPATs from other species, and its mRNA is highly expressed in testis and brain, tissues where long chain polyenoic acids are abundant. The applicant is now in a unique position to address, by biochemical and molecular biological approaches, the role of these two acyltransferases in the positional distribution of fatty acids in glycerophospholipids and in the regulation of lipid metabolism. There are three specific aims. 1. The applican will determine the fatty acyl-CoA binding site of the mitochondrial GPAT by in vitro mutagenesis and photo-affinity labeling. The investigator will generate cells either depleted of, or expressing different levels of, the mitochondrial GPAT. 2. Using these cells, the applicant will determine the positional fatty acid composition of glycerophospholipids to demonstrate the role of mitochondrial GPAT in the prevalence of saturated fatty acids at the sn-1 position. The investigator will also measure fatty acid oxidation and esterification to examine the role of mitochondrial GPAT in lipid metabolism. The applicant will positively identify and characterize the cloned AGPAT by complementing an E. coli mutant and analyzing fatty acid composition. She will also express this AGPAT in acyl-CoA. She will also determine the contribution of this AGPAT in the enrichment of long chain polyenoic acids at the sn-2 position of glycerophospholipids.

描述（改编自申请人的摘要）：磷脂酸，其中 用作甘油磷脂生物合成的骨架，合成 从从头开始，通过甘油-3-磷酸甘油的顺序酰化。 甘油-3-磷酸酰基转移酶（GPAT）酰化Sn-1的位置 形式1-酰基甘油-3-磷酸（溶物磷酸盐），这是第一个 承诺并假定是限制速率的，在甘油磷脂中步骤 合成。 1-酰基甘油-3-磷酸酰基转移酶（AGPAT）酰化 SN-2的位置产生1,2-二酰基甘油-3-磷酸（磷酸）。 GPAT和AGPA活性都在内质网中发现。 在 此外，在中发现了更喜欢饱和脂肪酰基辅酶A的GPAT 线粒体。 线粒体GPAT和 微粒体AGPAT可能在脂肪的不对称分布中起关键作用 IE的酸在SN-1位置饱和，在SN-2处不饱和 位置，这被认为在维护 甘油磷脂的功能和结构功能。 申请人 克隆鼠线粒体GPAT，表达了昆虫细胞中的蛋白质， 并纯化并重构GPAT活动。 最近，申请人 还克隆了与已知Agpats同源的全长哺乳动物cDNA序列 来自其他物种及其mRNA在睾丸和大脑中高度表达， 长链聚酸丰富的组织。 申请人现在是 通过生化和分子生物学来解决独特的位置 方法，这两个酰基转移酶在位置中的作用 脂肪酸在甘油磷脂中的分布和调节 脂质代谢。 有三个特定的目标。 1。应用程序将 通过IN确定线粒体GPAT的脂肪酰基-COA结合位点 体外诱变和照片亲和力标记。 调查员会 生成耗尽或表达不同水平的细胞 线粒体GPAT。 2。使用这些单元格，申请人将确定 甘油磷脂的位置脂肪酸组成，以证明 线粒体GPAT在饱和脂肪酸流行中的作用 SN-1位置。 研究者还将测量脂肪酸氧化和 酯化以检查线粒体GPAT在脂质中的作用 代谢。 申请人将积极识别并表征 克隆的Agpat通过补充大肠杆菌突变体并分析脂肪酸 作品。 她还将在酰基-COA中表达此AGPAT。 她也会 确定该AGPAT在长链富集中的贡献 在甘油磷脂的SN-2位置的聚烯酸。