Elucidating the mechanisms within and among enzymes that coordinate and regulate phospholipid acyl chain composition

项目摘要 细胞膜是生命的结构。膜的结构、动力学和功能受到 磷脂组合物。磷脂环境可以影响细胞的结构、动力学和功能。 蛋白质如转运蛋白和受体。维持和调节数以百计的 适当比例的磷脂种类涉及许多细胞机制。其中一些机制 在合成和重塑磷脂的酶中起作用。我们专注于 了解调节磷脂前体中酰基链分配的机制。 这样的知识可以促进通过影响膜结构来影响蛋白质功能的治疗， 解决与阳离子两亲性药物相关的磷脂病，并减轻促炎性 磷脂前体1-酰基甘油-3-磷酸（lysoPA）和磷脂酸盐可以发送的信号。 最近，我们发现酿酒酵母中不同酰基转移酶的复合缺失导致了 磷脂组成表型的分级。对该体内数据的代数分析表明， 两种主要的1-酰基甘油-3-磷酸O-酰基转移酶（AGPAT），其具有两种含有酰基的底物， 链，基于各自的酰基链长度唯一地配对底物。我们建议执行 详尽的体外试验，以确定这两个AGPAT，Slc 1和Lpt 1的动力学参数，使用两个- 通过lysoPA和酰基-CoA底物配对的两个阵列。如果发现选择性配对的证据， 各酵母AGPAT的最接近的人类同源物将在Sf 9昆虫细胞中表达。微粒体 将经历相同的二乘二阵列的衬底配对。这可能会建立一个新的 调节人体细胞中磷脂成分的机制。 其次，我们将采取更广泛的方法和测试的假设，有基板通道之间的 将脂肪酸依次掺入CDP-DAG的反应。CDP-DAG是磷脂前体 其上连接有头部基团。即使在相对简单的S.酿酒酵母，每个 脂肪酸和CDP-DAG之间的反应由多种同工酶介导。我们将使用膜 酵母双杂交试验，以测试22种酶之间的148种可能的物理相互作用， 介导脂肪酸和CDP-DAG之间的连续但分支的反应的蛋白质。如果特定 当发现指示通道作用的相互作用时，将类似地测定相应的人同源物。 调节磷脂组成的稳态机制可以通过转录丰度来实现。到 确定新的机制，酵母菌株与四个复合基因缺失基因型与梯度 磷脂表型将进行RNA测序。将进行统计分析，以确定 特定或成簇的转录物被单个或成簇的磷脂物质成比例地改变。平行 研究将首先从基因上去除已知调节磷脂产生的Opi 1转录因子。

PROJECT SUMMARY Cell membranes are the fabric of life. Membrane structure, dynamics, and function are influenced by phospholipid composition. The phospholipid milieu can influence the structure, dynamics, and function of proteins such as transporters and receptors. Maintaining and regulating the abundance of hundreds of phospholipid species at appropriate ratios involves a host of cellular mechanisms. Some of these mechanisms operate within and among the enzymes that synthesize and remodel phospholipids. We are focused on understanding the mechanisms that regulate the allocation of acyl chains among phospholipid precursors. Such knowledge may facilitate therapies to influence protein function via influencing membrane structure, to address the phospholipidosis associated with cationic amphiphilic drugs, and to attenuate pro-inflammatory signals that the phospholipid precursors 1-acylglycerol-3-phosphate (lysoPA) and phosphatidate can send. Recently, we found that compound deletion of different acyltransferases in Saccharomyces cerevisiae caused a gradation of phospholipid composition phenotypes. Algebraic analysis of this in vivo data suggested that the two major 1-acylglycerol-3-phosphate O-acyltransferases (AGPAT), which have two substrates containing acyl chains, uniquely pair substrates based on the respective acyl chain lengths. We propose to perform exhaustive, in vitro assays to determine kinetic parameters for these two AGPATs, Slc1 and Lpt1, using a two- by-two array of lysoPA and acyl-CoA substrate pairings. If evidence of selective pairing is found, the three closest human homologs for the respective yeast AGPATs will be expressed in Sf9 insect cells. Microsomes from these cells will undergo the same two-by-two array of substrate pairings. This may establish a novel mechanism for regulating phospholipid composition in human cells. Secondly, we will take a broader approach and test the hypothesis there is substrate channeling among the reactions that sequentially incorporate fatty acids into CDP-DAG. CDP-DAG is the phospholipid precursor onto which head groups are attached. Even in the relatively simple metabolic framework of S. cerevisiae, each reaction between fatty acids and CDP-DAG is mediated by multiple isoenzymes. We will use the membrane yeast two-hybrid assay to test for the 148 possible physical interactions among the 22 enzymes and binding proteins that mediate the sequential yet branched reactions between fatty acids and CDP-DAG. If specific interactions indicating channeling are found, the respective, human homologs will be similarly assayed. Homeostatic mechanisms that regulate phospholipid composition may do so via transcript abundance. To identify novel mechanisms, yeast strains with the four compound gene-deletion genotypes with a gradation of phospholipid phenotypes will undergo RNA sequencing. Statistical analysis will be developed to identify specific or clustered transcripts proportionately altered by single or clustered phospholipid species. Parallel studies will first genetically remove the Opi1 transcription factor known to regulate phospholipid production.