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

阐明协调和调节磷脂酰基链组成的酶内部和之间的机制

• 批准号: 9811977
• 负责人: Peter Michael Oelkers
• 金额: $ 46.8万
• 依托单位: UNIVERSITY OF MICHIGAN AT DEARBORN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9811977
• 关键词: Acyl Coenzyme A; Acyltransferase; Address; Affect; Algebra; Animal Model; Attenuated; Binding Proteins; Biochemical; Biological Assay; Cations; Cell Membrane Structures; Cell membrane; Cell physiology; Cells; Co-Immunoprecipitations; Complex; Data; Data Set; Environment; Enzymes; Event; Faculty; Fatty Acids; Gene Cluster; Gene Deletion; Gene Expression; Genotype; Glycerophospholipids; Growth; Head; Human; Individual; Inflammatory; Insecta; Ions; Isoenzymes; Kinetics; Knowledge; Length; Life; Lipids; MCHR1 gene; Mediating; Membrane; Mentors; Metabolic; Microsomes; Pathway interactions; Pharmaceutical Preparations; Phenotype; Phospholipids; Production; Proteins; Reaction; Role; Saccharomyces cerevisiae; Schedule; Series; Side; Signal Transduction; Statistical Data Interpretation; Structure; Substrate Specificity; Testing; Textiles; Transcript; Two-Hybrid System Techniques; Yeasts; alpha-glycerophosphoric acid; amphiphilicity; base; biological adaptation to stress; exhaustion; experimental study; in vitro Assay; in vivo; inorganic phosphate; novel; phosphatidate; preference; protein function; protein protein interaction; receptor; transcription factor; transcriptome; transcriptome sequencing; trend; undergraduate student

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.

项目摘要 细胞膜是生命的结构。膜结构，动力学和功能受到影响 磷脂组成。磷脂环境可以影响 蛋白质，例如转运蛋白和受体。维持和调节数百万 以适当比率的磷脂物种涉及多种细胞机制。其中一些机制 在合成和重塑磷脂的酶内部和之间运行。我们专注于 了解调节磷脂前体中酰基链分配的机制。 这样的知识可能会通过影响膜结构，促进疗法影响蛋白质功能 解决与阳离子两亲药有关的磷脂病，并减弱促炎 信号表明磷脂前体1-酰基甘油-3-磷酸（Lysopa）和磷酸酯可以发送。 最近，我们发现酿酒酵母中不同酰基转移酶的复合缺失引起 磷脂组成表型的渐变。对该体内数据的代数分析表明 两个主要的1-酰基甘油-3-磷酸O-酰基转移酶（AGPAT），它们具有两个含有酰基的底物 链，独特的基于各自的酰基链长度的底物。我们建议执行 详尽的体外测定，以确定这两个AGPATS SLC1和LPT1的动力学参数 lysopa和酰基-COA底物配对的阵列。如果找到了选择性配对的证据，则三个 相应的酵母Agpats最接近的人类同源物将在SF9昆虫细胞中表达。微粒体 从这些细胞中，将经历相同的两乘二次底物配对阵列。这可能建立小说 调节人类细胞中磷脂组成的机制。 其次，我们将采取更广泛的方法并检验假设，在 将脂肪酸顺序掺入CDP-DAG中的反应。 CDP-DAG是磷脂前体 固定了头部组。即使在酿酒酵母的相对简单的代谢框架中，每个框架都 脂肪酸和CDP-DAG之间的反应是由多种同工酶介导的。我们将使用膜 酵母两杂交测定法以测试22种酶之间的148种可能的物理相互作用和结合 介导脂肪酸和CDP-DAG之间的顺序但分支反应的蛋白质。如果具体 发现指示通道的相互作用，将类似地测定各自的人类同源物。 调节磷脂成分的稳态机制可以通过转录丰度进行。到 识别具有四个复合基因脱落基因型的新型机制，酵母菌菌株的分级 磷脂表型将进行RNA测序。将开发统计分析以识别 特异性或聚类的转录本通过单个或簇的磷脂物种成比例地改变。平行线 研究将首先遗传去除已知调节磷脂产生的OPI1转录因子。