Mutually Orthogonal Metabolic Probes for Multiplexed Imaging of de novo Phospholipid Biosynthesis

用于从头磷脂生物合成多重成像的相互正交代谢探针

• 批准号: 10086319
• 负责人: Brittany Marie White-Mathieu
• 金额: $ 6.49万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10086319
• 关键词: Affect; Anabolism; Azides; Biological Factors; Cell Line; Cell membrane; Cell model; Cell physiology; Cells; Cellular biology; Chemicals; Chemistry; Choline; Color; Comprehension; Coupled; Cyclooctenes; Development; Disease; Endoplasmic Reticulum; Enzymes; Ethanolamines; Evaluation; Exhibits; Fellowship; Fluorescent Dyes; Foundations; Genetic Diseases; Goals; Homeostasis; Human; Image; Impairment; In Vitro; Individual; Knock-out; Knowledge; Label; Lecithin; Lenz-Majewski syndrome; Limb structure; Lipids; Liver Failure; Mammalian Cell; Membrane; Membrane Biology; Metabolic; Molecular; Monitor; Mouse Cell Line; Mutation; Organism; Pathway interactions; Patients; Phosphatidylcholine Biosynthesis; Phosphatidylethanolamine; Phosphatidylserine Synthase; Phosphatidylserines; Phospholipids; Reaction; Regulation; Reporting; Research; Research Proposals; Role; Saccharomyces cerevisiae; Specificity; Testing; Therapeutic; Time; Validation; Work; Yeasts; base; craniofacial; cross reactivity; design; experimental study; fluorophore; functional group; gain of function mutation; human disease; imaging capabilities; in vivo; insight; live cell imaging; multiplexed imaging; mutant; novel; response; small molecule; targeted treatment; tool

PROJECT SUMMARY/ABSRACT Mutations in enzymes responsible for phospholipid biosynthesis and remodeling have been identified as key biological factors in a growing number of genetic disorders. For example, Lenz-Majewski syndrome, a disease associated with craniofacial and limb abnormalities, and intellectual impairment, is characterized by gain of function mutations in phosphatidylserine synthase 1 enzyme (PSS1) that results in the accumulation of phostphatidylserine (PS) in the endoplasmic reticulum (ER). While it is well established that the biosynthesis of PS is tightly coupled to that of phospholipids including phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the consequences that increased PS synthesis has on the biosynthesis and membrane content of PC and PE in patients with Lenz-Majewski syndrome are not known. This lack of knowledge limits our understanding of the disease since PC and PE account for over half of the cell's total phospholipids. In addition, changes in the relative concentrations of these lipids, especially in response to perturbations in the cellular content of other phospholipids, are associate with a variety of human diseases including liver failure. The over-arching goal of this proposal is to unveil the causal relationship between the biosynthesis and cellular content of PC and PE, in real-time with live cells, when PS biosynthesis is disturbed in Lenz-Majewski syndrome. Bioorthogonal choline and ethanolamine probes will be incorporated into PC and PE respectively through their de novo biosynthetic pathways to enable multiplexed, live-cell imaging of these phospholipids after tagging with a fluorescent dye. The proposed research is crafted into two Specific Aims to achieve this research goal. Specific Aim 1 focuses on the synthesis of bioorthogonal choline and ethanolamine probes, and evaluation of labeling efficiency and specificity of these probes for PC and PE respectively. Wild-type and knockout Saccharomyces cerevisiae yeast strains will elucidate the enzymatic incorporation (i.e. Kennedy biosynthesis) of these probes and the optimized labeling strategy will be transitioned into mammalian cells. Specific Aim 2 is designed to demonstrate the multiplexed imaging capabilities of the mutually orthogonal PC and PE probes in mammalian cell lines and in cellular models of Lenz-Majewski syndrome. Flux through the Kennedy biosynthetic pathway of PC and PE, and changes in membrane content of these phospholipids, in response to uncontrolled PS synthesis will provide a complete picture on changes in cell physiology during this disease. Completion of the research proposed in this fellowship will have a broad impact in cell and membrane biology, providing novel tools to visualize perturbations in local lipid composition and study the effects of these changes on cellular function in real-time. Specifically, this work provides the framework for the characterization of PC and PE biosynthesis in an ever-growing class of diseases characterized by mutations in enzymes associates with phospholipid biosynthesis.

项目概要/摘要 负责磷脂生物合成和重塑的酶的突变已被确定为关键 越来越多的遗传疾病中的生物因素。例如，伦茨-马波斯基综合征， 与颅面和肢体异常以及智力障碍相关，其特征在于获得 磷脂酰丝氨酸合成酶1（PSS 1）的功能突变，导致 磷脂酰丝氨酸（PS）在内质网（ER）。虽然已经很好地确定， PS与磷脂酰胆碱（PC）和磷脂酰乙醇胺（PA）紧密偶联 (PE)，PS合成增加对PC的生物合成和膜含量的影响， Lenz-Mastenski综合征患者的PE尚不清楚。这种知识的缺乏限制了我们对 因为PC和PE占细胞总磷脂的一半以上。此外， 这些脂质的相对浓度，特别是在响应其他细胞内容物的扰动时， 磷脂与包括肝衰竭在内多种人类疾病有关。的总体目标 这一建议旨在揭示PC和PE的生物合成与细胞含量之间的因果关系， 实时与活细胞，当PS生物合成被扰乱Lenz-Mastenski综合征。生物正交胆碱 和乙醇胺探针将分别通过它们的从头生物合成被并入PC和PE中。 在用荧光染料标记后，能够对这些磷脂进行多重活细胞成像。 为了实现这一研究目标，拟议的研究分为两个具体目标。具体目标1重点 生物正交胆碱和乙醇胺探针的合成，以及标记效率和 这些探针分别对PC和PE的特异性。野生型和敲除酿酒酵母 菌株将阐明这些探针的酶促掺入（即Kennedy生物合成）和优化的 标记策略将过渡到哺乳动物细胞中。具体目标2旨在证明 相互正交的PC和PE探针在哺乳动物细胞系中的多重成像能力， Lenz-Masterski综合征的细胞模型。通过PC和PE的肯尼迪生物合成途径， 这些磷脂的膜含量的变化，响应于不受控制的PS合成，将提供 在这种疾病中细胞生理变化的完整图片。完成本报告中提出的研究 奖学金将在细胞和膜生物学产生广泛的影响，提供新的工具来可视化扰动 在局部脂质组成，并研究这些变化对细胞功能的实时影响。具体来说， 工作提供了一个框架，在一个不断增长的类PC和PE生物合成的特点， 以磷脂生物合成相关酶突变为特征的疾病。