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: 10263358
• 负责人: Brittany Marie White-Mathieu
• 金额: $ 6.64万
• 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/10263358
• 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.

项目摘要/弃权 负责磷脂生物合成和重塑的酶的突变已被确定为钥匙 越来越多的遗传疾病中的生物因素。例如，Lenz-Majewski综合征，一种疾病 与颅面和肢体异常以及智力障碍相关的特征是 磷脂酰丝氨酸合酶1酶（PSS1）中的功能突变，导致积累 内质网中（ER）中的苯丙酰丝氨酸（PS）。虽然可以很好地确定 PS紧密耦合到包括磷脂酰胆碱（PC）和磷脂酰乙醇胺在内的磷脂的偶联 （PE），PS合成增加对PC和PC的生物合成和膜含量的后果 Lenz-Majewski综合征患者的PE尚不清楚。缺乏知识限制了我们对 由于PC和PE以来，该疾病占细胞总磷脂的一半以上。另外，变化 这些脂质的相对浓度，尤其是针对其他细胞含量的扰动 磷脂与包括肝衰竭在内的多种人类疾病有关。总体目标的目标 该建议是揭示PC和PE的生物合成与细胞含量之间的因果关系 当Lenz-Majewski综合征干扰PS生物合成时，实时使用活细胞。生物正交胆碱 和乙醇胺探针将通过新生生物合成分别纳入PC和PE 用荧光染料在标记后，可以对这些磷脂的多重生活成像进行多路复用的途径。 拟议的研究被制定为实现这一研究目标的两个具体目的。特定目标1重点 关于生物正交胆碱和乙醇胺探针的合成，以及标记效率的评估 这些探针分别对PC和PE的特异性。野生型和淘汰葡萄糖酿酒酵母酵母 菌株将阐明这些探针的酶促掺入（即肯尼迪生物合成）和优化 标记策略将过渡到哺乳动物细胞。特定目标2旨在证明 哺乳动物细胞系中相互正交PC和PE探针的多重成像功能以及 Lenz-Majewski综合征的细胞模型。通过PC和PE的肯尼迪生物合成途径的通量，以及 这些磷脂的膜含量的变化，响应不受控制的PS合成将提供 完整了解这种疾病期间细胞生理的变化。在此提出的研究完成 奖学金将对细胞和膜生物学产生广泛的影响，提供新颖的工具来可视化扰动 在局部脂质组成和研究这些变化对实时细胞功能的影响。具体来说，这是 工作为在不断增长的类别中表征PC和PE生物合成的框架 以与磷脂生物合成相关的酶突变为特征的疾病。