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

项目摘要/ABSRACT 负责磷脂生物合成和重塑的酶的突变被认为是关键 生物因素在越来越多的遗传疾病中起作用。例如，Lenz-Majewski综合征，一种疾病 与头面部和四肢畸形以及智力障碍有关，其特征是获得 导致磷脂酰丝氨酸合成酶1酶(PSS1)功能突变导致 内质网(ER)中的磷脂酰丝氨酸(PS)。虽然公认的是生物合成的 PS与包括磷脂酰胆碱(PC)和磷脂酰乙醇胺在内的磷脂紧密偶联 (PE)，PS合成增加对PC和PC的生物合成和膜含量的影响 Lenz-Majewski综合征患者中的PE尚不清楚。这种知识的缺乏限制了我们对 本病因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，尤其是在对其他细胞成分的扰动反应时 磷脂与包括肝功能衰竭在内的多种人类疾病有关。的总体目标是 这项建议旨在揭示PC和PE的生物合成与细胞含量之间的因果关系 当Lenz-Majewski综合征的PS生物合成受到干扰时，实时与活细胞一起工作。生物正交胆碱 乙醇胺探针将通过其从头合成的方式分别被引入到PC和PE中 在用荧光染料标记后，能够对这些磷脂进行多路、活细胞成像的途径。 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及其标记效率和稳定性的评价 这两种探针分别对PC和PE具有特异性。野生型和敲除型酿酒酵母 菌株将阐明这些探针的酶结合(即肯尼迪生物合成)和优化的 标记策略将过渡到哺乳动物细胞。《特定目标2》旨在展示 相互正交的PC和PE探针在哺乳动物细胞系和哺乳动物细胞系中的多重成像能力 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，以响应不受控制的PS合成将提供一个 关于这种疾病期间细胞生理学变化的完整图片。完成本报告中建议的研究 联谊会将对细胞和膜生物学产生广泛的影响，为可视化扰动提供新的工具 并实时研究这些变化对细胞功能的影响。具体地说，这 工作为PC和PE生物合成的表征提供了框架，在不断增长的类别 以酶突变为特征的疾病与磷脂的生物合成有关。

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.