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

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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.