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Deciphering phosphatidic acid homeostasis and signaling using optogenetic membrane editors

使用光遗传学膜编辑器破译磷脂酸稳态和信号传导

基本信息

批准号：
10729180
负责人：
JEREMY BASKIN
金额：
$ 34.99万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-15 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10729180
关键词：
Acyl Coenzyme A Agonist Binding Binding Proteins Bioinformatics Biology Biotinylation Cell membrane Cells Cullin Proteins Cytosol Data Directed Molecular Evolution Disease Dwarfism Enzymes Equilibrium Event Exhibits Fishes Generations Goals Head Heritability Homeostasis Hydrolysis In Vitro Infection Lecithin Light Lipid Binding Lipids Location Malignant Neoplasms Mediating Membrane Metabolism Molecular Molecular Conformation Musculoskeletal Diseases Mutation Nerve Degeneration Noise OS4 Gene Organelles Outcome Oxygen Pathologic Peptides Peripheral Phosphatidic Acid Phosphatidylinositols Phospholipase D Phospholipids Physiological Physiological Processes Predisposition Production Proteins Proteome Proteomics Regulation Research Role Signal Pathway Signal Transduction Signaling Molecule Sterols Syndrome Time Toxic effect Ubiquitination cancer type feeding improved lipid transfer protein lipid transport lipidome method development next generation optogenetics recruit spatiotemporal tool ubiquitin-protein ligase voltage

项目摘要

Project Summary/Abstract Phosphatidic acid (PA) is a multifunctional signaling lipid and central biosynthetic intermediate that is subject to strong homeostatic regulation, with its levels tightly controlled in space and time. Though many PA- metabolizing enzymes and PA transporters are characterized, it is not well understood how cells sense changes in PA levels and how homeostasis is achieved. To both elucidate mechanisms underlying the spatiotemporal regulation of PA metabolism and reveal a broader spectrum of effector proteins that propagate PA signaling, we posit that new strategies to rapidly perturb PA levels with organelle-level precision are required. We have begun to develop precision “membrane editing” tools for the rapid installation of physiologically active pools of PA on target organelles. An optogenetic phospholipase D (optoPLD) uses blue light to recruit a bacterial PLD to desired organelle membranes, where it generates transient pools of PA via phosphatidylcholine hydrolysis, and recent directed evolution efforts have yielded second-generation, super-active optoPLDs (superPLDs). The combination of superPLD-mediated membrane editing and organelle membrane proteomics via proximity biotinylation using a membrane-tethered TurboID, which we term a “feeding and fishing” (F+F) strategy, has afforded us a global view of rapid changes to the integral and peripheral membrane proteomes of the plasma membrane during conditions when its lipidome is edited using superPLD to transiently elevate PA levels. Beyond detecting known regulators of PA metabolism, we identified and validated new candidate proteins for sensing, transporting, and signaling the presence of PA in these membranes. Yet, several critical issues remain unaddressed, related to both method development and mechanistic understanding of hits from our screens. The overall objective of this proposal is to deploy new optogenetic and proteomics tools to understand how cells establish and maintain functionally distinct PA pools in different locations to balance biosynthetic and signaling needs. First, we will develop ultralow-background, next-generation optogenetic PLDs and apply them to elucidate roles for PA in mediating crosstalk between two major cell signaling pathways and discover new regulators of PA homeostasis. Second, we will elucidate roles for a new player implicated in the interorganelle transport of PA using a combination of cellular and in vitro studies. Third, we will elucidate the molecular details and functional importance of the interaction of PA with a newly discovered PA-binding protein whose mutation causes a heritable musculoskeletal disease. Collectively, our studies will yield widely useful tools for membrane editing and deciphering PA signaling and establish a mechanistic framework for understanding how cells exert spatiotemporal control over the levels and bioactivity of a pleiotropic lipid to maintain homeostasis and direct specific physiological and signaling events.

项目总结/文摘