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

• 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
• 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

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/摘要 磷脂酸（PA）是一种多功能的信号脂质和中心生物合成中间体， 强大的稳态调节，其水平在空间和时间上受到严格控制。虽然很多人都... 代谢酶和PA转运蛋白的特点，它是不很好地理解细胞如何感觉变化 以及体内平衡是如何实现的。为了阐明时空的内在机制， 调节PA代谢，并揭示了更广泛的传播PA信号的效应蛋白，我们 因此，需要新的策略来快速干扰PA水平与细胞器水平的精度。我们已经开始 开发精确的“膜编辑”工具，用于快速安装PA的生理活性池， 靶细胞器。光遗传磷脂酶D（optoPLD）使用蓝光将细菌PLD募集到所需的细胞中。 细胞器膜，在那里它通过磷脂酰胆碱水解产生短暂的PA池，最近 定向进化的努力已经产生了第二代超活性optoPLD（superPLD）。的 通过邻近将superPLD介导的膜编辑和细胞器膜蛋白质组学结合起来 使用膜系TurboID的生物素化，我们称之为“喂食和捕鱼”（F+F）策略， 为我们提供了一个快速变化的整体和外周膜蛋白质组的血浆 在使用superPLD编辑其脂质组以瞬时升高PA水平的条件下，超出 通过检测已知的PA代谢调节因子，我们鉴定并验证了用于传感的新的候选蛋白， 在这些膜中运输和发出PA存在的信号。然而，仍然存在几个关键问题， 未解决的问题，与方法开发和从我们的屏幕命中的机械理解有关。的 该提案的总体目标是部署新的光遗传学和蛋白质组学工具，以了解细胞如何 在不同位置建立和维持功能上不同的PA库以平衡生物合成和信号传导 需求首先，我们将开发超低背景，下一代光遗传学PLD，并应用它们来阐明 PA在介导两种主要细胞信号通路之间串扰中的作用并发现新的调节因子 PA稳态。其次，我们将阐明一个新的球员牵连在PA的细胞器间运输的作用 使用细胞和体外研究的组合。第三，我们将阐明分子细节和功能 PA与一种新发现的PA结合蛋白相互作用的重要性，这种蛋白的突变会导致 遗传性肌肉骨骼疾病总的来说，我们的研究将为膜编辑提供广泛有用的工具 并破译PA信号，建立一个机制框架，了解细胞如何发挥作用， 对多效性脂质的水平和生物活性的时空控制以维持体内平衡和指导 特定的生理和信号事件。