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.

项目摘要/摘要 磷脂酸（PA）是一种多功能信号脂质和中央生物合成中间体，是受试者 强大的体内稳态调节，其水平在空间和时间上受到严格控制。虽然很多 代谢酶和PA转运蛋白的表征，尚不清楚细胞的感觉如何变化 在PA水平以及如何实现稳态。两者都阐明了时空的基础机制 调节PA代谢并揭示了传播PA信号传播的效应蛋白的广泛范围，我们 认为需要使用细胞器级精度快速扰动PA水平的新策略。我们已经开始 开发精确的“膜编辑”工具，以快速安装PA的物理活跃池 目标细胞器。光遗传学磷脂酶D（Optopld）使用蓝光募集细菌PLD到所需的 细胞器膜，它通过磷脂酰胆碱水解生成PA的瞬时池，最近 定向的进化努力产生了第二代超级活跃的OPTPLD（SUPERPLDS）。 SuperPLD介导的膜编辑和细胞器膜蛋白质组学的组合通过接近度 使用膜螺旋涡轮增压的生物素化，我们称其为“喂食和捕鱼”（F+F）策略 为我们提供了对等离子体的积分和周围膜蛋白质组的快速变化的全球视野 膜在使用SuperPLD瞬时提高PA水平的脂质组进行编辑时。超过 检测PA代谢的已知调节剂，我们确定并验证了新的候选蛋白用于感测， 运输，并在这些膜中发出PA的存在。然而，仍然存在一些关键问题 未经后果，与方法开发和机械理解有关，对我们的屏幕播放。这 该建议的总体目的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护功能上不同的PA池，以平衡生物合成和信号传导 需要。首先，我们将开发超倒态的下一代光遗传学PLD，并将其应用于阐明 PA在介导两个主要细胞信号通路之间的串扰中的作用，发现了新的调节剂 PA稳态。其次，我们将阐明在PA的Interganelle运输中实现的新玩家的角色 结合细胞和体外研究。第三，我们将阐明分子细节和功能 PA与新发现的PA结合蛋白相互作用的重要性，该蛋白的突变导致A 可遗传的肌肉骨骼疾病。总的来说，我们的研究将产生广泛有用的膜编辑工具 并解密PA信令并建立一个机械框架，以了解细胞如何执行 对多效性脂质的水平和生物活性的时空控制，以维持稳态和直接 特定的生理和信号事件。