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)是一种多功能的信号转导脂质和中枢生物合成中间体。 到强大的动态平衡调节，其水平在空间和时间上受到严格控制。尽管很多私人助理- 代谢酶和PA转运体的特征，还不是很好地了解细胞感觉如何变化 在PA水平和如何实现动态平衡。为了阐明时空关系背后的机制 调节PA代谢并揭示传播PA信号的更广泛的效应蛋白，我们 假设需要新的策略来以细胞器级别的精度快速扰乱PA水平。我们已经开始了 开发用于快速安装PA生理活性池的精密“膜编辑”工具 靶细胞器。一种光遗传磷脂酶D(Oppld)利用蓝光招募细菌PLD到所需的 细胞器膜，在那里它通过磷脂酰胆碱水解产生暂时的PA池，最近 定向进化努力已经产生了第二代、超级活跃的光电PLD(SuperPLD)。这个 超PLD介导的膜编辑和细胞器膜蛋白质组学的邻近结合 使用膜系留的TurboID的生物素化，我们称之为“喂食和捕鱼”(F+F)策略，具有 为我们提供了血浆整体和外周膜蛋白质组快速变化的全局视角 在使用SuperPLD编辑其脂质体以瞬时提高PA水平的条件下，膜。超越 通过检测已知的PA代谢调节因子，我们确定并验证了新的候选传感蛋白， 在这些膜上运输PA，并发出PA存在的信号。然而，几个关键问题仍然存在。 未解决的问题，既与方法开发有关，也与对屏幕点击的机械理解有关。这个 这项提案的总体目标是部署新的光遗传学和蛋白质组学工具来了解细胞如何 在不同位置建立和维护不同功能的PA池，以平衡生物合成和信号传递 需要。首先，我们将开发超低本底的下一代光遗传PLD，并将其应用于阐明 PA在调节两条主要细胞信号通路之间的串扰中的作用和发现新的调节因子 PA动态平衡。其次，我们将阐明PA在细胞器间运输中所扮演的新角色 使用细胞和体外研究相结合的方法。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 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 破译PA信号并建立一个机制框架来了解细胞是如何发挥作用的 对多效性脂质水平和生物活性的时空控制以维持动态平衡和直接 特定的生理和信号事件。