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.

项目概要/摘要磷脂酸 (PA) 是一种多功能信号脂质和主要生物合成中间体尽管许多 PA- 具有强大的稳态调节能力，但其水平在空间和时间上受到严格控制。虽然代谢酶和 PA 转运蛋白已被表征，但尚不清楚细胞如何感知变化 PA 水平以及如何实现体内平衡，以阐明时空的潜在机制。 PA 代谢的调节并揭示了传播 PA 信号传导的更广泛的效应蛋白，我们认为需要新的策略以细胞器级的精度快速扰动 PA 水平，我们已经开始了。开发精确的“膜编辑”工具，用于在生物体上快速安装具有生理活性的 PA 池光遗传学磷脂酶 D (optoPLD) 使用蓝光将细菌 PLD 募集到所需的位置。细胞器膜，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）。 superPLD 介导的膜编辑和细胞器膜蛋白质组学通过邻近相结合使用膜束缚的 TurboID 进行生物素化，我们称之为“喂食和捕鱼”(F+F) 策略，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图使用 superPLD 编辑膜的脂质组以暂时提高 PA 水平。检测已知的 PA 代谢调节因子，我们识别并验证了用于传感的新候选蛋白，然而，仍然存在一些关键问题。尚未解决，与方法开发和对屏幕点击的机械理解有关。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来了解细胞如何在不同位置建立和维护功能不同的 PA 池，以平衡生物合成和信号传导首先，我们将开发超低背景的下一代光遗传学 PLD 并应用它们来阐明。 PA 在介导两个主要细胞信号通路之间的串扰中的作用并发现新的调节因子其次，我们将阐明参与 PA 细胞间运输的新参与者的作用。第三，我们将结合细胞和体外研究来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，该蛋白的突变导致总的来说，我们的研究将产生广泛有用的膜编辑工具。破译 PA 信号传导并建立一个机制框架来理解细胞如何发挥作用对多效性脂质的水平和生物活性进行时空控制，以维持稳态并直接特定的生理和信号事件。