Defining the mode of action and regulation of endogenous bio-active metabolites fundamental to cell biology.

定义细胞生物学基础内源性生物活性代谢物的作用模式和调节。

• 批准号: RGPIN-2020-05822
• 负责人: Murphy, John
• 金额: $ 2.19万
• 依托单位: University of Prince Edward Island
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=745830
• 关键词: Defining; mode; action; regulation; endogenous

OBJECTIVES: Metabolites form the basis of chemical reactions necessary for life and perform their roles by interacting with proteins and nucleic acids. Unfortunately, the protein interactions of many bio-active metabolites in cells is unknown, limiting our understanding of their mode of action. The long-term goal of my research program is to determine mode of action and regulation of bio-active metabolites produced by mammalian cells. The short-term objectives of my laboratory are to determine protein interactions and regulation of i) the redox molecule NAD+ and ii) several of its precursors in the kynurenine pathway (KP) of de novo NAD+ biosynthesis. NAD+ has longstanding roles in energy homeostasis but is re-emerging as a key contributor to pathways that generate antioxidants, enabling cells to survive oxidative stress. At the same time, intermediates of NAD+ synthesis in the KP have newly appreciated roles in the production of danger signals by the mammalian immune system. However, the mode of action and regulation of NAD+ and its precursors and how they contribute at the molecular level to antioxidant or immune functions are largely unknown. APPROACHES: My laboratory will use new technology that detects small molecule-protein interactions to examine mode of action of cellular metabolites and assess the role of these interactions in cell phenotype. To do this, we will use thermal proteome profiling (TPP) methods that measure protein solubility on a global scale (with and without a metabolite of interest) over a temperature gradient. Following the identification of proteins with shifts in thermal solubility - indicative of metabolite binding - they will be prioritized for further study to understand how they link metabolite to phenotype. Building on previous work, my laboratory's initial focus will be to establish quantitative TPP methods for endogenous metabolites for which we do not fully understand the mode of action: i) NAD+ and ii) KP intermediates. We will then examine the role of these newly identified protein interactions in antioxidant production and cell survival (for NAD+) or danger signal production by macrophages in vitro (for KP intermediates). Using molecular techniques, we will also examine how transcription control of enzymes in the KP regulate macrophage functions through the metabolite-protein interactions we identify. SIGNIFICANCE: Despite decades of research, the function of many bio-active metabolites in mammals is incompletely understood. A better grasp of the functionality of metabolites with emerging importance such NAD+ and KP intermediates will provide critical knowledge that advances fundamental understanding of metabolism. Moving forward, new techniques for probing molecular interactions in cells will further unlock advances that change the way we think about the biochemical processes sustaining life.

目的：代谢物形成生命所必需的化学反应的基础，并通过与蛋白质和核酸相互作用来发挥作用。不幸的是，许多生物活性代谢物在细胞中的蛋白质相互作用是未知的，限制了我们对其作用模式的理解。我的研究计划的长期目标是确定哺乳动物细胞产生的生物活性代谢物的作用和调节模式。我的实验室的短期目标是确定蛋白质相互作用和调节i）氧化还原分子NAD+和ii）其几种前体在犬尿氨酸途径（KP）的从头NAD+生物合成。NAD+在能量稳态中具有长期作用，但正在重新成为产生抗氧化剂的途径的关键贡献者，使细胞能够在氧化应激中生存。同时，KP中NAD+合成的中间体在哺乳动物免疫系统产生危险信号中具有新的作用。然而，NAD+及其前体的作用和调节模式以及它们如何在分子水平上对抗氧化剂或免疫功能做出贡献在很大程度上是未知的。方法：我的实验室将使用检测小分子-蛋白质相互作用的新技术来检查细胞代谢物的作用模式，并评估这些相互作用在细胞表型中的作用。为此，我们将使用热蛋白质组分析（TPP）方法，在温度梯度上测量全球范围内的蛋白质溶解度（有和没有感兴趣的代谢产物）。在鉴定出具有热溶解度变化的蛋白质（指示代谢物结合）后，将优先对它们进行进一步研究，以了解它们如何将代谢物与表型联系起来。 基于以前的工作，我的实验室的初步重点将是建立定量TPP方法的内源性代谢物，我们不完全了解的作用模式：i）NAD+和ii）KP中间体。然后，我们将研究这些新发现的蛋白质相互作用在抗氧化剂生产和细胞存活（NAD+）或危险信号生产的巨噬细胞在体外（KP中间体）的作用。使用分子技术，我们还将研究KP中酶的转录控制如何通过我们发现的代谢物-蛋白质相互作用来调节巨噬细胞功能。 意义：尽管经过数十年的研究，许多生物活性代谢物在哺乳动物中的功能仍不完全清楚。更好地掌握具有新兴重要性的代谢物的功能，如NAD+和KP中间体，将提供促进对代谢的基本理解的关键知识。展望未来，探测细胞中分子相互作用的新技术将进一步揭示改变我们对维持生命的生化过程的看法的进展。