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

目的：代谢产物构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内的蛋白质相互作用尚不清楚，这限制了我们对其作用模式的了解。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模式和调节。我的实验室的短期目标是确定蛋白质的相互作用和调控：1)氧化还原分子NAD；2)NAD生物合成的犬尿氨酸途径(KP)中的几个前体。NAD在能量动态平衡中具有长期的作用，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。与此同时，Kp中NAD合成的中间产物在哺乳动物免疫系统产生危险信号中的作用也得到了新的认识。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或免疫功能做出贡献，在很大程度上尚不清楚。 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图谱(TPP)方法，在全球范围内(有无感兴趣的代谢物)测量温度梯度上的蛋白质溶解度。在鉴定出热溶解度发生变化的蛋白质--代谢物结合的指示--之后，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。 在以前工作的基础上，我的实验室最初的重点将是为我们不完全了解其作用模式的内源性代谢物建立定量TPP方法：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体外巨噬细胞产生抗氧化剂和细胞存活(对于NAD)或危险信号产生(对于KP中间体)中的作用。利用分子技术，我们还将研究KP中酶的转录控制如何通过我们确定的代谢物-蛋白质相互作用来调节巨噬细胞的功能。 意义：尽管经过了几十年的研究，但许多生物活性代谢物在哺乳动物中的功能尚不完全清楚。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，将提供关键知识，促进对新陈代谢的基本理解。展望未来，探测细胞中分子相互作用的新技术将进一步揭开新技术的面纱，改变我们对维持生命的生化过程的看法。

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.