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

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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.