ABSTRACT Signaling metabolites control various cellular processes, including cell cycle, differentiation, and adaptations to environmental stimuli. Intracellular trafficking of signaling metabolites is crucial for maintaining cellular homeostasis and integrate metabolic and transcriptional responses. Defects in metabolite transport and distribution may lead to multiple diseases, including cancer, immunological, inflammatory, and metabolic disorders. Subcellular compartmentalization allows the same molecules to partake in distinct biological processes. Signaling metabolites generally act as second messengers for specific proteins or ligands for sensors and nuclear receptors (NR), ligand-activated transcription factors that sense environmental signals and drive cellular response. Because of their intrinsic reactivity, the intracellular levels of NR ligands, along with their subcellular localization, are tightly controlled and may oscillate greatly depending on nutritional states and pathophysiological conditions. Despite our understanding of their functions, our knowledge of how nuclear receptor ligands travel across organelles remains limited due to the lack of specific tools to target such mechanisms. We propose to integrate chemoproteomics, metabolomics, and cellular assays, to develop novel chemical tools to interrogate the protein interactomes of NR ligands and identify their intracellular chaperones. Leveraging these technologies, we intend to reveal the molecular and functional basis of intracellular trafficking of signaling metabolites and identify dedicated protein chaperones that bind NR ligands at their site of synthesis or entry into the cell, transport them to the nucleus, and deliver them to NRs. A driving finding of our preliminary work was the discovery of PGRMC2 as an intracellular heme chaperone that transports heme from mitochondria to the nucleus and regulates the transcriptional activity of heme-responsive transcription factors such as Rev- Erb and BACH1. We will use the experience acquired from this initial work to extend our studies to the identification of other transport mechanisms for known NR ligands, such as fatty acids, that activate PPARs, a family of ligand-activated transcription factors that regulate metabolism and systemic energy homeostasis. The second major goal of this proposal is to develop spatial- and time-resolved protein-metabolite maps, which we expect to go beyond the identification of intracellular trafficking mechanisms and have a broader impact on the field by providing a powerful strategy to study metabolite-protein crosstalk. Lastly, this project uniquely combines our multidisciplinary expertise in transcriptional regulation, metabolism, and chemical biology to lead the exploration of a new exciting findings in cell biology.