Project Summary Metabolites are central to orchestrating all cellular processes, yet functions of many metabolites remain unknown. The predominant mode of metabolite action is regulating protein activities. Hence, one way to understand a metabolite's role is to identify its protein targets. My research program investigates how organisms exploit metabolites to regulate protein activities and, consequently, cellular processes by untargeted identification of protein-metabolite complexes. We are especially interested in identifying enzyme-ligand pairings contributing to metabolic adaptations. I address this question from the perspective of a biochemist and an expert in mass-spectrometry. To obtain a snapshot of the protein- metabolite interactome in a single experiment, my group developed PROMIS; an original biochemical approach that relies on the co-fractionation of proteins and associated small-molecule ligands. The protein-metabolite interaction (PMI) networks are queried for novel regulatory interactions followed by in-detail functional analysis. One of the key findings coming from our work is uncovering the role of proteinogenic dipeptides in the regulation of central carbon metabolism in yeast and plants; specific dipeptides act at different points of flux control. These findings represent just the tip of the iceberg considering the hundreds of metabolites present in PMI datasets generated by my group and spanning four model organisms including gram-negative bacteria Escherichia coli, budding yeast Saccharomyces cerevisiae, plant Arabidopsis thaliana, and roundworm Caenorhabditis elegans. Over the next five years, the focus of my research program is to build on the experimental toolbox and wealth of PMI data to uncover the evolutionary conservation of these PMI networks and to functionally characterize previously unknown metabolite-protein pairings associated with processes essential to organismal health. To address these goals, I will lead projects under three broad themes (1) assembly and validation of the organism-specific and pan-organismal protein-metabolite interactome, (2) extending the analysis to the unknown compounds present in the protein-complexes and introducing computational methods to predict PMIs and (3) further functional characterization of the roles of dipeptides in regulating central carbon metabolism in Arabidopsis. The knowledge gained over the next five years in the proposed projects will expand our understanding of the regulatory roles of metabolites. The envisaged organism-specific and pan-organismal protein-metabolite interactomes will provide a solid foundation for us and others to uncover novel small-molecule regulators and their modes of action. Functional dissection of the dipeptide-enzyme interaction networks will contribute to the mechanistic understanding of the central question in biology of how organisms remodel their metabolism to changing environments. The outcome of my research may lead to innovation in ...