Glycolysis is a primary and central metabolic pathway of the carbohydrates that supplies the energy source and building block precursors to the living system. A strong correlation between deregulated glucose metabolism and various human diseases has been studied, however mechanisms that integrate the metabolic state with regulatory pathways in cellular systems are largely unexplored. Here we report the direct link between glycolysis and KEAP1-NRF2 transcriptional program via a novel reactive metabolite-induced posttranslational modification. We demonstrate that reactive dicarbonyl metabolite methylglyoxal, mainly generated from glycolysis in cells, is a signaling messenger of the activation of NRF2 transcriptional program. Methylglyoxal induces the formation of a methylimidazole crosslink between cysteine electrophile sensor and proximal arginine residues posttranslational modification between two monomeric KEAP1 which was firstly characterized by our study. In-vivo experiment of NRF2-dependent UV-damage mouse model with the PGK1 inhibitor showed therapeutic efficacy, demonstrating the physiological relevance of regulating glucose metabolism for activating NRF2 signaling cascade. In summary, our work presented herein highlights the role of reactive metabolite-induced posttranslational modifications in cell signaling. Furthermore, we developed the novel chemical proteomic technologies to unveil the underlying protein communication networks and their dynamic and temporal interactions with other proteins or ligands in live cells. Our P3 profiling technology was designed to profile high resolution protein proximity maps by mild and fast proximity biotin labeling with light activation. Application of our approach to KEAP1 identified potential protein-protein interaction partners including well-characterized KEAP1 binding protein PGAM5, demonstrating that our novel chemical proteomic tool is applicable to study challenging research targets such as redox signaling networks of the cell. We also designed SILAC surface mapping assay to study the protein-ligand interactions in live cells, and we confirmed our strategy with photoaffinity analogue probe of GNF-2 and its binding site in c-ABL that is well-characterized. Indeed, the results of our quantitative proteomic workflow with PGK1 and its novel inhibitor CBR-470-1 suggest that CBR-470-1 may inhibit PGK1 activity by interfering its interaction with glycolytic intermediates 1,3-BPG and 3PG. Overall, these new chemical proteomic technologies with photoreactive functionalities and quantitative mass spectrometry may provide an unbiased, global insight into the nature of protein communications and regulations in complex biological systems that lead to elucidate the network of reactive metabolite-induced posttranslational modifications in cell signaling.




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