Bioorthogonal Chemical Probe for Live Cell Imaging of Lactylation - PROJECT SUMMARY Protein lactylation is a recently discovered posttranslational modification (PTM), biochemically coupled to L- lactate and methylglyoxal cellular levels. Although our understanding of this PTM is still in its infancy, it is rational to assume it has important physiological and pathological implications. For example, emerging data show perturbed protein lactylation in conditions such as autoimmunity, inflammation, and cancer, where L-lactate levels fluctuate at the spatiotemporal scale. Therefore, it is crucial that we understand the spatiotemporal dynamics of lactylation in living systems to improve our perspective of cell biology and identify new clues to detect and treat diseases. State-of-the-art technologies such as isotope labeling coupled with mass spectrometry used for studying lactylation are invasive and generate data lacking the spatiotemporal dynamics of living systems, creating a critical need for alternative technologies. This proposal aims to develop a bioorthogonal chemistry- based live-cell imaging technology to study protein lactylation at the spatiotemporal scale. The proposed live- cell imaging technology will allow us to probe lactylation in real-time, connecting the spatiotemporal data with dynamic biological processes such as metabolite-PTM-epigenetic relationship, intracellular protein trafficking, cell development, cell differentiation, and cell migration. Our strategy is to design a fluoro-substituted L-lactic acid metabolic analog that reacts via a novel bioorthogonal fluorine-selenol substitution reaction to trigger aggregation-induced emission (AIE) in a nucleocytoplasmic-localizing imaging probe, allowing high-resolution imaging of intracellular lactylated proteins. We will achieve the goal of this project using three specific aims. In specific aim 1, we will apply enantioselective chemical synthesis to design a bioorthogonal fluoro-substituted L- lactic acid metabolic analog to tag intracellular proteins and determine if this analog is a substrate for protein lactylation. Specific aim 2 will develop a selenol-containing, nucleocytoplasmic-localizing, AIE imaging probe that fluoresces only after fluorine-selenol substitution reaction to eliminate background signals for improved resolution detection of fluoro-lactylated proteins. The final aim will investigate the metabolic analog and imaging probe in real-time imaging of lactylation in live cells. When completed, the proposed research will result in a live-cell imaging technology that captures the spatiotemporal dynamics of protein lactylation in the cytoplasm and nucleus and is easily adaptable for cytosolic and nuclear imaging of other PTMs, including acetylation and O- GlcNAcylation. Successful demonstration of the feasibility of this exploratory technology will expand the toolbox of bioorthogonal chemistry-based metabolic labeling, introducing new chemistry and strategy to improve the efficiency and resolution of bioorthogonal chemistry-based live-cell imaging. Importantly, the proposed technology will enable detailed investigations of highly dynamic intracellular PTMs to improve our understanding of cell biology, which is critical to detecting and treating diseases.
