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.

Grant Number: 1R21GM160866-01
NIH Institute/Center: NIH
Principal Investigator: Christian Agatemor