Living cells continually adapt to their environments. Observing these cellular mechanisms in real-time and space is challenging. A key sugar nutrient-sensing mechanism in mammalian cells is the reversible protein modification O-GlcNAc. O-GlcNAcylation (O-linked N-acetylglucosamine addition to serine and threonine protein residues) is derived from intracellular glucose and therefore alters protein (and cell) behavior as a response to changes in local cellular environment. Very few biological or chemical methods exist to study O-GlcNAcylation in living cells, limiting our understanding of dynamic O-GlcNAc protein modifications in changing physiological settings (a). Here, we present spatiotemporal tools to capture and perturb O-GlcNAc dynamics in living cells based on photochemistry (b) and spatial protein labeling (c). By harnessing these bioorthogonal chemical labeling reactions, our tools can identify O-GlcNAc changes in real-time as cells perform nutrient sensing and signaling activities. We identify key proteins that respond to cellular environment changes via spatiotemporal O-GlcNAc regulation, such mRNA splicing factors as well as the signal transduction factor NF-kB. Our real-time, intracellular tools reveal effects driven by O-GlcNAc cycling in situ and offer insight into cell regulatory events at the molecular level.

In situ methods for capturing dynamic O-GlcNAc protein glycosylation events

ACS Spring 2022

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