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Spatiotemporal proximity labeling tools to track GlcNAc sugar-modified functional protein hubs during cellular signaling

Yimin Liu, Zachary M. Nelson, Ali Reda, View ORCID ProfileCharlie Fehl
doi: https://doi.org/10.1101/2022.04.13.488185
Yimin Liu
1Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI, United States
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Zachary M. Nelson
1Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI, United States
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Ali Reda
1Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI, United States
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Charlie Fehl
1Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI, United States
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  • ORCID record for Charlie Fehl
  • For correspondence: charlie.fehl@wayne.edu
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Abstract

A fundamental mechanism that all eukaryotic cells use to adapt to their environment is dynamic protein modification with monosaccharide sugars. In humans, O-linked N-acetylglucosamine (O-GlcNAc) is rapidly added to and removed from diverse protein sites as a response to fluctuating nutrient levels, stressors, and signaling cues. Two aspects remain challenging for tracking functional O-GlcNAc events with chemical strategies: spatial control over subcellular locations and time control during labeling. The objective of this study was to create intracellular proximity labeling tools to identify functional changes in O-GlcNAc patterns with spatiotemporal control. We developed a labeling strategy based on the TurboID proximity labeling system for rapid protein biotin conjugation that we directed to O-GlcNAc protein modifications inside cells, a set of tools we called “GlycoID.” Localized variants to the nucleus and cytosol, nuc-GlycoID and cyt-GlycoID, labeled O-GlcNAc proteins and their interactomes in subcellular space. Labeling during insulin as well as serum stimulation revealed functional changes in O-GlcNAc proteins as soon as 30 minutes of signaling. We demonstrated using proteomic analysis that the GlycoID strategy captured O-GlcNAcylated “activity hubs” consisting of O-GlcNAc proteins and their associated protein-protein interactions. The ability to follow changes in O-GlcNAc hubs during physiological events like insulin stimulation poises these tools to be used for determining mechanisms of glycobiological cell regulation. Our functional O-GlcNAc datasets in human cells will be a useful resource for O-GlcNAc-driven mechanisms.

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Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted April 13, 2022.
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Spatiotemporal proximity labeling tools to track GlcNAc sugar-modified functional protein hubs during cellular signaling
Yimin Liu, Zachary M. Nelson, Ali Reda, Charlie Fehl
bioRxiv 2022.04.13.488185; doi: https://doi.org/10.1101/2022.04.13.488185
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Spatiotemporal proximity labeling tools to track GlcNAc sugar-modified functional protein hubs during cellular signaling
Yimin Liu, Zachary M. Nelson, Ali Reda, Charlie Fehl
bioRxiv 2022.04.13.488185; doi: https://doi.org/10.1101/2022.04.13.488185

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