Dataset Information

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

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 and removed on diverse protein sites as a response to fluctuating nutrient levels, stressors, and signaling cues. Two aspects elude methods to track O-GlcNAc response functions with chemical strategies: spatial control over subcellular locations and time control during labeling reactions. The objective of this study was to create intracellular proximity labeling tools to identify functional changes of O-GlcNAc patterns with spatiotemporal control. We developed a labeling strategy based on TurboID system for rapid protein biotin conjugation that was directed to O-GlcNAc protein modifications inside cells, a set of tools we call “GlycoID.” Localized variants to the nucleus and cytosol, nuc-GlycoID and cyt-GlycoID, label O-GlcNAc proteins and their interactomes in subcellular space. Labeling during insulin as well as serum stimulation revealed functional changes in O-GlcNAc proteins in as soon as 30 minutes of signaling. We demonstrate that the GlycoID strategy can capture 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 datasets in HeLa cells will be a useful functional resource for O-GlcNAc-associated physiology.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Mixed Disorder As Reaction To Stress

SUBMITTER: Charlie Fehl

LAB HEAD: Charlie Fehl

PROVIDER: PXD033062 | Pride | 2022-10-15

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	allSpectra.CID.ITMS.iso_0.apl	Other
	allSpectra.CID.ITMS.iso_1.apl	Other
	allSpectra.CID.ITMS.iso_10.apl	Other
	allSpectra.CID.ITMS.iso_11.apl	Other
	allSpectra.CID.ITMS.iso_12.apl	Other

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Publications

Spatiotemporal Proximity Labeling Tools to Track GlcNAc Sugar-Modified Functional Protein Hubs during Cellular Signaling.

Liu Yimin Y Nelson Zachary M ZM Reda Ali A Fehl Charlie C

ACS chemical biology 20220712 8

A fundamental mechanism that all eukaryotic cells use to adapt to their environment is dynamic protein modification with monosaccharide sugars. In humans, O-linked <i>N</i>-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 durin ...[more]

PMID: 35819414

Similar Datasets

Project description:Over the past decades, protein O-GlcNAcylation has been found to play a fundamental role in cell cycle control, metabolism, transcriptional regulation, and cellular signaling. Nevertheless, quantitative approaches to determine in vivo GlcNAc dynamics at a large-scale are still not readily available. Here, we have developed an approach to isotopically label O-GlcNAc modifications on proteins by producing 13C-labeled UDP-GlcNAc from 13C6-glucose via the hexosamine biosynthetic pathway. This metabolic labeling was combined with quantitative mass spectrometry-based proteomics to determine site-specific protein O-GlcNAcylation turnover rates. First, an efficient enrichment method for O-GlcNAc peptides was developed with the use of phenylboronic acid solid-phase extraction and anhydrous DMSO. The near stoichiometry reaction between the diol of GlcNAc and boronic acid dramatically improved the enrichment efficiency. Additionally, our kinetic model for turnover rates integrates both metabolomic and proteomic data, which increase the accuracy of the turnover rate estimation. Other advantages of this metabolic labeling method include in vivo application, direct labeling of the O-GlcNAc sites and higher confidence for site identification. Concentrating only on nuclear localized GlcNAc modified proteins, we are able to identify 159 O-GlcNAc sites on 74 proteins and determine turnover rates of 24 O-GlcNAc peptides from 21 proteins extracted from HeLa nuclei. In general, we found O-GlcNAcylation turnover rates are slower than those published for phosphorylation or acetylation. Nevertheless, the rates widely varied depending on both the protein and the residue modified. We believe this methodology can be broadly applied to reveal turnovers/dynamics of protein O-GlcNAcylation from different biological states and will provide more information on the significance of site-specific O-GlcNAcylation, enabling us to study the temporal dynamics of this critical modification in a site-specific manner for the first time.