Quantitative time-resolved chemoproteomics reveals that stable O -GlcNAc regulates box C/D snoRNP biogenesis

Abstract

-linked GlcNAcylation (-GlcNAcylation), a ubiquitous posttranslational modification on intracellular proteins, is dynamically regulated in cells. To analyze the turnover dynamics of -GlcNAcylated proteins, we developed a quantitative time-resolved-linked GlcNAc proteomics (qTOP) strategy based on metabolic pulse-chase labeling with an -GlcNAc chemical reporter and stable isotope labeling with amino acids in cell culture (SILAC). Applying qTOP, we quantified the turnover rates of 533-GlcNAcylated proteins in NIH 3T3 cells and discovered that about 14% exhibited minimal removal of -GlcNAc or degradation of protein backbones. The stability of those hyperstable-GlcNAcylated proteins was more sensitive to -GlcNAcylation inhibition compared with the more dynamic populations. Among the hyperstable population were three core proteins of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs): fibrillarin (FBL), nucleolar protein 5A (NOP56), and nucleolar protein 5 (NOP58). We showed that-GlcNAcylation stabilized these proteins and was essential for snoRNP assembly. Blocking -GlcNAcylation on FBL altered the 2'--methylation of rRNAs and impaired cancer cell proliferation and tumor formation in vivo.

Artificial Cysteine S‐Glycosylation Induced by Per‐O‐Acetylated Unnatural Monosaccharides during Metabolic Glycan Labeling

“…Chemoproteomic profiling identified hundreds to thousands of modified proteins for each unnatural sugar (Figure S8 A and Table S5) as well as hundreds of cysteine sites modified with un‐, mono‐, di‐, or triacetylated unnatural sugars (Figure A, Figure S8 B and Table S6). Based on the analysis of the Ac 4 GlcNAz‐treated lysates using a resolvable mass tag, the stoichiometry of the S‐glycosylation of the two proteins GAPDH and AK2 was estimated to be about 22 % and 25 %, respectively (Figure B). Ac 4 GlcNAz labeling in lysates was abolished by adding Ac 4 GlcNAc, which indicates that the nonspecific S‐glycosylation was independent of the azido group (Figure S9 A).…”

O-linked β-N-acetylglucosamine transferase plays an essential role in heart development through regulating angiopoietin-1

“…Since O-GlcNAcylation was first reported, more than one thousand proteins have been shown to be O-GlcNAcylated. O-GlcNAcylation affects diverse cellular and molecular processes, including protein-protein interactions, transcriptional regulation, autophagy, cell proliferation and apoptosis, and insulin sensitivity [3][4][5].…”

O-GlcNAc regulates gene expression by controlling detained intron splicing

“…Many signaling processes act at short time scales. Because compounds that rapidly and specifically reduce O-GlcNAc through inhibition of OGT were not available until recently, studies that interrogate OGT function have primarily focused on changes that occur long after an initial perturbation has been applied (∼24-48 h) (16,17). Much has been learned about OGT, but the long time frame of typical studies is a limitation: some of the observed pathway changes may be downstream of the initial changes, and important effects may have been missed due to compensatory changes that occur prior to interrogation.…”