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

• Published in: Proceedings of the National Academy of Sciences - PNAS 2017-08, Vol.114 (33), p.E6749-E6758
• Main Authors: Qin, Wei, Lv, Pinou, Fan, Xinqi, Quan, Baiyi, Zhu, Yuntao, Qin, Ke, Chen, Ying, Wang, Chu, Chen, Xing
• Format: Article
• Language: English
• Subjects: Amino acids; Biological Sciences; Biosynthesis; Cancer; Cell culture; Cell proliferation; DNA methylation; Fibrillarin; Labeling; Nucleoli; O-GlcNAcylation; Physical Sciences; PNAS Plus; Proteins; Proteomics; Ribonucleoproteins (small nucleolar)
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1702688114

O-linked GlcNAcylation (O-GlcNAcylation), a ubiquitous posttranslational modification on intracellular proteins, is dynamically regulated in cells. To analyze the turnover dynamics of O-GlcNAcylated proteins, we developed a quantitative time-resolved O-linked GlcNAc proteomics (qTOP) strategy based on metabolic pulse-chase labeling with an O-GlcNAc chemical reporter and stable isotope labeling with amino acids in cell culture (SILAC). Applying qTOP, we quantified the turnover rates of 533 O-GlcNAcylated proteins in NIH 3T3 cells and discovered that about 14% exhibited minimal removal of O-GlcNAc or degradation of protein backbones. The stability of those hyperstable O-GlcNAcylated proteins was more sensitive to O-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 O-GlcNAcylation stabilized these proteins and was essential for snoRNP assembly. Blocking O-GlcNAcylation on FBL altered the 2′-O-methylation of rRNAs and impaired cancer cell proliferation and tumor formation in vivo.