College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China.
Proc Natl Acad Sci U S A. 2017 Aug 15;114(33):E6749-E6758. doi: 10.1073/pnas.1702688114. Epub 2017 Jul 31.
-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.
-连接的 GlcNAc 化（-GlcNAcylation）是一种在细胞内蛋白质上普遍存在的翻译后修饰，在细胞中动态调节。为了分析 -GlcNAc 化蛋白质的周转动力学，我们开发了一种基于代谢脉冲追踪标记的定量时间分辨 -连接的 GlcNAc 蛋白质组学（qTOP）策略，该策略使用 -GlcNAc 化学报告器和细胞培养中的稳定同位素标记的氨基酸（SILAC）。应用 qTOP，我们定量测定了 NIH 3T3 细胞中 533 种 -GlcNAc 化蛋白质的周转率，发现约 14%的蛋白质 -GlcNAc 最小去除或蛋白质骨架降解。与更动态的群体相比，这些超稳定 -GlcNAc 化蛋白质的稳定性对 -GlcNAc 化抑制更为敏感。在超稳定群体中，有三个框 C/D 小核仁核糖核蛋白复合物（snoRNP）的核心蛋白：核仁蛋白 5A（NOP56）、核仁蛋白 5（NOP58）和核仁蛋白 5（FBL）。我们表明 -GlcNAcylation 稳定了这些蛋白质，并且对于 snoRNP 组装是必不可少的。阻断 FBL 上的 -GlcNAcylation 改变了 rRNAs 的 2'--甲基化，并损害了体内癌细胞的增殖和肿瘤形成。
