Combined Antibody/Lectin Enrichment Identifies Extensive Changes in the O-GlcNAc Sub-proteome upon Oxidative Stress.
Combined Antibody/Lectin Enrichment Identifies Extensive Changes in the O-GlcNAc Sub-proteome upon Oxidative Stress.
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DOI：
10.1021/acs.jproteome.6b00369
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发表时间：
2016-12-02
影响因子：
4.4
通讯作者：
Zachara NE
中科院分区：
文献类型：
--
作者：
Lee A;Miller D;Henry R;Paruchuri VD;O'Meally RN;Boronina T;Cole RN;Zachara NE
O-linked N-acetyl-β-D-glucosamine （O-GlcNAc）是一种动态的翻译后修饰，可以修饰和调节超过3000种核、细胞质和线粒体蛋白。在暴露于应激和损伤后，细胞和组织会增加许多蛋白质的O-GlcNAc修饰或o - glcn酰化，从而促进细胞应激反应和生存。本研究的目的是鉴定在急性氧化应激（H2O2）下O-GlcNAc酰化的差异蛋白，以深入了解O-GlcNAc促进生存的机制。我们通过使用细胞培养氨基酸稳定同位素标记（SILAC）和一种新的“g5 -凝集体”免疫沉淀策略实现了这一目标，该策略结合了四种o - glcnac特异性抗体（CTD110.6, RL2， HGAC39和HGAC85）和凝集素WGA。采用G5-lectibody色谱柱结合碱性反相（bRP）色谱和C18 hplc -MS/MS，对990个蛋白进行了鉴定和定量。鉴定的数百种蛋白在氧化应激下显示出与g5 -受体柱的关联增加（bbb250）或减少（>10），其中我们验证了24种蛋白的o - glcn酰化状态。对糖基化改变的蛋白质的分析表明，应激诱导的o - glcn酰化变化聚集在已知的调节细胞对损伤反应的途径中，包括蛋白质折叠、转录调节、表观遗传学和参与RNA生物发生的蛋白质。总之，这些数据表明，应激诱导的o - glcn酰化调节了许多不同的细胞途径，促进细胞和组织的存活。
O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) is a dynamic post-translational modification that modifies and regulates over 3,000 nuclear, cytoplasmic, and mitochondrial proteins. Upon exposure to stress and injury, cells and tissues increase the O-GlcNAc modification, or O-GlcNAcylation, of numerous proteins promoting the cellular stress response and thus survival. The aim of this study was to identify proteins that are differentially O-GlcNAcylated upon acute oxidative stress (H2O2) to provide insight into the mechanisms by which O-GlcNAc promotes survival. We achieved this goal by employing Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) and a novel “G5-lectibody” immunoprecipitation strategy that combines four O-GlcNAc-specific antibodies (CTD110.6, RL2, HGAC39, and HGAC85) and the lectin WGA. Using the G5-lectibody column in combination with basic Reversed Phase (bRP) chromatography and C18 RPLC-MS/MS, 990 proteins were identified and quantified. Hundreds of proteins identified demonstrated increased (>250) or decreased (>110) association with the G5-lectibody column upon oxidative stress, of which we validated the O-GlcNAcylation status of 24 proteins. Analysis of proteins with altered glycosylation suggest that stress-induced changes in O-GlcNAcylation cluster into pathways known to regulate the cells response to injury and include protein folding, transcriptional regulation, epigenetics, and proteins involved in RNA biogenesis. Together, these data suggest that stress-induced O-GlcNAcylation regulates numerous and diverse cellular pathways to promote cell and tissue survival.
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DOI：
10.1073/pnas.85.24.9595
发表时间：
1988-12-01
影响因子：
11.1
作者：
DONOFRIO, M;STARR, CM;HANOVER, JA
通讯作者：
HANOVER, JA
影响因子：
7
作者：
Everley, PA;Krijgsveld, J;Gygi, SP
通讯作者：
Gygi, SP
混合谱系白血病 5 (MLL5) 蛋白质稳定性由 O-GlcNac 转移酶 (OGT) 和泛素特异性蛋白酶 7 (USP7) 协同调节
影响因子：
3.7
作者：
Ding, Xiaodan;Jiang, Wei;Zhang, Yan
通讯作者：
Zhang, Yan
影响因子：
3.7
作者：
Bhuiyan, Tanja;Waridel, Patrice;Herr, Winship
通讯作者：
Herr, Winship
影响因子：
8
作者：
Bachmaier R;Aryee DN;Jug G;Kauer M;Kreppel M;Lee KA;Kovar H
通讯作者：
Kovar H