Reeves Erica K M, Gordish-Dressman Heather, Hoffman Eric P, Hathout Yetrib
Research Center for Genetic Medicine, Children's National Medical Center, NW, Washington, DC 20010, USA.
Proteome Sci. 2009 Jul 30;7:26. doi: 10.1186/1477-5956-7-26.
Prednisone, one of the most highly prescribed drugs, has well characterized effects on gene transcription mediated by the glucocorticoid receptor. These effects are typically occurring on the scale of hours. Prednisone also has a number of non-transcriptional effects (occurring on minutes scale) on protein signaling, yet these are less well studied. We sought to expand the understanding of acute effects of prednisone action on cell signaling using a combination of SILAC strategy and subcellular fractionations from C2C12 myotubes.
De novo translation of proteins was inhibited in both SILAC labeled and unlabeled C2C12 myotubes. Unlabeled cells were exposed to prednisone while SILAC labeled cells remained untreated. After 0, 5, 15, and 30 minutes of prednisone exposure, labeled and unlabeled cells were mixed at 1:1 ratios and fractionated into cytosolic and nuclear fractions. A total of 534 proteins in the cytosol and 626 proteins in the nucleus were identified and quantitated, using 3 or more peptides per protein with peptide based probability < or = 0.001. We identified significant increases (1.7- to 3.1- fold) in cytoplasmic abundance of 11 ribosomal proteins within 5 minutes of exposure, all of which returned to baseline by 30 min. We hypothesized that these drug-induced acute changes in the subcellular localization of the cell's protein translational machinery could lead to altered translation of quiescent RNAs. To test this, de novo protein synthesis was assayed after 15 minutes of drug exposure. Quantitative fluorography identified 16 2D gel spots showing rapid changes in translation; five of these were identified by MS/MS (pyruvate kinase, annexin A6 isoform A and isoform B, nasopharyngeal epithelium specific protein 1, and isoform 2 of Replication factor C subunit 1), and all showed the 5' terminal oligopyrimidine motifs associated with mRNA sequestration to and from inactive mRNA pools.
We describe novel approaches of subcellular proteomic profiling and assessment of acute changes on a minute-based time scale. These data expand the current knowledge of acute, non-transcriptional activities of glucocorticoids, including changes in protein subcellular localization, altered translation of quiescent RNA pools, and PKC-mediated cytoskeleton remodeling.
泼尼松是最常被处方的药物之一，其对由糖皮质激素受体介导的基因转录具有明确的作用。这些作用通常在数小时的时间尺度上发生。泼尼松对蛋白质信号传导也有许多非转录作用（在数分钟的时间尺度上发生），但对这些作用的研究较少。我们试图通过结合稳定同位素标记氨基酸细胞培养法（SILAC）策略和C2C12肌管的亚细胞分级分离来扩展对泼尼松作用于细胞信号传导的急性效应的理解。
在SILAC标记和未标记的C2C12肌管中，蛋白质的从头合成均受到抑制。未标记的细胞暴露于泼尼松，而SILAC标记的细胞保持未处理状态。在泼尼松暴露0、5、15和30分钟后，将标记和未标记的细胞按1:1的比例混合，并分级分离为细胞质和细胞核部分。使用每种蛋白质3个或更多肽段且基于肽段的概率≤0.001，共鉴定并定量了细胞质中的534种蛋白质和细胞核中的626种蛋白质。我们发现在暴露5分钟内，11种核糖体蛋白的细胞质丰度显著增加（1.7至3.1倍），所有这些在30分钟时均恢复到基线水平。我们推测细胞蛋白质翻译机制的亚细胞定位中这些药物诱导的急性变化可能导致静态RNA的翻译改变。为了验证这一点，在药物暴露15分钟后检测了从头蛋白质合成。定量荧光自显影片鉴定出16个二维凝胶斑点显示翻译的快速变化；其中5个通过串联质谱（MS/MS）鉴定（丙酮酸激酶、膜联蛋白A6同工型A和同工型B、鼻咽上皮特异性蛋白1以及复制因子C亚基1的同工型2），并且所有这些都显示出与mRNA进出无活性mRNA池的隔离相关的5'末端寡嘧啶基序。
我们描述了亚细胞蛋白质组学分析的新方法以及基于分钟时间尺度的急性变化评估。这些数据扩展了目前对糖皮质激素急性非转录活性的认识，包括蛋白质亚细胞定位的变化、静态RNA池翻译的改变以及蛋白激酶C介导的细胞骨架重塑。
