Proteomic profiling of glucocorticoid-exposed myogenic cells: Time series assessment of protein translocation and transcription of inactive mRNAs.
Proteomic profiling of glucocorticoid-exposed myogenic cells: Time series assessment of protein translocation and transcription of inactive mRNAs.
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DOI：
10.1186/1477-5956-7-26
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发表时间：
2009-07-30
期刊：
影响因子：
2
通讯作者：
Hathout Y
中科院分区：
文献类型：
--
作者：
Reeves EK;Gordish-Dressman H;Hoffman EP;Hathout Y
泼尼松是最常用的处方药之一，对糖皮质激素受体介导的基因转录有很好的作用。这些影响通常以小时为单位发生。泼尼松对蛋白质信号传导也有许多非转录作用（发生在分钟尺度上），但这些研究较少。我们试图扩大使用SILAC策略和C2 C12肌管亚细胞分级组合的泼尼松作用对细胞信号传导的急性效应的理解。在SILAC标记和未标记的C2 C12肌管中，蛋白质的从头翻译均受到抑制。未标记的细胞暴露于泼尼松，而SILAC标记的细胞保持未处理。在泼尼松暴露0、5、15和30分钟后，将标记的和未标记的细胞以1：1的比例混合，并分级分离成细胞溶质和细胞核级分。共鉴定并定量了细胞溶质中的534种蛋白质和细胞核中的626种蛋白质，每种蛋白质使用3种或更多肽，基于肽的概率≤ 0.001。我们确定了显着增加（1.7至3.1倍）的细胞质丰度的11个核糖体蛋白在5分钟内曝光，所有这些返回到基线的30分钟。我们假设，这些药物诱导的急性变化，在亚细胞定位的细胞的蛋白质翻译机器可能会导致改变翻译的静止RNA。为了测试这一点，在药物暴露15分钟后测定从头蛋白质合成。定量荧光分析鉴定了16个显示翻译快速变化的2D凝胶斑点;其中5个通过MS/MS鉴定（丙酮酸激酶、膜联蛋白A6亚型A和亚型B、鼻咽上皮特异性蛋白1和复制因子C亚基1的亚型2），并且所有斑点均显示5'末端寡嘧啶基序，这些基序与非活性mRNA库的mRNA螯合相关。我们描述了亚细胞蛋白质组学分析和评估急性变化的新方法，以分钟为基础的时间尺度。这些数据扩大了目前的知识急性，非转录活性的糖皮质激素，包括蛋白质亚细胞定位的变化，改变翻译的静止RNA池，和PKC介导的细胞骨架重塑。
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 ≤ 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.
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影响因子：
--
作者：
Liu J;Rothermund CA;Ayala-Sanmartin J;Vishwanatha JK
通讯作者：
Vishwanatha JK
影响因子：
7
作者：
Davuluri, RV;Suzuki, Y;Zhang, MQ
通讯作者：
Zhang, MQ
影响因子：
7
作者：
Gronborg, M;Kristiansen, TZ;Pandey, A
通讯作者：
Pandey, A
影响因子：
2.7
作者：
KLEENE, KC;DISTEL, RJ;HECHT, NB
通讯作者：
HECHT, NB
影响因子：
4.6
作者：
Almon, Richard R.;DuBois, Debra C.;Jusko, William J.
通讯作者：
Jusko, William J.