Proteins associated with STAMP - a new comodulator of glucocorticoid receptors

本项目的方法是使用共纯化蛋白的质谱鉴定来鉴定可以结合STAMP的因子，因此可能与STAMP调节EC50和gr调节基因表达的部分激动剂活性相关。该方法还有望识别参与其他尚未确定的STAMP作用的蛋白质。这项研究是与Sanford Markey博士（NIMH， NIH）合作进行的。由于内源性STAMP水平非常低（He and Simons Jr., 2007, Mol. Cell.）。医学杂志。， 27,1467 -1485)，我们过度表达了外源性flag标记的STAMP，我们证明它保留了完整的生物活性。瞬时转染产生高水平的FlagSTAMP和共纯化蛋白。然而，许多这些蛋白被怀疑是非特异性结合的，并且不在TIF2、SRC-1和GR的已知相关蛋白中。纯化的STAMP的定量Western blot分析表明，只有0.4%的STAMP与GR相关。因此，我们分离了293个稳定转染了FlagSTAMP的人胚胎肾细胞，并对其进行了表征，希望获得更低、更生理水平的STAMP，但仍足够高，可以从合理数量的细胞中纯化。这些细胞中的FlagSTAMP水平远低于瞬时转染的细胞。全细胞提取物通过质谱分析揭示了不同种类的相关蛋白。在添加糖皮质激素激动剂后，STAMP与GRs一起集中在细胞核中（He和Simons Jr., 2007, Mol. Cell.）。医学杂志。， 27, 1467-1485)。因此，我们目前正在用稳定转染的STAMP从糖皮质激素处理过的细胞的细胞核中纯化STAMP，并将共纯化的蛋白质阵列与糖皮质激素全细胞提取物的蛋白质阵列进行比较。三到五种最丰富或最有趣的共纯化蛋白将在与内源性蛋白的共ip筛选中进行检测，然后进行全细胞检测，以确定推定的stamp相关蛋白的可能生物活性。

The approach of this project is to use mass spectral identification of co-purified proteins to identify factors that can bind to STAMP and may therefore be relevant for STAMP modulation of the EC50 and partial agonist activity in GR-regulated gene expression. This method is also expected to identify proteins that are involved in other, yet unidentified actions of STAMP. This study is being conducted in collaboration with Dr. Sanford Markey (NIMH, NIH). Due to the very low levels of endogenous STAMP (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485), we have overexpressed an exogenous Flag-tagged STAMP, which we demonstrated retains full biological activities. Transient transfections gave high levels of FlagSTAMP and co-purified proteins. However, many of these proteins were suspected to be non-specifically bound and were not among the known associated proteins of TIF2, SRC-1, and GR. Quantitative Western blot analysis of the purified STAMP indicated that only 0.4% of the STAMP was associated with GRs. Therefore, 293 human embryonic kidney cells with stably transfected FlagSTAMP were isolated and characterized in hopes of obtaining lower and more physiological levels of STAMP but still elevated enough to permit purification from a reasonable number of cells. The level of FlagSTAMP in these cells is much lower than in the transiently transfected cells. Whole cell extracts revealed a different assortment of associated proteins by mass spectrum analysis. STAMP is concentrated in the nucleus, along with GRs, upon addition of a glucocorticoid agonist (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485). Therefore, we are currently purifying STAMP from the nuclei of glucocorticoid-treated cells with stably transfected STAMP and comparing the array of co-purified proteins to those from the whole cell extracts glucocorticoid. Three to five of the most abundant andor interesting co-purified proteins will be examined in co-IP screens with endogenous proteins before moving on to whole cell assays to determine possible biological activities of the putative STAMP-associated proteins. These studies should identify new proteins that participate in, or modify the activity of, STAMP modulation of the EC50 and partial agonist activity in GR-regulated gene expression. These results will increase our understanding of several physiologically relevant transcriptional properties of GR-steroid complexes that permit a continuum of responses and constitute new therapeutic targets for differential control of gene expression by steroid hormones during development, differentiation, homeostasis, and endocrine therapies. These combined findings contribute to our long-term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology. Other currently unknown activities of STAMP may also be unearthed.