Proteins associated with STAMP - a new comodulator of glucocorticoid receptors

该项目的方法是使用共纯化蛋白的质谱鉴定来鉴定可以与STAMP结合的因子，因此可能与STAMP调节gr调节基因表达中ec50%和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。例如，我们发现改变细胞中STAMP的水平可以以一种独立于类固醇的方式影响细胞的生长速度。此外，STAMP水平的变化似乎与卵巢癌的出现有关。这项质谱研究是与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Flag/STAMP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，对293个稳定转染Flag/STAMP的人胚胎肾细胞进行分离和表征，希望获得更低和更生理水平的STAMP，但仍然足够高，可以从合理数量的细胞中纯化。这些细胞中的Flag/STAMP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，我们从含有稳定转染STAMP的糖皮质激素处理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。在各种条件下，只有一种蛋白质被一致地观察到被增殖。目前正在对这种蛋白质进行更详细的研究。

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 percent 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. For example, we have found that changing the levels of STAMP in cells can affect their growth rate in a manner that is independent of steroid. In addition, changing levels of STAMP appear to be associated with associated with the appearance of ovarian cancers. This mass spectral 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 Flag/STAMP 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 Flag/STAMP 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 Flag/STAMP 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 have purified STAMP from the nuclei of glucocorticoid-treated cells containing stably transfected STAMP and compared the array of co-purified proteins to those from the whole cell extracts glucocorticoid. Only one protein is consistently observed to be copurified under a variety of conditions. This protein is currently being examined in greater detail. These studies should identify new proteins that not only participate in, or modify the activity of, STAMP modulation of the EC50 and percent partial agonist activity in GR-regulated gene expression but also affect cell growth. 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.