Proteins associated with STAMP - a new comodulator of glucocorticoid receptors

该项目的方法是使用共纯化蛋白质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调节基因表达的EC 50和部分激动剂活性百分比相关。 该方法还有望鉴定参与STAMP其他尚未鉴定的作用的蛋白质。 例如，我们发现改变细胞中STAMP的水平可以以一种不依赖于类固醇的方式影响它们的生长速率。 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的水平非常低（He和Simons Jr.，2007，Mol. Cell.生物学：27，1467-1485），我们已经过表达了外源Flag标记的STAMP，我们证明其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，这些蛋白质中的许多被怀疑是非特异性结合的，并且不属于已知的TIF 2，SRC-1和GR的相关蛋白质。纯化的STAMP的定量Western印迹分析表明，只有0.4%的STAMP与GR相关。 因此，分离并表征了具有稳定转染的Flag/STAMP的293个人胚胎肾细胞，希望获得更低和更生理水平的STAMP，但仍升高到足以允许从合理数量的细胞中纯化。 这些细胞中Flag/STAMP的水平比瞬时转染的细胞中低得多。 全细胞提取物通过质谱分析揭示了相关蛋白质的不同分类。 在加入糖皮质激素激动剂后，STAMP与GR一起沿着集中在细胞核中（He和Simons Jr.，2007，Mol. Cell.生物学，27，1467-1485）。 因此，我们已经从含有稳定转染的STAMP的糖皮质激素处理的细胞的细胞核中纯化了STAMP，并将共纯化的蛋白质阵列与来自全细胞提取物糖皮质激素的蛋白质阵列进行了比较。 只有一种蛋白质被一致地观察到在各种条件下被共纯化。 目前正在对这种蛋白质进行更详细的研究。 这些研究应该确定新的蛋白质，不仅参与，或修改的活动，STAMP调制的EC 50和百分比部分激动剂活性GR调节基因表达，但也影响细胞生长。 这些结果将增加我们对GR-类固醇复合物的几种生理相关转录特性的理解，这些特性允许连续的反应，并构成新的治疗靶点，用于在发育、分化、稳态和内分泌治疗期间通过类固醇激素对基因表达进行差异控制。 这些综合研究结果有助于我们在分子水平上定义类固醇激素的作用并了解其在人体生理学中的作用的长期目标。

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.