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学鉴定来确定可以与STAMP结合的因子，从而可能与EC50的STAMP调节以及GR调节的基因表达中的部分激动剂活性百分比相关。这种方法还有望识别与STAMP的其他尚未识别的作用有关的蛋白质。例如，我们已经发现，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它们的生长速度。此外，STAMP水平的变化似乎与卵巢癌的出现有关。这项质谱学研究是与Sanford Markey博士(NIMH，NIH)合作进行的。由于内源性STAMP水平非常低(他和小西蒙斯，2007，Mol.牢房。Biol.，27,1467-1485)，我们过表达了一个外源FLAG标记的印记，我们证明它保留了全部的生物活性。瞬时转染法获得高水平的Flag/StAMP和共纯化蛋白。然而，这些蛋白中的许多被怀疑是非特异性结合的，不属于已知的TIF2、SRC-1和GR的相关蛋白。对纯化的印记进行定量的Western印迹分析表明，只有0.4%的印记与GRS有关。因此，我们分离了293个稳定表达FLAG/STAMP的人胚胎肾细胞，并对其进行了鉴定，希望获得更低和更具生理性的STAMP水平，但仍能提高到足以从合理数量的细胞中进行纯化的水平。这些细胞中的FLAG/STAMP水平远低于瞬时转染组。通过质谱分析，全细胞提取物显示了不同种类的相关蛋白质。当添加糖皮质激素激动剂时，STAMP与GRs一起集中在细胞核中(他和Simons Jr.，2007，Mol.牢房。Biol.，27,1467-1485)。因此，我们从含有稳定表达的STAMP的糖皮质激素处理细胞的细胞核中提纯了STAMP，并将共纯化的蛋白质阵列与全细胞糖皮质激素提取液中的蛋白进行了比较。在不同的条件下，只有一种蛋白质被持续观察到被复制。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，这些蛋白质不仅参与或改变EC50的STAMP调节活性和GR调节基因表达中部分激动剂活性的百分比，而且还影响细胞生长。这些结果将增加我们对GR-类固醇复合体的几个生理相关转录特性的理解，这些复合体允许一系列反应，并成为类固醇激素在发育、分化、动态平衡和内分泌治疗过程中差异控制基因表达的新的治疗靶点。这些综合的发现有助于我们在分子水平上定义类固醇激素的作用，并了解它们在人类生理学中的作用。

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.