Proteins associated with STAMP - a new comodulator of glucocorticoid receptors

我们以前使用共纯化蛋白的质谱学鉴定来鉴定与STAMP结合的因子的方法，由于内源性STAMP的水平非常低而受挫。瞬时转染FLAG/STAMP真核表达载体后，FLAG/STAMP蛋白和共纯化蛋白的表达水平较高。然而，这些蛋白中的大多数似乎是非特异性结合的，并不在已知的TIF2、SRC-1和GR相关蛋白之列。对纯化的印记进行定量的Western印迹分析表明，在这些样本中只有0.4%的印记与GRS有关。通过质谱分析，稳定转基因的293人胚胎肾细胞确实提供了更低和更生理水平的STAMP和不同种类的相关蛋白。然而，没有一种共纯化的蛋白质似乎具有生理意义。 鉴于共纯化蛋白质的质谱鉴定结果有限，我们集中使用来自有标记和无标记细胞的微阵列数据来识别可能的结合蛋白。如其他文献(DK047039-04)所述，我们最近从野生型小鼠和内源性STAMP基因已被敲除的小鼠(KO小鼠)中制备了小鼠胚胎成纤维细胞(MEF)。对糖皮质激素诱导8小时的细胞进行了基因芯片分析。获得了近3000个基因的高质量数据，这些基因的表达水平在地塞米松处理后变化了1.5倍。野生型和KO MEF细胞之间表达变化最大的基因正在检查是否涉及其他已知的辅助因子。我们的假设是，STAMP与这些已知辅助因子的相互作用可能是STAMP KO MEF细胞中糖皮质激素调节基因表达显著变化的原因。那些在基础水平表达有重大差异的基因是介导STAMP明显的类固醇非依赖性效应的候选基因，如细胞生长和肿瘤形成。最初确定的辅因子将通过共免疫沉淀进一步检查印记结合情况，并在使用瞬时转基因细胞的基因诱导试验中进一步检查辅因子活性。 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GR调节基因表达中EC50和PAA的STAMP调节活性，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。

Our previous approach of using mass spectral identification of co-purified proteins to identify factors that bind to STAMP was thwarted by the very low levels of endogenous STAMP. Transient transfections of Flag/STAMP plasmid did give high levels of overexpressed Flag/STAMP and co-purified proteins. However, most of these proteins appeared 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 in these samples. Stably transfected 293 human embryonic kidney cells did afford lower and more physiological levels of STAMP and a different assortment of associated proteins by mass spectrum analysis. However, no co-purified protein appeared to be physiologically interesting. In view of the limited results using mass spectral identification of co-purified proteins, we have concentrated on using microarray data from cells with and without STAMP to identify possible binding proteins. As described elsewhere (DK047039-04), we have recently prepared mouse embryo fibroblasts (MEFs) from wild type mice and mice in which the endogenous STAMP gene has been knocked out (KO mice). Microarray analyses have been performed on cells that were induced by glucocorticoid steroid for 8 hr. High quality data were obtained for almost 3,000 genes, for which the level of expression changed by ≥ 1.5 fold after Dex treatment. Those genes with the largest change in expression between wild type and KO MEF cells are being examined for the involvement of other known cofactors. Our hypothesis is that STAMP interaction with these known cofactors may be responsible for the significant change in glucocorticoid-regulated gene expression in the STAMP KO MEF cells. Those genes for which there are major differences in basal level expression are candidates for mediating the apparent steroid-independent effects of STAMP, such as cell growth and tumor formation. Initially identified cofactors will be further examined for STAMP binding by co-immunoprecipitation and for cofactor activity in gene induction assays using transiently transfected cells. These studies should identify new proteins that not only participate in, or modify the activity of, STAMP modulation of the EC50 and PAA 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.