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水平很低，以及与瞬时转染的FLAG/STAMP的非特异性结合而受挫。出于这个原因，我们专注于使用来自有印记和没有印记的细胞的微阵列数据来识别可能的结合蛋白。如其他文献(DK047039-05)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调节STAMP明显的类固醇非依赖性效应的候选基因，例如细胞生长和肿瘤形成(他等人，2010，BMC癌症，10,128)。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。与此同时，具有相同遗传背景的STAMP KO小鼠(C57/BL6)已经被制备出来，并正在研究其表型异常。任何生理缺陷的鉴定和表征都将指向可能与STAMP结合的其他蛋白质。最后，对GR、TIF2和STAMP组合的转录特性的研究(在DK047039-05中描述)表明，GR、TIF2和STAMP可以形成一个三元复合体，其中不同的蛋白质表面不同地改变类固醇调节基因诱导的Amax、EC50和PAA。这些结果表明，与STAMP相关的蛋白质的影响可能比最初认为的更微妙和更有选择性。能够调节GR控制的基因表达的一个、两个或全部三个参数的能力增加了识别能够与STAMP结合的其他蛋白质的重要性。 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AMAX、EC50和PAA在GR调节的基因表达中的STAMP调节活性，而且还参与诸如细胞生长等受体无关的过程。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 and non-specific binding to transiently transfected Flag/STAMP. For this reason, we have concentrated on using microarray data from cells with and without STAMP to identify possible binding proteins. As described elsewhere (DK047039-05), 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 (He et al., 2010, BMC Cancer, 10, 128). 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. At the same time, STAMP KO mice in a homogeneous genetic background (C57/BL6) have been prepared and are being studied for phenotypic abnormalities. The identification and characterization of any physiological defects will point to other proteins that might bind STAMP. Finally, studies of the transcriptional properties of the combination of GR, TIF2, and STAMP (described in DK047039-05) indicate that GR, TIF2, and STAMP can form a ternary complex in which different protein surfaces unequally modify the Amax, EC50, and PAA of steroid-regulated gene induction. These results suggest that the effects of STAMP-associated proteins can be more subtle, and selective, than initially believed. The ability to modulate one, two, or all three parameters of GR-controled gene expression heightens the importance of identifying other proteins that can bind to STAMP. These studies should identify new proteins that not only participate in, or modify the activity of, STAMP modulation of the Amax, EC50 and PAA in GR-regulated gene expression but also receptor-independent processes such as 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.