Proteins associated with STAMP - a new comodulator of glucocorticoid receptors

与 STAMP 相关的蛋白质 - 糖皮质激素受体的新共调节剂

• 批准号: 8349789
• 负责人: S S SIMONS
• 金额: $ 19.63万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349789
• 关键词: Adverse effects; Agonist; Androgens; Binding; Binding Proteins; Biological Assay; Cells; Co-Immunoprecipitations; Complex; Data; Data Quality; Defect; Development; Embryo; Fibroblasts; Gene Expression; Gene Targeting; Genes; Genetic; Glucocorticoid Receptor; Glucocorticoids; Goals; Homeostasis; Human; Knock-out; Malignant Neoplasms; Mediating; Membrane Proteins; Molecular; Mus; NCOA2 gene; Organism; Physiological; Physiology; Process; Progesterone Receptors; Property; Protein Binding; Proteins; Role; Steroids; Time; Wild Type Mouse; cell growth; cofactor; gene induction; hormone therapy; new therapeutic target; receptor; response; steroid hormone; tumor

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.

我们先前使用共纯化蛋白质的质谱鉴定来鉴定与STAMP结合的因子的方法被非常低水平的内源性STAMP和与瞬时转染的Flag/STAMP的非特异性结合所阻碍。 出于这个原因，我们集中使用来自有和没有STAMP的细胞的微阵列数据来鉴定可能的结合蛋白。 如其他地方所述（DK 047039 -05），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）制备了小鼠胚胎成纤维细胞（MEF）。 对糖皮质激素类固醇诱导8小时的细胞进行了微阵列分析。获得了近3,000个基因的高质量数据，这些基因的表达水平在Dex处理后变化了1.5倍。 正在检查野生型和KO MEF细胞之间表达变化最大的那些基因是否涉及其他已知辅因子。 我们的假设是STAMP与这些已知辅因子的相互作用可能是导致STAMP KO MEF细胞中糖皮质激素调节的基因表达发生显著变化的原因。 在基础水平表达中存在主要差异的那些基因是用于介导STAMP的明显的类固醇非依赖性作用（例如细胞生长和肿瘤形成）的候选基因（He et al.，2010，BMC Cancer，10，128）。 将通过免疫共沉淀进一步检查最初鉴定的辅因子的STAMP结合，并使用瞬时转染细胞在基因诱导测定中检查辅因子活性。 与此同时，已经制备了具有同质遗传背景（C57/BL 6）的STAMP KO小鼠，并正在研究其表型异常。 任何生理缺陷的鉴定和表征将指向可能结合STAMP的其他蛋白质。 最后，对GR、TIF 2和STAMP组合的转录特性的研究（描述于DK 047039 -05中）表明，GR、TIF 2和STAMP可以形成三元复合物，其中不同的蛋白质表面不相等地修饰类固醇调节的基因诱导的Amax、EC 50和PAA。 这些结果表明，STAMP相关蛋白的作用可能比最初认为的更微妙和选择性。 调节GR控制的基因表达的一个、两个或所有三个参数的能力提高了鉴定可与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，不仅参与或修改的活性，STAMP调制的Amax，EC 50和PAA在GR调节的基因表达，而且受体的非依赖性过程，如细胞生长。 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。