Intracellular Mechanisms of Glucocorticoid Action

描述（由申请人提供）：核受体对启动子活性的调节需要组装大型多亚基复合物，这些复合物直接影响基础转录机制或修饰核心组蛋白以影响染色质结构和重塑。负责维持蛋白质结合到细胞应答启动子的高度有序动力学的机制尚未明确建立。我们最近开发了一种新的原位荧光恢复后光漂白（FRAP）检测，导致分子伴侣及其相关的辅助分子伴侣作为糖皮质激素受体（GR）的核迁移率因子的鉴定。该试验将被利用，以提供额外的机制的见解伴侣在GR的核动力学的作用。在这个建议中要测试的主要假设是，分子伴侣发挥了重要作用，类固醇受体的动态在靶位点的细胞核内，通过其调节受体的核流动性，染色质交换和激素交换。具体目标1旨在确定单个分子伴侣的作用和分子伴侣复合物组装的途径，介导其作为类固醇受体核迁移因子的活性。伴侣蛋白组装将在一种新的原位核迁移率测定和类固醇受体/伴侣蛋白复合物形成的影响的背景下进行操作，通过共免疫沉淀试验进行评估。具体目标2将确定分子伴侣是否参与GR和其他因子在细胞核内受体靶位点的动态交换。原位FRAP试验将用于确定特定的伴侣复合物是否需要GR和各种受体辅因子从功能靶基因的动态交换。染色质免疫沉淀试验也将与透化细胞一起使用，以检查分子伴侣对GR和辅因子募集以及靶基因染色质循环的影响。最后，特定目标3将确定是否需要分子伴侣来交换来自染色质结合GR的激素。将使用区分激素释放与激素交换的测定法在透化细胞中评估分子伴侣对激素交换对核GR的影响。分子伴侣对特定靶位点激素交换的影响将使用荧光GR配体来揭示。公共卫生相关性：Hsp 90抑制剂如格尔德霉素（GA）正在评估用于乳腺癌治疗，主要是由于它们在癌细胞中对hsp 90客户蛋白如雌激素受体、p53和各种激酶的选择性作用。然而，针对hspQO或其他分子伴侣的治疗性抗癌药物的开发没有考虑到这些蛋白质在染色质动力学或组蛋白修饰中的新发现的作用，这是本提案的主题。

DESCRIPTION (provided by applicant): The regulation of promoter activity by nuclear receptors requires the assembly of large multi-subunit complexes that either directly impact the basal transcriptional machinery or modify core histones to affect chromatin structure and remodeling. The mechanisms responsible for maintaining the highly ordered dynamics of protein binding to hormonally responsive promoters have not been definitively established. We have recently developed a novel in situ fluorescence recovery after photobleaching (FRAP) assay that led to the identification of molecular chaperones and their associated co-chaperones as nuclear mobility factors for the glucocorticoid receptor (GR). This assay will be exploited to provide additional mechanistic insights into the role of chaperones in nuclear dynamics of GR. The major hypothesis to be tested in this proposal is that molecular chaperones play a major role in steroid receptor dynamics at target sites within the nucleus via their regulation of receptor nuclear mobility, chromatin exchange and hormone exchange. Specific Aim 1 seeks to identify the role of individual chaperones and the pathway of chaperone complex assembly that mediates their activity as steroid receptor nuclear mobility factors. Chaperone protein assembly will be manipulated in the context of a novel in situ nuclear mobility assay and effects on steroid receptor/chaperone complex formation assessed by co-immunoprecipitation assays. Specific Aim 2 will determine whether molecular chaperones participate in the dynamic exchange of GR and other factors at receptor target sites within the nucleus. The in situ FRAP assay will be used to determine whether specific chaperone complexes are required for the dynamic exchange of GR and various receptor cofactors from a functional target gene. Chromatin immunoprecipitation assays will also be used with permeabilized cells to examine chaperone effects on GR and cofactor recruitment and cycling on the chromatin of target genes. Finally, Specific Aim 3 will determine whether molecular chaperones are required for the exchange of hormone from chromatin- bound GR. The effects of molecular chaperones on hormone exchange on nuclear GRs will be assessed in permeabilized cells using assays that distinguish hormone release from hormone exchange. Chaperone effects on hormone exchange at a specific target site will be revealed using a fluorescent GR ligand. Public Health Relevance: Hsp90 inhibitors such as geldanamycin (GA) are being evaluated for breast cancer therapy due primarily to the their selective action in cancer cells on hsp90 client proteins such as estrogen receptor, p53 and various kinases. However, the development of therapeutic anti-cancer drugs directed against hspQO or other chaperones has not taken into account the newly discovered roles for these proteins in chromatin dynamics or histone modification, which is the subject of this proposal.