Intracellular Mechanisms of Glucocorticoid Action

糖皮质激素作用的细胞内机制

基本信息

批准号：
8034952
负责人：
Donald B DeFranco
金额：
$ 1.66万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8034952
关键词：
Accounting Affect Antineoplastic Agents Binding Biological Assay Cell Nucleus Cells Chromatin Chromatin Structure Client Co-Immunoprecipitations Complex Disease Epigenetic Process Estrogen Receptors Fluorescence Recovery After Photobleaching Geldanamycin Gene Targeting Genetic Transcription Glucocorticoid Receptor Glucocorticoids Goals Histones Hormones In Situ Individual Label Ligands Mediating Molecular Chaperones Nuclear Nuclear Receptors Pathway interactions Phosphotransferases Photobleaching Play Progesterone Receptors Protein Binding Proteins RNA RNA Polymerase II Recovery Regulation Research Personnel Response Elements Reverse Transcriptase Polymerase Chain Reaction Role Site Steroid Receptors Stress-Induced Protein TP53 gene Testing Transcriptional Activation cancer cell cancer therapy chromatin immunoprecipitation chromatin modification cofactor histone modification inhibitor/antagonist insight malignant breast neoplasm novel promoter public health relevance receptor receptor binding therapeutic development transcription factor

项目摘要

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.

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生产络合物，这些复合物要么直接影响基础转录机械或修饰核心组蛋白以影响染色质结构和重塑。尚未确定为维持蛋白质与激素反应启动子的高度有序动力学的机制。我们最近在光漂白（FRAP）测定后开发了一种新型的原位荧光恢复，该测定结果导致分子伴侣及其相关的伴侣鉴定为糖皮质激素受体（GR）的核迁移率因子。该测定法将被利用，以提供有关伴侣在GR核动力学中作用的其他机械见解。在该提案中要检验的主要假设是，分子伴侣通过调节受体核迁移率，染色质和激素交换，在核内目标部位的类固醇受体动力学中起主要作用。特定目标1旨在确定单个伴侣的作用和伴侣复合体的途径，从而将其活性作为类固醇受体核迁移率因子介导。伴侣蛋白组装将在新型原位核迁移率分析的背景下进行操纵，并对通过共免疫沉淀测定法评估的类固醇受体/伴侣伴侣复合物的影响。具体目标2将确定分子伴侣是否参与核内受体靶位点的GR的动态交换和其他因素。原位FRAP分析将用于确定是否需要特定的伴侣复合物，以使GR和来自功能靶基因的各种受体辅因子的动态交换需要。染色质免疫沉淀测定也将与透化细胞一起使用，以检查伴侣对靶基因染色质的GR和辅因子募集的影响。最后，特定的目标3将确定是否需要分子伴侣从染色质结合GR交换激素。分子伴侣对激素交换对核GRS的影响将在透明的细胞中使用将激素释放与激素交换区分开的测定法。伴侣对特定目标部位激素交换的影响将使用荧光GR配体揭示。公共卫生相关性：HSP90抑制剂（例如Geldanamycin（GA））正在评估乳腺癌治疗的乳腺癌治疗，这主要是由于它们在HSP90客户蛋白上的选择性作用，例如雌激素受体，p53和各种激酶。但是，针对HSPQO或其他伴侣的治疗性抗癌药物的开发尚未考虑到这些蛋白质在染色质动力学或组蛋白修饰中的新作用，这是本提案的主题。