Mechanisms of Xenoestrogen Stress: A Proteomic and Functional Genomic Approach

异雌激素应激机制：蛋白质组学和功能基因组学方法

基本信息

批准号：
7450524
负责人：
P. Lee Ferguson
金额：
$ 20.28万
依托单位：
UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-15 至 2011-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7450524
关键词：
4-nonylphenol Address Affinity Affinity Chromatography Area Arts Binding Biological Assay Biological Models Biosensor Breast Cell Line Cells Complex Consensus DNA-Protein Interaction Dependence Development Diagnostic Disease Elements Endocrine Environmental Exposure Environmental Pollution Estradiol Estrogen Antagonists Estrogen Receptor Modulators Estrogen Receptors Estrogens Ethylenes Event Exposure to Functional disorder Future Gene Expression Gene Expression Regulation Gene Silencing Gene Targeting Genomics Goals Health Hormones Human Human Cell Line ICI 182780 Immunoprecipitation Impaired health In Vitro Incidence Individual Investigation Kinetics Lead Ligands Link Lung MCF7 cell Malignant Neoplasms Malignant neoplasm of lung Mammalian Cell Mass Spectrum Analysis Mediating Messenger RNA Molecular Molecular Profiling Nature Nuclear Nuclear Extract Optics Organism Outcome Patients Phenols Play Production Protein Isoforms Proteins Proteomics Receptor Activation Recruitment Activity Regulation Reporter Research Response Elements Risk Risk Assessment Role Signal Pathway Small Interfering RNA Stress Surface Plasmon Resonance Techniques Technology Testing Therapeutic Transcription Factor AP-1 Transcriptional Regulation Work base cancer cell cell type design environmental agent estrophilin experience functional genomics genetic regulatory protein human RIPK1 protein human disease improved method development nonylphenol novel pentabromodiphenyl ether phenyl ether promoter protein complex public health relevance receptor receptor binding reproductive response therapeutic development tool xenoestrogen

项目摘要

DESCRIPTION (provided by applicant): Xenoestrogens interfere with normal endocrine functioning and have been linked to reproductive and developmental abnormalities in wildlife species and causally associated with human disease. Estrogen receptors (ER) are primary targets of these compounds and elucidating their role in xenoestrogen-related dysfunction is the focus of this proposal. Our primary goal is to identify ER-specific suites of co-regulatory proteins in response to estradiol (E2) and the xenoestrogens DDE, nonylphenol, and PBDEs, and determine their effect on ER activation. We will test the hypothesis that varied transcriptional regulation of gene targets by E2 and xenoestrogens is dependent on assembly of specific ER protein complex formation. This premise will be tested in 4 specific aims which employ state-of-the-art proteomic and functional genomic technologies in appropriate cell lines (human breast and lung). Out first hypothesis is that ERs will recruit compound-specific suites of accessory proteins that correlate with downstream activity. To test this hypothesis, we will use a tandem-affinity purification (TAP) and/or promoter-affinity/immunoprecipitation strategy to isolate ER-interacting proteins following stimulation with E2 and xenoestrogens. From this, we will identify known and possibly novel proteins by a combination of MALDI-TOF/TOF and HPLC-QTOF mass spectrometry. We will also use reporter assays to determine the effect of each xenoestrogen on ER activity in these cell lines. In Specific Aim 2, we will use an alternate approach based on combination of surface plasmon resonance with mass spectrometry to identify ER coregulatory proteins and to quantitate the ligand-dependence of binding kinetics and affinity of the ER complexes with various response elements. Once co-accessory proteins are identified, we will test the hypothesis that (Specific Aim 3) a subset of these proteins are transcriptionally regulated in response to E2 and the contaminants and will be tested using quantitative RT- PCR. Specific Aim 4 will address whether ER-modulating proteins are differentially required for ligand-specific transcriptional activity by ERs. We will employ mRNA knockdown strategies (siRNA) to address the functional role of select co-regulatory proteins in ER activity by xenoestrogens. The long term goal of these studies is to elucidate mechanisms of gene regulation by environmental contaminants, and how these actions lead to diseases/dysfunction. The goal of our research is to increase our basic understanding of the molecular events crucial to development of adverse outcomes associated with xenoestrogen exposure. This work will lead to more appropriate risk assessment and future development of therapeutic strategies for endocrine-related diseases. PUBLIC HEALTH RELEVANCE: Individuals exposed to xenoestrogens may be at risk for impaired health. The rising incidence in certain cancers and other endocrine related diseases over the past few decades suggests exposure to environmental agents, such as xenoestrogens, may play a role in disease development and progression. Elucidation of the molecular mechanisms that may contribute to the production of hormonally relevant diseases will lead to improved patient diagnostics and therapeutics as well as establishing relationships between environmental exposures and adverse health conditions.

描述（由申请人提供）：异种雌激素会干扰正常的内分泌功能，并与野生动物物种的生殖和发育异常有关，并与人类疾病有因果关系。雌激素受体（ER）是这些化合物的主要靶点，阐明它们在异雌激素相关功能障碍中的作用是该提案的重点。我们的主要目标是识别响应雌二醇 (E2) 和异雌激素 DDE、壬基酚和 PBDE 的 ER 特异性共调节蛋白套件，并确定它们对 ER 激活的影响。我们将检验以下假设：E2 和异雌激素对基因靶标的不同转录调控依赖于特定 ER 蛋白复合物形成的组装。这一前提将在 4 个具体目标中进行测试，这些目标在适当的细胞系（人类乳腺和肺）中采用最先进的蛋白质组学和功能基因组技术。第一个假设是 ER 将招募与下游活动相关的化合物特异性辅助蛋白套件。为了检验这一假设，我们将使用串联亲和纯化 (TAP) 和/或启动子亲和/免疫沉淀策略来分离 E2 和异雌激素刺激后的 ER 相互作用蛋白。由此，我们将通过结合 MALDI-TOF/TOF 和 HPLC-QTOF 质谱法来鉴定已知的和可能新颖的蛋白质。我们还将使用报告基因检测来确定每种异雌激素对这些细胞系中 ER 活性的影响。在具体目标 2 中，我们将使用基于表面等离子共振与质谱相结合的替代方法来识别 ER 共调节蛋白，并定量 ER 复合物与各种响应元件的结合动力学和亲和力的配体依赖性。一旦确定了辅助蛋白，我们将测试以下假设：（具体目标 3）这些蛋白的一个子集响应 E2 和污染物进行转录调节，并将使用定量 RT-PCR 进行测试。具体目标 4 将解决 ER 配体特异性转录活性是否需要不同的 ER 调节蛋白。我们将采用 mRNA 敲低策略 (siRNA) 来解决选定的共调节蛋白在异种雌激素 ER 活性中的功能作用。这些研究的长期目标是阐明环境污染物的基因调控机制，以及这些行为如何导致疾病/功能障碍。我们研究的目标是增加我们对与异雌激素暴露相关的不良后果的发展至关重要的分子事件的基本了解。这项工作将导致更适当的风险评估和未来内分泌相关疾病治疗策略的发展。公共卫生相关性：接触异雌激素的个体可能面临健康受损的风险。过去几十年来，某些癌症和其他内分泌相关疾病的发病率不断上升，表明接触环境因素（例如异雌激素）可能在疾病的发生和进展中发挥作用。阐明可能导致激素相关疾病产生的分子机制将改善患者的诊断和治疗，并建立环境暴露与不良健康状况之间的关系。