Mechanisms of Xenoestrogen Stress: A Proteomic and Functional Genomic Approach

说明(申请人提供)：异种雌激素干扰正常的内分泌功能，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，阐明它们在异种雌激素相关功能障碍中的作用是本提案的重点。我们的主要目标是确定雌激素(E2)和外源雌激素DDE、壬基酚和PBDEs反应的ER特异性共调节蛋白套件，并确定它们对ER激活的影响。我们将检验这一假设，即E2和异种雌激素对基因靶标的不同转录调控依赖于特定ER蛋白复合体的组装形成。这一前提将在4个特定目标中进行测试，这些目标使用最先进的蛋白质组和功能基因组技术在适当的细胞系(人的乳房和肺)中。第一个假设是，内质网将招募与下游活动相关的附属蛋白的化合物特异性套件。为了验证这一假设，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在E2和异种雌激素刺激后与ER相互作用的蛋白。由此，我们将通过MALDI-TOF/TOF和HPLC-QTOF质谱学相结合的方法来鉴定已知的和可能的新蛋白质。我们还将使用报告分析来确定每种异种雌激素对这些细胞系中ER活性的影响。在具体目标2中，我们将使用一种基于表面等离子体共振和质谱学相结合的替代方法来鉴定ER共调节蛋白，并定量测定ER复合体与各种反应元件结合动力学和亲和力的配体依赖性。一旦鉴定出辅助蛋白，我们将检验这样的假设，即(特定目标3)这些蛋白的子集对E2和污染物的转录调控，并将使用定量RT-PCR进行测试。具体目标4将解决内质网调节蛋白是否对内质网的配体特异性转录活动有差异需求。我们将使用mRNA敲除策略(SiRNA)来研究选定的共调控蛋白在异种雌激素诱导的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对基因调控的机制，以及这些行为如何导致疾病/功能障碍。我们研究的目的是增加我们对分子事件的基本了解，这些分子事件对于与异种雌激素暴露相关的不良后果的发生至关重要。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：暴露于异种雌激素的个人可能面临健康受损的风险。在过去的几十年里，某些癌症和其他内分泌相关疾病的发病率不断上升，这表明暴露于环境因素，如异种雌激素，可能在疾病的发生和发展中发挥作用。阐明可能导致荷尔蒙相关疾病产生的分子机制将有助于改进患者的诊断和治疗，并建立环境暴露与不利健康状况之间的关系。

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.