Mechanisms of Xenoestrogen Stress: A Proteomic and Functional Genomic Approach

描述（由申请人提供）：异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，阐明其在雌激素相关功能障碍中的作用是本研究的重点。我们的主要目标是鉴定雌激素受体特异性的共调节蛋白对雌二醇（E2）和异种雌激素DDE、壬基酚和多溴二苯醚的反应，并确定它们对雌激素受体激活的影响。我们将验证E2和异种雌激素对基因靶点的不同转录调节依赖于特定ER蛋白复合物形成的组装这一假设。这一前提将在4个特定目标中进行测试，这些目标将在适当的细胞系（人类乳腺和肺）中使用最先进的蛋白质组学和功能基因组技术。我们的第一个假设是，内质网将招募与下游活性相关的化合物特异性附属蛋白。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E2和异种雌激素刺激后的er相互作用蛋白。由此，我们将通过结合MALDI-TOF/TOF和HPLC-QTOF质谱法鉴定已知的和可能新的蛋白质。我们还将使用报告基因测定法来确定每种异雌激素对这些细胞系内质网活性的影响。在Specific Aim 2中，我们将使用一种基于表面等离子体共振与质谱相结合的替代方法来鉴定内质网协同调节蛋白，并量化内质网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，我们将验证（Specific Aim 3）这些蛋白的一个子集在E2和污染物的反应中受到转录调节的假设，并将使用定量RT- PCR进行测试。Specific Aim 4将探讨内质网是否需要不同的内质网调节蛋白来实现配体特异性转录活性。我们将采用mRNA敲低策略（siRNA）来解决选择的共调节蛋白在雌激素内质网活性中的功能作用。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，以及这些作用如何导致疾病/功能障碍。我们研究的目的是增加我们对与异雌激素暴露相关的不良后果发展的关键分子事件的基本理解。这项工作将导致更适当的风险评估和内分泌相关疾病的治疗策略的未来发展。

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.