Mechanisms of Xenoestrogen Stress: A Proteomic and Functional Genomic Approach

描述（由申请方提供）：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受体特异性的协同调节蛋白的套件，响应雌二醇（E2）和异种雌激素DDE，壬基酚，和多溴联苯醚，并确定其对雌激素受体激活的影响。我们将测试的假设，不同的转录调控基因靶E2和外源性雌激素是依赖于特定的ER蛋白复合物的形成组装。这一前提将在4个特定目标中进行测试，这些目标采用最先进的蛋白质组学和功能基因组学技术在适当的细胞系（人乳腺和肺）中进行。我们的第一个假设是，ER将招募与下游活性相关的化合物特异性辅助蛋白。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，以分离ER相互作用的蛋白刺激后与E2和异种雌激素。由此，我们将通过MALDI-TOF/TOF和HPLC-QTOF质谱的组合来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来确定这些细胞系中每种异种雌激素对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体共振与质谱相结合的替代方法来鉴定ER辅调节蛋白，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。一旦鉴定出辅助蛋白，我们将检验以下假设：（特异性目的3）这些蛋白质的一个子集在转录上响应于E2和污染物而受到调节，并将使用定量RT-PCR进行检测。具体目标4将解决ER调节蛋白是否是ER的配体特异性转录活性所需的差异。我们将采用mRNA敲低策略（siRNA）来解决选择的协同调节蛋白在雌激素受体活性的功能作用。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外源性雌激素暴露相关的不良后果发展至关重要的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。 公共卫生相关性：暴露于异种雌激素的个人可能有健康受损的风险。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，这表明暴露于环境因素，如外源性雌激素，可能在疾病的发展和进展中发挥作用。阐明可能有助于产生与生殖有关的疾病的分子机制，将有助于改进患者的诊断和治疗，并建立环境暴露与不良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.