Proteomic Study of DNA Bound Androgen Receptor Complex

描述（由申请人提供）： 雄激素受体（AR）是核受体超家族的成员，核受体超家族是一大组对前列腺癌的发展至关重要的配体激活的转录因子，并且作为多蛋白复合物的一部分起作用，所述多蛋白复合物通过称为雄激素应答元件（战神）的特异性DNA序列与雄激素应答基因的启动子区域结合。AR转录复合物的成分的鉴定和表征对于控制前列腺癌的策略的发展是必不可少的。虽然一些蛋白质已被证明与AR的研究使用酵母双杂交系统和染色质免疫沉淀（CHIP），AR转录复合物尚未被纯化和直接检查。该提案将探索使用固定化启动子DNA片段捕获AR转录复合物的技术可行性，并应用一种新的基于质谱（MS）的蛋白质组学方法来检查这些捕获的AR复合物。 将使用两种策略来鉴定复合物中的蛋白质。第一个将比较在用雄激素刺激之前和之后在来自LNCaP核提取物的含有ARE的DNA模板上捕获的复合物，这导致AR从细胞质易位到细胞核以促进DNA结合。第二种策略将比较结合到三种含有ARE的DNA模板的AR复合物与在战神中含有消除AR结合的点突变的相同模板。次要目的是描述雄激素拮抗剂比卡鲁胺存在下AR转录复合物的特征，据报道比卡鲁胺可募集转录辅阻遏物，并描述蛋白体抑制剂MG-132参与AR介导的转录的特征。 一旦实施，该方法有可能揭示在各种生理条件下转录复合物组合物的差异，如逃避癌症治疗中的雄激素消融。它也可以适用于研究整个核受体家族（通过改变用于捕获的特定DNA结合位点），以确定与健康，发育和疾病相关的复合物的变化。

DESCRIPTION (provided by applicant): The androgen receptor (AR) is a member of the nuclear receptor superfamily, a large group of ligand-activated transcription factors crucial to the development of prostate cancer and functions as part of a multiprotein complex which binds to the promoter regions of androgen responsive genes via specific DNA sequences called androgen response elements (AREs). Identification and characterization of the constituents of the AR transcription complex is essential to the development of strategies for controlling prostate cancer. Although a number of proteins have been shown to interact with the AR from studies using the yeast two-hybrid system and chromatin immunoprecipitation (CHIP), the AR transcription complex has yet to be purified and directly examined. This proposal will explore the technical feasibility of capturing the AR transcription complex using immobilized promoter DNA segments and applying a novel mass spectrometry (MS)-based proteomic method to examine these captured AR complexes. Two strategies will be used to identify the proteins in the complex. The first will compare the complexes captured on ARE-containing DNA template from LNCaP nuclear extract before and after stimulation with androgen, which causes translocation of AR from cytoplasm to nucleus to facilitate DNA binding. A second strategy will compare AR complexes bound to the three ARE-containing DNA templates with the same templates containing point mutations in the AREs that abrogate AR binding. Secondary aims will characterize the AR transcription complex in the presence of the androgen antagonist bicalutamide which has been reported to recruit transcriptional corepressors and to characterize the involvement of the proteosome in AR mediated transcription using the proteosome inhibitor MG-132. Once implemented, this method has the potential to reveal differences in transcription complex compositions under various physiological conditions such as escape from androgen ablation in cancer therapy. It also can be adapted to study the entire nuclear receptor family (by changing the hormone-specific DNA binding sites used for capture) to identify changes in the complexes that correlate with health, development and disease.