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之一，核受体超家族是一大类配体激活的转录因子，在前列腺癌的发生发展中起关键作用，是多蛋白复合体的一部分，该复合体通过称为雄激素反应元件(Ares)的特定DNA序列与雄激素反应基因的启动子区域结合。鉴定和鉴定AR转录复合体的成分对于制定控制前列腺癌的策略至关重要。尽管使用酵母双杂交系统和染色质免疫沉淀(CHIP)的研究已经证明了一些蛋白质与AR相互作用，但AR转录复合体尚未得到纯化和直接检测。该提案将探索利用固定的启动子DNA片段捕获AR转录复合体的技术可行性，并应用一种新的基于质谱仪(MS)的蛋白质组学方法来检测这些捕获的AR复合体。 将使用两种策略来识别复合体中的蛋白质。第一个将比较在含有ARE的DNA模板上捕获的LNCaP核提取物在雄激素刺激前后的复合体，雄激素刺激导致AR从细胞质移位到细胞核，以促进DNA结合。第二种策略将比较与三个含Are的DNA模板结合的AR复合体与相同的模板，这些模板包含ARE中取消AR结合的点突变。二次AIMS将在雄激素拮抗剂比卡鲁胺存在的情况下表征AR转录复合体，已有报道比卡鲁胺招募转录共抑制物，并利用蛋白酶体抑制剂MG-132表征蛋白酶体参与AR介导的转录。 一旦实施，该方法有可能揭示不同生理条件下转录复合体组成的差异，例如在癌症治疗中逃脱雄激素消融。它还可以用于研究整个核受体家族(通过改变用于捕获的激素特异性DNA结合位点)，以确定与健康、发育和疾病相关的复合体中的变化。

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.