Proteolytic Regulation of Estrogen Receptor-alpha

描述（由申请人提供）：雌激素受体（ER α）的下调是雌激素控制细胞对刺激反应的基本调节机制。在乳腺癌细胞中，ER α的水平通常会不适当地升高或检测不到，这可能会影响疾病的自然史和治疗。我们的实验室和其他人已经发现，雌激素控制细胞ER α含量的最急性机制是26S蛋白酶体对受体蛋白的调节性破坏。最近的证据表明，蛋白酶体活性是至关重要的类固醇激素受体的转录激活。然而，ER α蛋白水解在雌激素依赖性转录控制中的具体作用尚不清楚。我们假设ER α蛋白水解和反式激活的调节是功能性偶联事件。而蛋白水解可以通过影响DNA上转录复合物形成的动力学来控制受体转录的幅度，转录复合物组装内的事件可以标记注定要被破坏的受体库。为了解决这一假设，目的1试图通过诱变和肽抑制剂研究来确定ER α N-末端中雌激素诱导的蛋白水解所需的序列元件;目的2提出使用染色质免疫沉淀分析来确定ER α蛋白水解的特定作用和ER α转录复合物组装中蛋白酶体功能的一般要求;目的3试图通过分析甲状腺激素的活性来阐明保护ER α免受蛋白水解的机制，甲状腺激素是ER α降解和转录的内源性抑制剂。通过本文提出的实验的成功完成，我们希望描绘连接ER α蛋白水解和转录的控制的调节事件。了解受体蛋白水解和转录之间的关系可能有助于识别ER α依赖性恶性肿瘤治疗干预的新靶点。

DESCRIPTION (provided by applicant): Down regulation of estrogen receptor (ERalpha) is a fundamental regulatory mechanism by which estrogen controls cellular response to stimulation. In breast cancer cells, the levels of ERalpha can often be inappropriately elevated or undetectable, which can affect both the natural history and treatment of the disease. Our laboratory and others have discovered that the most acute mechanism by which estrogen controls cellular ERalpha content is the regulated destruction of receptor protein by the 26S proteasome. Recent evidence suggests that proteasome activity is crucial for activation of transcription by steroid hormone receptors. The specific role that ERalpha proteolysis plays in the control of estrogen-dependent transcription, however, is unknown. We hypothesize that the regulation of ERalpha proteolysis and transactivation are functionally coupled events. Whereas proteolysis can control the magnitude of receptor transcription by affecting the kinetics of transcriptional complex formation on DNA, events within transcriptional complex assembly can reciprocally mark the pool of receptors destined for destruction. To address this hypothesis, Aim 1 seeks to identify a sequence element in the ERalpha N-terminus required for estrogen-induced proteolysis by mutagenesis and peptide inhibitor studies; Aim 2 proposes to define the specific role of ERa proteolysis and the general requirement for proteasome function in ERalpha transcriptional complex assembly using chromatin immunoprecipitation analyses; and, Aim 3 seeks to elucidate mechanism(s) that protect ERalpha from proteolysis by dissecting the activity of thyroid hormone, an endogenous inhibitor of ERalpha degradation and transcription. Through the successful completion of the herein proposed experiments, we hope to delineate the regulatory events that link the control of ERalpha proteolysis and transcription. Understanding the relationship between receptor proteolysis and transcription will likely contribute to the identification of novel targets for therapeutic intervention of ERalpha-dependent malignancies.