Structure and function of CSL, the transcriptional regulator in the Notch pathway

描述（由申请人提供）：Notch通路的异常激活是t细胞急性淋巴细胞白血病（T-ALL）发生的一个致病因素，T-ALL是儿童中最常见的白血病。在其他血源性癌症、实体瘤恶性肿瘤和病毒诱导肿瘤中也观察到致癌Notch信号。异常Notch信号在很大程度上与人类肿瘤相关，这强调了开发新型治疗方法的必要性，这种方法可以从药理学上操纵该途径。我们的长期目标是获得对Notch通路中转录调控如何实现的分子理解。保守转录因子CSL是Notch通路中转录的主要调控因子，其中心地位使其成为治疗干预的一个有吸引力的靶点。虽然在识别CSL的结合伙伴方面取得了进展，但这些复合物如何调节转录的结构细节却知之甚少。本提案的目的集中在表征csl介导的转录复合物与辅抑制因子和病毒蛋白。我们假设CSL的变构变化和/或CSL上重叠的共调节因子结合位点是CSL从抑制因子转化为转录激活因子的基础。为了检验我们的假设并实现我们的目标，我们将追求以下三个目标。(1)我们将基于CSL- notch - mastermind活性转录复合体建立CSL界面突变体库，分析转录共调控因子利用的CSL结合表面。(2)我们将确定协同抑制因子CIR、SMRT和SHARP与CSL相互作用所需的最小结构域，并研究这些配合物的x射线结构。(3)我们将描绘疱疹病毒蛋白EBNA2、RTA和LANA结合CSL所必需的结构域，并确定这些复合物的x射线结构。这一建议的完成将阐明构成CSL转录复合物与协抑制因子和疱疹病毒蛋白的分子细节，从而从根本上推进该领域的发展，并为合理开发针对CSL介导的转录复合物的药物提供结构基础。

DESCRIPTION (provided by applicant): Aberrant activation from the Notch pathway is a causative factor in the leukemogenesis of T-cell acute lymphoblastic leukemia (T-ALL), the most common leukemia found in children. Oncogenic Notch signaling is also observed in other blood-borne cancers, solid tumor malignancies, and virally induced tumors. The considerable extent to which aberrant Notch signaling is associated with human neoplasias emphasizes the need for developing novel therapeutics that pharmacologically manipulate the pathway. Our long-term goal is to gain a molecular understanding of how transcriptional regulation is achieved in the Notch pathway. The conserved transcription factor CSL is the primary regulator of transcription in the Notch pathway, and its centrality makes it an attractive target for therapeutic intervention. While progress has been made in identifying binding partners of CSL, the structural details as to how these complexes regulate transcription are poorly understood. The objective of this proposal focuses on characterizing CSL-mediated transcription complexes with corepressors and viral proteins. We hypothesize that allosteric changes in CSL and/or overlapping coregulator binding sites on CSL underlie the conversion of CSL from a repressor to an activator of transcription. To test our hypothesis and accomplish our objective the following three aims will be pursued. (1) We will analyze the CSL binding surfaces utilized by transcriptional coregulators by creating a library of CSL interfacial mutants based on the CSL-Notch-Mastermind active transcription complex. (2) We will determine the minimal domains necessary for the corepressors CIR, SMRT, and SHARP to interact with CSL and pursue X-ray structures for these complexes. (3) We will delineate the domains of the herpesvirus proteins EBNA2, RTA, and LANA necessary for binding CSL and determine X-ray structures for these complexes. Completion of this proposal will illuminate the molecular details that comprise CSL transcription complexes with corepressors and herpesvirus proteins, thereby fundamentally advancing the field, and provide a structural basis for rationally developing drugs that target CSL-mediated transcription complexes. "Lay language" - The abnormal action of the cellular Notch pathway results in human cancers. We are determining structural models for Notch pathway components in order to better understand pathway function, biology and tumorigenesis, which will have an impact on disease diagnosis, prevention, and treatment.