Oncogenic Notch Signaling: Structural Studies

描述(申请人提供)：本研究的长期目标是了解Notch受体正常激活的结构基础，确定T细胞急性淋巴细胞白血病/淋巴瘤(T-ALL)中发现的突变导致Notch病理生理激活的生化机制，并最终将这一知识用于T-ALL的治疗。 为了了解Notch蛋白的非共价相关亚基通常是如何结合在一起的，了解癌症相关突变如何增加Notch信号，并阐明Notch转录激活复合体是如何组装的，我们提出了以下具体目标： 目的1.确定Notch异二聚化结构域的结构。 目的2.确定肿瘤来源的异二聚化结构域突变如何激活Notch。 目的3.确定Notch受体激活转录的结构基础。 3A。解决Notch转录激活复合体或包含Notch ankyrin重复结构域的Key亚复合体的结构。 3B。确定阻止Notch转录激活复合体组装的小分子。 这项建议中提出的结构研究将填补我们目前对Notch信号的理解的一个主要空白。拟议的对Notch异二聚化结构域的核磁共振研究将揭示分子内界面的性质，该分子内界面对于保持Notch的静止构象至关重要，目前已知该界面包含在Notch依赖的人类T-ALL中频繁发现的激活突变。拟议的Notch转录激活复合体的X射线结晶学研究将揭示Notch的ankyrin重复结构域如何与CSL转录因子合作招募策划者共激活因子。这些进展将为突变研究确定候选残基，旨在探索特定的锚蛋白重复接触在协调策划者招募和与CSL结合方面的重要性。 最后，纯化的Notch、大师样多肽和CSL与同源DNA结合的三元复合体的可获得性为我们创造了一个机会，使我们有机会识别破坏该复合体的小分子，并在体内探索Notch信号，并作为T-ALL靶向治疗的化合物先导。由于缺口在分化和增殖中的广泛重要性，阻止缺口信号的干预不仅可能导致T-ALL的新的治疗形式，而且对缺口活性的操纵也可能在乳腺癌和其他癌症的治疗中具有普遍价值。

DESCRIPTION (provided by applicant): The long-term goal of the studies proposed here is to understand the structural basis for normal activation of Notch receptors, to define the biochemical mechanism underlying pathophysiologic activation of Notch by mutations found in T cell acute lymphocytic leukemia/lymphoma (T-ALL), and ultimately to harness this knowledge for the treatment of T-ALL. To understand how the non-covalently associated subunits of Notch proteins are normally held together, find out how cancer-associated mutations increase Notch signaling, and elucidate how the Notch transcriptional activation complex is assembled, we propose the following specific aims: Aim 1. Determine the structure of a Notch heterodimerization domain. Aim 2. Determine how tumor-derived heterodimerization domain mutations activate Notch. Aim 3. Determine the structural basis underlying activation of transcription by Notch receptors. 3A. Solve the structure of a Notch transcriptional activation complex or a key subcomplex containing the Notch ankyrin repeat domain. 3B. Identify small molecules that prevent assembly of the Notch transcriptional activation complex. The structural studies proposed in this proposal will fill a major gap in our current understanding of Notch signaling. The proposed NMR studies of a Notch heterodimerization domain will reveal the nature of an intramolecular interface that is crucial for maintaining Notch in its resting conformation, and that is now known to harbor activating mutations found frequently in Notch-dependent human T-ALLs. The proposed X- ray crystallographic studies of Notch transcriptional activation complexes will uncover how the ankyrin repeat domain of Notch cooperates with the CSL transcription factor to recruit the mastermind co-activator. These advances will identify candidate residues for mutational studies designed to probe the importance of specific ankyrin repeat contacts in coordinating mastermind recruitment, and in binding to CSL. Finally, the availability of purified ternary complexes of Notch, Mastermind-like polypeptides, and CSL bound to cognate DNA creates an opportunity for us to identify small molecules that disrupt the complex, with potential future utility in probing Notch signaling in vivo, and as compound leads for targeted therapy in T-ALL. Because of the broad importance of NOTCH in differentiation and proliferation, interventions that prevent NOTCH signaling may not only lead to new forms of treatment for T-ALL, but manipulation of NOTCH activity may also be of general value in management of breast and other cancers as well.