Structure and Mechanism in Intracellular Notch Signaling

描述(申请人提供)：多细胞生物体中的细胞对其环境的感知和反应对于发育中的组织和器官的形成、对营养水平和激素信号的变化的适应以及成年受损组织的再生是不可或缺的。这些传感系统中的损伤会导致疾病状态，如出生缺陷、癌症和退行性疾病。因此，了解细胞传感背后的分子机制不仅可以更好地了解生物过程，还可以深入了解人类疾病的原因，有助于诊断、治疗和治愈。我的实验室一直在研究一条这样的通路，即Notch信号通路。在Notch信号转导中，邻近细胞的局部信息通过Notch基因(S)编码的跨膜受体被感知，并被转导到细胞核中，激活下游基因的表达，导致细胞分化。Notch途径的一个独特特征是，当激活时，受体通过从膜上切割Notch胞内域(NICD)而转化为转录因子。切割后，NICD通过结合一种称为CSL的因子来激活转录。这种相互作用涉及NICD和CSL远距离区域之间的二价接触，并由~100个残基RAM接头片段介导，尽管该接头片段在很大程度上是无序的，但在协调共抑制因子的置换和辅助激活因子的招募方面发挥了关键作用。NiCd的活性也受到Deltex等许多胞质蛋白的强烈调控，尽管机制细节尚不清楚。在这个提案中，我们使用传统的生物物理、结构方法和溶液热力学来表征Notch信号的这些方面，但也将这些方法与使用哺乳动物细胞培养的Notch信号的功能分析结合起来。对于我的实验室来说，第二种方法是一个新的方向，但到目前为止，结果是值得的，给了生物物理学研究的结果一个功能背景，并将细胞培养研究集中在定量的、尖锐聚焦的机制问题上。我们还更加重视核磁共振和计算模拟。具体地说，我们正在确定Notch受体和Deltex之间相互作用的结构基础，正在确定破坏结合的表面取代，并正在确定破坏这种相互作用如何扰乱泛化、转录激活、受体内吞作用以及与假定的去泛素酶的相互作用。此外，我们正在使用核磁共振、AUC、模拟和突变来表征NICD的RAM区域。最后，我们正在确定RAM如何增强NICD和CSL之间保守的二价相互作用，以及这种二价相互作用如何热力学地将辅阻遏子解离与辅激活子结合结合起来。这里正在研究的系统是了解内在无序区域如何影响蛋白质结构和功能的理想系统。

DESCRIPTION (provided by applicant): Cells in multicellular organisms sense and respond to their environments is integral to the formation of tissues and organs in development, adaptation to changes in nutrient levels and hormone signals, and regeneration of damaged tissues in adults. Lesions in these sensing systems result in disease states such as birth defects, cancer, and degenerative diseases. Thus, understanding the molecular mechanisms behind cell sensing will not only provide a better understanding of biological processes, it will provide insights into the causes of human diseases, and will aid diagnosis, treatment, and cure. My laboratory has been studying one such pathway, the Notch signaling pathway. In Notch signaling, local information from neighboring cells is sensed through a transmembrane receptor encoded by the Notch gene(s), and is transduced into the nucleus to activate the expression of downstream genes, leading to cell differentiation. One unique feature of the Notch pathway is that upon activation, the receptor is converted to a transcription factor through cleavage of the Notch intracellular domain (NICD) from the membrane. Following cleavage, NICD activates transcription by binding a factor called CSL. This interaction involves bivalent contacts between distant regions of NICD and CSL, and is mediated by a ~100 residue RAM linker segment, which though largely disordered, plays a key role in orchestrating displacement of co-repressors, and recruitment of coactivators. NICD activity is also strongly modulated by a number of cytosolic proteins such as Deltex, although the mechanistic details are murky. In this proposal, we use traditional biophysical, structural methods, and solution thermodynamics to characterize these aspects of Notch signaling, but also combine these methods with functional assays for Notch signaling using mammalian cell culture. This second approach is a new direction for my laboratory, but results so far have been rewarding, giving results from biophysical studies a functional context, and focusing cell culture studies on quantitative, sharply focused mechanistic questions. We are also putting greater emphasis on NMR and computational simulations. Specifically, we are determining the structural basis of interaction between the Notch receptor and Deltex, are identifying surface substitutions that disrupt binding, and are determining how disrupting this interaction perturbs ubiqutination, transcriptional activation, receptor endocytosis, and interaction with a putative deubiquitinase. In addition, we are characterizing the RAM region of NICD using NMR, AUC, simulation, and mutagenesis. Finally, we are determining how RAM enhances the conserved bivalent interaction between NICD and CSL, and how this bivalent interaction thermodynamically couples corepressor dissociation with coactivator binding. The system under study here is ideal for understanding how intrinsically disordered regions influence protein structure and function.