Structure and Mechanism in Intracellular Notch Signaling

描述（由申请人提供）：多细胞生物体中的细胞感知并响应其环境，是发育中组织和器官形成、适应营养水平和激素信号变化以及成人受损组织再生的组成部分。这些传感系统中的病变导致疾病状态，例如出生缺陷、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，并有助于诊断，治疗和治愈。我的实验室一直在研究这样一种途径，Notch信号通路。在Notch信号传导中，来自邻近细胞的局部信息通过由Notch基因编码的跨膜受体被感知，并被转导到细胞核中以激活下游基因的表达，从而导致细胞分化。Notch途径的一个独特特征是在活化时，受体通过从膜切割Notch胞内结构域（NICD）而转化为转录因子。裂解后，NICD通过结合一种称为CSL的因子激活转录。这种相互作用涉及NICD和CSL的远端区域之间的二价接触，并且由~100个残基的RAM接头区段介导，该RAM接头区段虽然在很大程度上是无序的，但在协调共阻遏物的置换和共激活物的募集中起关键作用。NICD活性也受到许多胞质蛋白如Deltex的强烈调节，尽管其机制细节尚不清楚。 在这个建议中，我们使用传统的生物物理，结构方法，和溶液热力学来表征这些方面的Notch信号，但也联合收割机这些方法与功能检测Notch信号使用哺乳动物细胞培养。第二种方法是我实验室的一个新方向，但到目前为止，结果是有益的，从生物物理研究的结果功能背景，并集中在定量细胞培养研究，明确集中的机制问题。我们也更加重视核磁共振和计算机模拟。 具体来说，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，并确定破坏这种相互作用如何干扰泛素化，转录激活，受体内吞作用以及与推定的去泛素化酶的相互作用。此外，我们正在使用NMR，AUC，模拟和诱变来表征NICD的RAM区域。最后，我们正在确定RAM如何增强NICD和CSL之间的保守的二价相互作用，以及这种二价相互作用如何将辅阻遏物解离与辅激活物结合进行化学偶联。这里研究的系统是理解内在无序区域如何影响蛋白质结构和功能的理想选择。

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.