Affinity-directed tagging of protein binding partners in signaling

项目摘要 本提案的长期目标是提供对信令更全面的理解 以及影响它们的小分子。绘制细胞中的信息流对于 了解细胞内稳态的调节，疾病中的失调，以及药物对细胞的影响。 瞬时蛋白质-蛋白质相互作用和翻译后修饰（PTM）是蛋白质相互作用的关键组成部分。 信息流然而，识别这些相互作用的伙伴，特别是那些翻译后 修饰酶由于其短暂的性质和修饰酶中大量的PTM而仍然具有挑战性。 cell.目前采用蛋白质组学和亲和下拉的方法是探测蛋白质的有力工具。 蛋白质相互作用和PTM，但这些方法有很大的局限性。这项建议旨在 通过设计和优化一种新的催化标记装置NEDDylator来应对这些挑战， 用稳定、简单和正交的标记标记其底物， 蛋白质组学我们的假设是，NEDDylator技术将推广到示例性的泛素 连接酶、磷酸酶、激酶和影响它们的小分子，所有这些都参与调节细胞增殖。 死亡这些办法有三个方面： 具体目标1：NEDD化剂的定量和机理分析。限速步骤和 将在三种复合物中确定NEDD化的亲和力和产物抑制的局限性： E3-底物对，充分表征的人生长激素受体蛋白复合物，和复合物 药物达沙替尼和它的靶点ABL之间的关系。 具体目标2：设计用于活细胞的NEDDylator。完全正交的小分子 可诱导的NEDDylator将被设计用于细胞研究，蛋白质组学工作流程也将被简化。 具体目标3：在天然蛋白质组中使用NEDDylator详细阐述重要的E3信号通路。 NEDDylator将应用于细胞死亡和疾病中具有重要生物学意义的几种途径。 信息流将通过一个途径一步一步地追踪，该途径始于泛素连接酶， 细胞凋亡和坏死，以及cereblon，多发性骨髓瘤药物沙利度胺的泛素E3连接酶靶标。 将识别和确认E3基质，并附上NEDDylator，以查找其各自的 细胞结合伴侣。 拟议的研究将验证和扩展一种新的催化标记平台， 在提取物和细胞中发现相互作用的蛋白质。与现有技术相比，这项新技术 原位共价标记蛋白质，将能够发现瞬时以及高亲和力的相互作用。 从这些研究中获得的知识，无论是技术上还是生物学上，都可能产生重大影响。 对我们理解细胞中蛋白质及其结合伙伴之间的分子相互作用的影响。

Project Summary The long-term goal of this proposal is to provide a more comprehensive understanding of signaling pathways and small molecules that impinge them. Mapping information flow in cells is critical to understanding cellular regulation in homeostasis, dysregulation in disease, and the impact of drugs in cells. Transient protein-protein interactions and post-translational modifications (PTMs) are key components of the information flow. However, identification of these interacting partners and especially those for post-translational modifying enzymes remains challenging due to their ephemeral nature and the vast numbers of PTMs in the cell. Current methods employing proteomics and affinity pull-downs are powerful tools for probing protein- protein interactions and PTMs, but these approaches have significant limitations. This proposal aims to address these challenges by engineering and optimizing a new catalytic tagging device, the NEDDylator, which tags its substrates with a stable, simple, and orthogonal mark allowing robust and quantitative identification by proteomics. Our hypothesis is that the NEDDylator technology will be generalizable to exemplary ubiquitin ligases, phosphatases, kinases, and small molecules that affect them, all of which are involved in regulated cell death. The approaches are three-fold: Specific Aim 1: Quantitative and mechanistic analysis of the NEDDylator. The rate-limiting steps and limitations of affinity and product inhibition will be determined for NEDDylation in three complexes: a natural E3-substrate pair, the well-characterized human growth hormone receptor protein complex, and the complex between the drug dasatinib and its target ABL. Specific Aim 2: Engineer the NEDDylator for use in living cells. A fully orthogonal and small molecule inducible NEDDylator will be designed for cellular studies, and the proteomic workflow will also be simplified. Specific Aim 3: Elaborate important E3 signaling pathways using the NEDDylator in native proteomes. The NEDDylator will be applied to several pathways of important biological interest in cell death and disease. Information flow will be traced step-by-step through a pathway starting at ubiquitin ligases important for apoptosis and necrosis, and cereblon, a ubiquitin E3 ligase target of the multiple myeloma drug, thalidomide. E3 susbstrates will be identified and validated, and the NEDDylator will be attached to find their respective cellular binding partners. The proposed studies will validate and expand a novel catalytic tagging platform to dramatically augment the discovery of interacting proteins in extracts and cells. Compared to existing methods, this new technology covalently tags proteins in situ and will enable the discovery of transient as well as high-affinity interactions. The knowledge gained from these studies, both technically and biologically, will likely have a significant impact on our understanding of molecular interactions between proteins and their binding partners in cells.