Affinity-directed tagging of protein binding partners in signaling

描述(由申请人提供)：这项提案的长期目标是提供对信号通路和影响它们的小分子的更全面的了解。绘制细胞中的信息流对于了解细胞内稳态的调节、疾病中的调节失调以及药物在细胞中的影响至关重要。瞬时蛋白质相互作用和翻译后修饰(PTM)是信息流的关键组成部分。然而，由于它们的短暂性质和细胞中大量的PTM，识别这些相互作用的伙伴，特别是那些翻译后修饰酶的伙伴仍然具有挑战性。目前使用蛋白质组学和亲和力下拉图的方法是探测蛋白质-蛋白质相互作用和PTM的强大工具，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设备NEDDylator来应对这些挑战，NEDDylator使用稳定、简单和正交的标记来标记其底物，从而允许通过蛋白质组学进行稳健和定量的鉴定。我们的假设是，NEDDylator技术将推广到典型的泛素连接酶、磷酸酶、激酶和影响它们的小分子，所有这些都参与调控细胞死亡。这些方法有三个方面：具体目标1：对NEDDylator进行定量和机械分析。亲和力和产物抑制的限速步骤和限制将在三个复合体中确定：天然E3底物对，具有良好特性的人类生长激素受体蛋白质复合体，以及药物达沙替尼与其目标ABL之间的复合体。具体目标2：设计用于活细胞的NEDDylator。一种完全正交和小分子可诱导的NEDDylator将被设计用于细胞研究，蛋白质组学工作也将得到简化。具体目标3：利用NEDDylator在天然蛋白质组中阐述重要的E3信号通路。NEDDylator将被应用于几条在细胞死亡和疾病方面具有重要生物学意义的途径。信息流将通过一条途径逐步追踪，从对细胞凋亡和坏死至关重要的泛素连接酶开始，以及多发性骨髓瘤药物沙利度胺的泛素E3连接酶靶标雷布隆。E3底物将被识别和验证，NEDDylator将被连接以找到它们各自的细胞结合伙伴。拟议的研究将验证和扩展一种新的催化标记平台，以显着增强在提取物和细胞中发现相互作用的蛋白质。与现有的方法相比，这项新技术可以原位共价标记蛋白质，并将能够发现瞬时和高亲和力的相互作用。从这些研究中获得的知识，无论是技术上的还是生物学上的，都可能对我们的 了解细胞中蛋白质及其结合伙伴之间的分子相互作用。

DESCRIPTION (provided by applicant): 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 substrates 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.