Affinity-directed tagging of protein binding partners in signaling

描述（由申请人提供）：本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理解。绘制细胞内的信息流对于理解细胞内稳态调节、疾病失调以及药物对细胞的影响至关重要。瞬时蛋白质相互作用和翻译后修饰（PTMs）是信息流的关键组成部分。然而，由于这些相互作用伙伴的短暂性和细胞中大量的ptm，鉴定这些相互作用伙伴，特别是翻译后修饰酶的相互作用伙伴仍然具有挑战性。目前采用蛋白质组学和亲和下拉的方法是探测蛋白质相互作用和ptm的有力工具，但这些方法具有显着的局限性。本提案旨在通过设计和优化一种新的催化标记装置nedylator来解决这些挑战，nedylator可以用稳定、简单和正交的标记来标记其底物，从而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，所有这些都参与调节细胞死亡。具体目标1:NEDDylator的定量和机制分析。将确定三种络合物中neddyylation的限速步骤和亲和力和产物抑制的局限性：天然e3 -底物对，已被充分表征的人类生长激素受体蛋白复合物，以及药物达沙替尼与其靶ABL之间的复合物。具体目标2：设计用于活细胞的NEDDylator。一个完全正交和小分子诱导的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。特异性目标3：利用天然蛋白质组中的NEDDylator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。信息流将通过对细胞凋亡和坏死至关重要的泛素连接酶和小脑（多发性骨髓瘤药物沙利度胺的泛素E3连接酶靶点）开始的途径逐步追踪。将鉴定和验证E3底物，并连接NEDDylator以找到它们各自的细胞结合伙伴。提出的研究将验证和扩展一种新的催化标记平台，以显着增加提取物和细胞中相互作用蛋白的发现。与现有方法相比，这种新技术在原位对蛋白质进行共价标记，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，无论是技术上还是生物学上，都可能对我们的生活产生重大影响

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.