Affinity-directed tagging of protein binding partners in signaling

信号传导中蛋白质结合伴侣的亲和定向标记

• 批准号: 8628677
• 负责人: JAMES A WELLS
• 金额: $ 32.77万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-16 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8628677
• 关键词: Address; Affect; Affinity; Apoptosis; Apoptosis Inhibitor; Apoptotic; Binding; Biological; Biology; Caspase; Cell Death; Cell Extracts; Cells; Cellular Structures; Chemistry; Complex; Dasatinib; Development; Devices; Disease; Engineering; Enzymes; Foundations; Future; Generic Drugs; Goals; Homeostasis; Homologous Gene; Human; In Situ; Knowledge; Life; Link; Maps; Methods; Multiple Myeloma; Names; Nature; Necrosis; Pathway interactions; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Phosphotransferases; Post-Translational Protein Processing; Process; Protein Binding; Protein Engineering; Proteins; Proteome; Proteomics; Research; Role; Sea; Signal Pathway; Signal Transduction; Somatropin; System; Technology; Thalidomide; Therapeutic; Time; Ubiquitin; Ubiquitination; Walking; Work; biological systems; cell growth regulation; design; human GHR protein; interest; new technology; novel; protein complex; protein protein interaction; public health relevance; receptor; small molecule; tool; ubiquitin ligase; ubiquitin-protein ligase

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.

项目摘要 该提案的长期目标是对信号提供更全面的理解 撞击它们的途径和小分子。映射细胞中的信息流对于 了解稳态的细胞调节，疾病失调以及药物在细胞中的影响。 瞬态蛋白质 - 蛋白质相互作用和翻译后修饰（PTM）是关键组成部分 信息流。但是，确定这些相互作用的伙伴，尤其是那些用于翻译后的伙伴 由于其短暂的性质和大量的PTM，修饰酶仍然具有挑战性 细胞。使用蛋白质组学和亲和力下拉的当前方法是探测蛋白质的强大工具 蛋白质相互作用和PTM，但是这些方法具有重大局限性。该建议旨在 通过工程和优化新的催化标记设备Neddylator来应对这些挑战 用稳定，简单和正交标记标记其底物，允许通过 蛋白质组学。我们的假设是，Neddylator技术将可以推广到模范的泛素 影响它们的连接酶，磷酸酶，激酶和小分子，所有这些分子都参与了调节的细胞 死亡。这些方法是三个方面： 特定目的1：Neddylator的定量和机械分析。利率限制步骤和 在三个复合物中，将确定亲和力和产品抑制的局限性：自然 E3覆物对，特征良好的人类生长激素受体蛋白复合物，并且复合物 在dasatinib及其靶标ABL之间。 特定目标2：工程师的Neddylator用于活细胞。完全正交和小分子 可诱导的Neddylator将设计用于细胞研究，并且还将简化蛋白质组学工作流程。 特定目标3：使用天然蛋白质组中的neddylator详细说明重要的E3信号通路。 Neddylator将应用于在细胞死亡和疾病中具有重要生物学兴趣的几种途径。 信息流将通过从泛素连接酶开始的途径进行逐步追踪 多发性骨髓瘤药物Thalidomide的泛素E3连接酶靶标的凋亡和坏死，Cereblon。 将确定和验证E3 SUSBSTRATES，并将附上Neddylator以找到各自的 细胞结合伙伴。 拟议的研究将验证和扩展一个新型的催化标记平台，以显着增强 在提取物和细胞中发现相互作用的蛋白质。与现有方法相比，这项新技术 共价标记原位标记蛋白质，并能够发现瞬态和高亲和力相互作用。 从技术和生物学上讲，从这些研究中获得的知识可能会产生重大影响 关于我们对细胞中蛋白质及其结合伴侣之间分子相互作用的理解。