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提供对信令的更全面的理解 路径和撞击它们的小分子。映射单元中的信息流对于 了解动态平衡中的细胞调节，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 信息流。然而，确定这些互动合作伙伴，特别是那些翻译后的合作伙伴 修饰酶仍然具有挑战性，因为它们的本质是短暂的，而且在 手机。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的强大工具。 蛋白质相互作用和PTMS，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，该设备 用稳定、简单和正交的标记标记其底物，允许通过以下方式进行稳健和定量的识别 蛋白质组学。我们的假设是，NEDDylator技术将可推广到示范性泛素 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：NEDDylator的定量和机械分析。速率限制步骤和 NEDD化的亲和力和产物抑制的限制将在三个络合物中确定：天然的 E3底物对，特性良好的人生长激素受体蛋白复合体，以及该复合体 药物达沙替尼和其靶标ABL之间的关系。 具体目标2：设计用于活细胞的NEDDylator。一种完全正交的小分子 可诱导的NEDDylator将被设计用于细胞研究，蛋白质组工作流程也将得到简化。 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。 信息流将通过一条从泛素连接酶开始的途径逐步追踪，泛素连接酶对 多发性骨髓瘤药物沙利度胺的泛素E3连接酶靶点--细胞凋亡和坏死，以及雷公藤红素。 E3悬浮物将被识别和验证，NEDDylator将被附加以找到各自的 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，以显著增加 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 在原位共价标记蛋白质，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，都可能产生重大影响。 关于我们对细胞中蛋白质及其结合伙伴之间的分子相互作用的理解。