Understanding the effects of p97 protein-protein interactions on cellular functions using antibody-based inhibitors

使用基于抗体的抑制剂了解 p97 蛋白-蛋白相互作用对细胞功能的影响

基本信息

批准号：
10548223
负责人：
Ziwen Jiang
金额：
$ 7.18万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10548223
关键词：
Adaptor Signaling Protein Affinity Antibodies Binding Biological Biological Process Cell Cycle Progression Cell physiology Chromatin Complex DNA biosynthesis Defect Disease Engineering Event Experimental Models Foundations Future Genes Goals Immunoglobulin Fragments Individual Knock-out Licensing Factor Light Literature Mediating Membrane Metabolic Pathway Nuclear Translocation Organelles Outcome Phage Display Play Process Property Proteins Proteomics Regulation Replication Licensing Research Rest Role Signal Pathway Signal Transduction Substrate Specificity System Technology Testing Time Transfection Ubiquitin Variant Work antibody engineering antibody inhibitor biophysical properties cell type drug discovery immune activation inhibitor insight intercellular communication interest knock-down multicatalytic endopeptidase complex new therapeutic target p65 prevent protein protein interaction proteostasis therapeutic development therapy development valosin-containing protein

项目摘要

Project Summary/Abstract Protein-protein interactions (PPIs) connect various proteins to achieve critical cellular functions. For instance, the ubiquitously expressed protein valosin-containing protein (VCP/p97) is involved in multiple biological events via PPIs, maintaining protein homeostasis in different organelles. Considering the participation of multiple proteins in each process, it will be fundamentally beneficial to systematically decipher the p97 PPI network. Proteomics studies have demonstrated that the p97 PPI network is highly dynamic. However, a comprehensive set of p97 PPIs, their functions, and protein substrates has been elusive. The overall goal of this proposal is to dissect the p97 PPI network using engineered antibody inhibitors, providing fundamental insights on the modulation of specific cellular functions through the regulation of p97 PPI. Till now, at least 30 adaptor proteins of p97 have been discovered. In general, the roles of these adaptor proteins are described in broad terms (e.g., “membrane remodeling”). What are the specific substrates or differentiating functions of these adaptors, and do they work independently or together? Our central hypothesis is that, by developing p97/adaptor specific inhibitors, the distinctive functions of each adaptor can be defined within the p97 PPI network. FAS-associated factor 1 (FAF1), an important adaptor protein of p97, will be employed as the primary example to develop the technology for the modulation of p97 PPI. FAF1 is of particular interest, as it has been shown to be involved in the regulation of chromatin-associated degradation and NF-κB signaling. Notably, such processes are found to involve both p97 and FAF1, and it is not clear whether all functions of FAF1 are p97-dependent. To test the hypothesis, antibody fragments will be engineered for the p97-binding (UBX) domain on FAF1, blocking FAF1's interaction with p97 and leaving the rest of FAF1's and p97's activities intact. Unlike the gene knockdown/knockout strategy, the proposed strategy will directly probe the role of the PPI without requiring the deficiency of involved proteins, reflecting the real-time contribution of the interaction of interest. The role of p97-FAF1 interaction will be evaluated in both chromatin- associated degradation process and NF-κB signaling. These two processes involve different domains of FAF1 for regular function, facilitating the assessment of p97 substrate specificity during the p97-FAF1 interaction. The goal of this proposal is to establish an experimental model to dissect p97 PPIs, exploring the linkages between p97 PPIs and FAF1's various cellular functions. This approach is broadly applicable to dissect the role of each adaptor protein, shedding light on the entire p97 interactome. The outcome of the proposed research will include a validated approach to modulate each p97/adaptor complex and can be extended to other important PPIs. Overall, these studies may also provide a foundation for development of therapies aimed at modulating specific properties of FAF1 and p97 in cell-cycle progression and immune-cell activation.

项目概要/摘要蛋白质-蛋白质相互作用 (PPI) 连接各种蛋白质以实现关键的细胞功能。例如，普遍表达的含缬氨肽蛋白 (VCP/p97) 参与多种通过质子泵抑制剂（PPI）参与生物事件，维持不同器官的蛋白质稳态。每个过程中都有多种蛋白质，系统地破译 p97 PPI 将从根本上受益蛋白质组学研究表明 p97 PPI 网络是高度动态的。一套全面的 p97 PPI、它们的功能和蛋白质底物一直难以捉摸。该提案的总体目标是使用工程抗体抑制剂剖析 p97 PPI 网络，提供有关通过 p97 PPI 调节特定细胞功能的基本见解。迄今为止，已经发现了至少30种p97接头蛋白。概括地说，这些接头蛋白的作用。用广义术语描述（例如，“膜重塑”）。这些适配器的功能，它们是独立工作还是一起工作？我们的中心假设是，通过开发 p97/接头特异性抑制剂，每个适配器都可以在 p97 PPI 网络中定义，FAS 相关因子 1 (FAF1) 是一个重要的适配器。 p97蛋白将被用作开发p97 PPI调节技术的主要例子。 FAF1 特别令人感兴趣，因为它已被证明参与染色质相关的调节值得注意的是，这些过程被发现涉及 p97 和 FAF1，但事实并非如此。明确 FAF1 的所有功能是否都依赖于 p97。为了检验这一假设，将使用抗体片段。针对 FAF1 上的 p97 结合 (UBX) 结构域进行设计，阻断 FAF1 与 p97 的相互作用并留下与基因敲低/敲除策略不同，所提出的策略保留了 FAF1 和 p97 的其余活性。将直接探测 PPI 的作用，无需涉及蛋白质的缺失，实时反映 p97-FAF1 相互作用的作用将在染色质中进行评估。相关的降解过程和 NF-κB 信号传导这两个过程涉及 FAF1 的不同域。对于常规功能，有助于评估 p97-FAF1 相互作用期间 p97 底物特异性。该提案的目标是建立一个实验模型来剖析 p97 PPI，探索其中的联系 p97 PPI 和 FAF1 的各种细胞功能之间的关系这种方法广泛适用于剖析其作用。每个接头蛋白的结构，揭示了整个 p97 相互作用组的研究结果。将包括一种经过验证的方法来调节每个 p97/接头复合物，并且可以扩展到其他重要的领域总的来说，这些研究也可能为开发旨在调节的疗法奠定基础。 FAF1 和 p97 在细胞周期进程和免疫细胞激活中的特殊特性。