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.

项目摘要/摘要蛋白质 - 蛋白质相互作用（PPI）连接各种蛋白质以达到关键的细胞功能。为了实例，含蛋白质瓣膜蛋白的普遍表达的蛋白质（VCP/p97）参与了多个通过PPI的生物事件，维持不同细胞器中的蛋白质稳态。考虑参与在每个过程中多种蛋白质的蛋白质，它将从根本上有益于系统地破译P97 PPI 网络。蛋白质组学研究表明，p97 PPI网络具有高度动态性。但是，全面的P97 PPI，它们的功能和蛋白质底物是难以捉摸的。该提案的总体目标是使用工程抗体抑制剂剖析p97 PPI网络，通过调节p97 PPI，提供了对特定细胞功能调节的基本见解。到目前为止，已经发现了P97的至少30个适配器蛋白。通常，这些衔接蛋白的作用用广泛的术语描述（例如，“膜重塑”）。什么是特定的底物或区分这些适配器的功能，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/适配器特异性抑制剂，可以在p97 PPI网络中定义每个适配器。 FAS相关因子1（FAF1），一个重要的适配器 p97的蛋白质将被用作开发调节p97 PPI的技术的主要例子。 FAF1特别有趣，因为它已被证明与染色质相关的调节有关降解和NF-κB信号传导。值得注意的是，发现此类过程涉及P97和FAF1，这不是清除FAF1的所有功能是否依赖于p97。为了检验假设，抗体片段将是为FAF1上的P97结合（UBX）域设计，阻止了FAF1与P97的互动并离开 FAF1和P97的其余活动完好无损。与基因敲低/淘汰策略不同，拟议的策略将直接探测PPI的作用，而无需涉及蛋白质的缺乏，反映了实时利益相互作用的贡献。 P97-FAF1相互作用的作用将在两种染色质中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，支持P97-FAF1相互作用期间p97底物特异性的评估。该建议的目的是建立一个实验模型以剖析P97 PPI，探索联系在P97 PPI和FAF1的各种细胞函数之间。这种方法广泛适用于剖析角色每个适配器蛋白，散发出整个p97相互作用组的光。拟议研究的结果将包括一种经过验证的方法来调节每个P97/适配器复合物，并可以扩展到其他重要的 PPI。总体而言，这些研究还可能为开发旨在调节的疗法的发展提供基础 FAF1和P97在细胞周期进展和免疫细胞激活中的特定特性。