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

项目摘要/摘要 蛋白质-蛋白质相互作用(PPI)将各种蛋白质连接起来，以实现关键的细胞功能。为 例如，普遍表达的含有Valosin的蛋白(VCP/p97)参与了多个 通过PPI发生生物事件，维持不同细胞器中的蛋白质动态平衡。考虑到参与度 对于每个过程中的多个蛋白质，系统地破译p97 ppi将是根本上有益的。 网络。蛋白质组学研究表明，p97 PPI网络是高度动态的。然而，a P97 PPI的全面集合、它们的功能和蛋白质底物一直难以捉摸。 这项提案的总体目标是使用工程抗体抑制剂剖析p97 PPI网络， 提供关于通过调节p97 PPI调节特定细胞功能的基本见解。 到目前为止，已经发现了至少30个p97适配子蛋白。总体而言，这些接头蛋白的作用 用广义的术语来描述(例如，“膜重构”)。具体的底物或区别是什么？ 这些适配器的功能，它们是独立工作还是协同工作？ 我们的中心假设是，通过开发p97/Adaptor特异性抑制剂，p97/Adaptor的独特功能 每个适配器可以在p97 PPI网络内定义。Fas相关因子1(FAF1)--一个重要的适配器 将以p97蛋白为主要研究对象，开发p97蛋白的调控技术。 FAF1特别令人感兴趣，因为它已被证明参与了染色质相关的调节 降解和NF-κB信号转导。值得注意的是，这样的过程被发现同时涉及p97和FAF1，而不是 明确FAF1的所有功能是否都依赖于p97。为了检验这一假设，抗体片段将被 针对FAF1上的p97结合(UBX)结构域进行工程，阻止FAF1‘S与p97的相互作用并离开 其余的FAF1‘S和P97’S活动完好无损。与基因敲除/敲除策略不同，所提出的策略 将直接探测PPI的作用，而不需要涉及蛋白质的不足，反映了实时性 利益互动的贡献。P97-FAF1相互作用的作用将在染色质- 相关的降解过程和NF-κB信号转导。这两个过程涉及FAF1的不同结构域 对于正常功能，便于评估p97-FAF1相互作用过程中p97底物的特异性。 这项建议的目标是建立一个实验模型来剖析p97 ppi，探索两者之间的联系。 P97PPI与FAF1‘S之间的多种细胞功能。这种方法广泛适用于剖析角色 对每个接头蛋白进行编码，从而揭示整个p97相互作用体。拟议研究的结果 将包括一种有效的方法来调制每个p97/接头复合体，并可以扩展到其他重要的 PPI。总体而言，这些研究也可能为旨在调节的疗法的开发提供基础 FAF1和p97在细胞周期进程和免疫细胞激活中的特异性。

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.