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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.

项目总结/文摘