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-¿B 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-¿B 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.
