Student Theses and Dissertations
Date of Award
2025
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Tarun Kapoor
Keywords
Valosin-containing protein (VCP), protein degradation, cofactors, ubiquitin, proteostasis, structural biology
Abstract
Valosin-containing protein (VCP or p97 in mammals, Cdc48 in yeast) is an essential member of the AAA (ATPases associated with diverse cellular activities) protein superfamily. As its family name suggests, VCP function is implicated in a broad range of biological pathways including protein degradation, DNA repair, and organelle membrane remodeling. VCP acts as a mechanoenzyme by converting the energy from ATP hydrolysis into mechanical work to segregate its protein substrates from macromolecular complexes, membranes, or organelles and unfold them. This activity is regulated by interactions with cofactors, of which there are ~30 reported to engage VCP at distinct sites for the recruitment of substrates, localization to a subcellular compartment, and modulation of ATPase or unfoldase activity. VCP overexpression or mutation is linked to cancer and neurodegeneration, respectively, underscoring the critical role of VCP in maintenance of proper cell function. Decades of research have focused on the biochemical and structural principles of VCP function, especially pertaining to its role in the proteostasis network by unfolding polyubiquitylated substrates prior to their recycling or proteasomal degradation. For example, several groups have used structural biology approaches to determine structures of VCP in complex with cofactors and unfolded substrate. Through analysis of these assemblies, we have gained important insight into how substrate unfolding is initiated. However, structure determination is limited to a subset of cofactors that bind and recruit substrates and have a well-established biological role. The body of work contained in my thesis describes my efforts, in collaboration with others, to identify and characterize proteins that interact with VCP site-specifically with the goal of understanding how these interactions impact VCP's role in diverse pathways. Chapter 1 serves as an introduction to proteostasis and the pathways involved in maintaining functional levels of proteins in cells. I also provide examples of how proteins in the proteostasis network have been targeted for the treatment of diseases. Additionally, I discuss the role of ubiquitylation, a post-translational modification with diverse functional implications, in the proteostasis pathway and include examples of small molecules that target enzymes within the ubiquitylation cascade. Finally, I introduce VCP's structure, biochemical activities, regulation by cofactors, and roles in distinct cellular pathways. I describe studies that associate VCP overexpression or mutation with cancer and neurodegenerative diseases, respectively, and end Chapter 1 by summarizing reports that have identified candidate VCP binding partners using proteomics-based approaches. In Chapter 2, I describe my efforts to establish a method combining amber suppression, photo-crosslinking, and quantitative proteomics that identifies domain-specific VCP interactors. Interestingly, we identify profilin-1 (PFN1), an actin monomer-binding protein, as a top hit in one of our proteomics datasets. We examine the interaction between VCP and PFN1 using a biochemical pulldown assay and computational modeling. In collaboration with Eric Vitriol, we show that VCP can regulate actin polymerization in a PFN1-dependent manner. I also discuss progress toward characterization of a VCP-USP19 complex, which is another potential interaction discovered using our proteomics workflow. Chapter 3 contains a second project that developed from our chemical proteomics experiments, in which we observe VCPIP1, a deubiquitinase, bound to two distinct sites on VCP. Using cryo-electron microscopy, we determined structures of VCP-VCPIP1 complexes in the absence of added nucleotide or in the presence of an ATP analog and observe VCPIP1's catalytic site below VCP's central pore, poised to cleave ubiquitin from substrates following unfolding. We also show that VCP stimulates VCPIP1's deubiquitinase activity in a biochemical assay. Together, these data suggest a model in which the activities of VCP and a deubiquitinase can be coupled to remodel substrates for recycling or degradation. In Chapter 4 I turn my focus to a project that I afforded significant time and energy, in which I examined the biochemical and structural properties of WRN helicase in complex with DNA substrates. I describe multiple efforts toward structure determination of WRN-DNA assemblies and additional studies where I examined the mechanism of action for a clinical-stage small molecule WRN inhibitor. While I have accumulated a large body of work focused on WRN, this project has not progressed to a publication. In the final chapter, I provide conclusions formed from the findings presented in this thesis and highlight potential directions for future studies. For example, I propose additional chemical proteomics experiments that could provide insight into how the VCP interactome may be altered in distinct cell states, such as disease or stress. I also suggest experiments toward structure determination of specific VCP complexes relevant to the findings presented herein. Finally, I discuss optimizations that may improve efforts toward structure determination of WRN-DNA complexes. In summary, this thesis captures the biochemical and structural properties of VCP unfoldase complexes in the context of proteostasis and cytoskeleton organization and highlights my progress toward the characterization of WRN-DNA-inhibitor interactions.
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Recommended Citation
Vostal, Lauren E., "Biochemical and Structural Insights into VCP Unfoldase and WRN Helicase Complexes" (2025). Student Theses and Dissertations. 829.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/829
Comments
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy