Jones, N. H. | Search bioRxiv

bioRxiv2026

VPS4 and CHMP7 release centromeres from the nuclear envelope for post-mitotic positioning in daughter nuclei

Eukaryotic chromosomes occupy ordered configurations within the nucleus, an organization that must be re-established in daughter cells as the nuclear envelope reforms at the end of mitosis. The conserved enzyme VPS4 and ESCRT-III proteins mediate nuclear envelope reformation, yet their role in post-mitotic centromere positioning remains unclear. Here, we develop a chemical genetics approach to analyze the role of VPS4 in human cells. VPS4 inhibition prevents the clearance of CHMP7 from centromeres, which remain constrained in ring-like configurations established during mitosis. Without VPS4 activity, CHMP7, but not other ESCRT-III proteins, forms nuclear foci, nuclear envelope protein distribution is altered and inner nuclear membrane invaginations appear. Following these defects, DNA damage is observed in the vicinity of centromeres. Depletion of CHMP7, but not CHMP4B, suppresses this damage. We propose that VPS4-mediated turnover of CHMP7 releases centromeres from transient nuclear envelope contacts, ensuring their proper positioning after mitosis and maintaining genome integrity.

cell biology↗

bioRxiv2023

Allosteric activation of VCP, a AAA unfoldase, by small molecule mimicry

The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme activity, activator binding must be permissive to different conformational states needed for enzyme function. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, a AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP Activator 1 (VA1), a compound that dose-dependently stimulates VCP ATPase activity up to [~]3-fold. Our cryo-EM studies resulted in structures ([~]2.9-3.5 [A]-resolution) of VCP in apo and ADP-bound states, and revealed VA1 binding an allosteric pocket near the C-terminus in both states. Engineered mutations in the VA1 binding site confer resistance to VA1, and furthermore, modulate VCP activity to a similar level as VA1-mediated activation. The VA1 binding site can alternatively be occupied by a phenylalanine residue in the VCP C-terminal tail, a motif that is post-translationally modified and interacts with cofactors. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry. SignificanceThe loss of function of valosin-containing protein (VCP/p97), a mechanoenzyme from the AAA superfamily that hydrolyzes ATP and uses the released energy to extract or unfold substrate proteins, is linked to protein aggregation-based disorders. However, druggable allosteric sites to activate VCP, or any AAA mechanoenzyme, have not been identified. Here, we report cryo-EM structures of VCP in two states in complex with VA1, a compound we identified that dose-dependently stimulates VCPs ATP hydrolysis activity. The VA1 binding site can also be occupied by a phenylalanine residue in the VCP C-terminal tail, suggesting that VA1 acts through mimicry of this interaction. Our study reveals a druggable allosteric site and a mechanism of enzyme regulation.

biochemistry↗