ASPL-driven subunit exchange remodels VCP/p97 hexamers and is impaired by a multisystem proteinopathy mutation

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Valosin-containing protein (VCP/p97) is a crucial AAA+ ATPase involved in protein quality control and proteasomal degradation.
• Missense mutations in VCP cause multisystem proteinopathy (MSP), a group of debilitating disorders.
• The precise molecular mechanisms by which VCP mutations lead to MSP remain poorly understood.

Purpose of the Study:

• To investigate the dynamics of VCP hexamer assembly and subunit exchange in human cell lysates.
• To elucidate the role of the VCP-interactor ASPL in modulating VCP dynamics.
• To understand how MSP-associated VCP variants affect VCP assembly and subunit exchange.

Main Methods:

• Development of a single-molecule pull-down assay to quantify VCP assembly and subunit exchange dynamics.
• Analysis of VCP subunit exchange in both human cell lysates and purified complexes.
• Kinetic analysis of the interaction between ASPL and wild-type versus mutant VCP.

Main Results:

• MSP-associated VCP variant R155H co-assembles with wild-type VCP into heterohexamers.
• Subunit exchange is significantly reduced for purified VCP complexes compared to those in cell lysates.
• ASPL selectively mediates monomer exchange, efficiently remodeling wild-type VCP but not R155H-VCP multimers.
• ASPL associates faster and dissociates slower with wild-type VCP than with R155H-VCP.

Conclusions:

• ASPL-driven monomer exchange is essential for remodeling VCP molecular machines and maintaining proteostasis.
• Impaired ASPL-mediated exchange of MSP variant monomers likely stabilizes mutant VCP in assemblies.
• This VCP assembly defect presents a potential therapeutic target for MSP.