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Updated: May 26, 2026
07:58
In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination
Published on: January 2, 2026
Npl4 decodes polyubiquitin length and gates D1-D2 coupling in human VCP/p97
Nils Walter1, Laxmikanta Khamari1, Jingxuan Tang1
1University of Michigan.
Research Square
|May 25, 2026
Summary
The VCP/p97 chaperone uses Npl4-Ufd1 to degrade polyubiquitinated proteins, with Npl4 binding strength dependent on ubiquitin chain length. D1 ATP hydrolysis drives Npl4 exchange, gating substrate processing and implicating cofactor cycling in disease.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Biology
Background:
- The VCP/p97 chaperone complex is crucial for protein degradation via the proteasome.
- Understanding how p97 recognizes polyubiquitin chain length and how its ATPase domains (D1 and D2) coordinate substrate processing is essential.
Purpose of the Study:
- To elucidate the mechanism by which VCP/p97 decodes polyubiquitin chain length.
- To investigate the roles of D1 and D2 ATPase activities in cofactor exchange and substrate processing.
- To explore the impact of disease-associated VCP/p97 mutations on its function.
Main Methods:
- Development and application of single-molecule ubiquitin recognition and dynamics (smUbiRAD) assay.
- Biochemical analysis of p97-Ufd1-Npl4 complex formation and dynamics.
- Characterization of wild-type and mutant VCP/p97 variants.
Main Results:
- A sharp chain-length threshold for Npl4 binding was identified, with preferential long-lived engagement on tetra- and penta-ubiquitin.
- Ufd1 and p97 stabilize Npl4-ubiquitin complexes by suppressing Npl4 dissociation.
- D1 ATP hydrolysis, not D2, drives rapid Npl4 exchange in assembled complexes, gating substrate processing.
- Mutant VCP/p97 variants (R155H, A232E) exhibit increased affinity and accelerated Npl4 exchange, suggesting hyperactive cofactor cycling in disease.
Conclusions:
- VCP/p97 utilizes a chain-length-dependent mechanism for substrate selection via Npl4.
- D1 ATPase activity acts as a gatekeeper for cofactor turnover and subsequent D2-mediated substrate processing.
- Dysregulation of VCP/p97 cofactor cycling, particularly hyperactivation, is linked to multisystem proteinopathy.
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