Characterization of p97 mutations causing multisystem proteinopathy support a gain-of-function model for pathology

Abstract

Valosin‐containing protein (VCP, or p97) is an ATPase essential in numerous protein quality control (PQC) pathways, such as ER‐associated degradation. p97 functions as a segregase, extracting ubiquitylated proteins from membranes or complexes so they can be degraded by the proteasome. However, the complexity of native p97 PQC substrates has stymied the detailed biochemical study of this function. Previously, to address this problem, we developed an in vitro p97 substrate based on an ubiquitin fusion degradation (UFD) pathway substrate, Ub‐GFP, and showed that the unfolding of this substrate by p97 is dependent upon extensive substrate ubiquitylation, the p97 adaptors NPLOC4‐UFD1L, and ATP hydrolysis. Here, we make use of this system, employing an updated version of this substrate, to explore how mutations in p97 that cause multisystem proteinopathy (MSP) affect substrate processing. Previous studies have shown that MSP mutants have higher basal ATP rates than wild type yet cause deficiencies in many p97‐dependent pathways, creating controversy as to whether these dominantly inherited mutations cause disease through a gain‐of‐function or a loss‐of‐function. We have now analyzed seven distinct MSP mutants, all of which showed modestly improved unfolding of our model substrate over wild type p97, providing evidence that the increased ATPase activity leads to a gain‐of‐function. Furthermore, we showed evidence that p97 inhibitors may restore proper p97 function to MSP mutants, suggesting a potential treatment strategy for p97 diseases.

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