Related Experiment Video

Updated: Mar 2, 2026

In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination
07:58

In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

Published on: January 2, 2026

486

Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP•NPLOC4•UFD1L is enhanced by a mutation that causes

Emily E Blythe1, Kristine C Olson2, Vincent Chau2

  • 1Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.

Proceedings of the National Academy of Sciences of the United States of America
|May 18, 2017
PubMed

Insights

The p97 segregase unfolds proteins, a key step in cellular degradation. This activity, crucial for protein quality control, is enhanced by specific ubiquitin chains and may be overactive in certain human diseases.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • p97 (also known as VCP) is a crucial AAA+ ATPase segregase involved in diverse ubiquitin-dependent cellular processes.
  • Its role in extracting proteins from membranes and complexes for proteasomal degradation is fundamental but poorly understood due to substrate complexity.

Purpose of the Study:

  • To develop a system to directly observe and characterize the protein unfolding activity of p97.
  • To investigate the factors influencing p97 unfolding activity and its potential role in disease pathogenesis.

Main Methods:

  • Development of a soluble substrate (Ub-GFP) modified with K48-linked ubiquitin chains for in vitro p97 activity assays.
  • Characterization of wild-type (WT) p97 and a disease-associated mutant's unfolding activity.

Main Results:

  • Demonstrated that WT p97 can unfold proteins in an ATP-hydrolysis-dependent manner.
  • Showed that p97 activity is reliant on its adaptors (NPLOC4-UFD1L) and substrate ubiquitination, with branched ubiquitin chains providing maximal stimulation.
  • Identified a disease-associated p97 mutant that exhibits enhanced unfolding activity, suggesting a link between excessive activity and pathogenesis.

Conclusions:

  • This study establishes a novel in vitro system for dissecting p97's fundamental unfolding mechanism.
  • The findings provide mechanistic insights into p97 function in protein degradation and suggest that dysregulated unfolding activity contributes to human diseases like inclusion body myopathy.

Keywords:
AAA ATPaseCdc48Npl4ProteasomeUfd1

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