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Published May 30, 2017 | Supplemental Material + Submitted + Published
Journal Article Open

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


p97 is a "segregase" that plays a key role in numerous ubiquitin (Ub)-dependent pathways such as ER-associated degradation. It has been hypothesized that p97 extracts proteins from membranes or macromolecular complexes to enable their proteasomal degradation; however, the complex nature of p97 substrates has made it difficult to directly observe the fundamental basis for this activity. To address this issue, we developed a soluble p97 substrate—Ub-GFP modified with K48-linked ubiquitin chains—for in vitro p97 activity assays. We demonstrate that WT p97 can unfold proteins and that this activity is dependent on the p97 adaptor NPLOC4-UFD1L, ATP hydrolysis, and substrate ubiquitination, with branched chains providing maximal stimulation. Furthermore, we show that a p97 mutant that causes inclusion body myopathy, Paget's disease of bone, and frontotemporal dementia in humans unfolds substrate faster, suggesting that excess activity may underlie pathogenesis. This work overcomes a significant barrier in the study of p97 and will allow the future dissection of p97 mechanism at a level of detail previously unattainable.

Additional Information

© 2017 National Academy of Sciences. Contributed by Raymond J. Deshaies, April 24, 2017 (sent for review April 14, 2017; reviewed by Peter K. Jackson and Robert T. Sauer). Published ahead of print May 16, 2017. We thank Willem den Besten, David Sherman, and Jing Li for assistance with cloning and protein purification; Arthur Horwich for the GroEL expression plasmid and antibody; Rati Verma and David Sherman for comments on the manuscript; the entire laboratory of R.J.D. for helpful discussion; and Tom Rapoport for communicating results before publication. Fluorescence measurements were carried out in the Beckman Institute Laser Resource Center and the Caltech Biophysical Facility. R.J.D. is an Investigator of the Howard Hughes Medical Institute (HHMI), and this work was supported by HHMI. Author contributions: E.E.B. and R.J.D. designed research; E.E.B. performed research; K.C.O. and V.C. contributed new reagents/analytic tools; E.E.B. and R.J.D. analyzed data; and E.E.B. and R.J.D. wrote the paper. Reviewers: P.K.J., Stanford University; and R.T.S., Massachusetts Institute of Technology. Conflict of interest statement: R.J.D. is a founder and a shareholder of Cleave Biosciences, which is developing CB-5083 for therapy of cancer. The other authors declare that no competing interests exist. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1706205114/-/DCSupplemental.

Attached Files

Published - PNAS-2017-Blythe-E4380-8.pdf

Submitted - 129528.full.pdf

Supplemental Material - pnas.201706205SI.pdf


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