CaltechAUTHORS
  A Caltech Library Service

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

Blythe, Emily E. and Olson, Kristine C. and Chau, Vincent and Deshaies, Raymond J. (2017) Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP•NPLOC4•UFD1L is enhanced by a mutation that causes multisystem proteinopathy. Proceedings of the National Academy of Sciences of the United States of America, 114 (22). E4380-E4388. ISSN 0027-8424. PMCID PMC5465906. https://resolver.caltech.edu/CaltechAUTHORS:20170516-101734172

[img] PDF - Published Version
See Usage Policy.

1222Kb
[img] PDF - Supplemental Material
See Usage Policy.

495Kb
[img] PDF - Submitted Version
See Usage Policy.

4Mb

Use this Persistent URL to link to this item: https://resolver.caltech.edu/CaltechAUTHORS:20170516-101734172

Abstract

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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1073/pnas.1706205114 DOIArticle
http://www.pnas.org/content/114/22/E4380PublisherArticle
http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1706205114/-/DCSupplementalPublisherSupporting Information
http://dx.doi.org/10.1101/129528DOIDiscussion Paper
http://www.biorxiv.org/content/early/2017/04/21/129528OrganizationDiscussion Paper
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465906/PubMed CentralArticle
ORCID:
AuthorORCID
Blythe, Emily E.0000-0001-6363-2644
Deshaies, Raymond J.0000-0002-3671-9354
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.
Funders:
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Subject Keywords:AAA ATPase; Cdc48; Npl4; Ufd1; Proteasome
Issue or Number:22
PubMed Central ID:PMC5465906
Record Number:CaltechAUTHORS:20170516-101734172
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170516-101734172
Official Citation:Emily E. Blythe, Kristine C. Olson, Vincent Chau, and Raymond J. Deshaies Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP•NPLOC4•UFD1L is enhanced by a mutation that causes multisystem proteinopathy PNAS 2017 114 (22) E4380-E4388; published ahead of print May 16, 2017, doi:10.1073/pnas.1706205114
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:77491
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:16 May 2017 17:38
Last Modified:03 Oct 2019 17:58

Repository Staff Only: item control page