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Title: Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism

Abstract

This work presents that the 26S proteasome is a large cellular assembly that mediates the selective degradation of proteins in the nucleus and cytosol and is an established target for anticancer therapeutics. Protein substrates are typically targeted to the proteasome through modification with a polyubiquitin chain, which can be recognized by several proteasome-associated ubiquitin receptors. One of these receptors, RPN13/ADRM1, is recruited to the proteasome through direct interaction with the large scaffolding protein RPN2 within the 19S regulatory particle. To better understand the interactions between RPN13, RPN2, and ubiquitin, we used human proteins to map the RPN13-binding epitope to the C-terminal 14 residues of RPN2, which, like ubiquitin, binds the N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain of RPN13. We also report the crystal structures of the RPN13 PRU domain in complex with peptides corresponding to the RPN2 C terminus and ubiquitin. Through mutational analysis, we validated the RPN2-binding interface revealed by our structures and quantified binding interactions with surface plasmon resonance and fluorescence polarization. In contrast to a previous report, we find that RPN13 binds ubiquitin with an affinity similar to that of other proteasome-associated ubiquitin receptors and that RPN2, ubiquitin, and the deubiquitylase UCH37 bind to RPN13 withmore » independent energetics. In conclusion, these findings provide a detailed characterization of interactions that are important for proteasome function, indicate ubiquitin affinities that are consistent with the role of RPN13 as a proteasomal ubiquitin receptor, and have major implications for the development of novel anticancer therapeutics.« less

Authors:
 [1];  [1];  [2];  [3];  [1]
  1. Univ. of Utah, Salt Lake City, UT (United States). Department of Biochemistry
  2. Colorado State Univ., Fort Collins, CO (United States). Department of Biochemistry and Molecular Biology
  3. Brookhaven National Lab. (BNL), Upton, NY (United States). Biology Department
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1376133
Report Number(s):
BNL-114068-2017-JA
Journal ID: ISSN 0021-9258
Grant/Contract Number:  
SC0012704; AC02-76SF00515
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 292; Journal Issue: 23; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; proteasome; RPN13; RPN2; ubiquitin; crystallography; surface plasmon resonance; fluorescence polarization

Citation Formats

VanderLinden, Ryan T., Hemmis, Casey W., Yao, Tingting, Robinson, Howard, and Hill, Christopher P. Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism. United States: N. p., 2017. Web. doi:10.1074/jbc.M117.785287.
VanderLinden, Ryan T., Hemmis, Casey W., Yao, Tingting, Robinson, Howard, & Hill, Christopher P. Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism. United States. doi:10.1074/jbc.M117.785287.
VanderLinden, Ryan T., Hemmis, Casey W., Yao, Tingting, Robinson, Howard, and Hill, Christopher P. Tue . "Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism". United States. doi:10.1074/jbc.M117.785287. https://www.osti.gov/servlets/purl/1376133.
@article{osti_1376133,
title = {Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism},
author = {VanderLinden, Ryan T. and Hemmis, Casey W. and Yao, Tingting and Robinson, Howard and Hill, Christopher P.},
abstractNote = {This work presents that the 26S proteasome is a large cellular assembly that mediates the selective degradation of proteins in the nucleus and cytosol and is an established target for anticancer therapeutics. Protein substrates are typically targeted to the proteasome through modification with a polyubiquitin chain, which can be recognized by several proteasome-associated ubiquitin receptors. One of these receptors, RPN13/ADRM1, is recruited to the proteasome through direct interaction with the large scaffolding protein RPN2 within the 19S regulatory particle. To better understand the interactions between RPN13, RPN2, and ubiquitin, we used human proteins to map the RPN13-binding epitope to the C-terminal 14 residues of RPN2, which, like ubiquitin, binds the N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain of RPN13. We also report the crystal structures of the RPN13 PRU domain in complex with peptides corresponding to the RPN2 C terminus and ubiquitin. Through mutational analysis, we validated the RPN2-binding interface revealed by our structures and quantified binding interactions with surface plasmon resonance and fluorescence polarization. In contrast to a previous report, we find that RPN13 binds ubiquitin with an affinity similar to that of other proteasome-associated ubiquitin receptors and that RPN2, ubiquitin, and the deubiquitylase UCH37 bind to RPN13 with independent energetics. In conclusion, these findings provide a detailed characterization of interactions that are important for proteasome function, indicate ubiquitin affinities that are consistent with the role of RPN13 as a proteasomal ubiquitin receptor, and have major implications for the development of novel anticancer therapeutics.},
doi = {10.1074/jbc.M117.785287},
journal = {Journal of Biological Chemistry},
number = 23,
volume = 292,
place = {United States},
year = {Tue Apr 25 00:00:00 EDT 2017},
month = {Tue Apr 25 00:00:00 EDT 2017}
}

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Cited by: 4 works
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Works referenced in this record:

Protein production by auto-induction in high-density shaking cultures
journal, May 2005


PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

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