TY - JOUR
T1 - Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit
AU - Chojnacki, Michal
AU - Mansour, Wissam
AU - Hameed, Dharjath S
AU - Singh, Rajesh K
AU - El Oualid, Farid
AU - Rosenzweig, Rina
AU - Nakasone, Mark A
AU - Yu, Zanlin
AU - Glaser, Fabian
AU - Kay, Lewis E
AU - Fushman, David
AU - Ovaa, Huib
AU - Glickman, Michael H
N1 - W.M. is supported in part by an ISF council for Higher Education Outstanding Minority (VATAT) fellowship, M.C. is supported through the EU Seventh Framework Programme ( FP7A-PEOPLE-2011-ITN ), M.A.N. is supported by a Fulbright postdoctoral fellowship and the Aly Kaufman Fellowship Trust at the Technion . This work was supported in part by a grant from the Netherlands Foundation for Scientific Research (NWO) to H.O. and a grant from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada (to L.E.K) and by NIH grants GM065334 and R21NS093454 (to D.F.) and GM095755 (to D.F. and M.H.G.), a USA-Israel Binational Science Foundation grant (to D.F. and M.H.G.), and an Israel Science Foundation grant 909-14 to M.H.G. H.O. and F.E.O. declare competing financial interests as co-founder and shareholder of UbiQ Bio BV.Copyright © 2017 Elsevier Ltd. All rights reserved.
PY - 2017/4/20
Y1 - 2017/4/20
N2 - Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (UbPT), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb conjugates. Our work demonstrates the value of UbPT, and we expect that its future uses will help define and investigate the ubiquitin interactome.
AB - Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (UbPT), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb conjugates. Our work demonstrates the value of UbPT, and we expect that its future uses will help define and investigate the ubiquitin interactome.
KW - Binding Sites
KW - Cross-Linking Reagents/chemistry
KW - Molecular Docking Simulation
KW - Nuclear Magnetic Resonance, Biomolecular
KW - Polyubiquitin/chemistry
KW - Proteasome Endopeptidase Complex/chemistry
KW - Protein Binding
KW - Protein Structure, Tertiary
KW - Protein Subunits/chemistry
KW - Saccharomyces cerevisiae/metabolism
KW - Saccharomyces cerevisiae Proteins/chemistry
KW - Ubiquitin/chemistry
KW - Ubiquitination/radiation effects
KW - Ultraviolet Rays
U2 - 10.1016/j.chembiol.2017.02.013
DO - 10.1016/j.chembiol.2017.02.013
M3 - Article
C2 - 28330605
SN - 2451-9448
VL - 24
SP - 443-457.e6
JO - Cell Chemical Biology
JF - Cell Chemical Biology
IS - 4
ER -