TY - JOUR
T1 - Development of Diubiquitin-Based FRET Probes to Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes
AU - Geurink, Paul P.
AU - Van Tol, Bianca D.M.
AU - Van Dalen, Duco
AU - Brundel, Paul J.G.
AU - Mevissen, Tycho E.T.
AU - Pruneda, Jonathan N.
AU - Elliott, Paul R.
AU - Van Tilburg, Gabriëlle B.A.
AU - Komander, David
AU - Ovaa, Huib
N1 - Funding Information:
We thank Dris El Atmioui and Henk Hilkman for SPPS and Prof. Dr. Kees Jalink for help with FLIM. This work was supported by the European Research Council (ERC grant no. 281699) and the Netherlands Organization for Scientific Research, (NWO VICI grant no. 724.013.002) to H.O. Work in the D.K. lab was funded by the Medical Research Council (U105192732), the European Research Council (Grant no. 309756), and the Lister Institute for Preventive Medicine, as well as the Marie Curie Initial Training Network "UPStream" (T.E.T.M.) and an EMBO Long-Term Fellowship (J.N.P.).
Publisher Copyright:
© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
PY - 2016/5/3
Y1 - 2016/5/3
N2 - Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the absolute quantification of chain cleavage specificity of DUBs by means of Michaelis-Menten kinetics. Each probe is equipped with a FRET pair consisting of Rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by SPPS and NCL. Our synthetic strategy includes the introduction of N,N′-Boc-protected 5-carboxyrhodamine as a convenient building block in peptide chemistry. We demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine DUBs in a high-throughput manner.
AB - Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the absolute quantification of chain cleavage specificity of DUBs by means of Michaelis-Menten kinetics. Each probe is equipped with a FRET pair consisting of Rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by SPPS and NCL. Our synthetic strategy includes the introduction of N,N′-Boc-protected 5-carboxyrhodamine as a convenient building block in peptide chemistry. We demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine DUBs in a high-throughput manner.
KW - FRET
KW - deubiquitinating enzymes
KW - native chemical ligation
KW - solid-phase synthesis
KW - ubiquitin conjugates
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U2 - 10.1002/cbic.201600017
DO - 10.1002/cbic.201600017
M3 - Article
C2 - 26996281
AN - SCOPUS:84962623766
SN - 1439-4227
VL - 17
SP - 816
EP - 820
JO - ChemBioChem
JF - ChemBioChem
IS - 9
ER -