Development of Diubiquitin-Based FRET Probes to Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes

Article abstract of DOI:10.1002/cbic.201600017

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.

Full text of DOI:10.1002/cbic.201600017

DOI: 10.1002/cbic.201600017
Communications
Development of Diubiquitin-Based FRET Probes To
Quantify Ubiquitin Linkage Specificity of Deubiquitinating
Enzymes
Paul P. Geurink,*[a] Bianca D. M. van Tol,[a] Duco van Dalen,[a] Paul J. G. Brundel,[a]
Tycho E. T. Mevissen,[b] Jonathan N. Pruneda,[b] Paul R. Elliott,[b] Gabriꢀlle B. A. van Tilburg,[a]
David Komander,[b] and Huib Ovaa*[a]
Deubiquitinating enzymes (DUBs) are proteases that fulfill cru-
cial roles in the ubiquitin (Ub) system, by deconjugation of Ub
from its targets and disassembly of polyUb chains. The specif-
icity 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 spe-
cificity of DUBs by means of Michaelis–Menten kinetics. Each
probe is equipped with a FRET pair consisting of Rhoda-
mine110 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-car-
boxyrhodamine as a convenient building block in peptide
chemistry. We demonstrate the value of our probes by quanti-
fying the linkage specificities of a panel of nine DUBs in a
high-throughput manner.
particular combination of which provides specificity for the
protein target or polyUb chain topology. Removal of Ub from
its targets and disassembly of polyUb chains are catalyzed by
deubiquitinating enzymes (DUBs). About 100 human DUBs
have been identified;[2] some exhibit Ub linkage specificity.
DUB action can rescue proteins from proteasomal degradation
and alter Ub signaling functions through chain remodeling in
a linkage-specific manner.[1] The synthesis of diubiquitin (diUb)
has made it possible to study processing by DUBs.[3] In order
to determine specificity, a DUB can be incubated with either
a native diUb molecule[4] or with a diUb activity-based probe[5]
of a given linkage. However current methods do not allow fast
and absolute quantification of DUB linkage specificity, and fur-
thermore cannot separate this specificity into binding affinity
and catalytic turnover rate (Km and kcat, respectively, in Michae-
lis–Menten kinetics).
The application of FRET pairs has proved useful in the study
of DUB activity, Ub chain conformation, and Ub-interacting
proteins.[6] In order to investigate chain cleavage specificity
across all isopeptide linkages, we developed a full chemical
synthesis of all seven isopeptide-linked diUb FRET pairs. These
pairs carry a novel dye-pair suitable for FRET and compatible
with solid phase peptide synthesis (SPPS). We determined Km
and kcat values of linkage-specific DUBs that are used in Ub
chain restriction analysis,[7] in order to obtain insight into their
catalytic action.
Ubiquitin (Ub), a 76 amino acid protein, is a post-translational
modifier that is crucial for a wide range of cellular processes,
including protein degradation, trafficking, and signaling.[1] Ub
is generally attached via its C-terminal carboxylate to the side-
chain amine of a lysine residue in the target protein, thereby
forming an isopeptide bond. Target proteins are frequently
modified with a polyUb chain, in which multiple Ub modules
are successively linked at the N terminus (linear polyUb) or any
of the seven internal lysine residues (isopeptide-linked polyUb:
K6, K11, K27, K29, K33, K48, and K63). The type of polyUb chain
largely determines its signaling function.[1]
In the FRET-based assay (Figure 1) the reagents consist of
two Ub modules, one equipped with a donor fluorophore and
the other with an acceptor; these are specifically linked by
a native isopeptide bond to each of the seven lysine residues.
We reasoned that the best position for fluorophore attachment
would be the N termini of both Ub modules, because the dis-
tance between the N termini ranges from 30 to 50 ꢁ, based on
available crystallographic data (Table S1 in the Supporting In-
formation), an ideal distance for FRET. Because the fluoro-
phores need to be compatible with all synthetic steps (see
below), we developed a new FRET pair by using 5-carboxy-
rhodamine110 (Rho) as the donor and 5-carboxytetramethyl-
rhodamine (TAMRA) as the acceptor. Fluorescein, the more
commonly used FRET donor, was initially tried but proved in-
compatible with the desulfurization step in the final synthesis
step (see below) and was therefore replaced by Rho. Upon ad-
dition of a DUB, the diUb FRET pair is cleaved, thereby result-
ing in loss of the FRET signal and hence an increase in donor
emission.
Ubiquitination is mediated by the concerted action of three
enzymes, E1 (activating), E2 (conjugating), and E3 (ligase), the
[a] Dr. P. P. Geurink, B. D. M. van Tol, D. van Dalen, P. J. G. Brundel,
G. B. A. van Tilburg, Prof. Dr. H. Ovaa
Department of Cell Biology, The Netherlands Cancer Institute
Plesmanlaan 121, 1066 CX Amsterdam (The Netherlands)
E-mail: p.geurink@nki.nl
h.ovaa@nki.nl
[b] T. E. T. Mevissen, Dr. J. N. Pruneda, Dr. P. R. Elliott, Dr. D. Komander
Medical Research Council Laboratory of Molecular Biology
FrancisCrick Avenue, Cambridge CB2 0QH (UK)
Supporting information for this article can be found under http://
dx.doi.org/10.1002/cbic.201600017.
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons At-
tribution-NonCommercial-NoDerivs License, which permits use and distribu-
tion in any medium, provided the original work is properly cited, the use is
non-commercial and no modifications or adaptations are made.
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Figure 1. Principle of the FRET-based diUb cleavage assay. Upon cleavage of the diUb FRET pair by a DUB, the FRET signal is lost.
Scheme 1. A) Problems encountered with unprotected Rho in peptide chemistry. B) Synthesis of N,N’-Boc-protected Rho. a) Ac2O, H2SO4, 1208C (99%);
b) EtOH, EDC, CH2Cl2 (94%); c) NaOEt, EtOH; d) Tf2O, pyridine, CH2Cl2 (57%); e) BocNH2, Pd2dba3, Xantphos, Cs2CO3, dioxane, 1008C (82%); f) NaOH, THF
(95%).
A major problem in the use of Rho (but also TAMRA) in SPPS
(Scheme 1A) is that when Rho is attached to an amine in a
globally side-chain-protected peptide, the 1-carboxylate
moiety of Rho is in an open conformation and can react upon
further extension of the peptide chain (Scheme 1A, 1!2). In
addition, the coupling of Rho is generally difficult because of
the poor solubility and intrinsic reactivity of the aniline moiet-
ies. We therefore prepared N,N’-Boc-protected Rho. When this
molecule is coupled to a peptide, the dual Boc protection
locks the 1-carboxylate in the closed lactone form, thus
making it unreactive (Scheme 1A, 3!4). We modified the
method reported by Grimm and Lavis[8] to prepare N,N’-Boc-
protected Rho 10 (Scheme 1). 5-Carboxyfluorescein (5) was
converted in four steps into ditriflate 8. Buchwald–Hartwig
coupling with BocNH2 resulted in the formation of N,N’-Boc-
protected Rho (9). Use of ethyl ester protection of the 5-car-
boxylate allowed selective liberation of the 5-carboxylate with-
out affecting the Boc groups, thereby resulting in 10. In con-
trast to unprotected Rho, 10 is very soluble in organic solvents,
can easily be coupled under standard peptide coupling condi-
tions, and can be prepared on a multi-gram scale.
The seven diUb FRET pairs 17a–g were constructed by
native chemical ligation (NCL) between Rho-Ub-thioester 14
and TAMRA-Ub containing a g-thioLys building block[3,9] 16
(Scheme 2). The individual Ub modules where synthesized by
linear SPPS on hyper-acid-labile trityl resin.[3] DiBoc-protected
Rho (10) was coupled to Ub1ꢀ75 (11) on resin to result in 12,
which was subsequently cleaved from the resin under mild
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Scheme 2. Synthesis of the seven isopeptide-linked diUb FRET pairs. a) 10, PyBOP, DIPEA, NMP; b) 20% HFIP/CH2Cl2; c) HCl·H-Gly-S(CH2)2CO2Me, EDC, HOBt,
CH2Cl2; d) TFA/H2O/phenol/iPr3SiH (90.5:5:2.5:2); e) TAMRA, PyBOP, DIPEA, NMP; f) TFA/H2O/phenol/iPr3SiH (90.5:5:2.5:2); g) MPAA, 6m Gnd·HCl, pH 7.2;
h) TCEP, GSH, VA-044, 6m Gnd·HCl, pH 7.0.
acidic conditions without affecting the global protection
scheme. Methyl-3-(glycylthio)-propionate was coupled to the li-
berated C-terminal carboxylate to give 13. Global deprotection
under strong acidic conditions followed by cation exchange
and RP-HPLC purification gave Rho-Ub-thioester 14. It is of
note that the Boc groups on Rho are concomitantly removed
during the global deprotection, thereby restoring its fluores-
cent properties. TAMRA-Ub modules containing g-thioLys on
each of the respective lysine positions (16a–g) were prepared
by coupling the 5-carboxy isomer of TAMRA to the Ub1–76 poly-
peptides 15a–g, followed by global deprotection and purifica-
tion (Figure S1 in the Supporting Information). Methionine-
1 was replaced by the isostere norleucine to prevent oxidation
of the thioether moiety. NCL reactions between 14 and 16a–g,
followed by desulfurization under radical conditions,[10] purifi-
cation by RP-HPLC, and gel filtration gave the final seven diUb
FRET pairs 17a–g in good yield and purity.
The purities of 17a–g were confirmed by LCMS analysis (Fig-
ure 2A, B, and Supporting Information) and gel analysis (Fig-
ure 2C). Upon excitation at 466 nm, the emission spectra of
the diUb FRET pairs and Rho-Ub and TAMRA-Ub revealed that
all seven molecules show a clear FRET signal (Figure 2D). We
performed fluorescence lifetime imaging microscopy (FLIM) to
determine FRET efficiencies of all the FRET pairs (Figure 2E,
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Figure 2. Characterization of diUb FRET pairs 17a–g. A) Analytical HPLC and B) MS of Lys6-linked diUb FRET pair 17a. C) SDS-PAGE analysis. D) Emission spec-
tra recorded at lex =466 nm. E) FRET efficiencies (E) determined by FLIM.
Table S3); these were found to be 0.45–0.60, depending on the
linkage, thus demonstrating efficient FRET in all these mole-
cules.
with reported data.[4a,b] We then incubated OTUD2 with Lys11-
and Lys27-linked diUb FRET pairs (17b and 17c, respectively)
and the corresponding unlabeled diUbs. We also included
a 1:1 mixture of the FRET pair and the unlabeled diUb for both
linkages. SDS-PAGE analysis (Figures 3A, S4 and S5) showed
that both the FRET pair and the unlabeled diUb substrates
were equally processed, thus we concluded that the attached
fluorophores do not affect DUB activity.
DUB-mediated cleavage of our new diUb FRET pairs was first
assessed by incubation with USP7 and OTUD2, two well-stud-
ied DUBs from the two largest DUB families and for which
cleavage of unlabeled diUbs has been reported. Reactions
were analyzed by SDS-PAGE (Figures S2 and S3) which showed
that the diUb selectivity of both DUBs was in good agreement
We next applied our FRET reagents for the quantification of
diUb linkage-specificity for nine DUBs derived from three dif-
ferent DUB families; each was shown to display a distinct spe-
cificity (Table 1).[4b] We incubated the DUBs with all diUb FRET
pairs at a fixed concentration (0.5 mm, to keep initial fluores-
cence constant for all samples) with an increasing concentra-
tion of the unlabeled diUb (0–28 mm). The enzyme concentra-
tion was chosen such that the reaction proceeded linearly for
at least 20 min (Supporting Information). The total amount of
processed diUb was then determined by monitoring the in-
crease in donor fluorescence over time; from this the rates of
initial velocity were calculated and fitted to the Michaelis–
Menten equation (Figure 3B and Supporting Information),
from which Km and, kcat were determined.
Table 1 shows the data for all combinations of DUB and
diUb substrate for which activity could be measured. Overall,
the individual diUb linkage types cleaved by each DUB were
consistent with published qualitative data.[4] As expected from
earlier findings, the unspecific DUB USP21 showed similar ac-
tivity towards most linkages.[4a] The virus-derived DUB vOTU,
which is also considered to be unspecific, showed some inter-
esting results: the catalytic efficiency (kcat/Km) for Lys6 was two
times higher than for Lys48, and four times higher than for
Figure 3. A) SDS-PAGE analysis of Lys11-linked diUb cleavage. OTUD2 was in-
cubated with unlabeled diUb, FRET pair 17b, or a 1:1 mixture; samples were
taken after 10, 30, 60, and 180 min. B) Michaelis–Menten kinetics of TRABID
for Lys29-, Lys33- and Lys63-linked diUb, as determined by the FRET assay.
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Communications
Acknowledgements
Table 1. Kinetic characterization of DUBs that are used in Ub chain re-
striction analysis[7] for the diUb FRET pairs 17a–g.[a]
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 Re-
search Council (Grant no. 309756), and the Lister Institute for Pre-
ventive Medicine, as well as the Marie Curie Initial Training Net-
work “UPStream” (T.E.T.M.) and an EMBO Long-Term Fellowship
(J.N.P.).
DUB
Linkage
Km [mm]
kcat [sꢀ1
]
kcat/Km [mꢀ1 sꢀ1
]
AMSH
Lys63
Lys63
Lys11
Lys48
Lys48
Lys63
Lys6
Lys11
Lys63
Lys29
Lys33
Lys63
Lys11
Lys27
Lys29
Lys6
Lys11
Lys48
Lys63
Lys6
Lys11
Lys33
Lys48
Lys63
45.4
2.4
0.0027
0.17
1.5
n.d.
0.66
n.d.
59
70048
78818
AMSH*[b]
Cezanne
OTUB1
19.4
@50
38.6
@50
@50
52.4
57.9
40.1
19.6
54.0
87.9
@50
@50
3.6
OTUB1*[b]
OTUD1
OTUD3
17158
4020
n.d.
0.0085
0.0061
0.053
0.028
0.034
4.4
162
105
1317
1431
627
TRABID
OTUD2
vOTU
Keywords: deubiquitinating enzymes · FRET · native chemical
ligation · solid-phase synthesis · ubiquitin conjugates
50253
n.d.
n.d.
0.87
0.22
1.0
0.091
0.12
0.087
0.13
0.067
0.16
242031
63110
119952
66266
60633
62735
60814
39181
60558
3.4
8.4
1.4
2.1
1.4
2.1
1.7
2.7
[1] D. Komander, M. Rape, Annu. Rev. Biochem. 2012, 81, 203–229.
[2] F. E. Reyes-Turcu, K. H. Ventii, K. D. Wilkinson, Annu. Rev. Biochem. 2009,
78, 363–397.
[3] F. El Oualid, R. Merkx, R. Ekkebus, D. S. Hameed, J. J. Smit, A. de Jong, H.
Hilkmann, T. K. Sixma, H. Ovaa, Angew. Chem. Int. Ed. 2010, 49, 10149–
10153; Angew. Chem. 2010, 122, 10347–10351.
[4] a) A. C. Faesen, M. P. A. Luna-Vargas, P. P. Geurink, M. Clerici, R. Merkx,
W. J. van Dijk, D. S. Hameed, F. El Oualid, H. Ovaa, T. K. Sixma, Chem.
Biol. 2011, 18, 1550–1561; b) T. E. T. Mevissen, M. K. Hospenthal, P. P.
Geurink, P. R. Elliott, M. Akutsu, N. Arnaudo, R. Ekkebus, Y. Kulathu, T.
Wauer, F. El Oualid, S. M. V. Freund, H. Ovaa, D. Komander, Cell 2013,
154, 169–184; c) M. S. Ritorto, R. Ewan, A. B. Perez-Oliva, A. Knebel, S. J.
Buhrlage, M. Wightman, S. M. Kelly, N. T. Wood, S. Virdee, N. S. Gray,
N. A. Morrice, D. R. Alessi, M. Trost, Nat. Commun. 2014, 5, 4763.
[5] a) M. P. C. Mulder, F. El Oualid, J. ter Beek, H. Ovaa, ChemBioChem 2014,
15, 946–949; b) J. F. McGouran, S. R. Gaertner, M. Altun, H. B. Kramer,
B. M. Kessler, Chem. Biol. 2013, 20, 1447–1455; c) G. Li, Q. Liang, P.
Gong, A. H. Tencer, Z. Zhuang, Chem. Commun. 2014, 50, 216–218;
d) N. Haj-Yahya, H. P. Hemantha, R. Meledin, S. Bondalapati, M. Seenaiah,
A. Brik, Org. Lett. 2014, 16, 540–543.
[6] a) S. Ohayon, L. Spasser, A. Aharoni, A. Brik, J. Am. Chem. Soc. 2012, 134,
3281–3289; b) R. A. Horton, E. A. Strachan, K. W. Vogel, S. M. Riddle,
Anal. Biochem. 2007, 360, 138–143; c) Y. Ye, G. Blaser, M. H. Horrocks,
M. J. Ruedas-Rama, S. Ibrahim, A. A. Zhukov, A. Orte, D. Klenerman, S. E.
Jackson, D. Komander, Nature 2012, 492, 266–270.
[7] M. K. Hospenthal, T. E. T. Mevissen, D. Komander, Nat. Protoc. 2015, 10,
349–361.
[8] J. B. Grimm, L. D. Lavis, Org. Lett. 2011, 13, 6354–6357.
[9] a) K. S. A. Kumar, M. Haj-Yahya, D. Olschewski, H. A. Lashuel, A. Brik,
Angew. Chem. Int. Ed. 2009, 48, 8090–8094; Angew. Chem. 2009, 121,
8234–8238; b) K. K. Pasunooti, R. Yang, S. Vedachalam, B. K. Gorityala,
C.-F. Liu, X.-W. Liu, Bioorg. Med. Chem. Lett. 2009, 19, 6268–6271; c) R.
Merkx, G. de Bruin, A. Kruithof, T. van den Bergh, E. Snip, M. Lutz, F.
El Oualid, H. Ovaa, Chem. Sci. 2013, 4, 4494–4498.
[10] Q. Wan, S. J. Danishefsky, Angew. Chem. Int. Ed. 2007, 46, 9248–9252;
Angew. Chem. 2007, 119, 9408–9412.
[11] M. A. Michel, P. R. Elliott, K. N. Swatek, M. Simicek, J. N. Pruneda, J. L.
Wagstaff, S. M. V. Freund, D. Komander, Mol. Cell 2015, 58, 95–109.
USP21
[a] Values in italics were obtained by extrapolation beyond the highest
substrate concentration. [b] Activated versions of AMSH and OTUB1 were
created by fusing them to their activators (STAM and UBE2D2, respective-
ly).[11]
Lys11 and Lys63 diUbs; this can largely be attributed to differ-
ences in kcat rather than Km. Another interesting result was for
AMSH. In agreement with earlier findings, this DUB had abso-
lute specificity for Lys63 diUb,[4c] although the overall efficiency
was rather low. Remarkably, the recently reported fusion of
AMSH with its natural activator STAM2[11] resulted in a more
than 1000-fold increase in catalytic efficiency, which can be at-
tributed to increases in both affinity and catalytic turnover.
Taken together, these data show that our quantitative assess-
ment of the DUB linkage specificity is in accordance with re-
ported data and that new insights can be obtained from the
kinetic parameters.
In summary, the set of all seven isopeptide-linked diUb FRET
pairs allows absolute quantification of DUB linkage specificity.
Our synthetic strategy, which includes a convenient N,N’-Boc-
protected Rho building block, allows efficient preparation of
these reagents in large quantities. The assay requires low
amounts of material, can easily be automated, and can be
used in high-throughput small-molecule screening or for the
assessment of (di)Ub binding domains.[6c] Overall, we believe
that our diUb FRET probes will be of great value in ongoing
efforts to crack the ubiquitin code and that the FRET pair pre-
sented here will facilitate FRET-pair synthesis in general.
Manuscript received: January 12, 2016
Final article published: && &&, 0000
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P. P. Geurink,* B. D. M. van Tol,
D. van Dalen, P. J. G. Brundel,
T. E. T. Mevissen, J. N. Pruneda, P. R. Elliott,
G. B. A. van Tilburg, D. Komander,
H. Ovaa*
The magnificent seven: All seven iso-
peptide-linked diubiquitin conjugates
equipped with a Rhodamine-TAMRA
FRET pair were prepared. The synthesis
includes the use of a highly convenient
N,N’-Boc-protected Rhodamine building
block. These probes enable the absolute
quantification of the ubiquitin linkage
specificity of deubiquitinating enzymes
by means of Michaelis–Menten kinetics.
&& – &&
Development of Diubiquitin-Based
FRET Probes To Quantify Ubiquitin
Linkage Specificity of
Deubiquitinating Enzymes
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ꢂ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!