Synthesis of Branched Triubiquitin Active-Site Directed Probes

Article abstract of DOI:10.1021/acs.orglett.9b02406

Active-site directed probes are powerful tools for studying the ubiquitin conjugation and deconjugation machinery. Branched ubiquitin chains have emerged as important proteasome-targeting signals for aggregation-prone proteins and cell cycle regulators. By implementing a new synthetic strategy for the electrophilic warhead, we herein report on the generation and reactivity of a series of branched triubiquitin active-site directed probes. These new tools can be used to dissect the molecular basis of branched chain assembly and disassembly.

Full text of DOI:10.1021/acs.orglett.9b02406

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Synthesis of Branched Triubiquitin Active-Site Directed Probes
Jiaan Liu,† Yanfeng Li,† Kirandeep K. Deol,† and Eric R. Strieter*,†,‡
†Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, United States
‡Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003, United States
S
* Supporting Information
ABSTRACT: Active-site directed probes are powerful tools for
studying the ubiquitin conjugation and deconjugation machinery.
Branched ubiquitin chains have emerged as important proteasome-
targeting signals for aggregation-prone proteins and cell cycle
regulators. By implementing a new synthetic strategy for the
electrophilic warhead, we herein report on the generation and
reactivity of a series of branched triubiquitin active-site directed
probes. These new tools can be used to dissect the molecular basis
of branched chain assembly and disassembly.
the molecular details underlying the linkage selectivity of
DUBs.34,39,40
biquitin (Ub) chains provide highly nuanced mecha-
1,2 Once a protein is modified
Unisms of cellular regulation.
Despite the impressive advances in Ub ABPs, there is still a
need for additional tools. Recent studies have shown that
branched Ub chains provide a unique signal for targeting
proteins to the proteasome for degradation. During mitosis, for
instance, the cullin-RING E3 ligase APC/C (anaphase
promoting complex/cyclosome) collaborates with the E2
conjugating enzymes UBE2C and UBE2S to assemble
Lys11/Lys48-linked branched chains on several cell cycle
regulators.41 In response to proteotoxic stress, nascent and
aggregation-prone proteins are also modified with Lys11/
Lys48-linked branched Ub chains to promote their clearance.42
In this case, branched chains are synthesized, in part, by the
HECT E3 ligase UBR5. UBR5 can also introduce Lys48
branch points on pre-existing Lys63 chains to redirect a
substrate to the proteasome for degradation.43 How E3s
assemble branched chains is unclear.44,45 Access to branched
chain ABPs would help address this problem. Branched chain
ABPs can also be used to understand how this architecture
affects the activity of DUBs.46,47
Our approach focused on a two-step sequence starting from
two different Ub variants. The idea was to site-specifically
couple a set of di-Ubs to a Ub C-terminally modified with an
α-bromo-vinylketone (BVK) moiety to afford tri-Ub deriva-
tives containing a native isopeptide bond on one side of the
branch point and a Michael acceptor on the other (Scheme 1).
Linkage-specific enzymes provide a powerful set of tools to
assemble dimers with amino acid substitutions in specific
subunits.48 With Cys introduced at specific sites in lieu of Lys,
with Ub, Ub itself can serve as the substrate to afford
polymeric Ub chains. During this elongation process, one of
seven amino groups of Ub (Lys6, Lys11, Lys27, Lys29, Lys33,
Lys48, and Lys63) or its amino terminus (Met1) can be
conjugated to the C-terminus of another ubiquitin mole-
cule.3−5 Moreover, a single Ub moiety can be modified at more
than one site, creating a branch point. Thus, in addition to
chain length, there is tremendous variability in chain
architecture. Effector proteins within the Ub signaling network,
i.e., deubiquitinases (DUBs)6−8 and “reader” proteins contain-
ing Ub-binding domains,9 are capable of exploiting subtle
differences in the orientation and spacing of Ub subunits
present in different chains to control the net flux through
biochemical pathways. With the immense diversity of chains
and the number of enzymes involved in Ub signaling, however,
there are still many questions that remain unresolved regarding
the assembly, recognition, and removal of specific chain types.
Active-site directed probes (also referred to as activity-based
probes; ABPs) have been particularly effective as tools for
dissecting the Ub conjugation and deconjugation
machiner.10,11 The basic design of an ABP includes a
recognition element that directs the probe to its target and
an electrophilic warhead that reacts with an enzyme active site
to afford a covalent adduct.12,13 The classic Ub-based ABPs use
monoUb as the recognition element.14−21 These ABPs have
been instrumental in the discovery of DUBs22−27 and
elucidating how substrate engagement induces conformational
changes in DUBs that are conducive to catalysis.28−34 More
recently, di- and tri-Ub-based probes have been added to
toolbox.35−39 The utility of these probes has been illustrated by
biochemical and structural studies that have helped illuminate
Received: July 11, 2019
© XXXX American Chemical Society
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Scheme 1. Strategy for Generating Branched Tri-Ub ABPs
Figure 2. Representative SDS−PAGE and MS analysis of tri-Ub
ABPs. (A) Coomassie-stained SDS−PAGE gel showing the formation
of probe 11a using Ub-BVK and di-Ub 10a. ESI-MS analysis of
purified 11a (observed deconvoluted m/z ratio for [M + H]+ is
25845.8; calculated m/z is 25846.6). The di-Ub species present along
with Ub-BVK 9 is formed by self-condensation of Ub-MESNa 7 while
concentrating the sample. (B) SDS−PAGE gel showing the formation
of 11b starting from Ub-BVK and di-Ub 10b. ESI-MS analysis of
purified 11b (observed deconvoluted m/z ratio for [M + H]+ is
25817.7; calculated m/z is 25817.1).
To synthesize the Ub-BVK derivative we turned to previous
work37 in which a latent form of BVK was coupled to Ub-
MESNA prior to deprotection under acidic conditions (4 mM
p-TsOH, 54% (v/v) TFA). In our hands, however, we
observed extensive hydrolysis during the deprotection of the
ethylene glycol ketal, resulting in the formation of Ub1−75
,
thereby precluding the generation of sufficient quantities of the
tri-Ub probe. To overcome these challenges, we sought to
devise a slightly different synthetic route to Ub-BVK.
Our synthesis commenced with a cross-metathesis reaction
between N-allylphthalimide (2) and methyl vinyl ketone using
the Hoveyda−Grubbs second-generation catalyst and Lewis
acid Cy2BCl (Figure 1).49 This reaction reproducibly furnished
3 in approximately 40% yield. After α-bromination, we
subjected ketone 4 to ketalization conditions with 1-(2-
nitrophenyl)-1,2-ethanediol (5). We reasoned the photolability
of the o-nitrophenyl group would enable deprotection under
milder conditions relative to those used for the ethylene glycol-
based ketal. With the photolabile ketal in place, the amino
group was unveiled using methyl hydrazine to give 6 and
coupled to Ub1−75-MESNa (7) to obtain 8. We found that the
aminolysis reaction occurs readily with only 30-fold excess of
amine 6 relative to Ub1−75-MESNa, minimizing the loss of
material.
Irradiation of the resulting photolabile Ub-BVK derivative
(8) was examined at different pHs. Reactions were monitored
by MALDI-TOF mass spectrometry (MS) (Figure S3). Under
all conditions, we detected significant amounts of Ub-BVK (9).
Although hydrolysis still occurs, the amount of Ub1−75 is still
markedly less than what we observed with ethylene glycol as a
protecting group. Moreover, compared to the deprotection of
the ethylene glycol ketal, which affords at least four products
Figure 1. Synthesis of Ub-BVK and tri-Ub ABPs. Di-Ub variants 10a
and 10b contain a K48C substitution in the proximal Ub subunit and
a native isopeptide bond at position-6 or -63, respectively. 10c
contains a K6C substitution in the proximal and a native isopeptide
bond at position-63 (Lys63-linked). 10d is a Lys48-linked dimer with
a K63C substitution in the proximal subunit. 11b* is a branched tri-
Ub probe where the distal Ub attached to position-48 is N-terminally
biotinylated. 11d* is a branched tri-Ub probe where the distal Ub
attached to position-63 is N-terminally biotinylated.
we can couple dimers to Ub-BVK using standard Cys thiolate
substitution chemistry.
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Figure 4. Labeling of HECT E3 ligases with branched tri-Ub probes.
Coomassie-stained SDS−PAGE gel showing the formation of a
covalent complex between the branched ABPs 11a, 11b, and 11d*
and active, but not inactive, UBE3C (A) and AREL1 (B).
capable of rapidly cleaving the Lys48 linkage within branched
trimers. In accord, we found that OTUB1*a more active
version with the E2 UBE2D2 genetically fused to the N-
terminus51reacts with the branched probes 11a and 11b
(Figure 3A). The catalytic Cys residue is critical for covalent
complex formation, as higher molecular weight bands are not
observed with the inactive C91A variant (Figure 3A).
OTUB1* also displays selectivity for the electrophilic warhead
at position-48. When other non-Lys48 residues are replaced
with the warhead (e.g., Lys6 in 11c or Lys63 in 11d*),
complex formation is not observed (Figures 3A and S7). In
addition, the time course assay shows the kinetics of covalent
complex formation are on par with the overall cleavage rate
(Figure 3B).
We also examined the reactivity of OTUD1 toward the
branched probes. In vitro, it has been shown that OTUD1
displays moderate selectivity toward Lys63-linked chains.52 We
found that OTUD1 cleaves the native Lys63 linkage in probes
11b and 11b*, leaving behind a di-Ub ABP with a warhead at
position-48 (Figure 3C). By contrast, when the warhead is at
position-63 (11d*) a covalent adduct is readily observed.
Together, these results demonstrate that certain DUBs can
selectively react with branched ABPs, similar to what has been
observed with di-Ub probes.
Figure 3. Labeling DUBs with branched tri-Ub probes. (A)
Coomassie-stained SDS−PAGE gel showing the reaction between
tri-Ub probes 11a−c and either active or inactive (C91A) OTUB1*.
Probes were incubated with OTUB1* for 5 min at room temperature.
(B) SYPRO Ruby-stained SDS−PAGE gel showing the cleavage of
native K48/K63 tri-Ub (12) by OTUB1* over time at 37 °C (left side
of the gel). Right side shows the labeling of OTUB1* with the 11b
probe at 37 °C. (C) SYPRO Ruby-stained SDS−PAGE gel showing
the labeling of OTUD1 with different tri-Ub probes. Reactions were
performed at 37 °C.
with Ub-BVK as a minor species, removal of the photolabile
ketal resulted in Ub-BVK as the major product.
With Ub-BVK in hand, we sought to assemble branched tri-
Ub probes. We started with di-Ub variants containing K6C,
K48C, or K63C substitutions in the proximal subunit (10a−
d). Upon mixing Ub-BVK with the Cys-containing dimers, we
observed robust trimer formation (Figure 2). MS analysis
confirmed the generation of each probe (Figures 2 and S6).
Next, we investigated the reactivity of the branched tri-Ub
probes toward cysteine-dependent DUBs. OTUB1 is Lys48-
specific DUB50 in the ovarian tumor (OTU) subfamily and is
Lastly, we investigated the reactivity of branched probes
toward E3 ligases bearing active-site Cys residues. Two HECT
E3 ligases were chosen: UBE3C and AREL1. The purpose here
was not to assess whether the E3s display selectivity toward
C
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certain probes since these enzymes synthesize heterotypic Ub
chains containing a mixture of linkages.53−55 Instead, we
wanted to measure the efficiency of labeling for future
structural studies aiming to identify cryptic Ub binding sites
that are important for chain assembly. With both UBE3C and
ARE1L, we detected the formation of a covalent adduct
between probes 11a, 11b, and 11d*, and the catalytic domains
of UBE3C and AREL1 only when the catalytic Cys is present
(Figures 4A,B and S8). In general, the labeling efficiency is
higher with UBE3C compared to AREL1 (Figure S9).
Interestingly, there does appear to be some selectivity. Probe
11c containing the warhead at position-6 and an isopeptide
bond at position-63 does not react with either UBE3C or
ARE1L even though it does label nonselective DUBs, e.g.,
USP7, to some extent (Figure S10). This observation may
reflect the linkage preferences of UBE3C and ARE1L
considering Lys6 and Lys63 are not produced by these
enzymes to a significant degree.53−55 These results demon-
strate that, in addition to DUBs, the branched probes can be
used to generate complexes with HECT ligases.
In conclusion, we report on the synthesis and reactivity of a
new set of Ub ABPs based on branched trimers. To generate
these probes, we devised an alternative synthetic approach to
the Michael acceptor linking one arm of the branched trimer to
the proximal subunit. The advances made include a facile
cross-metathesis reaction and photodeprotection of a ketal. We
found that like other Ub ABPs the branched probes readily
react with E3 ligases and DUBs containing active-site Cys
residues. These probes will be valuable tools for uncovering
cryptic Ub binding sites on E3s and DUBs that lead to linkage
selectivity in both chain assembly and disassembly.
REFERENCES
■
(1) Hershko, A.; Ciechanover, A. Annu. Rev. Biochem. 1998, 67,
425−479.
(2) Oh, E.; Akopian, D.; Rape, M. Annu. Rev. Cell Dev. Biol. 2018,
34, 137−162.
(3) Komander, D.; Rape, M. Annu. Rev. Biochem. 2012, 81 (1), 203−
229.
(4) Swatek, K. N.; Komander, D. Cell Res. 2016, 26 (4), 399−422.
(5) Yau, R.; Rape, M. Nat. Cell Biol. 2016, 18 (6), 579−586.
(6) Mevissen, T. E. T.; Komander, D. Annu. Rev. Biochem. 2017, 86
(1), 159−192.
(7) Clague, M. J.; Urbe, S.; Komander, D. Nat. Rev. Mol. Cell Biol.
2019, 20, 338−352.
́
(8) Clague, M. J.; Barsukov, I.; Coulson, J. M.; Liu, H.; Rigden, D. J.;
Urbe, S. Physiol. Rev. 2013, 93 (3), 1289−1315.
(9) Husnjak, K.; Dikic, I. Annu. Rev. Biochem. 2012, 81, 291−322.
(10) Hewings, D. S.; Flygare, J. A.; Bogyo, M.; Wertz, I. E. FEBS J.
2017, 284 (10), 1555−1576.
(11) Witting, K. F.; Mulder, M. P. C.; Ovaa, H. J. Mol. Biol. 2017,
429 (22), 3388−3394.
(12) Cravatt, B. F.; Wright, A. T.; Kozarich, J. W. Annu. Rev.
Biochem. 2008, 77 (1), 383−414.
(13) Sanman, L. E.; Bogyo, M. Annu. Rev. Biochem. 2014, 83 (1),
249−273.
(14) Hershko, A.; Rose, I. A. Proc. Natl. Acad. Sci. U. S. A. 1987, 84
(7), 1829−1833.
(15) Pickart, C. M.; Rose, I. A. J. Biol. Chem. 1986, 261 (22),
10210−10217.
(16) Borodovsky, A.; Kessler, B. M.; Casagrande, R.; Overkleeft, H.
S.; Wilkinson, K. D.; Ploegh, H. L. EMBO J. 2001, 20, 5187.
(17) Borodovsky, A.; Ovaa, H.; Kolli, N.; Gan-Erdene, T.;
Wilkinson, K. D.; Ploegh, H. L.; Kessler, B. M. Chem. Biol. 2002, 9
(10), 1149−1159.
(18) Mulder, M. P. C.; Witting, K.; Berlin, I.; Pruneda, J. N.; Wu, K.
P.; Chang, J. G.; Merkx, R.; Bialas, J.; Groettrup, M.; Vertegaal, A. C.
O.; et al. Nat. Chem. Biol. 2016, 12 (7), 523−530.
(19) Whedon, S. D.; Markandeya, N.; Rana, A. S. J. B.; Senger, N.
A.; Weller, C. E.; Turecek, F.; Strieter, E. R.; Chatterjee, C. J. Am.
Chem. Soc. 2016, 138 (42), 13774.
(20) Ekkebus, R.; Van Kasteren, S. I.; Kulathu, Y.; Scholten, A.;
Berlin, I.; Geurink, P. P.; De Jong, A.; Goerdayal, S.; Neefjes, J.; Heck,
A. J. R.; et al. J. Am. Chem. Soc. 2013, 135 (8), 2867−2870.
(21) Sommer, S.; Weikart, N. D.; Linne, U.; Mootz, H. D. Bioorg.
Med. Chem. 2013, 21 (9), 2511−2517.
(22) Love, K. R.; Catic, A.; Schlieker, C.; Ploegh, H. L. Nature
Chemical Biology; Nature Publishing Group, 2007; pp 697−705.
(23) Keusekotten, K.; Elliott, P. R.; Glockner, L.; Fiil, B. K.;
Damgaard, R. B.; Kulathu, Y.; Wauer, T.; Hospenthal, M. K.; Gyrd-
Hansen, M.; Krappmann, D.; et al. Cell 2013, 153, 1312−1326.
(24) Kwasna, D.; Rehman, S. A. A.; Natarajan, J.; Matthews, S.;
Madden, R.; De Cesare, V.; Weidlich, S.; Virdee, S.; Ahel, I.; Gibbs
Seymour, I.; et al. Mol. Cell 2018, 70, 150−164.
(25) Haahr, P.; Borgermann, N.; Guo, X.; Typas, D.; Achuthankutty,
D.; Hoffman, S.; Shearer, R.; Sixma, T.; Mailand, N. Mol. Cell 2018,
70, 165−174.
(26) Hewings, D. S.; Heideker, J.; Ma, T. P.; AhYoung, A. P.; El
Oualid, F.; Amore, A.; Costakes, G. T.; Kirchhofer, D.; Brasher, B.;
Pillow, T.; et al. Nat. Commun. 2018, 9 (1), 1162.
(27) Hermanns, T.; Pichlo, C.; Woiwode, I.; Klopffleisch, K.;
Witting, K. F.; Ovaa, H.; Baumann, U.; Hofmann, K. Nat. Commun.
2018, 9 (1), 799.
(28) Johnston, S. C.; Riddle, S. M.; Cohen, R. E.; Hill, C. P. EMBO
J. 1999, 18 (14), 3877−3887.
(29) Hu, M.; Li, P.; Li, M.; Li, W.; Yao, T.; Wu, J.-W.; Gu, W.;
Cohen, R. E.; Shi, Y. Cell 2002, 111, 1041.
(30) Misaghi, S.; Galardy, P. J.; Meester, W. J. N.; Ovaa, H.; Ploegh,
H. L.; Gaudet, R. J. Biol. Chem. 2005, 280 (2), 1512−1520.
(31) Boudreaux, D. A.; Maiti, T. K.; Davies, C. W.; Das, C. Proc.
Natl. Acad. Sci. U. S. A. 2010, 107 (20), 9117−9122.
ASSOCIATED CONTENT
■
S
* Supporting Information
̌
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b02406.
Synthetic protocols, protein expression information,
enzyme assays, and characterization of crucial inter-
mediates (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: estrieter@chem.umass.edu.
ORCID
Eric R. Strieter: 0000-0003-3447-3669
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a research grant from the National
Institutes of Health (RO1GM110543) and an NSF graduate
research fellowship to K.K.D. (GRFP1451512). Mass spectral
data were acquired at the University of Massachusetts Mass
Spectrometry core facility. The Orbitrap Fusion was funded by
NIH SIG S10OD010645. We thank Dr. Steve Eyles (UMass
Amherst) for assistance with mass spectrometry.
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(32) Sahtoe, D. D.; VanDijk, W. J.; ElOualid, F.; Ekkebus, R.; Ovaa,
H.; Sixma, T. K. Mol. Cell 2015, 57 (5), 887−900.
(33) VanderLinden, R. T.; Hemmis, C. W.; Schmitt, B.; Ndoja, A.;
Whitby, F. G.; Robinson, H.; Cohen, R. E.; Yao, T.; Hill, C. P. Mol.
Cell 2015, 57 (5), 901−911.
(34) Mevissen, T. E. T.; Kulathu, Y.; Mulder, M. P. C.; Geurink, P.
P.; Maslen, S. L.; Gersch, M.; Elliott, P. R.; Burke, J. E.; Van Tol, B. D.
M.; Akutsu, M.; et al. Nature 2016, 538, 402−405.
(35) McGouran, J. F.; Gaertner, S. R.; Altun, M.; Kramer, H. B.;
Kessler, B. M. Chem. Biol. 2013, 20 (12), 1447−1455.
(36) Mulder, M. P. C.; El Oualid, F.; Ter Beek, J.; Ovaa, H.
ChemBioChem 2014, 15, 946−949.
(37) Li, G.; Liang, Q.; Gong, P.; Tencer, A. H.; Zhuang, Z. Chem.
Commun. 2014, 50, 216.
(38) Haj-Yahya, N.; Hemantha, H. P.; Meledin, R.; Bondalapati, S.;
Seenaiah, M.; Brik. Org. Lett. 2014, 16 (2), 540−543.
(39) Paudel, P.; Zhang, Q.; Leung, C.; Greenberg, H. C.; Guo, Y.;
Chern, Y.-H.; Dong, A.; Li, Y.; Vedadi, M.; Zhuang, Z.; et al. Proc.
Natl. Acad. Sci. U. S. A. 2019, 116, 7288.
(40) Flierman, D.; Van Der Heden Van Noort, G. J.; Ekkebus, R.;
Geurink, P. P.; Mevissen, T. E. T.; Hospenthal, M. K.; Komander, D.;
Ovaa, H. Cell Chem. Biol. 2016, 23 (4), 472−482.
(41) Meyer, H. J.; Rape, M. Cell 2014, 157 (4), 910−921.
(42) Yau, R. G.; Doerner, K.; Castellanos, E. R.; Haakonsen, D. L.;
Werner, A.; Wang, N.; Yang, X. W.; Martinez-Martin, N.; Matsumoto,
M. L.; Dixit, V. M.; et al. Cell 2017, 171 (4), 918−933.
(43) Ohtake, F.; Tsuchiya, H.; Saeki, Y.; Tanaka, K. Proc. Natl. Acad.
Sci. U. S. A. 2018, 115, E1401−E1408.
(44) Liu, C.; Liu, W.; Ye, Y.; Li, W. Nat. Commun. 2017, 8, 1−15.
(45) Deol, K. K.; Lorenz, S.; Strieter, E. R. Front. Physiol. 2019,
DOI: 10.3389/fphys.2019.00835.
(46) Wertz, I. E.; Newton, K.; Seshasayee, D.; Kusam, S.; Lam, C.;
Zhang, J.; Popovych, N.; Helgason, E.; Schoeffler, A.; Jeet, S.; et al.
Nature 2015, 528 (7582), 370−375.
(47) Ohtake, F.; Saeki, Y.; Ishido, S.; Kanno, J.; Tanaka, K. Mol. Cell
2016, 64 (2), 251−266.
(48) Michel, M. A.; Komander, D.; Elliott, P. R. Enzymatic Assembly
of Ubiquitin Chains. In The Ubiquitin Proteasome System. Methods in
Molecular Biology; Humana Press, Inc.: New York, 2018; pp 73−84.
(49) Vedrenne, E.; Dupont, H.; Oualef, S.; Elkaïm, L.; Grimaud, L.
Synlett 2005, 4, 670−672.
̈
(50) Edelmann, M. J.; Iphofer, A.; Akutsu, M.; Altun, M.; di Gleria,
K.; Kramer, H. B.; Fiebiger, E.; Dhe-Paganon, S.; Kessler, B. M.
Biochem. J. 2009, 418 (2), 379−390.
(51) Wiener, R.; Dibello, A. T.; Lombardi, P. M.; Guzzo, C. M.;
Zhang, X.; Matunis, M. J.; Wolberger, C. Nat. Struct. Mol. Biol. 2013,
20 (9), 1033−1039.
(52) Ritorto, M. S.; Ewan, R.; Perez-Oliva, A. B.; Knebel, A.;
Buhrlage, S. J.; Wightman, M.; Kelly, S. M.; Wood, N. T.; Virdee, S.;
Gray, N. S.; et al. Nat. Commun. 2014, 5, 1−11.
(53) Michel, M. A.; Elliott, P. R.; Swatek, K. N.; Simicek, M.;
Pruneda, J. N.; Wagstaff, J. L.; Freund, S. M. V.; Komander, D. Mol.
Cell 2015, 58 (1), 95−109.
(54) Kristariyanto, Y. A.; Abdul Rehman, S. A.; Campbell, D. G.;
Morrice, N. A.; Johnson, C.; Toth, R.; Kulathu, Y. Mol. Cell 2015, 58
(1), 83−94.
(55) Kristariyanto, Y. A.; Choi, S.-Y.; Rehman, S. A. A.; Ritorto, M.
S.; Campbell, D. G.; Morrice, N. A.; Toth, R.; Kulathu, Y. Biochem. J.
2015, 467 (2), 345−352.
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