Inhibiting the protein ubiquitination cascade by ubiquitin-mimicking short peptides

Article abstract of DOI:10.1021/ol3027736

Short heptapeptides were identified to function as ubiquitin (UB) mimics that are activated by E1 and form thioester conjugates with E1, E2, and HECT type E3 enzymes. The activities (k_cat/K_1/2) of E1 with the UB-mimicking peptides ar

Full text of DOI:10.1021/ol3027736

ORGANIC
LETTERS
2
012
Vol. 14, No. 22
760–5763
Inhibiting the Protein Ubiquitination
Cascade by Ubiquitin-Mimicking
Short Peptides
5
†
,§
†,§
†
†
Bo Zhao, Chan Hee J. Choi, Karan Bhuripanyo, Eric B. Villhauer, Keya Zhang,
†
‡
Hermann Schindelin, and Jun Yin*
,†
Department of Chemistry, University of Chicago, Chicago, Illinois 60637, United States,
and Rudolf Virchow Center for Experimental Biomedicine and Institute for Structural
Biology, University of W u€ rzburg, Josef-Schneider-Str. 2, 97080 W u€ rzburg, Germany
junyin@uchicago.edu
Received October 8, 2012
ABSTRACT
Short heptapeptides were identified to function as ubiquitin (UB) mimics that are activated by E1 and form thioester conjugates with E1, E2, and
HECT type E3 enzymes. The activities (kcat/K1/2) of E1 with the UB-mimicking peptides are 130À1,400-fold higher than the equally long peptide with
the native C-terminal sequence of UB. By forming covalent conjugates with E1, E2, and E3 enzymes, the UB-mimicking peptides can block the
transfer of native UB through the cascade.
3
UB is covalently attached to cellular proteins through
the tandem action of E1, E2, and E3 enzymes to regulate
1
many key biological processes. E1 catalyzes the conden-
enzymes to form UB∼E2 conjugates. In the last step, E3s
bridge the transfer of UB from E2 to the modification
4
targets. Similarly, UB-like proteins (UBLs) such as
sation reaction between the C-terminal carboxylate of UB
and ATP to form a UB-AMP intermediate that subse-
quently reacts with a catalytic Cys residue of E1 to afford
Nedd8 and SUMO are transferred by their own sets of
5
E1ÀE2ÀE3 cascades to cellular targets. Together UB and
UBLs carry a broad band of signals for the regulation of
6
a UB∼E1 conjugate (“∼”designates the thioester linkage)
cell biology.
The enzymatic cascades for protein modification by UB
or UBLs have been the intense focus of drug discovery
2
Supporting Information (SI), Figure S1). UB bound to
(
E1 is then transferred to the catalytic Cys residue of the E2
7
efforts. The identification of the compound MLN4924 as
a potent inhibitor of the Nedd8 E1 demonstrates the drugg-
†
University of Chicago.
University of W €u rzburg.
These authors contributed equally.
8
ability of the E1 enzymes. MLN4924 is currently in clinical
‡
§
9
trials for the treatment of myeloma and lymphoma. Several
(
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inhibitors of the E1 enzyme specific for UB have been
developed that inactivate the catalytic Cys residue of E1 or
4
(
1
0
disrupt UB binding to E1. These inhibitors can attenuate
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(
3
(
1
0.1021/ol3027736 r 2012 American Chemical Society
Published on Web 11/07/2012

7
1
76
covering LRLRGG . Phage selection of the library
with Ube1, the human E1, identified UB variants with
quite different C-terminal sequences from the wild type
Table 1. Kinetic Parameters of ATP-PP Exchange Catalyzed by
i
Ube1 with the UB-Mimicking Peptides
(
wt) UB (SI, Figure S2). Interestingly these UB variants
12
K
1/2
k
cat
À1
k
cat/K1/2
share similar reactivities as the wt UB with Ube1. As
shown by the sequence alignments of the phage selected
UB clones, except for Arg72 and Gly75 that have a strong
preference for wt residues, positions 71 and 73 in the UB
variants are predominantly occupied with bulky aromatic
side chains such as Phe, Tyr, and Trp, instead of Leu
(Figure S2). Position 74 of the UB variants may also have
aromatic or positively charged His side chains to replace the
wt Arg residue. It has previously been reported that short
peptides corresponding to the C-terminal sequences of the
wt UB can be activated by the E1 enzymes and transferred
À1
À1
(μM)
(min
)
(μM min )
wtUB (full length)
1.4 ( 0.5
88 ( 17
60
C-terminal peptides of wtUB and variants
70
76
a
a
À5
P1 ( VLRLRGG
)
76
À
À
7.7 Â 10
1.0 Â 10
2.3 Â 10
70
À2
À2
À1
P2 ( VWRFHGG
)
342 ( 17
426 ( 11
141 ( 5
3.5 ( 0.6
9.7 ( 1.4
15 ( 2.7
7
0
76
P3 ( VQRYWGG
)
70
76
P4 ( VYRFYGG
)
1.1 Â 10
a
K
1/2 and kcat could not be determined for P1 due to its low activity.
of the UB transfer cascades are also valid targets for inhibitor
design. Recent screening efforts have identified compounds
1
3
through the E1ÀE2ÀE3 cascade for protein modification.
1
1
that bind to E2 or E3 and block UB transfer. Here we
report the identification of short peptides that mimic UB and
form covalent conjugates with the E1ÀE2ÀE3 cascade
We were thus interested in assaying if the C-terminal
peptides of the UB variants from phage selection are more
reactive with the E1 enzyme than the C-terminal peptide
of wt UB.
(Table 1). Once these peptides are charged to the cascade
enzymes, they can effectively block UB transfer through the
cascade. The development of these UB-mimicking peptides
provides a new way to inhibit protein modification by UB.
We identified the UB-mimicking peptides in a study
profiling the specificity of the E1 enzymes with the C-terminal
We synthesized peptides corresponding to the C-terminal
sequences of wtUB (P1, VLRLRGG), and UB var-
iants e27 (P2, VWRFHGG), e40 (P3, VQRYWGG) and
e25 (P4, VYRFYGG), and measured the ATP-PP ex-
i
change kinetics of the peptides catalyzed by Ube1 (Figure S2
2a
and Table 1). The P1 peptide UB could not saturate
1
2
sequence of UB by phage display. We constructed a
UB library with randomized UB C-terminal residues
Ube1 at a concentration as high as 500 μM in the ATP-PPi
exchange reaction, so only k /K could be measured.
cat 1/2
(
8) (a) Brownell, J. E.; Sintchak, M. D.; Gavin, J. M.; Liao, H.;
In contrast, the P2ÀP4 peptides displayed a much higher
Bruzzese, F. J.; Bump, N. J.; Soucy, T. A.; Milhollen, M. A.; Yang, X.;
Burkhardt, A. L.; Ma, J.; Loke, H. K.; Lingaraj, T.; Wu, D.; Hamman,
K. B.; Spelman, J. J.; Cullis, C. A.; Langston, S. P.; Vyskocil, S.; Sells,
T. B.; Mallender, W. D.; Visiers, I.; Li, P.; Claiborne, C. F.; Rolfe, M.;
Bolen, J. B.; Dick, L. R. Mol. Cell 2010, 37, 102. (b) Soucy, T. A.; Smith,
P. G.; Milhollen, M. A.; Berger, A. J.; Gavin, J. M.; Adhikari, S.;
Brownell, J. E.; Burke, K. E.; Cardin, D. P.; Critchley, S.; Cullis, C. A.;
Doucette, A.; Garnsey, J. J.; Gaulin, J. L.; Gershman, R. E.; Lublinsky,
A. R.; McDonald, A.;Mizutani, H.; Narayanan, U.; Olhava, E. J.; Peluso,
S.; Rezaei, M.; Sintchak, M. D.; Talreja, T.; Thomas, M. P.; Traore, T.;
Vyskocil, S.;Weatherhead, G. S.;Yu, J.;Zhang, J.;Dick, L. R.;Claiborne,
C. F.; Rolfe, M.; Bolen, J. B.; Langston, S. P. Nature 2009, 458, 732.
affinity for Ube1 with K values of 141À426 μM. These
1/2
peptides were 130À1,400-fold more active than P1 in the
ATP/PP exchange reactions based on the k /K values
i
cat 1/2
(Table 1). Despite the higher activities of the phage selected
peptides with Ube1, P2ÀP4 were still 545À6,000 fold less
active than full length UB, largely due to the much lower
K1/2 of UB with Ube1 (1.4 μM) (Table 1). The high affinity
of UB with Ube1 can be attributed to the multiple binding
(
9) (a) Milhollen, M. A.; Traore, T.; Adams-Duffy, J.; Thomas,
interfaces between UB and E1 besides the UB C-terminus
(
M. P.; Berger, A. J.; Dang, L.; Dick, L. R.; Garnsey, J. J.; Koenig, E.;
Langston, S. P.; Manfredi, M.; Narayanan, U.; Rolfe, M.; Staudt, L. M.;
Soucy, T. A.; Yu, J.; Zhang, J.; Bolen, J. B.; Smith, P. G. Blood 2010, 116,
14
SI, Figure S3A). 7-mer peptides with the C-terminal
sequences of the UB variants e6, e19, e26, e46, and e47
from phage selection (Figure S2) were not reactive with
Ube1 based on ATP-PP exchange. These peptides have the
1
515. (b) Soucy, T. A.; Dick, L. R.; Smith, P. G.; Milhollen, M. A.;
Brownell, J. E. Genes Cancer 2010, 1, 708.
10) (a) Kitagaki, J.; Yang, Y.; Saavedra, J. E.; Colburn, N. H.; Keefer,
i
(
L. K.; Perantoni, A. O. Oncogene 2009, 28, 619. (b) Ungermannova, D.;
Parker, S. J.; Nasveschuk, C. G.; Wang, W.; Quade, B.; Zhang, G.; Kuchta,
R. D.; Phillips, A. J.; Liu, X. PLoS One 2012, 7, e29208. (c) Xu, G. W.; Ali,
M.; Wood, T. E.; Wong, D.; Maclean, N.; Wang, X.; Gronda, M.; Skrtic,
M.; Li, X.; Hurren, R.; Mao, X.; Venkatesan, M.; Beheshti Zavareh, R.;
Ketela, T.; Reed, J. C.; Rose, D.; Moffat, J.; Batey, R. A.; Dhe-Paganon, S.;
Schimmer, A. D. Blood 2010, 115, 2251. (d) Yang, Y.; Kitagaki, J.; Dai,
R. M.; Tsai, Y. C.; Lorick, K. L.; Ludwig, R. L.; Pierre, S. A.; Jensen, J. P.;
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K. H.; Weissman, A. M. Cancer Res. 2007, 67, 9472.
second to last Gly (Gly75 of wt UB) replaced with larger
residues that may disrupt peptide binding to the E1 enzyme.
Since the peptides P2ÀP4 can be activated by Ube1 as
in the case of wt UB, we refer to them as “UB-mimicking
peptides”.
We modeled structures of the UB-mimicking peptides
14
bound toUba1basedon theUB-Uba1 complex and used
the Protein Interfaces, Surfaces, and Assemblies (PISA)
(11) (a) Aghajan, M.; Jonai, N.; Flick, K.; Fu, F.; Luo, M.; Cai, X.;
15
Ouni, I.; Pierce, N.; Tang, X.; Lomenick, B.; Damoiseaux, R.; Hao, R.;
Del Moral, P. M.; Verma, R.; Li, Y.; Li, C.; Houk, K. N.; Jung, M. E.;
Zheng, N.; Huang, L.; Deshaies, R. J.; Kaiser, P.; Huang, J. Nat.
Biotechnol. 2010, 28, 738. (b) Ceccarelli, D. F.; Tang, X.; Pelletier, B.;
Orlicky, S.; Xie, W.; Plantevin, V.; Neculai, D.; Chou, Y. C.; Ogunjimi,
A.; Al-Hakim, A.; Varelas, X.; Koszela, J.; Wasney, G. A.; Vedadi, M.;
Dhe-Paganon, S.; Cox, S.; Xu, S.; Lopez-Girona, A.; Mercurio, F.;
Wrana, J.; Durocher, D.; Meloche, S.; Webb, D. R.; Tyers, M.; Sicheri,
F. Cell 2011, 145, 1075. (c) Orlicky, S.; Tang, X.; Neduva, V.; Elowe, N.;
Brown, E. D.; Sicheri, F.; Tyers, M. Nat. Biotechnol. 2010, 28, 733.
server to analyze peptide binding with Uba1 (Figure S3).
PISA calculations suggested that the wt P1 peptide and the
P2, P3, and P4 peptides have similar interface areas with E1,
but the binding energy of P1 is 1.5À2 kcal/mol less than that
(13) (a) Jonnalagadda, S.; Ecker, D. J.; Sternberg, E. J.; Butt, T. R.;
Crooke, S. T. J. Biol. Chem. 1988, 263, 5016. (b) Madden, M. M.; Song, W.;
Martell, P. G.; Ren, Y.; Feng, J.; Lin, Q. Biochemistry 2008, 47, 3636.
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(12) Zhao, B.; Bhuripanyo, K.; Schneider, J.; Zhang, K.; Schindelin,
H.; Boone, D.; Yin, J. ACS Chem. Biol. 2012, DOI: 10.1021/cb300339p.
(15) Krissinel, E.; Henrick, K. J. Mol. Biol. 2007, 372, 774.
Org. Lett., Vol. 14, No. 22, 2012
5761

Figure 1. Reactivities of UB-mimicking peptides with E1 and E2
enzymes. (A) Transfer of biotin-labeled peptides to the E1
enzyme Ube1. Biotin-labeled UB was used as a control in the
reactions. (B) Transfer of biotin-labeled peptides P2, P3, and P4
from E1 to the E2 enzymes Ubc1, UbcH5a, and UbcH7.
Figure 2. Transfer of UB-mimicking peptides P3 and P4 to the
E3 enzymes. (A) Biotin-labeled P3 and P4 can be transferred to
the HECT domain of E6AP. (B) Biotin-labeled P3 and P4 cannot
be transferred to CHIP, a U-box E3 for self-modification.
of the P2ÀP4 peptides with E1 (SI, Table S1). The differ-
ences in the binding energies of the peptides with E1
calculated by PISA match the differences in ΔG calculated
based on the K1/2 values of the peptides in the ATP-PPi
exchange reactions surprisingly well (Table S1).
We next tested if the peptides P2ÀP4 can form thioester
conjugates with E1 and found that biotin-labeled P2, P3
and P4 peptides are all transferred to E1 to form biotin-
peptide∼E1 thioesters (Figure 1A). By contrast biotin
labeled peptide P1 with the sequence of wtUB cannot
be transferred to E1 at any detectable level. This result
and P4 peptides can be transferred from UbcH7 to the
HECT domain of E6AP to form peptide∼HECT conjugates
1
6
(
Figure 2A). However, P3 and P4 cannot be transferred
17
from E2 to CHIP, a U-box E3, for self-modification by the
peptides (Figure 2B). It was previously found that the Cys
thiol is more reactive with UB∼E2 conjugates than the amino
1
8
group of Lys since the thiol is a better nucleophile. We thus
rationalized that the HECT E3 is more reactive than the
U-box E3 in the peptide transfer reaction because the catalytic
Cys residue of HECT is more reactive than the Lys residues of
the U-box E3 to attack the peptide∼E2 conjugates.
matches the ATP-PP assay and suggests that P2ÀP4
i
So far we have shown that peptides P2ÀP4 can be
activated by E1 to form thioester conjugates with the E1,
E2, and HECT E3s. We next analyzed whether peptide
loading onto the various components of the E1ÀE2ÀE3
cascade would inhibit UB transfer through the cascade. In
this way, the UB-mimicking peptides may serve as mechanism-
based inhibitors to block protein ubiquitination. To test
this idea, we incubated the E1 enzyme Ube1 with peptides
P1ÀP4 at concentrations of 10, 20, and 50 μM in the
presence of ATP to allow peptide activation by E1 and the
formation of peptide∼E1 thioester conjugates. After a pre-
incubation, UB with an N-terminal HA tag (HA-UB) was
are more reactive with E1 than the P1 peptide. We also
found that P2ÀP4 can be transferred to E2 enzymes such
as Ubc1, UbcH5a, and UbcH7 to form peptide∼E2
conjugates (Figure 1B).
Since peptides P3 and P4 showed strong activities with
the E2 enzymes, we assayed the transfer of these peptides
from E2 to E3. E3s are divided into two main classes based
on the mechanisms of UB transfer reactions they catalyze
1
b
(
Figure S1). The HECT type E3s have catalytic Cys re-
sidues to attack the thioester bond of the UB∼E2 conjugates
to form UB∼HECT conjugates before passing the UB to
the modified proteins. In contrast, the RING or U-box E3s
bind both the UB∼E2 conjugate and target proteins to
facilitate the direct transfer of UB to the modified proteins.
In the absence of substrate proteins, the RING and U-box
E3s can be autoubiquitinated at Lys residues. Western blots
of the peptide transfer reactions showed that biotinylated P3
(
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P. M.; Huibregtse, J. M.; Pavletich, N. P. Science 1999, 286, 1321.
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Nature 2011, 474, 105.
(
(
5
762
Org. Lett., Vol. 14, No. 22, 2012

enzymes followed by the addition of UB. Western blot
analysis of the reaction showed that P3 and P4 peptides
significantly inhibited the transfer of UB to CHIP E3 at a
concentration of 50 μM since the formation of polyubi-
quitinated CHIP was significantly decreased after prein-
cubation of the peptides (Figure 3B). We next tested if P3
and P4 can inhibit UB transfer to the HECT domain of
E6AP. After incubation of P3 and P4 with Ube1, UbcH7,
and the E6AP HECT, we added HA-UB to initiate UB
transfer to the HECT domain. Western blot analysis of the
transfer reactions showed that UB transfer to the HECT
domain was blocked at a concentration as low as 5 μM of
the P3 or P4 peptide (Figure 3C). The reason that the
peptides showed higher activities to inhibit UB transfer to
HECT E3 could be that the peptides could charge onto the
catalytic Cys residue of the HECT domain and directly
block UB loading on HECT (Figure 2A). However, the
peptides could not be transferred to the Lys residues of the
CHIP E3 to block UB conjugation (Figure 2B). Overall,
our results suggest that the UB-mimicking peptides can
block UB transfer through the E1ÀE2ÀE3 cascade and
inhibit protein ubiquitination.
In this study we developed the UB-mimicking peptides
P2ÀP4 that are two to three orders of magnitude more
reactive toward the E1 enzyme than the P1 peptide with the
C-terminal sequence of wtUB. The E1 enzyme activates
the UB-mimicking peptides to form thioester conjugates
with E1, E2, and HECT type E3s. The loading of the UB-
mimicking peptides on enzymes of the ubiquitination
cascade blocks the formation of UB thioester intermedi-
ates with these enzymes and inhibits UB transfer through
the cascade. The mechanism of the UB-mimicking pep-
tides in inhibiting UB transfer is quite unique in that they
do not target a specific enzyme in the cascade, but instead
block UB transfer at every step of the E1ÀE2ÀE3 cascade.
In future studies, we plan tomeasure the inhibitory proper-
ties of the UB-mimicking peptides in cell-based assays. We
expect these peptides to attenuate global protein ubiquiti-
nation in the cell rather than inhibit UB transfer through
specific E2 or E3 enzymes. Furthermore since UBL pro-
teins have their own cascade enzymes for the modification
of cellular proteins, we can potentially use the same
strategy to identify UBL-mimicking peptides that block
UBL transfer through the cascades.
Figure 3. Inhibition of UB transfer through the E1ÀE2ÀE3
cascade by the UB-mimicking peptides. (A) Inhibition of the
formation of UB∼Ube1 conjugate by peptides P2, P3, and P4.
(
B) Inhibition of CHIP polyubiquitination by increasing con-
centrations of the P3 and P4 peptides. (C) Inhibition of UB
transfer to the HECT domain of E6AP by increasing concentra-
tions of P3 and P4 peptides. 5 μM HA-UB was added in each
reaction after preincubation with the peptides.
added to test if UB can still be loaded on the E1 enzyme.
While P1 with the wtUB sequence cannot prevent the forma-
tion of UB∼E1 thioester conjugates, peptides P2, P3, and P4
can completely block the formation of the UB∼Ube1 con-
jugate at a concentration as low as 10 μM (Figure 3A).
These results suggest that the formation of a peptide∼E1
conjugate can inhibit UB loading onto the E1 enzyme.
We also assayed the competitive inhibition of UB by the
peptides during UB transfer to Ube1. In this assay increas-
ing concentrations of peptides were incubated with 1 μM
UB for reaction with Ube1. The amount of UB∼E1
conjugate formed in the reactions was measured by Wes-
tern blot. As shown in Figure S4 in the SI, P2ÀP4 peptides
can inhibit the formation of the UB∼E1 conjugate with
Acknowledgment. This work was supported by a lab
startup grant from the University of Chicago, and a
National Science Foundation CAREER award (1057092)
to J.Y., and by the Deutsche Forschungsgemeinschaft
(SCHI 425/6-1 and FZ 82) to H.S. This work was also
funded in part by the Chicago Biomedical Consortium
with support from the Searle Funds at The Chicago
Community Trust.
IC values of 266, 172, and 220 μM, respectively, while the
50
P1 peptide cannotsignificantly inhibit UB loading onE1 at
a concentration as high as 1 mM. Although the IC values
5
0
of the P2ÀP4 peptides are still high for direct competition
with UB in the reaction with E1, the UB-mimicking
peptides can effectively block UB loading on E1 once they
occupy the catalytic Cys residue of E1.
We further tested the activity of UB-mimicking peptides
to inhibit UB transfer to E2 and E3. We incubated Ube1,
UbcH5a, and CHIP with P3 and P4 at concentrations of
Supporting Information Available. Supporting figures
and detailed experimental methods. This material is available
free of charge via the Internet at http://pubs.acs.org.
5À100 μM to allow peptide transfer to the E1 and E2
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 22, 2012
5763