Facile synthesis of native and protease-resistant ubiquitylated peptides

Article abstract of DOI:10.1002/cbic.201402135

The reversible post-translational modification of eukaryotic proteins by ubiquitin regulates key cellular processes including protein degradation and gene transcription. Studies of the mechanistic roles for protein ubiquitylation require quantities of hom

Full text of DOI:10.1002/cbic.201402135

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DOI: 10.1002/cbic.201402135
Facile Synthesis of Native and Protease-Resistant
Ubiquitylated Peptides
Caroline E. Weller, Wei Huang, and Champak Chatterjee*[a]
This paper is dedicated to Professor Niels H. Andersen on the occasion of his 70th birthday.
The reversible post-translational modification of eukaryotic
proteins by ubiquitin regulates key cellular processes including
protein degradation and gene transcription. Studies of the
mechanistic roles for protein ubiquitylation require quantities
of homogenously modified substrates that are typically inac-
cessible from natural sources or by enzymatic ubiquitylation in
vitro. Therefore, we developed a facile and scalable methodol-
ogy for site-specific chemical ubiquitylation. Our semisynthetic
strategy utilized a temporary ligation auxiliary, 2-(aminooxy)-
ethanethiol, to direct ubiquitylation to specific lysine residues
in peptide substrates. Mild reductive removal of the auxiliary
after ligation yielded ubiquitylated peptides with the native
isopeptide linkage. Alternatively, retention of the ligation auxili-
ary yielded protease-resistant analogues of ubiquitylated pep-
tides. Importantly, our strategy was fully compatible with the
presence of protein thiol groups, as demonstrated by the syn-
thesis of peptides modified by the human small ubiquitin-relat-
ed modifier 3 protein.
poses a significant challenge to generating useful quantities of
site-specifically ubiquitylated proteins for in vitro investiga-
tions. Hence, multiple chemical strategies have been devel-
oped to generate native isopeptide-linked ubiquitylated pep-
tides,[10] as well as disulfide,[11] triazole,[12] and hydroxamate-
linked analogues of ubiquitylated proteins.[13] Until now, meth-
ods to conjugate Ub with its targets by a native linkage have
employed ligation auxiliaries such as g- and d-thiolysine,[10b,14]
or a photolytically removable auxiliary based on the o-nitro-
benzyl scaffold.[10a] When incorporated at the desired Lys ubiq-
uitylation site these auxiliaries permit native chemical liga-
tion[15] with a Ub C-terminal a-thioester to generate an isopep-
tide linkage, after which the auxiliaries can be chemically or
photolytically removed. However, the complex multistep syn-
thesis of these ligation auxiliaries, the requisite desulfurization
of thiolysine analogues post-ligation, and the slow kinetics of
ligation with the photocleavable auxiliary ultimately limit their
broad applicability. Therefore, a readily achievable ligation aux-
iliary that exhibits good ligation kinetics and is removable
under conditions that do not affect native Cys residues would
greatly expand the scope of ubiquitylated and SUMOylated
proteins accessible for mechanistic studies.
The 76-residue protein ubiquitin (Ub) is highly conserved in
eukaryotes[1] and regulates almost every aspect of cellular func-
tion.[2] Ubiquitin and the family of structurally related small
ubiquitin-like modifier proteins (SUMOs) play vital roles in cel-
lular processes ranging from histone-mediated gene activa-
tion[3] or silencing[4] to 26S proteasome-mediated protein deg-
radation.[5] Elucidating the functional consequences of ubiqui-
tylation and SUMOylation of the many hundreds of known
protein substrates in human cells is a major challenge for
modern cell biology.[6] Such studies require the ability to follow
the dynamic regulation and subcellular localization of ubiquity-
lated and SUMOylated proteins in vivo,[7] as well as the ability
to investigate the direct biochemical and biophysical conse-
quences of these post-translational modifications in vitro.[8]
Protein modification by Ub is a multistep process involving
a family of E1, E2, and E3 ligase enzymes that utilize the
hydrolysis of ATP to activate the C terminus of Ub, and then
conjugate it with specific Lys e-amines in protein substrates by
means of isopeptide linkages.[2] More than 600 different E3
ligases, a majority of which remain uncharacterized, are in-
volved in site-specific protein ubiquitylation in humans.[9] This
With this goal in mind, we turned our attention to a deriva-
tive of the 2-(aminooxy)ethanethiol auxiliary first reported by
Kent and co-workers.[16] The 2-(aminooxy)ethanethiol group
was shown to facilitate native chemical ligation at sterically un-
hindered Gly-Gly and Gly-Ala sites in short peptide sequences.
Because Ub, and indeed most ubiquitin-like proteins, termi-
nates in a C-terminal Gly-Gly sequence (Figure S1 in the Sup-
porting Information),[17] we envisioned that the auxiliary could
be applied for peptide ubiquitylation as depicted in Scheme 1.
A facile three-step synthetic scheme afforded the suitably
protected auxiliary 1, in multigram quantities and 46% overall
yield (Scheme 2A and Figures S2–S4) for application in Fmoc-
[a] C. E. Weller, W. Huang, Prof. Dr. C. Chatterjee
Department of Chemistry, University of Washington
Box 351700, Seattle, WA 98195 (USA)
E-mail: chatterjee@chem.washington.edu
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201402135.
Scheme 1. Site-specific peptide ubiquitylation.
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(Mtt) protecting group at the Lys e-amine targeted for ubiqui-
tylation (Scheme 2B).[19] After chain assembly, the Lys side
chain was selectively deprotected with 1% TFA in dichlorome-
thane, and the free e-amine was subsequently coupled with
bromoacetic acid. The auxiliary was readily introduced through
direct nucleophilic displacement of bromine with a 1m solu-
tion of 1 in DMSO overnight at room temperature. Importantly,
the unreacted auxiliary could be filtered away from the solid
phase and reused at least four times without appreciable
losses in incorporation. Finally, TFA-mediated cleavage from
the resin afforded the auxiliary-containing peptide 2, in 38%
purified yield based on the initial resin loading (Figure S5).
Heterologously expressed human Ub(1–75)-a-thioester 3
was obtained by thiolysis of the corresponding GyrA intein
fusion with mercaptoethanesulfonic acid (MES, Figure S6).[10a]
Ligation was initiated by mixing 2.5 mm of 2 and 0.5 mm of 3
in 6m guanidinium hydrochloride (Gn·HCl), 100 mm Na2HPO4,
and 10 mm tris(2-carboxyethyl)phosphine (TCEP) at pH 7.5 at
room temperature. Although auxiliary-mediated ligation onto
a secondary amine had previously been shown to be slower
than ligation to the primary amine in Cys,[20] the enhanced nu-
cleophilicity of the aminooxy group at pH 7.5 led to complete
acyl transfer within 12 h (Figure S7).[21] The ligation product
was then purified by reversed-phase (RP-) HPLC to afford the
auxiliary-containing branched protein QKUb(aux)E (4, Figure S8).
In contradiction to previous results with short peptides,[16]
our initial attempts to cleave the ligation auxiliary from 4 re-
ductively with activated Zn in acidic HPLC buffers or water
proved largely unsuccessful (Table 1, entries 1 and 2). The in-
clusion of 6m Gn·HCl during the reduction step overcame this
issue and led to better removal of the b-mercaptoethanol
group, resulting in the native isopeptide linkage at Lys
(Figure 1 and Table 1, entry 3). The requirement for a denatur-
ant to facilitate efficient electron transfer from Zn to the low-
energy NÀO bond in 4 is consistent with previous biophysical
studies that showed that Ub retains its tertiary structure in
solution at ꢀpH 1 (Figure S9).[22] However, this might be a limi-
tation for some Ub targets.
Scheme 2. Synthesis and application of the ligation auxiliary.
A) a) BrCH2CH2Br, Et3N, DMF, 18 h, 258C, 64%; b) TrtSH, NaH, DMF, 2 h, 258C,
79%; c) H2NNH2, CHCl3, 2.5 h, 258C, 90%. B) a) i: 1% TFA, 1% TIS in CH2Cl2;
ii: BrCH2COOH, DIC, DMF; b) 1, 1m in DMSO; c) reagent K; d) Ub(1–75)-a-
thioester (3), 6m Gn·HCl, 100 mm Na2HPO4, 10 mm TCEP, pH 7.5; e) 6m
Gn·HCl, Zn, pH 3.0, 378C, 24 h. PG: protecting group. Trt: trityl. Mtt: 4-meth-
yltrityl. Boc: tert-butyloxycarbonyl. Fmoc: 9-fluorenylmethoxycarbonyl. TFA:
trifluoroacetic acid. TIS: triisopropylsilane. DIC: N,N’-diisopropylcarbodiimide.
Reagent K: TFA/thioanisole/H2O/phenol/ethane-1,2-dithiol (82.5:5:5:5:2.5,
v/v/v/v/v).
We next sought to identify suitable reagents and conditions
to maximize the yields of reduced products (Table 1, entries 4–
7 and Figure S10). Several methods have been reported for the
based (9-fluorenylmethoxycarbonyl-based) solid-phase peptide
synthesis. In order to test the utility of the auxiliary for peptide
ubiquitylation, we first consid-
ered an internal peptide se-
quence from Ub itself. There
Table 1. Reductive removal of the ligation auxiliary.
exist seven Lys residues at posi-
tions 6, 11, 27, 29, 33, 48, and 63
in Ub, and all seven are found to
be modified by ubiquitin in
vivo.[18] Therefore, we first chose
a short N-terminally acetylated
and C-terminally amidated tri-
peptide, QKE, containing Lys63
of Ub, as a test substrate for
Product
QKUb
Reagent
pH
T [8C]
t [h]
Yield [%][a]
1
2
3
4
5
6
7
8
9
E
Zn, AcN/H2O (1:1)
Zn, H2O
1
1
1
1
7
3
3
3
3
3
22
22
22
37
37
37
37
37
37
37
24
24
24
24
17
24
24
24
24
24
5
12
28
54
5
31
80
75
80
60
QKUbE
QKUbE
QKUbE
QKUbE
QKUbE
QKUbE
QKSuE
KAKUbI
KAKSuI
Zn, 6m Gn·HCl
Zn, 6m Gn·HCl
Raney nickel[b]
In, 6m Gn·HCl
Zn, 6m Gn·HCl
Zn, 6m Gn·HCl
Zn, 6m Gn·HCl
Zn, 6m Gn·HCl
auxiliary-mediated
ubiquityla-
10
tion. The peptide was assembled
on Rink amide resin with use of
an orthogonal 4-methyltrityl
[a] Yields were calculated from purified product weights and relative signal intensities in ESI-MS. [b] As per the
procedure reported by McGinty et al.[20]
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with the ubiquitin C-terminal hydrolase L3 (UCH-L3)
and the Sentrin-specific protease 1 (SENP1), cysteine
proteases that remove Ub and SUMO, respectively,
from diverse cellular targets (Figure 2A).[27]
Complete hydrolysis of the isopeptide linkage was
observed in both cases, and the full-length Ub(1–76)
and SUMO-3(2–92) proteins were observed after 3.5
and 8 h, respectively (Figure 2B, C and Figure S20).
Because the C-terminal Gly in the full-length proteins
could only be derived from conjugation at Lys29
(Scheme 1), these results indicated that auxiliary-
mediated ligation of Ub and SUMO to the KAKI pep-
tide had occurred at the desired Lys29 side chain.
Complete hydrolysis of KAKUbI by UCH-L3 suggests
Figure 1. Synthesis of QKUbE. A) C18 analytical RP-HPLC chromatogram of reduced and
purified QKUbE. B) ESI-MS of reduced QKUbE. m/z calcd: 8992.0 Da [M+H]+; found:
(8992.2Æ1.8) Da.
conversion of N,N-disubstituted hydroxylamines into
the corresponding amines, including reduction with
Raney Ni[23] and indium metal.[24] However, we ob-
served that treatment of 4 with activated Zn in 6m
Gn·HCl at pH 3.0 and 378C for 24 h yielded the best
results with minimal side-products (Table 1, entry 7).
A small amount of hydrolyzed Ub(1–75) was the
major side-product observed during auxiliary remov-
al. An N-to-S acyl shift of the amide backbone that
would yield a thioester susceptible to hydrolysis has
previously been demonstrated at Gly-Cys junctions[25]
and with N-methylated Cys at low pH and elevated
temperatures.[26] However, for 4 the hydrolysis prod-
uct is more likely a result of direct nucleophilic attack
by water on the protonated tertiary amide. Evidence
of this was provided by our observation that alkyla-
tion of the auxiliary thiol post-ligation, which pre-
cludes an N-to-S acyl shift, did not significantly affect
the small degree of hydrolysis (Figure S11).
Figure 2. Testing the site of Ub and SUMO linkage. A) Hydrolysis of KAKUbI and KAKSuI by
UCH-L3 and SENP1, respectively. B) ESI-MS of KAKUbI assayed with UCH-L3. m/z calcd:
8565.8 Da [M+H]+; found: (8566.4Æ0.8) Da. C) ESI-MS of KAKSuI assayed with SENP1. m/z
calcd: 13394.6 Da [M+H]+; found: (13394.6Æ2.1) Da.
With a robust methodology for peptide ubiquityla-
tion in hand we proceeded to test its scope for pep-
tide SUMOylation with a recombinant human SUMO-
3(2–91)-a-thioester and the peptide QK(aux)E (Figures
S12–S13). Ligation proceeded with similar kinetics as
for Ub and was complete in 12 h (Figures S14–S15). A previ-
ously reported ligation of SUMO to a secondary amine re-
quired seven days;[10a] this highlights the advantage of employ-
ing the aminooxy group for acyl transfer. As expected, reduc-
tive removal of the pendant auxiliary group afforded the iso-
peptide-linked QKSuE in good yield while retaining Cys47 in
SUMO (Table 1, entry 8 and Figure S16).
that our chemical strategy does not interfere with the correct
folding of Ub in the final ubiquitylated product. Furthermore,
X-ray crystal structures of Ub bound to UCH-L3 reveal exten-
sive protein–protein interactions that may only exist in the
native folded form of Ub (Figure S21).[28] Thus, our methodolo-
gy yields site-specifically ubiquitylated and SUMOylated pep-
tides that adopt a native fold and are suitable for in vitro bio-
chemical studies.
We further explored the substrate scope of ligation with a
KAKI peptide sequence, which contains both Lys27 and Lys29
of Ub (Figure S17). Installation of the ligation auxiliary at the
Lys29 position in KAKI led to similar yields for ubiquitylation
and SUMOylation of this peptide (Table 1, entries 9 and 10,
and Figures S18–S19). However, unlike the N-terminally acety-
lated QKE peptide, the reaction of Ub(1–75)- and SUMO-3(2–
91)-a-thioesters with KAKI could formally proceed through nu-
cleophilic attack variously by the peptide N terminus, by the
Lys27 e-amine, or by the auxiliary alkoxyamine. In order to test
the precise site of ligation, KAKUbI and KAKSuI were assayed
When subjecting semisynthetic ubiquitylated proteins to
assays in complex mixtures, such as cell lysates, one must con-
tend with the presence of approximately 100 deubiquitylating
enzymes (DUBs) that might hydrolyze the isopeptide linkage.[29]
In thinking of this problem, we noted that retention of the li-
gation auxiliary in the final ubiquitylated peptide yields an N-
alkylated Gly76. The enhanced proteolytic stability of polymeric
N-substituted glycines, also known as peptoids, and peptoid–
peptide hybrid molecules has previously been demonstrat-
ed.[30] Therefore, we wondered if simply retaining the ligation
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auxiliary in the final ubiquitylated product would inhibit pro-
teolysis of the isopeptide bond. To our delight, incubation of
the unreduced ligation product containing the auxiliary group,
QKUb(aux)E, with UCH-L3 for 3.5 h at 378C led to no observable
hydrolysis of the isopeptide linkage (Figure 3A, lane 3). In com-
parison, the reduced ligation product QKUbE was completely
hydrolyzed to produce Ub(1–76) under identical conditions
deubiquitylation of QKUbE by UCH-L3; however, Ub has no in-
trinsic thiol groups, and the mass of this species corresponded
to an enzyme-bound Ub(1–76) acyl intermediate (Figure S26).
In conclusion, we have demonstrated the successful applica-
tion of a synthetically facile and readily removable ligation aux-
iliary to the ubiquitylation and SUMOylation of various peptide
targets. Our methodology significantly advances current ubiq-
uitylation strategies, because it is
orthogonal to the presence of
Cys residues in proteins. When
employed in combination with
native chemical ligation at Cys,
this should greatly expand the
substrate scope of protein tar-
gets. Furthermore, post-ligation
the auxiliary mimics an N-substi-
tuted Gly76 and inhibits several
deubiquitylating enzymes. Our
preliminary results also indicate
Figure 3. Protease-resistant ubiquitylated peptide. A) Coomassie-stained 18% SDS-PAGE gel of DUB activity to-
wards QKUbE and QKUb(aux)E. Lane 1: UCH-L3+QKUbE. Lane 2: UCH-L3(C95A)+QKUbE. Lane 3: UCH-L3+QKUb(aux)E.
Lane 4: UCH-L3(C95A)+QKUb(aux)E. Lane 5: UCH-L3+QKUb(aux)E+50 mm DTT, 958C, 5 min. An asterisk indicates the
Ub(1–76)-UCH-L3 acyl enzyme intermediate. B) ESI-MS of the observed UCH-L3-QKUb(aux)E adduct. m/z calcd:
35247.5 Da [M+H]+; found: (35252.5Æ5.6) Da.
that the auxiliary group has utili-
ty as probe in pull-down
a
assays to identify ubiquitin C-ter-
minal hydrolases. Finally, the
mild conditions for removal of
the auxiliary after ligation do not
(Figure 3A, lane 1). To test the scope of DUB resistance, we
also assayed KAKUb(aux)I with the ubiquitin-specific proteases
Usp2 and Usp5, which are structurally vastly different from
UCH-L3.[31] We observed that the ligation auxiliary also inhibit-
ed the removal of Ub from peptide targets by these enzymes
(Figure S22).
inhibit protein recognition by specific proteases such as UCH-
L3 and SENP1, and this is promising for studies of the bio-
chemical effects of ubiquitylation and SUMOylation.
Experimental Section
See the Supporting Information.
Recently, N-methylation of the isopeptide bond was demon-
strated to be a means to inhibit UCH-L3 activity.[32] This re-
quired the challenging chemical synthesis of an N-methylated
branched polypeptide and a final desulfurization step to gen-
erate wild-type Ub. We demonstrate that N-alkylation of Gly76
offers an alternate and facile means to access both protease-
resistant and wild-type isopeptide-linked ubiquitylated pep-
tides by a single synthetic route.
Acknowledgements
This work was supported by the National Institute of General
Medical Sciences and the National Institutes of Health (grant
1R01GM110430-01), the Department of Chemistry and the Royal-
ty Research Fund at the University of Washington, Seattle. C.E.W.
gratefully acknowledges an ARCS Foundation Fellowship and an
NSF Graduate Research Fellowship (grant number DGH-1256082).
Interestingly, non-reducing denaturing polyacrylamide gel
electrophoresis of the products obtained from incubating
QKUb(aux)E with UCH-L3 in the presence of ꢀ6 mm dithiothreitol
(DTT) revealed a new higher-molecular-weight band (Fig-
ure 3A, lane 3). Reversed-phase liquid chromatography of the
assay products followed by electrospray ionization mass spec-
trometry (LC-ESI-MS) revealed a species corresponding to disul-
fide-linked QKUb(aux)E and UCH-L3 (Figure 3B). This higher-mo-
lecular-weight species was also observed upon incubation of
KAKUb(aux)I with UCH-L3 (Figure S23). In keeping with a disulfide
linkage, the adduct was not observed when assay products
were boiled with 50 mm DTT prior to gel electrophoresis (Fig-
ure 3A, lane 5), or when the active site mutant UCH-L3(C95A)
was employed in assays (Figure 3A, lane 4 and Figure S24). Di-
sulfide formation is, however, not the sole mechanism for
UCH-L3 resistance because unlinked QKUb(aux)E remained intact
whereas QKUbE was effectively hydrolyzed (Figure S25). A
higher-molecular-weight band was also observed during the
Keywords: conjugation · peptides · protein modifications ·
SUMO · ubiquitin
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Received: March 28, 2014
Published online on May 18, 2014
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