A chemical probe for lysine malonylation

Article abstract of DOI:10.1002/anie.201300252

Tag! You′re it! MalAM-yne is a chemical reporter for malonylation of lysines within proteins (see scheme), a newly identified posttranslational modification. MalAM-yne is cell-permeable and metabolically incorporated into proteins in living cells. Subsequent bioorthogonal tag conjugation allows the fluorescent visualization of cellular malonylation and profiling of malonylated proteins. Copyright

Full text of DOI:10.1002/anie.201300252

Angewandte
Chemie
DOI: 10.1002/anie.201300252
Protein Modifications
A Chemical Probe for Lysine Malonylation**
Xiucong Bao, Qian Zhao, Tangpo Yang, Yi Man Eva Fung,* and Xiang David Li*
Protein posttranslational modifications (PTMs) play funda-
mental roles in regulating normal cell physiology and disease
otide (NAD)-dependent hydrolase, which had been previ-
ously classified as a deacetylase but was found to have much
stronger activity toward malonyllysines over acetyllysines
[1]
pathogenesis. Extensive studies on their cellular functions
and mechanisms have advanced our understanding on some
well-known PTMs such as phosphorylation, methylation, and
acetylation. However, the biological roles played by many
[
3b]
(Figure 1a). Given the distinct chemical nature of malo-
nyllysine with a negatively charged carboxylate group, it has
been proposed that lysine malonylation could result in
[
2]
[2a,c]
newly identified PTMs still remain poorly understood.
significant changes in protein structure and function.
Lysine malonylation, a covalent modification of the side
chain of lysine, with a malonyl group incorporated at the e-
amine (Figure 1a), was recently identified as a novel PTM
Characterization of the biological functions of malonyla-
tion requires identification of malonylated protein substrates.
Use of an anti-malonylysine antibody led to the identification
of several malonylated proteins, including metabolic enzy-
[
3]
using two different approaches. One involved an antibody-
[
3a]
[4]
mes and histones, which implies a potential
role for this PTM in metabolic and epigenetic
regulations. Although the antibody has been
used to detect malonylated proteins, immuno-
bloting methods are not ideal for monitoring
the dynamics of malonylation. We still lack
a more general and unbiased method to profile
new malonylated substrates and examine their
dynamic regulation. Herein, we present the
development of an alkyne-functionalized
chemical probe for efficient metabolic label-
ing, robust fluorescent visualization, and mass-
spectrometry-based identification of malony-
lated proteins.
Figure 1. a) The hypothesized enzymatic reactions for lysine (de)malonylation. b) Chem-
ical formulas of Mal-yne and MalAM-yne. c) Strategy for detection and identification of
malonylated protein substrates using chemical probes.
Alkyne-carrying chemical probes that can
be metabolically incorporated into proteins
allow for subsequent copper(I) ion-catalyzed
click chemistry to conjugate the labeled pro-
[5]
based affinity purification of malonylated peptides in con-
teins with azide-fluorescent dyes or affinity purification tags.
They have therefore been used to detect and identify a variety
[
3a]
jugation with mass spectrometry. The other was through
a careful analysis of the enzymatic activity and structural
features of sirtuin 5 (Sirt5), a nicotinamide adenine dinucle-
[
6]
[7]
[8]
of PTMs, including acetylation, lipidation, glycosylation,
[9]
and AMPylation. Isotopic sodium malonate has been used
in cell culture to enhance lysine malonylation in bacteria and
[
3a]
human cells. This result indicated that malonate can be
used for metabolic labeling of malonylated proteins. Inspired
by these studies, we designed and synthesized a malonate
analogue, 2-propargyl malonate (Mal-yne; Figure 1b and
Supporting Information, Scheme S1), as a potential chemical
probe for lysine malonylation (Figure 1c).
[
+]
[+]
[
*] X. Bao, Q. Zhao, T. Yang, Dr. Y. M. E. Fung, Prof. X. D. Li
Department of Chemistry,
The University of Hong Kong Institution
Pokfulam Road, Hong Kong (China)
E-mail: eva.fungym@hku.hk
xiangli@hku.hk
+
We first examined whether Mal-yne could be metabol-
ically incorporated into cellular proteins. A stock solution of
Mal-yne (in PBS at pH 7.4) was used for metabolic labeling of
HeLa S3 cells. After harvesting the cells, the whole-cell
lysates were subjected to azide–alkyne click chemistry to
conjugate the Mal-yne labeled proteins to a rhodamine dye.
The labeled proteins were then resolved by SDS-PAGE and
visualized by in-gel fluorescent imaging (Figure 1c). Dose-
and time-dependent analyses revealed that a wide range of
proteins was labeled by Mal-yne at the optimal concentration
of 10–20 mm for 4–6 hours (Supporting Information, Fig-
ure S1). This concentration is comparable to that used in the
[
] These authors contributed equally to this work.
[
**] We thank Prof. Chi-Ming Che for generous support on the mass
spectrometer, Prof. Dan Yang for advice and support, Prof. Quan
Hao for useful discussions, and Di Hu for technical assistance.
X.D.L. acknowledges a seed fund from The University of Hong Kong
(
201111159240) and Hung Hing Ying Physical Science Research
Fund (20373739). Y.M.E.F. thanks the Small Project Funding from
The University of Hong Kong (201109176193) and the Special
Equipment Grant from the University Grants Committee of the
Hong Kong Special Administrative Region, China (Project Code:
SEG_HKU02).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201300252.
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metabolic labeling of malonylated proteins with isotopic
chemistry. In-gel fluorescent analysis showed that the fluo-
rescence signal of the probe-labeled proteins faded rapidly
(Figure 3a), indicating that the Mal-yne group can be
[
3a]
malonate (20 mm for 24 hours),
as well as acetylated
proteins with alkyne-derived chemical reporters (2.5–10 mm
[
6]
for 6–8 hours). However, we noticed that malonate, with
two negative charges at physiological pH, may have limited
permeability across cell membranes. As a result, a relatively
high concentration of Mal-yne had to be used to achieve
optimal metabolic labeling. To test this hypothesis, we
synthesized a new probe MalAM-yne (Figure 1b) that
masks the carboxylates of Mal-yne with two acetoxymethyl
(
AM) groups (Supporting Information, Scheme S1). We
expected that this uncharged probe could readily permeate
the cell membrane, and the subsequent cleavage of AM
groups by nonspecific cellular esterases would rapidly release
Mal-yne inside living cells for metabolic labeling. When
compared with Mal-yne, at a much lower concentration of
MalAM-yne (50 mm), labeled a variety of proteins in as little
as one hour (Figure 2), showing an improvement of the
Figure 3. Analysis of the dynamics of malonylation using MalAM-yne.
In the a) absence or b) presence of a Sirt5 inhibitor (suramin),
HeLa S3 cells were labeled with MalAM-yne (200 mm) for one hour and
chased with bis(acetoxymethyl) malonate (MalAM; 200 mm) for time
indicated. The cell lysates were reacted with rhodamine azide and
analyzed by in-gel fluorescent scanning. Coomassie-blue staining
shows the protein loading for each lane.
Figure 2. Assessment of chemical probes Mal-yne and MalAM-yne for
metabolic labeling of cells. HeLa S3 cells were incubated with 20 mm
Mal-yne for six hours or MalAM-yne at the indicated concentrations for
one hour. The cell lysates were reacted with rhodamine azide and
analyzed by in-gel fluorescent scanning. Coomassie-blue staining
shows the protein loading for each lane.
efficiently removed from the protein substrates. To examine
whether this removal was regulated by an endogenous
demalonylase, we pre-incubated the cells with suramin,
[
10]
a Sirt5 inhibitor.
This treatment not only enhanced the
labeling efficiency. Importantly, the profile of the proteins
labeled with MalAM-yne was similar to that with Mal-yne
labeling of some proteins by MalAM-yne (Supporting
Information, Figure S3), it also significantly reduced the
rate of Mal-yne removal from the labeled proteins (Fig-
ure 3b). These results suggest that MalAM-yne modified
proteins could be substrates for Sirt5. Taken together, the
rapid incorporation and removal of the chemical probe reveal
the fast dynamics of protein malonylation.
(
Supporting Information, Figures S1,S2), suggesting the two
probes are metabolically equivalent. We therefore focused on
MalAM-yne for further studies on lysine malonylation.
Like most PTMs, lysine malonylation is a reversible
biochemical process. While the enzymes that place malonyl
groups on lysine side chains remain unknown, Sirt5 has been
identified as a demalonylase for the removal of this modifi-
To assess the ability of our chemical probe for identifica-
tion of malonylation substrates, we first tested if MalAM-yne
could selectively enrich known malonylated proteins.
HeLa S3 cells were labeled with MalAM-yne (200 mm) for
one hour. The labeled proteins were conjugated to biotin
through click chemistry and isolated using streptavidin-
coated beads. Western blot analysis using antibodies against
[3]
cation. To determine whether the protein labeling by
MalAM-yne is also reversible, we used a pulse-chase
[
8a]
assay
to monitor the removal of a propargyl malonate
group. HeLa S3 cells were pulse-labeled with MalAM-yne for
one hour, washed, and chased with media containing bis-
[3a]
(
acetoxymethyl) malonate (MalAM). The cells were har-
five known malonylated substrates,
including PGK1,
vested and lysed at different time points. The labeled proteins
were conjugated with a rhodamine azide dye through click
HSPA9, HSP90b, GAPDH, and NSUN2, confirmed their
specific enrichment by the probe (Figure 4a). To identify new
2
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processes, but are also involved in other diverse
cellular functions (Supporting Information, Fig-
ure S4).
To further examine if MalAM-yne modifies
the lysine residues of proteins, we focused on
HSP90b that has a known malonylation site on
[
3a]
lysine residue 399 (K399). We overexpressed
streptavidin-tagged HSP90b in HEK293T cells.
The cells were labeled with MalAM-yne (200 mm)
for one hour, and the tagged protein was then
purified with Strep-Tactin resin. Following diges-
tion with trypsin in solution, the resulting peptides
were separated by HPLC and analyzed with an
LTQ-Orbitrap mass spectrometer. To identify the
peptides that were modified with MalAM-yne, the
MS/MS spectra were anlyzed by protein sequence
alignment using the SEQUEST search engine,
followed by manual verification to ensure the
Figure 4. Western-blot analysis shows the enrichment of a) known and b) potential
substrates of malonylation. HeLa S3 cells were incubated with MalAM-yne (200 mm)
for one hour. The labeled proteins were then conjugated to biotin through click
chemistry. Following incubation with streptavidin beads, the enriched proteins were
eluted for western-bloting analysis.
malonylated substrates, we performed a proteomic analysis of
the MalAM-yne labeled proteins using an LTQ-Orbitrap
mass spectrometer. Out of the 17 currently known malony-
correct assignments. As shown in Figure 5, we identified
Malyne
a peptide with a 2-propargylmalonylated lysine (K
)
3
93
Malyne
405
residue, EMLQQSK
ILKVIR , with a mass shift of
[3a]
lated proteins, 14 of them were identified by MalAM-yne
labeling in both independent experiments (Supporting Infor-
mation, Table S1). In addition, we identified another 361
MalAM-yne enriched proteins as new candidates of malony-
lation substrates (Supporting Information, Table S2). We
subsequently verified the identification of five new malony-
lated proteins (Supporting Information, Table S3), including
PSMD2, EF2, HSPA1, PRDX1, and CDC37, by Western blot
analysis of the affinity-purified proteins (Figure 4b). A
bioinformatics survey revealed that the newly identified
malonylated proteins are mainly associated with metabolic
124.0160 Da, corresponding to the addition of a Mal-yne
group at K399. In the MS/MS spectrum of this peptide, we
Malyne
also found that many of the peptide fragments with the K
residue have satellite peaks, with a mass loss of 44 Da
(Figure 5), which corresponds to the loss of CO . This unique
2
Malyne
mass signature verified the identification of the K
site,
because this thermal decarboxylation process has also com-
monly been observed in the collision-induced dissociation
[3a]
(CID) MS/MS spectra of lysine-malonylated peptides. This
result indicated that MalAM-yne can be used to identify
malonylation sites (Supporting Information, Figure S5) in live
[3a]
cells under physiological conditions. The validation of this
known malonylated peptide also inspired us to use the probe
to identify novel malonylation sites. We thus studied one of
the newly identified malonylated protein candidates, PRDX1.
Malyne
LC-MS/MS analysis of this protein revealed a K
con-
PG-
1
69
Malyne
taining
peptide,
HGEVCPAGWK
1
90
SDTIKPDVQK (Supporting Information, Figure S6), indi-
cating that a novel malonylation site had been identified on
this protein. These experiments collectively demonstrate that
MalAM-yne can directly target Lys residues for the detection
and identification of lysine malonylation.
To explore the scope of MalAM-yne to study protein
malonylation, we performed metabolic labeling of different
cell types with MalAM-yne. In-gel fluorescent analysis
showed robust labeling of a wide variety of proteins in all
the cell types that were tested (Supporting Information,
Figure S6), ranging from bacteria to human cells, indicating
that lysine malonylation is evolutionarily conserved in these
species. Additionally, the different protein-labeling patterns
of various human cell lines suggest a diverse role and the
dynamic nature of lysine malonylation in mammalian cells.
In summary, we have developed the first chemical probe,
MalAM-yne, to study protein lysine malonylation. MalAM-
yne is permeable across the cell membrane and allows for
efficient metabolic labeling, robust fluorescent detection, and
proteomic profiling of malonylated proteins in a variety of
cells. MalAM-yne based fluorescent detection also enabled
Figure 5. The CID MS/MS spectrum of a triply charged tryptic-digest
3
93
Malyne
405
peptide, EMLQQSK
ILKVIR , from MalAM-yne modified
HSP90b.
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Angewandte
Communications
the dynamic analysis of malonylation levels and patterns,
which therefore is a complementary method to the antibody-
based methods. Moreover, when combined with methods to
inhibit or activate the functions of a protein, such as RNA
interference or the use of chemical inhibitors or activators,
our probe that unbiasedly labels malonylated proteins could
be used to determine cellular substrates of the demalonylase,
Sirt5. It is also worth mentioning that the probe, as a mimic of
malonate, could be subjected to a variety of metabolic
pathways. Therefore, the further identification of malonyla-
tion sites and functional validation of these substrates is an
important next step, and will be reported in due course.
M. Z. Wang, L. Ho, E. Verdin, Trends Endocrinol. Metab. 2012,
3, 467 – 476.
2
[
3] a) C. Peng, Z. Lu, Z. Xie, Z. Cheng, Y. Chen, M. Tan, H. Luo, Y.
Zhang, W. He, K. Yang, B. M. Zwaans, D. Tishkoff, L. Ho, D.
Lombard, T. C. He, J. Dai, E. Verdin, Y. Ye, Y. Zhao, Mol. Cell.
Proteomics 2011, 10, M111 012658; b) J. Du, Y. Zhou, X. Su, J. J.
Yu, S. Khan, H. Jiang, J. Kim, J. Woo, J. H. Kim, B. H. Choi, B.
He, W. Chen, S. Zhang, R. A. Cerione, J. Auwerx, Q. Hao, H.
Lin, Science 2011, 334, 806 – 809.
4] Z. Xie, J. Dai, L. Dai, M. Tan, Z. Cheng, Y. Wu, J. D. Boeke, Y.
Zhao, Mol. Cell. Proteomics 2012, 11, 100 – 107.
5] E. M. Sletten, C. R. Bertozzi, Angew. Chem. 2009, 121, 7108 –
[
[
[
7
133; Angew. Chem. Int. Ed. 2009, 48, 6974 – 6998.
6] Y. Y. Yang, J. M. Ascano, H. C. Hang, J. Am. Chem. Soc. 2010,
32, 3640 – 3641.
1
Received: January 11, 2013
Revised: February 4, 2013
Published online: && &&, &&&&
[7] a) B. R. Martin, B. F. Cravatt, Nat. Methods 2009, 6, 135 – 138;
b) G. Charron, M. M. Zhang, J. S. Yount, J. Wilson, A. S.
Raghavan, E. Shamir, H. C. Hang, J. Am. Chem. Soc. 2009,
131, 4967 – 4975; c) G. Charron, J. Wilson, H. C. Hang, Curr.
Opin. Chem. Biol. 2009, 13, 382 – 391.
Keywords: click chemistry · lysine malonylation ·
metabolic labeling · posttranslational modifications ·
protein modifications
.
[
8] a) B. W. Zaro, Y. Y. Yang, H. C. Hang, M. R. Pratt, Proc. Natl.
Acad. Sci. USA 2011, 108, 8146 – 8151; b) N. J. Agard, C. R.
Bertozzi, Acc. Chem. Res. 2009, 42, 788 – 797.
[
9] M. Grammel, P. Luong, K. Orth, H. C. Hang, J. Am. Chem. Soc.
2
011, 133, 17103 – 17105.
[
1] C. T. Walsh, Posttranslational Modifications of Proteins:
Expanding Natureꢀs Inventory, Roberts and Co. Publishers,
Greenwood Village, CO, 2006.
2] a) H. Lin, X. Su, B. He, ACS Chem. Biol. 2012, 7, 947 – 960;
b) C. A. Olsen, Angew. Chem. 2012, 124, 3817 – 3819; Angew.
Chem. Int. Ed. 2012, 51, 3755 – 3756; c) W. He, J. C. Newman,
[
10] a) A. Schuetz, J. Min, T. Antoshenko, C. L. Wang, A. Allali-
Hassani, A. Dong, P. Loppnau, M. Vedadi, A. Bochkarev, R.
Sternglanz, A. N. Plotnikov, Structure 2007, 15, 377 – 389;
b) A. S. Madsen, C. A. Olsen, J. Med. Chem. 2012, 55, 5582 –
[
5
590.
4
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Angew. Chem. Int. Ed. 2013, 52, 1 – 5
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Chemie
Communications
Protein Modifications
X. Bao, Q. Zhao, T. Yang, Y. M. E. Fung,*
X. D. Li*
&&&& — &&&&
A Chemical Probe for Lysine Malonylation
Tag ! You’re it! MalAM-yne is a chemical
reporter for malonylation of lysines within
proteins (see scheme), a newly identified
posttranslational modification. MalAM-
yne is cell-permeable and metabolically
incorporated into proteins in living cells.
Subsequent bioorthogonal tag conjuga-
tion allows the fluorescent visualization
of cellular malonylation and profiling of
malonylated proteins.
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!