Chemical Probes Reveal Sirt2's New Function as a Robust "eraser" of Lysine Lipoylation

Article abstract of DOI:10.1021/jacs.9b06913

Lysine lipoylation, a highly conserved lysine post-translational modification, plays a critical role in regulating cell metabolism. The catalytic activity of a number of vital metabolic proteins, such as pyruvate dehydrogenase (PDH), depends on lysine lipoylation. Despite its important roles, the detailed biological regulatory mechanism of lysine lipoylation remains largely unexplored. Herein we designed a powerful affinity-based probe, KPlip, to interrogate the interactions of lipoylated peptide/proteins under native cellular environment. Large-scale chemical proteomics analysis revealed a number of binding proteins of KPlip, including sirtuin 2 (Sirt2), an NAD⁺-dependent protein deacylase. To explore the potential activity of Sirt2 toward lysine lipoylation, we designed a single-step fluorogenic probe, KTlip, which reports delipoylation activity in a continuous manner. The results showed that Sirt2 led to significant delipoylation of KTlip, displaying up to a 60-fold fluorescence increase in the assay. Further kinetic experiments with different peptide substrates revealed that Sirt2 can catalyze the delipoylation of peptide (DLAT-PDH, K259) with a remarkable catalytic efficiency (k_cat/K_m) of 3.26 × 10³ s^-1 M^-1. The activity is about 400-fold higher than that of Sirt4, the only mammalian enzyme with known delipoylation activity. Furthermore, overexpression and silencing experiments demonstrated that Sirt2 regulates the lipoylation level and the activity of endogenous PDH, thus unequivocally confirming that PDH is a genuine physiological substrate of Sirt2. Using our chemical probes, we have successfully established the relationship between Sirt2 and lysine lipoylation in living cells for the first time. We envision that such chemical probes will serve as useful tools for delineating the roles of lysine lipoylation in biology and diseases.

Full text of DOI:10.1021/jacs.9b06913

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Article
Chemical Probes Reveal Sirt2’s New Function
as a Robust “Eraser” of Lysine Lipoylation
Yusheng Xie, Lanfang Chen, Rui Wang, Jigang Wang, Jingyu Li, Wei XU,
Yingxue Li, Shao Q. Yao, Liang Zhang, Quan Hao, and Hongyan Sun
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b06913 • Publication Date (Web): 23 Oct 2019
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Chemical Probes Reveal Sirt2’s New Function as a Robust “Eraser” of
Lysine Lipoylation
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Yusheng Xie,1,2† Lanfang Chen,3† Rui Wang,4† Jigang Wang,5 Jingyu Li,4 Wei Xu,4 Yingxue Li,4 Shao
Q. Yao,6 Liang Zhang,2,4* Quan Hao,3* and Hongyan Sun1,2*
1Department of Chemistry and COSDAF (Centre of Super-Diamond and Advanced Films), City University of Hong
Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, China
2Key Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen Research Institute of City University of
Hong Kong, Shenzhen, 518057, China
3School of Biomedical Sciences, University of Hong Kong, Hong Kong, China
4Department of Biomedical Science, College of Veterinary Medicine and Life Sciences, City University of Hong Kong, 83
Tat Chee Avenue, Kowloon, Hong Kong, China
5Department of Pharmacology, National University of Singapore, Singapore
6Department of Chemistry, National University of Singapore, Singapore
KEYWORDS: Chemical probes, Lysine lipoylation, Sirtuins, Photo-crosslinking, Proteomic profiling
ABSTRACT: Lysine lipoylation, a highly conserved lysine PTM, plays a critical role in regulating cell metabolism. The catalytic
activity of a number of vital metabolic proteins, such as pyruvate dehydrogenase (PDH), depends on lysine lipoylation. Despite
its important roles, the detailed biological regulatory mechanism of lysine lipoylation remains largely unexplored. Herein we
designed a powerful affinity-based probe KPlip to interrogate the interactions of lipoylated peptide/proteins under native
cellular environment. Large-scale chemical proteomics analysis revealed a number of binding proteins of KPlip, including
Sirt2, an NAD+-dependent protein deacylase. To explore the potential activity of Sirt2 toward lysine lipoylation, we designed
a single-step fluorogenic probe KTlip, which reports delipoylation activity in a continuous manner. The results showed that
Sirt2 led to significant delipoylation of KTlip, displaying up to 60-fold fluorescence increase in the assay. Further kinetic
experiments with different peptide substrates revealed that Sirt2 can catalyze the delipoylation of peptide (DLAT-PDH, K259)
with a remarkable catalytic efficiency (kcat/Km) of 3.26 × 103 s-1M-1. The activity is about 400-fold higher than that of Sirt4, the
only mammalian enzyme with known delipoylation activity. Furthermore, overexpression and silencing experiments demon-
strated that Sirt2 regulates the lipoylation level and the activity of endogenous PDH, thus unequivocally confirming that PDH
is a genuine physiological substrate of Sirt2. Using our chemical probes, we have successfully established the relationship
between Sirt2 and lysine lipoylation in living cells for the first time. We envision that such chemical probes will serve as useful
tools for delineating the roles of lysine lipoylation in biology and diseases.
Posttranslational modifications (PTMs) of lysine residues are
highly prevalent in living organisms and play important
roles in regulating diverse biological processes such as gene
transcription, DNA repair, chromatin structure modulation
and metabolism.1 Notable examples of lysine PTMs include
methylation, acetylation, lipidation, ubiquitination, sumoy-
lation, and others.1b Recently the discovery of numerous
new lysine acylations, such as succinylation (Ksucc),
crontonylation (Kcr), 2-hydroxyisobutyrylation (Khib), β-
hydroxybutyrylation (Kbhb), has further expanded the
landscapes of lysine PTMs.2 Deciphering the regulatory
mechanisms of these new lysine PTMs is important to fur-
ther elucidate their biological functions. Research in this
field has therefore seen tremendous development and at-
tracted increasing attention in recent years.
Lysine lipoylation (Klip) is a highly conserved lysine PTM
found in bacteria, virus and mammals.3 It plays a critical role
in regulating cell metabolism. Klip is reported to occur on
several essential metabolic multimeric complexes, includ-
ing the branched-chain α-ketoacid dehydrogenase (BCKDH),
the α-ketoglutarate dehydrogenase (KDH) complex, the py-
ruvate dehydrogenase (PDH) complex and the glycine
cleavage complex (GCV).3b, c Klip is required as an essential
cofactor for maintaining the activity of these enzyme com-
plexes.4 Malfunction of these metabolic complexes, on the
other hand, can lead to numerous diseases.3e, 5 For instance,
dysregulation of PDH activity has been linked to many dis-
eases including metabolic disorders, cancer, Alzheimer’s
disease and viral infection.6 Notwithstanding
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Figure 1. (A) Chemical structures of probe KPlip, its negative control probe KPlip-C and Competitor. (B) Synthetic route of
probe KPlip via solid phase peptide synthesis approach (SPPS). (C) Overall scheme of the chemical proteomic experiments
to identify high confidence binding proteins of KPlip. A series of chemical proteomic experiments were performed, including
KPlip/KPlip-C with live cells/cellular lysates and competition experiments. (D) Gene ontology analysis of the 67 proteins
identified by KPlip reveals their association with a number of biological processes, e.g. protein deacetylation process. The
names of the four proteins belonging to the functional cluster of “histone/protein deacetylation” are shown. (E) Scheme show-
ing PDH activity is regulated by delipoylation.
the important roles of lysine lipoylation in biology, its regu-
latory mechanisms, in particular the enzymes that catalyze
the removal (“erasers”) of this PTM, are still poorly under-
stood. In 2013, Denu et al. screened the in vitro deacylation
activity of sirtuins against histone peptides with various
acyl modifications including lipoylation.7 However, there
remains a lack of knowledge of lysine lipoylation regulation,
such as detailed enzymatic activity and in vivo substrate
specificity. Elucidating the regulatory mechanism of lysine
lipoylation will help understand its roles in biology and var-
ious diseases.
In a recent seminal work, Cristea et al. discovered that Sirt4
could interact with PDH complex using immunoenrichment
methods.3c The study revealed that Sirt4 is the first mam-
malian enzyme that can modulate PDH activity through
delipoylation in living cells. However, it was noted that the
delipoylation activity of Sirt4 in vitro was rather weak, es-
pecially when compared with the deacetylation activity of
sirtuins.3c, d, 7-8 This raises an intriguing question--whether
there are other enzymes that can erase Klip more efficiently
in native cellular environment.
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To address this question, we endeavored to develop robust streptavidin beads, resolved by SDS-PAGE, digested with
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chemical tools to aid in understanding the biological func-
tions of lysine lipoylation. Specifically in this study, we have
successfully developed the first affinity-based probe KPlip
capable of interrogating the lipoylated peptide/protein in-
teractions under native cellular environments. This chemi-
cal probe allows us to identify potential regulators of lysine
lipoylation, thus uncovering new biology related to lipoy-
lation. Furthermore, we have developed a fluorogenic probe
KTlip to detect delipoylation activity in a continuous man-
ner. The probe enables us to quickly and reliably examine
whether a given protein possesses delipoylation activity.
trypsin, and finally analyzed by label-free LC-MS/MS quan-
titation (Supplementary Data 6). High-specificity binders of
KPlip were defined as those showing substantial signal de-
crease (≥ 3-fold) in the presence of the competitor. This led
to a total of 588 proteins identified (Supplementary Data
2B). We compared the proteins identified in both the probe-
selectivity and competition experiments, leading to a high-
confidence list of 67 KPlip-binding proteins. (Supplemen-
tary Data 2C).
9
We then performed the Gene Ontology (GO) analysis of the
high-confidence KPlip-binding proteins (Supplementary
Data 3). It revealed that a variety of biological processes,
such as cellular lipid catabolic process and acyl-CoA meta-
bolic process (Supplementary Data 3), were significantly
enriched. Specifically, “histone/protein deacetylation” list
caught our attention, because they may include proteins
that regulate Klip modification. These proteins are summa-
rized in Figure 1D. It is interesting to note that Sirt2, a mem-
ber of the sirtuin family of NAD+-dependent HDAC,11 is
among the “histone/protein deacetylation” list. The sirtuin
proteins have been reported to mediate the hydrolysis of
various acyl groups of lysine, including Ksucc, Kmal, and
others.12 The fact that Sirt2 is among the Klip-interacting
proteomes prompted us to investigate its role in regulating
lysine lipoylation. It was also noted that Sirt4 was not iden-
tified in our protein list. A plausible reason is the instability
and the low abundance of Sirt4 in human proteome (Paxdb,
www.paxdb.org)
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Results
Chemical Proteomic profiling using probe KPlip. Pro-
filing the interaction between an enzyme and its substrate
is challenging, as their interactions are usually weak, transi-
ent and dynamic.9 Affinity-based-probes (AfBPs) provide a
powerful tool to overcome those hindrances by utilizing
photo-cross-linking to convert transient protein-ligand in-
teractions into covalent chemical linkages.10 Chemical pro-
teomics using AfBPs coupled to mass spectrometry is a ro-
bust strategy that facilitates elucidation of complex molec-
ular mechanisms by studying protein-ligand interactions
within signaling networks.10b, c To identify potential regula-
tors of Klip, we first designed and synthesized an AfBP,
KPlip (Fig. 1A), on the basis of a peptide sequence derived
from BCKDH protein (K105, QSDKlipASVT). In the probe de-
sign, the N-terminal Gln residue was replaced by a photo-
crosslinker. To synthesize KPlip, the peptide was firstly as-
sembled by standard Fmoc solid phase peptide synthesis
(SPPS) method. The Mtt group in the peptide was then
deprotected to allow the installation of a lipoyl group. Sub-
sequently a photo-cross-linker with diazirine was intro-
duced to the peptide (Fig. 1B). KPlip was further purified by
HPLC and characterized by LC-MS. For comparison study,
we also synthesized a control probe KPlip-C without the
lipoyl moiety (Fig. 1A).
In-gel labeling of KPlip. To further validate the interac-
tion between Klip and Sirt2, we first examined the labeling
efficiency of KPlip with recombinant Sirt2. The enzyme was
first incubated with either KPlip or a negative control probe
KPlip-C followed by 15 min of UV irradiation. Upon click
chemistry with rhodamine azide (Rh-N3), the reaction mix-
tures were resolved by SDS-PAGE and analyzed by in-gel
fluorescence scanning. The results (Fig. 2A) showed that
Sirt2 was labeled by KPlip efficiently, whereas no labeling
was observed in the control probe KPlip-C, suggesting that
Sirt2 interacts with the probe KPlip by specifically recog-
nizing the Klip moiety. Time-dependent experiments
showed that 2-min UV irradiation resulted in intense label-
ing (Fig. 2B). The fluorescence intensity of the labeled Sirt2
band increased when the concentrations of the probe in-
creased, demonstrating that probe KPlip labeled the target
protein in a concentration-dependent manner (Fig. 2C).
Further selectivity study revealed that probe KPlip labeled
Sirt2 much more strongly than other sirtuins did (Fig. 2D),
suggesting that Sirt2 recognizes lipoylated lysine more
preferentially than other sirtuins do. To examine whether
lipoic acid of KPlip forms disulfide bond with cysteine resi-
dues in Sirt2, excessive reducing reagent TCEP was added
to the solution containing Sirt2 and KPlip prior to UV irra-
diation. No diminished effect of Sirt2 labeling was observed
(Fig. 2E, Fig. S26C & D), suggesting the labeling of Sirt2 by
probe KPlip was not redox-based. Moreover, the labeling of
Sirt2 can be inhibited by a lipoylated peptide competitor
(VQSDKlipASVT, Fig. 1A) verifying that the interaction be-
tween Sirt2 and lipoylated peptide is selective and direct
(Fig. 2F).
With the two probes in hand, we next performed chemical
proteomics experiments (Fig. 1C), aiming to pull down po-
tential interacting proteins of KPlip in cellular environment.
To minimize “false hits” produced by non-specific protein
binding, probe KPlip-C was used in parallel as a negative
control. Briefly, probes (10 µM) were incubated with live
HEK 293 cells or lysates for 2 h. Following ultraviolet (UV)
irradiation, the KPlip/ KPlip-C labeled proteomes were
clicked with biotin-azide, enriched by affinity purification,
resolved by SDS-PAGE, and finally analyzed by LC-MS/MS.
Upon removal of non-specific binding proteins identified
from the control probe KPlip-C, only the proteins which
were positively identified in both lysate (Supplementary
Data 4) and live-cell (Supplementary Data 5) experiments
were chosen as potential targets. This led to a probe-selec-
tivity list of 419 protein candidates (Supplementary Data
2A).
Next, we performed a series of competition experiments to
further identify the high-specificity binders of KPlip (Fig.
1C). HEK 293 cell lysates were preincubated with DMSO or
a lipoylated peptide competitor (VQSDKlipASVT, Fig. 1A) at
100 µM. Subsequently the lysates were labeled with probe
KPlip (10 µM) by UV irradiation. The probe-labeled prote-
omes were then conjugated with biotin-azide, enriched by
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Pull down validation of KPlip. We first performed live to incubate with live HEK 293 cells for 2 h. The treated cells
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cell labeling with KPlip. As shown in Fig. 2G, several major
bands were fluorescently labeled. Probe KPlip-C and DMSO
control showed very faint or no labeling, indicating that
probe KPlip recognized these proteins in specific manner.
We next validate the physiological interaction of Klip and
Sirt2 using pull down experiments. 10 μM KPlip was used
were subsequently UV-irradiated and subjected to click re-
action with biotin-azide (Bio-N3). The cells were then lysed
and subjected to enrichment with streptavidin beads. The
enriched proteins were finally analyzed by western blotting
with anti-Sirt2. As shown in Fig. 2G, Sirt2 was successfully
pulled
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Figure 2. (A) Recombinant Sirt2 was labeled by probe KPlip but not by control probe KPlip-C. (B) Time-dependent UV irra-
diation labelling of KPlip with recombinant Sirt2. (C) Concentration-dependent labeling of KPlip with recombinant Sirt2 after
15 min of UV irradiation. (D) Labeling profiles of KPlip with recombinant Sirt1, Sirt2, Sirt3, Sirt5 and Sirt6. (E) Labeling of
Sirt2 by KPlip was not affected by reducing reagent TCEP. (F) Labeling of Sirt2 by KPlip was inhibited in a dose-dependent
manner by a lipoylated peptide competitor. A-F: enzyme loading: 0.33 μg/lane. (G) In-gel labelling of live HEK293 cells with
probe KPlip. The potential Sirt2 band was labeled with an asterisk. Protein loading: 16 μg/lane. The corresponding pull-down
(PD)/ Western blot (WB) results with anti-Sirt2 were shown at the bottom. Results show that endogenous Sirt2 can be pulled
down with probe KPlip but not probe KPlip-C.
down by KPlip. In contrast, Sirt2 was not observed in the
absence of probe or with the control probe (KPlip-C) (Fig.
2G). These results unambiguously proved that endogenous
Sirt2 can be pulled down by lipoylated peptides under na-
tive cellular environment. In addition, we also performed
pull-down assay to validate the binding of HDAC8/BRMS1L
to Klip. The results show that both proteins can be effec-
tively pulled down by KPlip (Fig. S28). This is consistent
with the LC-MS/MS results. These experiments indicate that
KPlip is a reliable tool for chemical proteomics to dissect
proteins interacting with lipoylated lysine.
reporting delipoylation activity, but at the same time it may
face the problem of substrate recognition of enzyme be-
cause the size of Klip is larger than that of Kac. In this study,
we designed a fluorogenic probe KTlip for profiling
delipoylation activity in vitro. As shown in Fig.3A, the probe
consists of a recognition group, Klip and an O-NBD moiety.
We hypothesize that when enzymes hydrolyze the lipoyl
group, the released amine will attack the O-NBD, yielding N-
NBD and turn on the fluorescence. (Fig. 3A, Scheme. S9).
Such a probe can report the delipoylation activity of en-
zymes continuously and reliably.
Delipoylation study with probe KTlip. After confirming
the interactions of Sirt2 and lipoylated peptide in cellular
context, we next investigate whether Sirt2 is capable of re-
moving lipoyl modification. To this end, we set out to de-
velop fluorescent probes to detect delipoylation activity.
Compared with mass spectrometry,13 radioisotopes,14 spe-
cific antibodies and HPLC,15 fluorescent probes possess
prominent advantages in detecting enzyme activity, such as
high sensitivity and simple procedure.16 Until now, no sin-
gle-step fluorescent probe has been developed to report
delipoylation activity. In fact, it is difficult to design single-
step fluorescent probes for detecting deacylation activity
because the aliphatic amide structure in Kacyl group does
not allow conjugation to a fluorophore.16d We have previ-
ously designed fluorescent probes to detect deacetylation
activity of HDACs through intramolecular reaction strat-
egy.9b We reasoned that this approach would be useful for
We first synthesized probe KTlip following the synthetic
route (Scheme. S2). We then examined the capacity of
HDACs to recognize and remove the lipoyl group of KTlip
by fluorimeter assay. Briefly, KTlip was incubated with var-
ious HDACs at 37 0C in HEPES buffer (pH 8.0). The fluores-
cence of the enzymatic reactions was then measured ac-
cordingly. As shown in Fig. 3C, Sirt2 showed the strongest
fluorescence increment, with 60-fold fluorescence increase.
Sirt1 showed much lower fluorescence signal, whereas
Sirt3, Sirt5, Sirt6 and HDAC8 did not show noticeable fluo-
rescence increase. The control group without cofactor NAD+
displayed negligible fluorescence, indicating the reaction
occurred through enzymatic catalysis. Further HPLC and
MS analysis confirmed that the molecular weight of the
newly generated peak corresponded to the expected tan-
dem delipoylated/exchanged product (Fig. S14 A & B). It
was noted that no delipoylated product was observed under
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Figure 3. (A) Schematic illustration of the fluorescence “turn-on” mechanism of probe KTlip through a delipoylation reaction.
(B) Synthetic scheme of lipoylated peptides KAlip-1 to KAlip-11 by SPPS. (C) Fluorescence measurement of KTlip (10 μM)
with sirtuins and HDAC8 (40 ng/μL) (ex = 480 nm). The enzymatic reaction product displayed green fluorescence (inset). (D)
Time-dependent fluorescence measurement of KTlip (10 μM) with Sirt2 (40 ng/μL), ex = 480 nm, em = 545 nm. (E) Lin-
eweaver-Burk plot analysis of KTlip with Sirt2. (F) HPLC analysis of the enzymatic reaction of representative peptide KAlip-
1 (40 μM) with Sirt2 (80 ng/µL). The reaction was monitored at 280 nm. The retention time of the newly generated peak
matched well with that of the peptide without lipoyl group. (G) Sirt2 delipolyated recombinant DLAT protein in the presence
of cofactor NAD+.
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Table 1. Delipoylation study of Sirt2 with various lipoylated peptides.
Peptide
Protein/Lysine
residue
Peptide sequence
Delipoylation
producta
kcat (s-1)
Km
(μM)
kcat/Km (s-1
M-1)
KAlip-1
KAlip-2
Histone, H3K9
Dps, K10
KQTARK(lip)STGGWW
VKSK(lip)ATNLWW
59%
97%
0.01 ±
0.0003
~
9.7 ±
0.2
~
1.06 × 103
~
KAlip-3
KAlip-4
KAlip-5
KAlip-6
KAlip-7
KAlip-8
KAlip-9
KAlip-10
KAlip-11
Pyruvate kinase,
K105
SDPIIK(lip)GSGTWW
SGASEK(lip)DIVHSGWW
EIETDK(lip)ATIGWW
EIETDK(lip)AVVTWW
EIETDK(lip) TSVQWW
VQSDK(lip)ASVTWW
EALPKK(lip) TGGPWW
ALESVK(lip)AASEWW
VEAMK(lip)LMNEWW
98%
100%
100%
100%
65%
~
~
~
~
~
GDH, K503
PDH, E2, K259
PDH, E3, K97
KDH, E2, K110
BCKDH, E2, K105
TNF-α, K20
~
0.061 ±
0.002
18.9 ±
0.6
3.26 × 103
~
~
~
~
~
~
1.72 × 103
~
100%
100%
71%
0.045 ±
0.0002
26.2 ±
3.3
~
~
Glycine cleavage
system, K107,
~
~
~
Biotin carboxyl car-
rier protein, K122,
94%
0.023 ±
0.0002
13.5 ±
1.4
1.67 × 103
a HPLC yield of delipoylated product was monitored at 280 nm after 40 mins.; ~ indicates data was undetermined.
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the enzymatic reaction condition for HDAC8 (Fig. S16). Af- Sirt2 remove Klip from lipoylated DLAT protein. To
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ter enzymatic reaction with Sirt2, a shift of peak absorption
from 380 nm to 480 nm was clearly observed. (Fig. S14C).
Through detailed kinetic study, the first order rate constant
of the reaction was determined to be 0.013 min-1 (Fig. 3D).
The Km value of KTlip obtained from the fluorescent
method matched well with that from the traditional HPLC
method (Fig. 3E & Fig. S15), underscoring that probe KTlip
can serve as a useful tool for detecting enzymatic delipoy-
lation activity. These data revealed that Sirt2 displays ro-
bust activity to remove lipoyl group in vitro.
test whether Sirt2 delipoylates protein substrates, recombi-
nant DLAT protein from mammalian cells was used for a
delipoylation assay. The Klip modification in DLAT was first
confirmed using Western blot with a lipoic acid antibody
(Fig. 3G). Subsequently DLAT was incubated with Sirt2 in
the presence of cofactor NAD+ and the lipoylation level of
DLAT was examined by Western blot (Fig. 3G). Negative
control with no NAD+ or no Sirt2 were included as well.
Comparing with the control groups, a substantial reduction
was observed in Klip level for DLAT treated with Sirt2 in the
presence of NAD+. These results indicated that Sirt2 has
delipoylation activity towards DLAT protein.
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Delipoylation study with various lipoylated peptides.
Encouraged by the above results, we next investigated
whether sirtuins exhibit substrate specificity in recognizing
different peptide sequences.17 To this end, we designed and
synthesized a total of 11 lipoylated peptides (KAlip-1 to
KAlip-11, Fig. 3B). The peptide sequences were selected
from reported lipoylated proteins (PDH, KDH, BCKDH and
GCV) and non-lipoylated proteins (e.g. histone) (Table S1).
The peptides were synthesized with standard Fmoc solid
phase synthesis approach and characterized by LC-MS. The
delipoylation activity was then determined by HPLC assay.
Sirt2 catalyzed the removal of lipoyl group for all the pep-
tide tested (Table1 & Fig. 3F & S19-24). Compared with non-
putative peptide, putative lipoylated peptides appear to be
more efficiently hydrolyzed by Sirt2. For example, the
delipoylated product of KAlip-1 (derived from histone) af-
ter 40 min was determined to be 59%, which is less than
that of putative lipoylated substrates such as KAlip-5 and
KAlip-6 (derived from PDH). It was noted that near abso-
lute delipoylation was observed for KAlip-5 and KAlip-8
within just 15 mins, signifying that Sirt2 catalyzed the re-
moval of lipoyl group with high efficiency. Furthermore,
control experiments without the addition of NAD+ and en-
zyme unambiguously proved that the hydrolysis reaction is
attributed to enzymatic activities rather than other environ-
mental factors (Fig. S19-24). In addition, the newly gener-
ated peak of KAlip-1 with Sirt2 showed the same retention
time as the unmodified peptide (Fig. 3F). We also further ex-
amined HDAC8’s delipoylation activity using KAlip-5 and
KAlip-1, no delipoylated product was observed (Fig. S17).
The enzymatic delipoylation results by fluorescent probe
KTlip and lipoylated peptide KAlip-5 and KAlip-1 together
indicated that HDAC8 has no delipoylation activity.
Figure 4. (A) Schematic diagram illustrating PDH catalyzes
the conversion of pyruvate to acetyl-CoA, which is linked to
TCA cycle. We hypothesize that PDH activity might be inhib-
ited by Sirt2 through delipoylation. (B) Relative PDH activ-
ity comparison between Hela-S3 cells overexpressing Sirt2
and wild-type cells. PDH activity was measured by a com-
mercial colorimetric assay. (C) Western blot analysis of en-
dogenous lipoylated DLAT of PDH in cells over-expressing
Sirt2 versus wild-type cells. DLAT is used as loading control.
Sirt2 delipoylates PDH in living cells. Having shown
that Sirt2 displayed strong delipoylation activity in vitro, we
next investigated whether Sirt2 could delipoylate the phys-
iological substrate in living cells. Lipoylated PDH is respon-
sible for converting pyruvate into acetyl-CoA, a central me-
tabolite that enters into TCA cycle.3c, 4 On the other hand, the
delipoylation of PDH impairs its activity (Fig. 4A). Although
the cellular localization of Sirt2 was conventionally anno-
tated to be in cytosol/nucleus, recent literature suggests
that Sirt2 could localize to mitochondria and regulate the
functions of mitochondrial proteins.18 In line with this, Sirt2
was detected in the mitochondrial fraction isolated from
HeLa S3 cells overexpressing Sirt2 (Fig. S29). Thus, we next
endeavored to investigate whether Sirt2 can delipoylate
PDH and regulate its function in living cells. To conduct the
test, we overexpressed Sirt2 in Hela S3 cells by transfecting
cells with pCMV4a-Sirt2 vector. After transfection, the cells
were harvested and lysed. Western blot experiments con-
firmed effective Sirt2 overexpression (Fig. 4C). The ob-
tained cell lysates were then subjected to PDH activity assay.
As shown in Fig 4B, PDH activity in cells overexpressing
Sirt2 was significantly decreased comparing to that in wild
type cells. In line with this, the lipoylation level of DLAT (E2
To have a more detailed assessment of the enzymatic
delipoylation reaction, we performed time-dependent ex-
periments and steady-state kinetics studies of KAlip-1, 5, 8
and 10 with Sirt2 (Fig. S25, Table 1 & S2). Interestingly, ki-
netics data (Table 1) revealed that the catalytic efficiency
(kcat/Km) for delipoylation activity of Sirt2 is of the same or-
der of magnitude as that of the deacetylation of Sirt1-2,12c
the desuccinylation/demalonylation of Sirt5 and the de-
myristoylation of Sirt6 in vitro (Table S2).12b, c The catalytic
efficiency of Sirt2 toward the putative lipoylated peptides
(KAlip-5, 8, 10) was slightly higher than toward the non-
putative H3K9 lipoyl peptide (KAlip-1). Strikingly, the cat-
alytic efficiency of Sirt2 (kcat/Km) to delipoylate DLAT K259
of PDH complex (KAlip-5) is approximately 426-fold higher
than that of Sirt4 (Table S2).3c Taken together, these in vitro
enzymology data demonstrated that Sirt2 is a robust
delipoylating enzyme.
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subunit of PDH) was decreased in cells overexpressing Sirt2, belled by KPlip and KPlip-C. Tris(2-carboxyethyl)phos-
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whereas the total level of DLAT remained unchanged (Fig.
4C). Furthermore, we performed siRNA knock-down exper-
iments to examine the regulatory effect of endogenous Sirt2
on the lipoylation of PDH. Effective siRNA-mediated knock-
down of Sirt2 was confirmed (Fig. S30B). Notably, Sirt2
knockdown led to an elevated lipoylation level in DLAT (Fig.
S30B). Concomitantly, increased PDH activity was also ob-
served (Fig. S30A). These results together prove that Sirt2
can effectively catalyze DLAT delipoylation and modulate
PDH activity in cells.
phine (0.4 mM), Tris(3-hydroxypropyltriazolylme-
thyl)amine (40 μM) and CuSO4 (0.4 mM) were then added.
The reaction was kept at room temperature for 2 h.
Affinity Enrichment of Biotinylated Proteins. After click
chemistry reaction of KPlip or KPlip-C using biotin-azide
(Bio-N3), chilled acetone was added to precipitate the pro-
teins. The pellets were then washed with cold methanol and
air-dried. The obtained pellets were re-dissolved in PBS
buffer containing less than 0.5% sodium dodecyl sulfate
(SDS) by heating and vortexing. Streptavidin magnetic
beads were then added and shaken gently at room temper-
ature for 4 h. The beads were collected by a magnet and
washed with 0.1% SDS/PBS once and PBS twice. The beads
were re-dissolved in 1 x SDS loading buffer and proteins
were released/denatured by boiling at 95 °C for 20 min.
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In conclusion, we have successfully developed a panel of
chemical probes to investigate the regulatory mechanism of
lysine lipoylation. The developed AfBP probe KPlip is capa-
ble of capturing Klip-interacting proteins in both living cells
and cell lysates. Notably, chemical proteomics experiments
with KPlip identified Sirt2 as a novel binder of lipoylated
substrate. KTlip is the first single-step fluorescent probe
developed for rapid profiling of delipoylation activity. The
enzymology data obtained from both KTlip and lipoylated
peptides demonstrated the robust delipoylation ability of
Sirt2 in vitro. It is noteworthy that the delipoylation activity
of Sirt2 is far superior to that of Sirt4, the only identified
mammalian delipoylating enzyme. Through our chemical
probes, we proved the novel function of Sirt2 to remove
lipoyl group with high catalytic efficiency. Furthermore, we
also showed that Sirt2 could effectively catalyze DLAT
delipoylation and downregulate PDH activity in cells. It is
noted that a recent report showed that Sirt3 could enhance
PDH activity through deacetylating the E1 subunit.19 This
suggests that sirtuins might play a complex role in the dy-
namic regulation of PDH activity through different deacyla-
tion mechanisms. With the probes developed in this study,
we envision that they will provide useful tools to further ad-
vance our understanding of lipoylation and other acylation
in biology and diseases.
In-gel digestion by trypsin. The pull-down sample was
separated on 12% SDS-PAGE gel and stained with Ruby pro-
tein staining method. The gel was cut into different pieces.
The collected gel was washed by 100 mM ammonium bicar-
bonate buffer, reduced by DTT (5 mM) at 50-60 °C for 30
minutes, alkylated by iodoacetamide (25 mM) at room tem-
perature for 45 minutes, dehydrated by 95% ethanol, di-
gested by sequencing grade modified trypsin at 37 °C for
overnight. After that, the digested solution was collected.
The gel was further extracted twice using 50% acetonitrile
containing 5% formic acid. The combined solution was
dried by speed vacuum for LC-MS/MS analysis.
LC-MS/MS Analysis. Upon extraction from the gel and de-
salting, the obtained peptides were redissolved in 12 μl of
0.1% formic acid and separated by an Easy-nLC 1200 sys-
tem coupled to a Q Exactive HF (Thermo Scientific). 5 μl of
peptides were injected and separated on a reverse phase
C18 column (75 μm × 15 cm) at a flow rate of 250 nL/min.
Mobile phase A (0.1% formic acid in ultrapure water) and
mobile phase B (0.1% formic acid and 80% acetonitrile in
ultrapure water) were used to establish a linear gradient of
7–25% mobile phase B in 50 min. Peptides were then ion-
ized by electrospray at 1.5 kV. The mass spectrometer was
operated in positive ion mode at a resolution of 120,000,
with a full MS spectrum (m/z = 350-1800) using an auto-
matic gain control (AGC) target of 3×106. Higher-energy col-
lisional dissociation (HCD) fragmentation was conducted
with the 12 most intense ions (normalized collision energy
27). MS/MS spectra were acquired with an AGC target of
1×105 at a resolution of 30,000. The dynamic exclusion time
was set to 30 s.
Materials and Methods
General Information. Sirtuins including Sirt1, Sirt2, Sirt3,
Sirt5 and Sirt6, were recombinantly expressed and purified
according to previous reports.12b, c, 17 Pyruvate Dehydrogen-
ase E2 (DLAT) (NM_001931) human recombinant protein
was from ORIGENE. Streptavidin Magnetic Beads were pur-
chased from New England Biolabs. In-gel fluorescence scan-
ning experiments were performed with a FLA-9000 Fujifilm
scanner. Antibody of Sirt2 (D4S6J) was from Cell Signaling.
Anti-bodies of DLAT (ab172617), lipoic acid (ab58724),
HDAC8 (ab187139), BRMS1L (ab107171) and Hsp60
(ab128567) were from Abcam. IRDye 680RD Donkey anti-
Rabbit IgG (Secondary antibody) was purchased from LI-
COR Biosciences. Immobilon-FL poly(vinylidene difluoride)
membrane for western blotting was purchased from Merck
Millipore. Western blotting was carried out with a C600 Az-
ure biosystem. Sirt2 siRNA (AM16708) was from Thermo
Fisher Scientifc. The plasmid pCMV4a-SIRT2-Flag was pur-
chased from Addgene (Plasmid #102623).20 The sequenc-
ing grade modified trypsin was purchased from Promega.
Proteomics Analysis. The raw data were created by XCal-
ibur 4.0.27 (Thermo Scientific) software and processed
with Proteome Discoverer (PD) software suite 2.2 (Thermo
Scientific), against UniProt human protein database (down-
loaded on 20171010) in Sequest HT node. The precursor
and fragment mass tolerances were set to 10 ppm and 0.02
Da, respectively. Reversed database searches were used to
evaluate false discovery rate (FDR) of protein and peptide
identifications. A maximum of two missed cleavage sites of
trypsin was allowed. Carbamidomethylation (C) was set as
static modification, and oxidation (M) and acetyl (N-termi-
nus) were set as variable modifications. False discovery rate
Cu(I)-Catalyzed Cycloaddition/Click Chemistry. Briefly,
20 μM azide reagent was added to the protein samples la-
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(FDR) of peptide spectrum matches (PSMs) and peptide Determination of the first-order rate constant k. It was
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identification were determined using the Percolator algo-
rithm at 1% based on q-value. The abundance values of pro-
teins were obtained via a label-free quantification method
using Proteome Discoverer 2.2.21 Minora Feature alignment
and feature mapping were applied to calculate the abun-
dance of peptides in the MS1 scan. Lists of protein identifi-
cations and quantification are attached in Supplementary
Data2.
calculated by fitting the fluorescence data to the following
equation:
Fluorescence intensity = 1 – exp (-kt).
Enzymatic reaction with lipoylated peptides. The lipoy-
lated peptides KAlip-1 to 11 was incubated with sirtuin and
cofactor NAD+ at 37 °C in 20 mM HEPES buffer (pH 8.0) con-
taining 150 mM NaCl, 1 mM MgCl2 and 2.7 mM KCl. The re-
action volume was set as 50 μL. At each specific reaction
time point, the reaction mixtures were quenched by adding
250 μL of methanol. The reactions were vortexed and cen-
trifuged. Supernatant was collected and then analyzed by
reverse phase HPLC. The new peak generated was collected
for ESI-MS or MALDI-TOF-MS analysis directly.
9
Bioinformatic analysis. For protein functional associa-
tions and protein–protein interaction analysis, we used the
web-based analytic tools. Gene ontology analysis was per-
formed in Metascape 3.0 (http://metascape.org) to explore
GO terms and enrichment analysis of target proteins.22
Terms with a p-value< 0.01, a minimum count of 3 and en-
richment factor >1.5 (the enrichment factor is the ratio be-
tween the observed counts and the counts expected by
chance) was set as the cut-off criteria. Subsequently, we
constructed the protein interaction biological networks of
identified proteins based on the STRING 11.0 database
(http://www.string-db.org) under the standard settings.23
In addition, the network data was exported to simple tabu-
lar text. The text was used to build protein interaction net-
works, which were then visualized and evaluated in Cyto-
scape software (v.3.7.1).24
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Kinetic study with lipoylated peptides. To determine the
values of kcat and Km, Purified Sirt2 with 400 μM NAD+ was
incubated with different concentrations of lipoylated pep-
tide (0-120 μM) in 20 mM HEPES buffer (pH 8.0) containing
150 mM NaCl, 1 mM MgCl2 and 2.7 mM KCl at 37 °C for 10
min (KAlip-1 and KAlip-10) or 5 min (KAlip-5 and KAlip-8).
The reactions were quenched by adding 250 μL of methanol
and then applied for HPLC analysis with a linear gradient of
5% to 85% B (acetonitrile) for 30 min. The generated
delipoylated product was quantified based on the peak area
monitored at 280 nm. The Km and kcat values were calculated
by curve-fitting Vinitial/[E] versus [S]. The experiments were
conducted in duplicate.
In-Gel Fluorescence Scanning. After click chemistry reac-
tion with rhodamine-azide (Rh-N3), chilled acetone was
added to the reaction mixtures. The reaction was then
placed in -20 °C freezer for 1 h to precipitate the proteins.
The pellets were washed with cold methanol and air-dried.
Finally, they were re-dissolved in 1 x SDS loading buffer by
heating at 95 °C for 10 min, and then resolved by SDS-PAGE.
The labeled proteins were visualized using a FLA-9000 Fu-
jifilm scanner (Ex = 532 nm).
PDH activity assay. To overexpress Sirt2 in cells,
pCMV4aSirt2-Flag vector were transfected into Hela-S3
cells using LipofectamineTM 2000 (Invitrogen). The activity
of PDH was assessed by measuring absorbance at 450 nm
using a microplate assay kit (Pyruvate dehydrogenase En-
zyme Activity Microplate Assay, Abcam, ab109902). 1000
μg of cell protein extracts were used for PDH immunocap-
ture in each well. The experiments were performed in du-
plicate.
Western Blotting. To perform Western blotting experi-
ments, samples were first separated by SDS−PAGE and then
transferred to an Immobilon-FL polyvinylidene difluoride
(PVDF) membrane. The samples were blocked with 5%
non-fat milk in TBST (0.1% Tween in Tris-buffered saline)
at room temperature for 1.5 h. Subsequently the membrane
was incubated with primary antibody (anti-sirt2/anti-lipoic
acid/anti-DLAT) at 4 °C overnight. After incubation, the
membrane was washed with TBST (3 × 5 min) gently at
room temperature. Following that the membrane was incu-
bated with the secondary antibody (IRDye 680RD Donkey
anti-Rabbit IgG) at room temperature for another 1 h. It was
then washed with TBST (3 × 5 min). Finally, the membrane
was applied to fluorescence scanning.
Mitochondria isolation. The mitochondrial fraction was
isolated according to the manufacturer’s instructions using
a mitochondria isolation kit (Thermo Fisher, Cat. No.
89874). The experiments were performed in duplicate.
ASSOCIATED CONTENT
Supporting Information. The Supporting Information is avail-
able free of charge on the ACS Publications website at DOI:
Corresponding Author
L. Zhang, liangzhang.28@cityu.edu.hk; Q. Hao, qhao@hku.hk;
H.Sun, hongysun@cityu.edu.hk,
Absorption and fluorescence study of probe KTlip. The
probe KTlip was incubated with sirtuin and NAD+ at 37 °C
in 20 mM HEPES buffer (pH 8.0) containing 150 mM NaCl, 1
mM MgCl2 and 2.7 mM KCl. The enzymatic reaction volume
was 50 μL. When the enzymatic reaction was complete, the
reaction was applied for absorption and fluorescence meas-
urement. The parameters set for absorbance measurement
were: UV-Visible light, collection region: 300– 550nm. The
parameters set for fluorescence measurement were: ex =
480 nm, slit width: 5 nm, collection region: 510 – 600 nm.
Author Contributions
†These authors contributed equally to this work
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We are grateful for the financial support from the Research
Grants Council of Hong Kong ((Nos. 11304118, 11302415 and
C7037-14G)), the National Natural Science Foundation of China
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Journal of the American Chemical Society
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(No. 21572190 and 21778044), Shenzhen Science and Tech-
nology Program (JCYJ20170413141047772,
JCYJ20180507181659781), the Synthetic Biology Research &
Development Programme (SBP) of National Research Founda-
tion (SBP-P4 and SBP-P8) of Singapore.
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ABBREVIATIONS
HDAC, histone deacetylase; Sirt2, Sirtuin 2; KPlip: activity
based protein profiling probe for lipoylated lysine proteome
profiling; KTlip: fluorescent turn-on probe for delipoylation
activity profiling; KAlip: lipoylated peptides for delipoylation
activity profiling
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(8) Pannek, M.; Simic, Z.; Fuszard, M.; Meleshin, M.; Rotili, D.; Mai,
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mitochondrial deacylase Sirtuin 4 reveal isoform-specific acyl
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