Site-Selective Synthetic Acylation of a Target Protein in Living Cells Promoted by a Chemical Catalyst/Donor System

Article abstract of DOI:10.1021/acschembio.9b00102

Cell biology is tightly regulated by post-translational modifications of proteins. Methods to modulate post-translational modifications in living cells without relying on enzymes or genetic manipulation are, however, largely underexplored. We previously reported that a chemical catalyst (DSH) conjugated with a nucleosome-binding ligand can activate an acyl-CoA and promote site-selective lysine acylation of histones in test tubes. In-cell acylation by this catalyst system is challenging, however, mainly due to the low cell permeability of acyl-CoA and the propensity of DSH to form inactive disulfide. Here, we report a new catalyst system effective for in-cell acylation, comprising a cell-permeable acyl donor and pro-drugged DSH. Using E. coli dihydrofolate reductase and trimethoprim as a model protein and ligand pair, the catalyst system enabled site-selective acylation of the target protein in living cells. The findings will lead to the development of useful chemical biology tools and new therapeutic strategies capable of synthetically modulating post-translational modifications.

Full text of DOI:10.1021/acschembio.9b00102

Letters
pubs.acs.org/acschemicalbiology
Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX
Site-Selective Synthetic Acylation of a Target Protein in Living Cells
Promoted by a Chemical Catalyst/Donor System
Wataru Hamajima,† Akiko Fujimura,† Yusuke Fujiwara, Kenzo Yamatsugu,*
Shigehiro A. Kawashima,* and Motomu Kanai*
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
S
* Supporting Information
ABSTRACT: Cell biology is tightly regulated by post-
translational modifications of proteins. Methods to modulate
post-translational modifications in living cells without relying
on enzymes or genetic manipulation are, however, largely
underexplored. We previously reported that a chemical
catalyst (DSH) conjugated with a nucleosome-binding ligand
can activate an acyl-CoA and promote site-selective lysine
acylation of histones in test tubes. In-cell acylation by this
catalyst system is challenging, however, mainly due to the low
cell permeability of acyl-CoA and the propensity of DSH to
form inactive disulfide. Here, we report a new catalyst system
effective for in-cell acylation, comprising a cell-permeable acyl
donor and pro-drugged DSH. Using E. coli dihydrofolate reductase and trimethoprim as a model protein and ligand pair, the
catalyst system enabled site-selective acylation of the target protein in living cells. The findings will lead to the development of
useful chemical biology tools and new therapeutic strategies capable of synthetically modulating post-translational
modifications.
enzymes is required for treating cancer cells with a loss of
rotein post-translational modifications (PTMs) increase
Pthe functional diversity of the proteome by the covalent function mutations in KAT genes.7
To date, several chemical biology approaches for producing
addition or removal of functional groups or proteins. These
modifications include phosphorylation, glycosylation, ubiquiti-
nation, sumoylation, methylation, and acetylation, all of which
are involved in almost all aspects of normal cell biology and
pathogenesis. Understanding the functions and regulations of
each PTM is thus important in the study of cell biology as well
as the development of disease-treating drugs. Among them,
acetylation of lysine residues has emerged as one of the most
abundant protein PTMs.1 Extensive studies have shown that
acetylation of lysine residues in histones, the major protein
component of chromatin, is a critical regulatory element in
activating gene transcription. Proteomic studies have revealed
that more than 1000 nonhistone proteins, involved in diverse
cellular processes, are also modified by acetylation.2 In addition
to the acetyl group, longer acyl chains including propionyl,
butyryl, crotonyl, and myristoyl or acidic acyl groups, such as
malonyl, succinyl, and glutaryl, have been identified in histones
and nonhistone proteins.3,4 Two different groups of enzymes,
lysine acetyltransferases (KATs) and lysine deacetylases
(KDACs), regulate lysine acylation levels.4,5 So far, five
KDAC inhibitors have been approved for the treatment of
cancer, especially T-cell lymphoma,6 indicating that increasing
lysine acylation is a promising anticancer strategy. However, as
the effects of KDAC inhibitors depend on the balance between
KATs and KDACs, an alternative strategy to bypass these
proteins with a desired PTM have been developed. The genetic
code expansion approach uses orthogonal aminoacyl-tRNA
synthetase (aaRS)/tRNA pairs for site-specific incorporation of
unnatural amino acids, including natural amino acids with
PTMs, into proteins both in vitro and in cells.8 Native chemical
ligation (NCL) enables ligation between the thiol group of an
N-terminal cysteine residue of a polypeptide and the C-
terminal thioester of a synthetic peptide, to generate
recombinant proteins with specific PTMs in the N-terminal
region.9 Expressed protein ligation (EPL) connects a
polypeptide carrying a C-terminal thioester that can be
prepared using the self-splicing protein intein with a synthetic
peptide; this can produce recombinant proteins with specific
PTMs in the C-terminal region in vitro and in cells.10−12
Recombinant proteins with site-specific PTMs can also be
generated by direct transformation of specific residues in
proteins through converting them to a dehydroalanine residue,
followed by radical-mediated carbon−carbon bond forma-
tion.13,14 These approaches, however, require genetic manip-
ulation to introduce the protein PTMs in cells, and thus it is
challenging to use therapeutically.
Received: February 6, 2019
Accepted: May 20, 2019
© XXXX American Chemical Society
A
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Figure 1. Schematic illustration of enzymatic lysine acylation and synthetic lysine acylation by DSH catalyst system.
A chemical catalyst system, which can promote a desired
protein PTM without genetic manipulation, may have
potential for therapeutic use.15 We reported two chemical
catalyst systems that synthetically modulate histone acylation
states without relying on enzymes.16,17 One, a novel
nucleophilic catalyst DSH, activates chemically stable thio-
esters, including acyl-CoA, under physiological conditions
through a dynamic thiol-thioester exchange and intramolecular
nucleophilic activation. The Hamachi group has developed
ligand-directed chemistry for selective labeling of endogenous
proteins.18−22 Similarly, a DSH catalyst conjugated with a
suitable targeting ligand can introduce acyl groups into specific
lysine residues on target proteins.16 For example, the LANA-
DSH catalyst, in which a DSH catalyst is conjugated with a
LANA (latency-associated nuclear antigen) N-terminal pep-
tide, binds to the acidic patch of nucleosomes and efficiently
promotes histone- and site-selective acylation of the proximate
H2BK120 residue in test tubes.16 However, in-cell acylation by
the DSH catalyst system has not been achieved yet. Critical
problems may include the low cell permeability of acyl-CoA
and the formation of disulfide dimers from DSH under aerobic
oxidation. In this study, we developed a new DSH catalyst
system for site-specific lysine acylation in living cells using E.
coli dihydrofolate reductase (eDHFR) and trimethoprim
(TMP) as a model protein and ligand pair.
RESULTS AND DISCUSSION
■
Figure 1 shows a comparison between endogenous enzymatic
protein acylation and synthetic protein acylation by a chemical
catalyst system. In living cells, KATs catalyze lysine acylation.
B
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Scheme 1. (a) Chemical structures of TMP-DSH and (b) Synthesis of TMP-DSH
KATs form a ternary complex (KAT/acyl-CoA/target protein)
before catalysis can occur. KATs enhance the reactivity of the
amino groups of lysine residues, which enables direct acyl
transfer from acyl-CoA to a lysine residue on the target
protein.23 In contrast, the chemical catalyst DSH accepts an
acyl group from acyl-CoA through dynamic thiol−thioester
exchange (A).16 The acyl group is then activated by the
generation of an acylpyridinium intermediate (B) through S-
to-N transfer; this intermediate then reacts with the proximal
lysine to produce an acylated lysine. Conjugation of the DSH
catalyst with a ligand for a target protein allows for the ligand-
dependent protein- and lysine residue-selective acylation. So
far, synthetic lysine acylation by the DSH catalyst has been
limited to reactions in test tubes, due to at least two problems:
first, acyl-CoA is a membrane-impermeant molecule and
cannot access DSH in the cells if subjected from outside of
the cells; second, DSH is easily oxidized to form a disulfide
dimer outside the cell, and the dimer is likely less membrane-
permeable than the monomer due to its increased molecular
size. Additionally, high concentrations of various proteins and
nucleophiles (e.g., glutathione) in the cell may reduce the
catalytic activity of DSH.
To develop a DSH catalyst system that functions in living
cells, we studied synthetic acylation of eDHFR using TMP as a
ligand, whose interaction is orthogonal to mammalian
systems.24 Because DSH transfers an acyl group only to
C
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Figure 2. In vitro acetylation of eDHFR-GFP using TMP-DSH. (a) eDHFR-GFP (5 μM) was reacted with TMP-DSH (S, M, L, 50 μM) in the
presence of acetyl-CoA (1 mM) and Tris(2-carboxyethyl)phosphine (TCEP, 200 μM) at 25 °C for 2 h, and lysine acetylation was detected by
immunoblotting using antiacetyl lysine antibody. Proteins were visualized by Coomassie Brilliant Blue (CBB) staining. (b) A mixture of eDHFR-
GFP (0.5 μM) and HEK293T cell extract was incubated with TMP-M-DSH (50 μM) and acetyl-CoA (1 mM) in the presence of TCEP (200 μM)
at 25 °C for 2 h. Proteins were visualized by Oriole staining. (c) eDHFR-GFP (5 μM) was treated with acetyl-CoA (1 mM) and TCEP (200 μM)
with and without TMP-M-DSH (50 μM) at 25 °C for 1−16 h. Acetylation yield of six lysine residues in eDHFR at 16 h is shown in the left panel.
Each number on the x-axis indicates the position of a lysine residue in eDHFR. Acetylation yield of K32 in eDHFR at 1, 2, 4, 8, and 16 h is shown
in the right panel. The list of chemical yields is shown in Table S1. Error bars represent range of two independent experiments (*not detected). (d)
eDHFR(WT or K32R)-GFP (5 μM) was reacted with TMP-M-DSH (50 μM) in the presence of acetyl-CoA (1 mM) and TCEP (200 μM) at 25
°C for 2 h, and lysine acetylation was detected by immunoblotting using antiacetyl lysine antibody. Proteins were visualized by CBB staining. (e)
The X-ray structure of the eDHFR-MTX ligand complex (PDB: 3DAU). Lysine residues are indicated in magenta.
proximal lysines, the linker length between TMP and DSH
should affect the acylation efficiency of eDHFR. Therefore, we
designed three types of TMP-DSH catalysts, in which TMP
and DSH were connected by a linker with one, two, or three
ethylene glycol unit(s) (refer to TMP-S-DSH 1, TMP-M-DSH
2, or TMP-L-DSH 3, respectively; Scheme 1a). The three
catalysts were synthesized through the synthetic routes shown
in Scheme 1b. Monodemethylated TMP (4) was O-alkylated
with linkers of different lengths to afford TMP-linker
conjugates with protected amino termini. The tert-butox-
ycarbamate group or the azido group was converted to an
amino group through either acidic treatment or hydrogenation,
respectively, to afford TMP-linker-amines (5, 7, and 8). These
amines were condensed with carboxylic acid 6 containing S-
protected DSH, and subsequent removal of the trityl group
under acidic conditions provided the TMP-DSH catalysts of
varying linker length (1, 2, and 3).
We conducted in vitro acetylation experiments to evaluate
which TMP-DSH catalyst is optimal for acetylation of eDHFR.
Recombinant eDHFR-GFP protein was prepared as a
substrate. When eDHFR-GFP was incubated with acetyl-
CoA only, acetylation of eDHFR-GFP was barely detected by
antiacetyl lysine antibody (Figure 2a). The addition of TMP-
M-DSH efficiently promoted eDHFR-GFP acetylation (Figure
2a). Interestingly, neither TMP-S-DSH nor TMP-L-DSH
promoted eDHFR-GFP acetylation efficiently (Figure 2a),
indicating that linker length between TMP and DSH is
important and that TMP-M-DSH is the optimal catalyst. To
evaluate the selectivity between eDHFR and other proteins, we
mixed eDHFR-GFP with a purified HEK293T cell extract
containing cytoplasmic and nucleoplasmic proteins and used
the mixture as a substrate. Remarkably, only a single band,
corresponding to eDHFR-GFP, was detected on the
immunoblotting when both TMP-M-DSH and acetyl-CoA
were added to the mixture (Figure 2b), indicating that TMP-
M-DSH can promote eDHFR-selective acetylation.
Next, we examined site-selectivity of the acetylation among
the six lysine residues (K32, K38, K58, K76, K106, and K109)
in eDHFR. After the reaction, unreacted lysine residues were
thoroughly propionylated. The resulting chemically modified
eDHFR-GFP was digested by trypsin and Glu-C peptidases,
and the peptide fragments were evaluated by liquid
chromatography-tandem mass spectrometry (LC−MS/MS).
We found that K32 was exclusively acetylated by TMP-M-
DSH and acetyl-CoA, while acetyl-CoA only did not promote
acetylation at any residues (Figure 2c, Table S1). Time-course
analysis of the reaction progress showed that the yield of the
K32 acetylation product increased linearly until ∼4 h and
reached a plateau at 8−16 h (Figure 2c, maximum yield =
93.72
1.76%). When K32 in eDHFR was mutated to
arginine, no acetylation band was detected by the immuno-
blotting, confirming that K32 is the sole acetylation site in
eDHFR-GFP by using the TMS-M-DSH/acetyl-CoA system
(Figure 2d). On the basis of the X-ray structure of the eDHFR-
MTX ligand complex (MTX and TMP bind to the same site of
eDHFR), K32 is proximate to the ligand binding pocket
D
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Figure 3. Structural modifications of acyl-donor and DSH catalyst for target protein- and lysine-selective acylation in living cells. (a) eDHFR-GFP-
transfected HEK293T cells were incubated with or without acetyl-CoA (10 mM) or NAC-Ac (10 mM), and TMP-M-DSH (25 μM) or TMP-M-
DSSMe (25 μM), at 37 °C for 5 h. Acetylation yield of K32 in eDHFR is shown. (b) Chemical structures of acetyl-CoA (9), NAC-Ac (10), NAC-
EtMal (11), NAC-N3Ac (12), and TMP-M-DSSMe (13). (c) TMP-M-DSH (50 μM) in HEPES buffer (25 mM, pH 7.6) was incubated at 37 °C.
Ratios of the disulfide dimer at 0, 0.5, 1, 2, and 4 h were determined by HPLC (UV, 254 nm; upper). TMP-M-DSSMe (50 μM) in HEPES buffer
(25 mM, pH 7.6) was incubated at 37 °C. After 4 h, glutathione (5 mM) was added and incubated for a further 0.5 h. Yields at 0, 0.5, 1, 2, 4, and
4.5 h determined by HPLC (UV, 254 nm; lower). Error bars represent range of two independent experiments. (d) eDHFR-GFP-transfected
HEK293T cells were incubated with or without NAC-Ac (10 mM) and/or TMP-M-DSSMe (25 μM) at 37 °C for 5 h. Acetylation yield of six
lysine residues in eDHFR is shown. Each number on the x-axis indicates the position of a lysine residue in eDHFR. (e) eDHFR-GFP-transfected
HEK293T cells were incubated with or without TMP-M-DSSMe (25 μM) and NAC-EtMal (10 mM) or NAC-N3Ac (10 mM) at 37 °C for 5 h.
Acylation yield of K32 in eDHFR was shown. (f) HEK293T cells were transfected with or without eDHFR(WT or K32R)-GFP expression
plasmids and treated with or without NAC-EtMal (10 mM) and TMP-M-DSSMe (25 μM) at 37 °C for 5 h. Malonylated lysines (Mal-Lys) and
eDHFR-GFP (GFP) were detected by immunoblotting. Proteins were visualized by CBB staining. For LC−MS/MS data, error bars represent the
range of two independent experiments. The list of chemical yields is shown in Tables S2−S4 (*not detected).
E
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(Figure 2e). This indicates that eDHFR-bound TMP-M-DSH
can reach K32, but not other lysine residues, and thus TMP-
M-DSH selectively acetylates K32 in eDHFR.
acetylcysteamine (NAC-EtMal 11 in Figure 3b) as a nonacetyl
acyl donor. The ethyl ester moiety would be cleaved by
esterase in cells, and thus NAC-EtMal would function as a
malonyl donor. In fact, a previous study showed that NAC-
EtMal can cross cell membranes and promote lysine
malonylation in a nonenzymatic manner.25 Treatment of
HEK293T cells expressing eDHFR-GFP with NAC-EtMal
only (10 mM) for 5 h did not significantly increase eDHFR-
K32 malonylation (Figure 3e, Table S4). Remarkably, addition
of TMP-M-DSSMe promoted both eDHFR-K32 malonylation
and eDHFR-K32 ethyl malonylation (Figure 3e, Table S4).
The yield of malonylated eDHFR-K32 was about one-third of
that of ethyl malonylated eDHFR-K32, suggesting that esterase
activity in HEK293T cells was not sufficient to cleave all ethyl
ester moieties. We also synthesized S-azidoacetyl-N-acetylcys-
teamine (NAC-N3Ac 12 in Figure 3b) as a non-natural acyl
donor. Like acetylation and ethyl malonylation, TMP-M-
DSSMe promoted eDHFR-K32 azidoacetylation in living cells
(Figure 3e, Table S4).
To evaluate whether TMP-M-DSSMe selectively promoted
acylation of eDHFR in living cells, we first conducted
immunoblotting analysis using antimalonyl lysine antibody.
As expected, TMP-M-DSSMe with NAC-EtMal efficiently
promoted acylation of eDHFR-GFP, especially at K32 (Figure
3f). Since antibodies often can have sequence bias, and perhaps
other modification sites are not efficiently detected, we also
quantitatively evaluate the protein and lysine residue selectivity
of our catalyst system in living cells by determining and
comparing the yield of acylation in several proteins using LC−
MS/MS. We transfected not only eDHFR-GFP but also
HaloTag-GFP into HEK293T cells. After the acylation
reaction, eDHFR-GFP and HaloTag-GFP were immunopreci-
pitated from cell extracts by anti-GFP antibodies. In addition,
endogenous Hsp90 was immunoprecipitated by Hsp90
antibodies. These three purified proteins were subjected to
LC-MS/MS analysis to quantify the yield of acylation at several
lysine residues. TMP-M-DSSMe promoted eDHFR-K32
acylation (∼30% yield), while acylation at K38, K58, and
K76 of eDHFR or acylation at any lysine residues in HaloTag
or Hsp90 was not detected at all (Figure S3a−c). These data
indicate that TMP-M-DSSMe selectively promoted acylation
of eDHFR in living cells.
Finally, we applied our ligand-DSH system to other proteins
to show its substrate generality. We designed and synthesized
new catalysts targeting HaloTag as well as endogenous Hsp90
proteins. Cl-DSSMe (compound 16 in Figure S3d) is expected
to bind with HaloTag;26 PU-DSSMe (compound 17 in Figure
S3d) is expected to bind with Hsp90.27,28 None of the catalysts
or acyl donors showed toxicity to HEK293T cells (Figure
S4a,b). We conducted acylation reactions using these new
catalysts and quantified the yield of acylation in eDHFR,
HaloTag, and endogenous Hsp90 in living cells. Cl-DSSMe
promoted HaloTag-K160 acylation (∼25% yield), while
acylation at K71, K117, and K124 of HaloTag or acylation
at any lysine residues in eDHFR or Hsp90 was not detected
(Figure S3a−c). Similarly, PU-DSSMe promoted Hsp90-K58
acylation (∼25% yield), while acylation at K74 and K84 of
Hsp90 or acylation at any lysine residues in eDHFR or
HaloTag was not detected (Figure S3a−c). Importantly, both
K160 of HaloTag and K58 of Hsp90 are located near the
ligand-binding pockets (Figure S3e,f). Consistently, click-
chemistry based experiments using an alkyne-containing acyl
donor (NAC-PA 18 in Figure S5a) also suggested that each
Then, we studied synthetic acetylation in living cells.
HEK293T cells expressing eDHFR-GFP were treated with
TMP-M-DSH and acetyl-CoA; however, acetylation of
eDHFR at K32 (eDHFR-K32) was barely observed (Figure
3a, Table S2). This is probably due to the inability of acetyl-
CoA to pass through the cell membrane. Since DSH can, in
principle, accept an acetyl group from any thioesters through
thiol-thioester exchange, we used N,S-diacetylcysteamine
(NAC-Ac 10 in Figure 3b),25 a thioester that lacks the adenyl
phosphopantothenate group of acetyl-CoA and is likely cell-
membrane-permeable. Intriguingly, treatment of HEK293T
cells expressing eDHFR-GFP with TMP-M-DSH and NAC-Ac
promoted eDHFR-K32 acetylation (Figure 3a, Table S2).
These data indicate that NAC-Ac can be used as a cell-
membrane-permeable acetyl donor for the DSH catalyst.
Next, we modified the structure of the DSH catalyst to
further improve the acetylation yield. DSH is easily oxidized to
form a disulfide dimer outside the cells (e.g., in medium). In
fact, HPLC analysis of TMP-M-DSH showed that DSH was
oxidized within 30 min at 37 °C in 25 mM HEPES buffer (pH
7.6; Figure 3c). This dimer is less likely to permeate the cell
membrane due to its increased molecular size. To prevent
oxidation of DSH outside cells, we synthesized TMP-M-
DSSMe 13, in which the thiol group of DSH was protected as
a disulfide with methanethiol (Figure 3b). Since the inside of
the cells is under reductive conditions, mainly due to the
presence of glutathione (GSH) in a high concentration
(millimolar range), it was expected that TMP-M-DSSMe
would be reduced and converted to active TMP-M-DSH inside
the cells. As expected, TMP-M-DSSMe was stable in 25 mM
HEPES buffer (pH 7.6) and was readily reduced to TMP-M-
DSH with 5 mM GSH (Figures 3c, S1). We then compared
the activity of TMP-M-DSH and TMP-M-DSSMe in living
cells. Notably, TMP-M-DSSMe promoted eDHFR-K32
acetylation about twice as much as TMP-DSH (Figure 3a,
Table S2), suggesting that oxidation of DSH outside the cells
indeed reduced efficacy of the DSH catalyst, and that DSSMe
can be used as a prodrug of DSH for intracellular reactions. As
the case with the reactions in test tubes, TMP-M-DSSMe
promoted acetylation of eDHFR specifically at K32 in living
cells (Figure 3d, Table S3). The acetylation of eDHFR at K32
was not promoted by Bz-DSSMe (14) or DSSMe (15; Figure
S2a,b), indicating that the TMP ligand was essential. By
comparing eDHFR-GFP in cells and recombinant eDHFR-
GFP in Western blotting experiments, we roughly estimated
the intracellular concentration of eDHFR-GFP to be ∼37 μM
(condition A in Figure S2c). In addition, by reducing the
amount of the plasmids for transfection, we also generated cells
that contain 9−19 μM or 5−9 μM eDHFR-GFP (condition B/
C in Figure S2c). We conducted acetylation experiments using
these three conditions and found that the acetylation yield of
eDHFR-K32 was comparable among these conditions (Figure
S2c). We also changed the concentration of NAC-Ac and
found that the acetylation yield of eDHFR-K32 depends on the
concentration of NAC-Ac (Figure S2d).
Since ligand-conjugated DSH can also promote a variety of
nonacetyl and non-natural acylations in test tubes,16 we tried
to address whether TMP-M-DSSMe could activate other
thioester acyl donors and transfer those acyl groups to eDHFR
at K32 in living cells. We synthesized S-ethyl malonyl-N-
F
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ting, the lysates were boiled with SDS sample buffer and analyzed by
CBB staining and Western blotting using the indicated antibodies. For
LC−MS/MS analysis, the lysates were incubated with GFP-Trap M
or MA magnetic beads (ChromoTek, gta-20 or gtma-20) at 4 °C for 1
h. After the incubation, the beads were washed with CRB buffer twice
and with 100 mM NH4HCO3 twice, before being resuspended in 20
μL of 100 mM NH4HCO3. A total of 20 μL of MeOH/propionic
anhydride (3/1) and 14 μL of 28% ammonia solution were added to
the suspension. After 30 min of incubation at RT, the solvents were
removed by a Speed-Vac evaporator. The beads were suspended in 50
μL of 0.01% protease MAX (Promega, V2072) in 50 mM NH4HCO3
aq. To the sample, 1 μg trypsin gold (Promega, V5280) was added,
and the mixture was incubated at 37 °C for 30 min. To this sample,
50 μL of 50 mM NH4HCO3 aq., 1 μg of Glu-C (Promega, V1651),
and 0.5 μL of 1% protease MAX were added, and the mixture was
incubated at 37 °C for 3 h. After the addition of 25 μL of 5% formic
acid to the supernatant, the solvent was removed using a Speed-Vac
evaporator. The dried sample was dissolved in 0.1% formic acid and
centrifuged (21 000g, 10 min). The supernatant was used for LC−
MS/MS analysis (see LC−MS/MS analysis section in the Supporting
Information).
catalyst can promote acylation of the target protein, although
the acyl donor itself slightly reacted with nontarget proteins
(Figure S5b,c). On the basis of these results, we concluded that
our catalyst system can selectively acylate target proteins in
living cells, and that the location of the acylation may be
explained by the proximate effects of the catalysts.
In this study, we utilized a well-established ligand−receptor
pair, TMP and eDHFR, and developed a chemical catalyst
system that enables synthetic and site-selective lysine acylation
in living cells. We demonstrated that NAC-acyl compounds are
cell-membrane-permeable acyl donors that can be activated by
the DSH catalyst in living cells. In addition, we found that pro-
drugged DSH, DSSMe, is a better catalyst for in-cell reactions
(Figure 1). Currently, the stoichiometry of acylation
introduced by our catalyst system is moderate (10−40%),
probably due to the existence of a high concentration of
glutathione in living cells, which could compete in the acyl
transfer reaction from the acyl donors to the DSH catalyst.
Therefore, developing a glutathione-resistant catalyst system is
an important next step in improving the efficacy of our catalyst
system. Interestingly, the stoichiometry of acylation depends
on the types of acyl donors. Investigation of the kinetics of the
acyl transfer from donors to lysine residues and optimization of
the acyl donors may also improve our system. We also
demonstrated that our catalyst system can divergently target
other proteins depending on the ligand, including endogenous
Hsp90. This report is the first example of selectively
introducing biologically significant PTM to targeted proteins
by means of chemical catalysis in living cells. As an important
next step, we will apply our findings to physiologically relevant
acetylated proteins, such as histones. We have previously
developed the LANA-DSH catalyst, which binds to nucleo-
somes and efficiently promotes histone- and site-selective
acylation of the proximate histone H2B lysine-120 (K120) in
test tubes.16 On the basis of our findings here, a combination
of a LANA-DSSMe catalyst and NAC-acyl donors may be able
to promote H2B−K120 acylation in living cells. Promotion of
efficient in-cell histone acylation and gene expression by a
chemical catalyst system may ameliorate the functional decline
caused by abnormal chromatin epigenetic modifications. Since
target selectivity of our catalyst system depends on ligands for
targets, we anticipate that increasing the repertoire of protein
ligands will endow our system to be a general chemical biology
strategy for manipulating PTMs in living cells. Chemical
catalyst systems are potentially applicable to living individuals,
since they can modify the target lysine residues of intracellular
proteins without genetic manipulation. Future investigations
will be directed at improving DSH catalyst systems for living
individuals by utilizing drug delivery technologies.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschem-
bio.9b00102.
Synthetic protocols, spectroscopic characterizations,
experimental details, Figures S1−S5, and Tables S1−
S8 (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: yamatsugu@mol.f.u-tokyo.ac.jp.
*E-mail: skawashima@mol.f.u-tokyo.ac.jp.
*E-mail: kanai@mol.f.u-tokyo.ac.jp.
■
ORCID
Kenzo Yamatsugu: 0000-0002-4260-414X
Shigehiro A. Kawashima: 0000-0002-2946-6627
Motomu Kanai: 0000-0003-1977-7648
Author Contributions
†These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank all members of our laboratory, especially R. M.
Newlon, for their valuable discussion and reading of this
manuscript. This work was supported by JSPS KAKENHI
Grant Number JP16H01300 (S.A.K.), JP26460142 (K.Y.),
JP18H04536 (K.Y.), JP17H01522 (M.K.), and JP17K19479
(M.K.).
METHODS
■
Cell Culture. HEK293T cells were grown in Dulbecco’s modified
Eagle’s medium with 10% fetal bovine serum, GlutaMAX (1×), 100
U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in an
atmosphere with 5% CO2.
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