Development of an activity-based probe for acyl-protein thioesterases

Article abstract of DOI:10.1371/journal.pone.0190255

Protein palmitoylation is a dynamic post-translational modification (PTM) important for cellular functions such as protein stability, trafficking, localization, and protein-protein interactions. S-palmitoylation occurs via the addition of palmitate to cys

Full text of DOI:10.1371/journal.pone.0190255

RESEARCH ARTICLE
Development of an activity-based probe for
acyl-protein thioesterases
Megan Garland1,2, Christopher J. Schulze2, Ian T. Foe2, Wouter A. van der Linden2,
Matthew A. Child2¤*, Matthew Bogyo1,2,3,4
*
1 Cancer Biology Program, Stanford University School of Medicine, Stanford, California, United States of
America, 2 Department of Pathology, Stanford University School of Medicine, Stanford, California, United
States of America, 3 Department of Chemical and Systems Biology, Stanford University School of Medicine,
Stanford, California, United States of America, 4 Department of Microbiology and Immunology, Stanford
University School of Medicine, Stanford, California, United States of America
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¤ Current address: Department of Life Science, Imperial College London, South Kensington, London, United
Kingdom.
* mbogyo@stanford.edu (MB); m.child@imperial.ac.uk (MAC)
Abstract
Protein palmitoylation is a dynamic post-translational modification (PTM) important for cellu-
lar functions such as protein stability, trafficking, localization, and protein-protein interac-
tions. S-palmitoylation occurs via the addition of palmitate to cysteine residues via a
thioester linkage, catalyzed by palmitoyl acyl transferases (PATs), with removal of the palmi-
tate catalyzed by acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases
(PPTs). Tools that target the regulators of palmitoylation–PATs, APTs and PPTs–will
improve understanding of this essential PTM. Here, we describe the synthesis and applica-
tion of a cell-permeable activity-based probe (ABP) that targets APTs in intact mammalian
cells and the parasite Toxoplasma gondii. Using a focused library of substituted chloroiso-
coumarins, we identified a probe scaffold with nanomolar affinity for human APTs (HsAPT1
and HsAPT2) and synthesized a fluorescent ABP, JCP174-BODIPY TMR (JCP174-BT).
We use JCP174-BT to profile HsAPT activity in situ in mammalian cells, to detect an APT in
T. gondii (TgPPT1). We show discordance between HsAPT activity levels and total protein
concentration in some cell lines, indicating that total protein levels may not be representative
of APT activity in complex systems, highlighting the utility of this probe.
OPEN ACCESS
Citation: Garland M, Schulze CJ, Foe IT, van der
Linden WA, Child MA, Bogyo M (2018)
Development of an activity-based probe for acyl-
protein thioesterases. PLoS ONE 13(1): e0190255.
https://doi.org/10.1371/journal.pone.0190255
Editor: Floyd Romesberg, Scripps Research
Institute, UNITED STATES
Received: October 29, 2017
Accepted: November 12, 2017
Published: January 24, 2018
Copyright: © 2018 Garland et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Introduction
Funding: The work was funded by a National
Institutes of Health grant R01 GM111703 to MB
and the Howard Hughes Medical Institute Medical
Research Fellows Program (to MG) and by the
Stanford University Medical School Medical
Scientist Training Program (to MG). The funders
did not have any role in the study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Post-translational modification (PTM) of proteins enables diversification of function beyond
the raw coding capacity of the genome. PTMs alter the functional state of proteins, often serv-
ing as switch-like modifications that regulate transduction of intracellular signals [1]. As such,
PTMs play integral roles in a range of processes including the maintenance of homeostasis [2],
the cellular response to stress [3], and even host-pathogen interactions [4]. Proteins responsi-
ble for the addition and removal of PTMs, so-called writers and erasers, regulate the specificity
of these modifications.
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Activity-based probe for acyl-protein thioesterases
Competing interests: The authors have declared
S-palmitoylation is the post-translational covalent addition of palmitic acid (a saturated
16-carbon acyl chain) onto a protein-associated cysteine residue via a thioester linkage [5,6].
One function of protein palmitoylation is to generate a lipid anchor, with the acyl chain insert-
ing into the lipid bilayer of cell membranes to retain the palmitoylated protein close to the
lipid membrane surface. In addition to its function in membrane protein organization and
localization, palmitoylation affects protein stability, trafficking and protein-protein interac-
tions [5,7]. Unlike other lipid-based PTMs such as myristoylation, palmitoylation is reversible
and considered to be dynamic [8]. The addition of palmitate onto protein substrates is cata-
lyzed by a family of enzymes called palmitoyl acyl transferases (PATs), with its removal coordi-
nated by acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs) [5].
APTs are located in the cytosolic compartment, while PPTs localize to the lysosome and are
thought to play a critical role in the turnover of palmitoylated proteins.
that no competing interests exist.
Modern chemical proteomic techniques and small-molecule tools have enabled functional
studies of this important signaling-associated PTM. Mass-spectrometry-based global profiling
of palmitoylated proteins has been achieved through metabolic incorporation of orthogonal
palmitic acid analogs in live cells [8,9]. In addition to this systems-based approach, small-mol-
ecules have been used to dissect the function of the enzymes involved in the regulation of pal-
mitoylation. For example, the small-molecule inhibitor palmostatin B (palmo B) has been used
to specifically link the activity of human acyl protein thioesterase 1 (HsAPT1) with the onco-
genic phenotype of H-RasG12V-transformed fibroblasts [10], and a small-molecule acyl pro-
tein thioesterase 2 (APT2) inhibitor was shown to rescue mislocalization of the scaffolding
protein Scribble and increase Scribble S-palmitoylation upon transformation with the tran-
scription factor Snail [11]. Using a similar small-molecule approach, we demonstrated that the
Toxoplasma gondii orthologue of HsAPT1 (TgPPT1) is the target of a substituted chloroiso-
coumarin [12] and its inhibition in extracellular T. gondii tachyzoites enhanced their ability to
invade host cells, highlighting a critical role for this PTM in establishing the initial point of
contact between the parasite and its host cell. Additionally, a general reporter substrate for
depalmitoylases was recently used to uncover signaling pathways that regulate dynamic APT
activity in situ [13]. Although these small-molecules have proven to be valuable tools, many
questions remain about the full repertoire of depalmitoylases in cells, as well as their subcellu-
lar localization and preferred substrates.
Activity-based probes (ABPs) are small-molecules functionalized with a reporter or affinity-
based tag that can be used to directly monitor the enzyme activity of a given target or set of tar-
gets in complex proteomes, live cells and organisms [14]. The ABP labels target enzymes by for-
mation of an activity-dependent covalent bond. For example, fluorophosphonate rhodamine
(FP-rho) is a fluorescent ABP that exhibits broad-spectrum reactivity for serine hydrolases. This
probe has been used to profile the activity of serine hydrolases in numerous experimental setups
[15,16]. ABPs can be used for a wide range of chemical biology approaches, and covalent small-
molecule inhibitors can often be converted into ABPs.
Previously, we found that a substituted chloroisocoumarin inhibitor covalently inhibits the
T. gondii depalmitoylase TgPPT1 [12]. We therefore performed a screen of structurally related
chloroisocoumarins to identify lead scaffolds for the human depalmitoylases HsAPT1 and
HsAPT2. This led to the development of a new fluorescent probe for depalmitoylase activity.
We demonstrate that this new tool is both selective for this enzyme class and cell-permeant,
capable of labeling of both HsAPT1 and HsAPT2 in vitro and in live mammalian cells. It also
specifically targets TgPPT1 in live T. gondii tachyzoites. In a proof-of-concept demonstration
of its utility, we tested the relationship between HsAPT activity and the metastatic potential of
oncogenically transformed mammalian cells. We observed an overall increase in HsAPT activ-
ity in oncogenically transformed cell lines as compared to primary cell lines. Additionally, we
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Activity-based probe for acyl-protein thioesterases
compared three pairs of cancer cell lines derived from the same tissue type with both high and
low metastatic potential. In two of the three pairs, we observed an inverse correlation between
ABP-labeled HsAPT activity and the reported metastatic potential of the cell lines. Further,
total protein levels did not always correlate with APT activity measured by the ABP, indicating
that tools to specifically measure enzyme activity may provide relevant information about the
regulation of their activity that cannot be obtained from measures of total protein levels.
Results
Substituted chloroisocoumarins inhibit HsAPT1 and HsAPT2
We recently performed a phenotypic screen to identify small-molecule modulators of host cell
invasion by the apicomplexan parasite T. gondii [12,17]. The small-molecules in this directed
screen were expected to covalently modify protein targets in an activity-dependent manner
[18]. Unexpectedly, this screen not only identified inhibitors of parasite invasion, but also
compounds that enhanced host cell invasion. Enhancer compounds were all structurally simi-
lar substituted chloroisocoumarins [12]. Using an alkynylated version of the lead enhancer
compound (JCP174), we identified the phenotypically relevant target of the enhancer pheno-
type as the previously uncharacterized thioesterase TgPPT1 [12]. TgPPT1 is the orthologue of
the human depalmitoylase HsAPT1, sharing 33% identity by BLAST analysis. On the basis of
this similarity, we reasoned that substituted chloroisocoumarins would likely inhibit the activ-
ity of the human depalmitoylases HsAPT1 and HsAPT2 and potentially serve as novel scaffolds
to generate selective covalent probes. Therefore, we screened a focused library of substituted
chloroisocoumarins to identify compounds that inhibited HsAPT1 and HsAPT2 activity. We
expressed recombinant HsAPT1 and HsAPT2 with an N-terminal His6 tag in E. coli and puri-
fied the proteins using nickel affinity chromatography (to a final purity of >95%, as shown in
S1 Fig). To measure the enzymatic activity of recombinant HsAPT1 and HsAPT2, we used the
previously reported substrate, 4-nitrophenyl octanoate (4-NPO), in which enzymatic cleavage
of the ester linkage can be measured by UV/Vis spectroscopy upon release of 4-nitrophenolate
[10] (Fig 1A). Using this activity readout, we performed our screen with recombinant HsAPT1
and HsAPT2 using a focused library of 100 substituted chloroisocoumarins (Fig 1B). Com-
pounds that produced >50% inhibition of either HsAPT1 or HsAPT2 activity relative to the
DMSO control at 10 μM were then re-screened across a range of concentrations to determine
in vitro IC50 values (Fig 1C and 1D). Compounds with IC50 values <10 μM for both HsAPT1
and HsAPT2 were scored as hits. According to these criteria, our screen achieved a hit rate of
7% (Table 1). The chloroisocoumarin JCP174 exhibited the greatest potency towards both
HsAPTs, with IC50 values of 1.7 μM and 0.75 μM against HsAPT1 and HsAPT2, respectively
(Table 1). We next tested the potency of JCP174 for both enzymes with increasing preincuba-
tion times from 30 minutes to 6 hours. The potency of JCP174 increased with longer pre-incu-
bation times for both targets, indicating an irreversible covalent mechanism (S1 Table). In
contrast, the published inhibitor of HsAPTs, palmo B, exhibited decreased potency with longer
preincubation times, as expected given its reversible mechanism of inhibition and stability in
aqueous solutions [10] (S1 Table).
To determine the selectivity of JCP174 and assess engagement with HsAPT1 and HsAPT2
in live mammalian cells, we performed ABP competition labeling experiments. As chloroiso-
coumarins are reported to be serine hydrolase inhibitors [12,19–21], with dichloroisocou-
marin (DCI) being the prototypical member of this inhibitor class, we selected the broad-
spectrum serine hydrolase ABP, FP-rho [16] for these experiments. We first determined
whether this broad-spectrum ABP, FP-rho, labeled HsAPT1 and HsAPT2. To identify ABP-
labeled protein species representing HsAPT1 and HsAPT2 in gel-based assays, we used
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Activity-based probe for acyl-protein thioesterases
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Activity-based probe for acyl-protein thioesterases
Fig 1. Chloroisocoumarins inhibit HsAPT1 and HsAPT2. A, 4-NPO biochemical esterase activity assay schematic. 4-nitrophenyl octanoate is
converted to 4-nitrophenolate (yellow) via esterase-mediated hydrolysis and measured at 401 nm by UV-Vis spectroscopy. B, Histogram of the results of
the 4-NPO assay screen with a substituted chloroisocoumarin library. Residual esterase activity of HsAPT1 and HsAPT2 is shown for each compound.
Structures of potent compounds are highlighted. Ã indicates dichloroisocoumarin (DCI). C, D, Dose-response curves for the chloroisocoumarin hits
against HsAPT1 (C) and HsAPT2 (D) plotting residual activity versus molar concantration (M). E, ABP competition labeling with the pan-serine
reactive ABP FP-rho. U-2OS cells were pre-treated with DMSO control, 50 μM ML348, or 50 μM ML349 before labeling with 0.5 μM FP-rho for 5
minutes. Samples were resolved by SDS-PAGE and visualized for fluorescent probe signal with a flatbed scanner. Carets indicate HsAPT1 and HsAPT2.
F, ABP competition labeling with the pan-serine reactive ABP FP-rho. Intact cells were pre-treated with different concentrations of JCP174 or
palmostatin B (Palmo B) before labeling with FP-rho. Samples were prepared as in (E). Carets indicate doublet of interest at ~25 kDa identified as
HsAPT1 and HsAPT2.
https://doi.org/10.1371/journal.pone.0190255.g001
previously published selective and reversible inhibitors of HsAPT1 and HsAPT2, ML348 and
ML349 [22]. As these piperazine amides are highly selective for binding to either HsAPT1
(ML348) or HsAPT2 (ML349), competition for target engagement between FP-rho and these
compounds allowed us to identify each enzyme in the labeling pattern of this broad-spectrum
probe. We pretreated mammalian U-2 OS cell lysates with 50 μM each of ML348 and ML349,
or DMSO control for 1 hour, then labeled with FP-rho for 5 minutes. This short probe labeling
time was chosen due to the reversible mechanism of these selective inhibitors, which also pre-
cludes their use as ABPs. Labeling was assessed by measuring fluorescent signal associated
with the resolved proteins species in-gel using a flatbed scanner. Compared to the DMSO con-
trol, in which FP-rho strongly labels two closely migrating species of approximate 25 kDa
molecular weight, ML348 competed for labeling of the lower protein of the doublet, while
ML349 competed for the higher protein in this doublet (Fig 1E). These data identify HsAPT1
as the lower band of the doublet and HsAPT2 as the upper band in the doublet at 25 kDa (Fig
1E), in accordance with previous reports [22]. To determine whether JCP174 could also com-
pete for labeling of HsAPT1 and HsAPT2, we pretreated U-2 OS cells with JCP174 for 1 hour,
washed the cells and prepared cell lysates that were then labeled with FP-rho to assess residual
activity of the hydrolase targets. JCP174 also competed with FP-rho for the labeling of both
HsAPT1 and HsAPT2 (Fig 1F). Further, palmo B [10] competed for the same two species (Fig
1F). These data demonstrate that JCP174, the most potent hit from our screen, acts via an irre-
versible covalent mechanism and effectively inhibits HsAPT1 and HsAPT2.
The ABP JCP174-BODIPY TMR labels HsAPT1 and HsAPT2 in vitro
Following the identification of JCP174 as the lead inhibitor for HsAPT1 and HsAPT2, we
sought to convert this compound into an ABP. We previously synthesized an alkynylated ver-
sion of JCP174 (JCP174-alk) [12]. Functionalization of JCP174 with an alkyne tag did not
Table 1. IC50 values of substituted chloroisocoumarins for rHsAPT1 and rHsAPT2 in the 4-NPO esterase assay.
Compound
HsAPT1 IC50 (μM)
HsAPT2 IC50 (μM)
DCI
1.7
0.31
1.8
2.7
0.32
2.9
Palmostatin B
JCP154
JCP174
1.7
0.75
5.2
JCP342
1.5
JCP343
1.4
9.1
JCP345
7.1
8.5
JCP362
1.2
0.95
0.50
1.8
JCP174-alk
JCP174-BODIPY TMR
0.93
3.0
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Activity-based probe for acyl-protein thioesterases
reduce its potency for either HsAPT1 or HsAPT2, with IC50 values of 0.925 μM and 0.498 μM
respectively (Fig 1C and 1D, Table 1). We then used JCP174-alk to synthesize a fluorescent
ABP in a one-step click reaction with an azide-BODIPY TMR fluorophore (543 nm excitation,
569 nm emission; Fig 2A, S2 Fig). Modification of JCP174-alk with the BODIPY TMR tag only
moderately reduced its potency, with IC50 values for HsAPT1 and HsAPT2 of 3.0 μM and
1.8 μM, respectively (Table 1). This confirmed that functionalization with the fluorophore did
not abolish affinity towards the target enzymes (S3 Fig).
We first determined if JCP174-BODIPY TMR (JCP174-BT), was able to covalently label
recombinant HsAPT1, HsAPT2, and TgPPT1. We observed dose-dependent competition with
the parent compound, JCP174, and palmo B in an ABP competition assay with JCP174-BT
(Fig 2B and 2C). We also observed that covalent modification of TgPPT1 by the parent com-
pound JCP174 resulted in a downward shift in the migration of the species when resolved by
SDS-PAGE (Fig 2C). Even though the protein is a single band by silver stain, we observed two
fluorescent bands upon probe labeling. This may be due to the fact that chloroisocoumarins
have two sites of attack by serine and histidine residues, rendering two different migratory spe-
cies. At higher concentrations of JCP174 treatment, only the lower migration species is
observed, indicating that this species may be the final version after cyclization and loss of chlo-
rine (Fig 2C). A change in the migration of TgPPT1 following covalent modification by small-
molecules has been previously reported [23]. To confirm the specificity of JCP174-BT for the
active site of TgPPT1 and activity-dependence of probe labeling, we compared in-gel labeling
of the wild-type TgPPT1 (TgPPT1WT) versus a catalytically dead TgPPT1 mutant where the
active site serine was mutated to an alanine (TgPPT1S128A) [12]. JCP174-BT covalently modi-
fied TgPPT1WT, but did not label the active site mutant, TgPPT1S128A (Fig 2C). Together,
these data show that the JCP174-BT probe retains potency for HsAPT1 and HsAPT2, and
covalently modifies the active site serine.
JCP174-BT labels depalmitoylases in intact parasites and mammalian cells
After verifying the ability of the probe to label recombinant enzymes in vitro, we tested its abil-
ity to label native depalmitoylases in intact cells. We observed strong labeling of the two species
that resolved as a closely migrating doublet around 25 kDa (Fig 3A) that we previously identi-
fied as HsAPT1 and HsAPT2 (see Fig 1E). To further confirm that the lower migrating species
of the probe-labeled doublet was HsATP1, we performed an immunoprecipitation (IP) using
an anti-HsAPT1 antibody (ProteinTech). We observed a strongly labeled doublet around 25
kDa in the input, with the lower species depleted in the unbound IP supernatant, and this
same lower molecular weight species was present in the IP elution (Fig 3B). We confirmed
HsAPT1 to be the fluorescently labeled species by western blot (Fig 3B). These data confirmed
that JCP174-BT covalently modified native HsAPT1 in live mammalian cells. While it is highly
likely that the labeled upper band is APT2, we were unable to find an antibody that would
allow direct immunoprecipitation to confirm this. Furthermore, both the APT1 and APT2
inhibitors failed to compete for any of the species labeled by the JCP174-BT probe likely
because of the reversible nature of the molecules and the slower rate of covalent modification
by JCP174-BT compared to FP-rho.
In addition to labeling HsAPT1 and HsAPT2 in intact cells, we tested whether JCP174BT
could label other depalmitoylases in situ. We labeled live extracellular T. gondii tachyzoites
with increasing concentrations of JCP174-BT, washed the parasites to remove excess probe,
lysed them, and resolved soluble lysates by SDS-PAGE. We observed two strongly labeled spe-
cies around 31 kDa in size (Fig 3C). To determine which of the labeled species visualized in
the lysate is TgPPT1, we used a previously generated T. gondii TgPPT1 knockout strain
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Activity-based probe for acyl-protein thioesterases
Fig 2. JCP174-BT labels HsAPT1, HsAPT2 and TgPPT1 in vitro. A, Structure of JCP174-BODIPY TMR (JCP174-BT) composed of chloroisocoumarin small-
molecule JCP174 (red) and BODIPY TMR fluorophore (yellow). B, C, In-gel ABP competition labeling with JCP174-BT. (B) Purified recombinant HsAPT1 or HsAPT2
was pre-treated with different concentrations of JCP174, palmostatin B, or DMSO (0) before labeling with JCP174-BT. Samples were resolved by SDS-PAGE and
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Activity-based probe for acyl-protein thioesterases
fluorescent probe signal visualized using a flatbed scanner (upper panel), with loading assessed via silver stain (lower panel). (C) Purified recombinant TgPPT1 WT and
active site mutant (S128A) were treated with JCP174, palmostatin B or DMSO (0) and labeled with JCP174-BT. Samples were analyzed as in (B).
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(ΔTgPPT1) [12]. Comparison of the probe labeling of wild-type or ΔTgPPT1 parasite strains
revealed that the doublet at ~31 kDa in the wild-type strain was absent in the ΔTgPPT1 knock-
out strain, confirming the identity of these two species as TgPPT1 (Fig 3D). This was also con-
sistent with the doublet species observed for recombinant enzyme preparations of TgPPT1
labeled with JCP174-BT (Fig 2C). Together, these data show that JCP174-BT is cell-permeant
and covalently modifies TgPPT1 and HsAPT1 in intact cells.
JCP174-BT profiles APT activity in intact mammalian cells and T. gondii
parasites
To demonstrate the utility of JCP174-BT to profile APT activity, we compared its labeling to
that of the broad spectrum ABP FP-rho in intact T. gondii tachyzoites and T. gondii lysates. We
treated intact wild-type or ΔTgPPT1 parasites with the same concentration of FP-rho or
JCP174-BT. We also labeled T. gondii lysates with the same concentration of FP-rho. When
visualized by in-gel fluorescence, JCP174-BT showed strong labeling of TgPPT1 in intact cells,
and this labeling was lost in the ΔTgPPT1 parasites (Fig 4A). However, FP-rho was unable to
label TgPPT1 in intact parasites suggesting that it likely is not cell-permeant (Fig 4A).
JCP174-BT predominantly labeled TgPPT1 and one other protein of approximately 55 kDa,
while FP-rho predominantly labeled four species in cellular lysates. These data demonstrate
the advantage of JCP174-BT to label intracellular depalmitoylases in intact tachyzoites.
To further demonstrate the utility of JCP174-BT, we performed the same comparison with
human U-2 OS cells. Comparison of intact cell labeling with JCP174-BT, FP-rho, or lysate
labeling with FP-rho revealed different fluorescent banding patterns (Fig 4B). Similar to the
studies in T. gondii cells, FP-rho did not significantly label any species when used on intact
cells but did show robust labeling of serine hydrolases, including HsAPT1 and HsAPT2 when
added to cellular lysates (Fig 4B). In contrast, JCP174-BT labeled APT1 and APT2 in intact
cells, highlighting its utility over a more broad-spectrum ABP (Fig 4B).
Palmitoylation and APTs have been linked to cancer phenotypes, with reports of small-
molecule inhibition of APTs able to partially revert the H-RasG12V-dependent oncogenic
phenotype [10]. Additionally, increased metastatic potential of cancers correlates with
increased incorporation of exogenous palmitate onto oncogenic signaling molecules [24].
Therefore, after confirming that JCP174-BT was cell-permeant and capable of labeling
HsAPT1 and HsAPT2 in intact mammalian cells, we sought to profile HsAPT activity in can-
cer cell lines. As palmitoylation is a dynamic PTM important in oncogenic signaling through
proteins such as Ras, we hypothesized that HsAPT activity would decrease in cancer cell lines
of higher metastatic potential, with the decreased activity of depalmitoylases leading to a
greater number of palmitoylated oncogenic signaling molecules. To test this, we profiled
HsAPT activity in three matched cancer cell line pairs representing cell lines derived from car-
cinoma of the breast, ovaries, and prostate. These matched pairs each contained one cancer
cell line with high metastatic or invasive potential and one with low metastatic potential
[24,25]. We labeled intact cells with JCP174-BT, and imaged labeling profiles in-gel as before.
While there was no uniform trend among all three matched cell lines, in-gel fluorescence
showed decreased HsAPT activity in cell lines of higher metastatic potential for two of the
three matched pairs (Fig 4C and S4 Fig). We next wanted to determine whether HsAPT activ-
ity observed via JCP174-BT labeling correlated with total HsAPT protein expression in each of
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Activity-based probe for acyl-protein thioesterases
Fig 3. JCP174-BT labels HsAPT1, HsAPT2 and TgPPT1 in intact cells. A, Intact U-2 OS mammalian cells were labeled with different concentrations of
JCP174-BT, washed to remove unbound probe, and lysed. Lysates were resolved by SDS-PAGE with fluorescent probe labeling visualized using a flatbed
scanner. Carets indicate doublet of interest ~25 kDa previously identified as HsAPT1 and HsAPT2. B, Intact U-2 OS mammalian cells were labeled with
JCP174-BT, washed to remove unbound probe, and lysed. HsAPT1 was immunoprecipitated from the lysate, with input, supernatant, and elution samples
resolved by SDS-PAGE. Fluorescent probe signal was visualized via flatbed scanner (upper panel). HsAPT1 identity was confirmed by western blot (lower
panel). The red caret indicates HsAPT1 species on the fluorescent scan. C, Intact T. gondii tachyzoites were labeled with different concentrations of JCP174-BT,
washed to remove unbound probe and lysed. Lysates were resolved by SDS-PAGE with fluorescent probe labeling visualized using a flatbed scanner. Carets
indicate bands of interest ~31 kDa. D, Intact wild-type T. gondii or ΔTgPPT1 tachyzoites were labeled with JCP174-BT as in (C). Carets indicate species
corresponding to TgPPT1 in labeling of wild-type tachyzoites.
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the cell lines. Using the Cancer Cell Line Encyclopedia (CCLE) RMA-normalized mRNA
expression data for HsAPT1 and HsAPT2 in the matched cancer cell lines (S2 Table) [26], we
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Activity-based probe for acyl-protein thioesterases
Fig 4. JCP174-BT profiles APT activity in intact mammalian cells and T. gondii parasites. A, Intact wild-type T. gondii or ΔTgPPT1 tachyzoites were
labeled with JCP174-BT or FP-rho, washed to remove unbound probe and lysed. T. gondii tachyzoite lysate was labeled with FP-rho. Lysates were resolved
by SDS-PAGE with fluorescent probe labeling visualized using a flatbed scanner. Carets indicate species corresponding to TgPPT1 in labeling of wild-type
tachyzoites. Asterisk (Ã) indicates m-Cherry expressed in ΔTgPPT1 parasites. B, Intact U-2 OS cells were labeled with JCP174-BT or FP-rho, washed to
remove unbound probe, and lysed. U-2 OS lysate was labeled with FP-rho. Lysates were resolved via SDS-PAGE. Fluorescent signal was visualized with a
flatbed scanner (fluorescence scan), with the coomassie stain of the same gel shown to indicate loading. Carets indicate the species corresponding to
HsAPT1 and HsAPT2. C, Intact mammalian cells were labeled with JCP174-BT and processed as in (A). Pairs of oncogenic cell lines from derived from
three tissue types (carcinomas of the breast, ovary and prostate) were chosen to contrast low metastatic/aggressive potential (MCF7, OVCAR-3, and
LNCaP) versus high metastatic potential (MDA-MB-231, SKOV-3, and PC-3) [25]. For each oncogenic pair, wedges indicate low to high metastatic
potential. Fluorescent signal was visualized with a flatbed scanner (top panel). Total HsAPT1 protein level was visualized by western blot (middle panel).
GAPDH was used as a loading control (lower panel).
https://doi.org/10.1371/journal.pone.0190255.g004
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Activity-based probe for acyl-protein thioesterases
confirmed the presence of HsAPT1 and HsAPT2 in each cell line tested. Via western blot, we
observed higher total protein expression of HsAPT1 in each of the three cell lines with lower
metastatic potential (Fig 4C and S4 Fig). Further analysis of HsAPT1 in the matched cancer cell
lines indicated discordance between total protein expression of HsAPT1, as measured by western
blot, and activity of HsAPT1, measured via JCP174-BT labeling (Fig 4C). The lack of correlation
between protein- and activity-levels was most apparent for the MCF7/MDA-MB-231 breast can-
cer-derived matched oncogenic pair. Here, we observed greater HsAPT activity in the cell line
with higher metastatic potential (MDA-MB-231), which was confirmed by FP-rho labeling of
lysates (S4 Fig). We observed a stronger labeling of HsAPT1 in the MCF7 cell line, while
HsAPT1 and HsAPT2 displayed similar activity in the MDA-MB-231 cell line. Slight discrepan-
cies in the labeling patterns of JCP174-BT and FP-rho may be due to the fact that FP-rho labeling
was performed in cell lysates, allowing this probe to access all serine hydrolases, while some ser-
ine hydrolases may be retained in inaccessible compartments in intact cell labeling. Together,
these data suggest that protein expression of APTs may not be representative of APT activity
within the cell and highlights the utility of JCP174-BT to profile activity of these enzymes in situ.
Discussion
Acyl-protein thioesterases are key regulatory enzymes for dynamic palmitoylation, but tools to
study them remain limited. We have successfully expanded the toolset available with the devel-
opment of an ABP that exhibits selectivity for acyl protein thioesterase activity in two species
and present proof-of-concept studies that demonstrate its ability to probe the activity of these
enzymes in vitro and in a diverse range of cell types.
Using an alkynylated covalent inhibitor scaffold previously identified as an inhibitor of the
T. gondii APT orthologue TgPPT1, we synthesized a probe via a single-step copper-catalyzed
cycloaddition reaction with an azido-fluorophore. The probe labels recombinant protein in
vitro and can access and selectively label APTs in live, intact cells. With a selective ABP tool
now available for the study of depalmitoylases, it is worthwhile noting the wider potential
future applications, such as for fluorescent polarization target-based small-molecule screens
[27] and screening in complex lysates [28]. In particular, further functionalization of the probe
with a fluorescence quencher group (such as QSY 21) would facilitate selective imaging and
subcellular localization of the active pool of these enzymes in cells and potentially for in vivo
models of health and disease [14].
APTs have been implicated in cancer, with the broad-spectrum APT inhibitor palmo B able
to partially reverse the oncogenic phenotype of H-RasG12V-transformed fibroblasts [10]. A
simple interpretation of these data is that APT activity contributes to the maintenance of an
oncogenic cell state, and therefore might correspondingly be higher in cancer cells. We used
our probe to more broadly test this, and found that APT activity in cancer cell lines was indeed
higher. However, for matched pairs of cancer cell lines, overall metastatic potential inversely
correlated with APT activity, indicating that although inhibition of this enzyme class may
reduce oncogenic phenotypes, it may also promote overall increased metastatic propensity due
to higher global palmitoylation of key signaling molecules. Further studies will be required to
test this hypothesis, but our study serves to highlight the power of this probe for tracking
depalmitoylase activity in intact cell systems.
Methods
Protein expression and purification
HsAPT1 (26.8 kDa) and HsAPT2 (26.9 kDa) were cloned into pET28a expression vectors
bearing an N-terminal His6 tag using 5’ Nde1 and 3’ BamHI (HsAPT1) or HindIII (HsAPT2)
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Activity-based probe for acyl-protein thioesterases
restriction sites. Constructs were transformed into chemically competent E. coli BL21(DE3)
pLysS cells (Lucigen, Middleton, WI) for expression. Expression was induced with 1 mM iso-
propyl β-d-1-thiogalactopyranoside (IPTG) for 4 hours. Cell pellets were resuspended in lysis
buffer (50 mM TRIS, 500 mM NaCl, 10% glycerol, 15 mM imidazole, pH 8.8) and membranes
were disrupted via sonication on ice. Lysates were clarified by centrifugation at 16,500 rpm for
30 minutes. N-terminally His6-tagged HsAPT constructs were purified with Ni2+ metal affinity
chromatography. Purified protein was eluted with high imidazole elution buffer (50 mM TRIS,
150 mM NaCl, 300 mM imidazole, pH 8.8), aliquoted, rapidly frozen in liquid nitrogen, and
stored at −80˚C. TgPPT1WT and TgPPT1S128A were expressed and purified as in Child et al.
[12].
4-NPO esterase activity assay
The 4-nitropenhyl octanoate (4-NPO) activity assay was modified from Dekker et al. [10].
Briefly, 50 μL 20 mM HEPES, 150 mM NaCl, 0.01% v/v Triton-X 100, pH 7.4 buffer with the
indicated concentrations of inhibitor or buffer control was added to 96-well plates. To each
well, 30 μL of recombinantly expressed enzyme diluted to 1 μM in 20 mM HEPES, 150 mM
NaCl, 0.01% v/v Triton-X 100, pH 7.4 buffer was added. The inhibitor and enzyme reaction
was incubated at 37˚C for 30 minutes before 20 μL of 4-NPO (3.0 mM) was added to the reac-
tion in 20 mM HEPES, 150 mM NaCl, 0.25% v/v Triton-X 100, pH 7.4 buffer. The reaction
progress was measured using a Cytation 3 plate reader at 401 nm at 1-minute intervals for 40
minutes.
Cell culture
Cell lines were a generous gift from the laboratory of S. Contag. SKOV3 (ATCC HTB-77) and
U-2 OS (ATCC HTB-96) cells were cultured in McCoy’s media supplemented with 10% (v/v)
fetal calf serum, 10 U/mL penicillin G, and 100 μg/mL streptomycin. PC-3 (ATCC CRL-1435)
and LnCaP (ATCC CRL-1740) cells were cultured in RPMI supplemented with 10% (v/v) fetal
calf serum, 10 U/mL penicillin G, and 100 μg/mL streptomycin. OVCAR-3 (ATCC HTB-161)
cells were cultured in RPMI supplemented with 20% (v/v) fetal calf serum, 10 U/mL penicillin
G, and 100 μg/mL streptomycin. MDA-MB-231 (ATCC HTB-26) and MCF7 (ATCC HTB-22)
cells were cultured in Dulbecco’s modified medium (DMEM) supplemented with 10% (v/v)
fetal calf serum, 10 U/mL penicillin G, and 100 μg/mL streptomycin, and 2 mM L-glutamine.
All cells were incubated at 37˚C and 5% CO2.
FP-rhodamine competition labeling assay
U-2 OS cells were seeded at a density of 300,000 cells per well into a 6-well plate and allowed to
adhere overnight. Cells were pre-treated with different concentrations of JCP174 or palmosta-
tin B for 1 hour at 37˚C. Cells were washed with PBS and lysates were prepared by incubating
pellets in TRIS-buffered saline (TBS, 50 mM TRIS, 150 mM NaCl, pH 7.4) with 0.5% NP40 for
30 minutes on ice. Lysates were clarified via centrifugation at 13,000 rpm for 30 minutes, and
total protein concentration was measured via bicinchoninic acid (BCA) assay. For labeling
with FP-rhodamine, 20 μg total protein was incubated with 0.5–1 μM FP-rhodamine (30×
stock in DMSO) in a total volume of 15 μL for 5–30 minutes at 37˚C. Reactions were quenched
with 1× SDS-PAGE loading buffer (4× stock), boiled for 5 minutes, and resolved by
SDS-PAGE. Fluorescent signal was visualized in the rhodamine channel with a flatbed
scanner.
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Activity-based probe for acyl-protein thioesterases
JCP174-BT competition labeling assay with recombinant enzyme
Recombinantly expressed HsAPT1, HsAPT2, and TgPPT1WT or TgPPT1S128A (100 ng) was
resuspended in TBS buffer with 0.5% NP40 to a total volume of 19.8 μL. Indicated concentra-
tions of inhibitor were added (100× stock in buffer), with the enzyme/inhibitor reaction incu-
bated at 37˚C for 60 minutes. After pre-incubation, JCP174-BT (100× stock in DMSO) was
added to a final concentration of 1 μM. Samples were incubated at 37˚C for 30 minutes. Reac-
tions were quenched with 1× SDS-PAGE loading buffer (4× stock), boiled for 5 minutes, and
resolved by SDS-PAGE. Fluorescent signal was visualized in the 4,4-difluoro-4-bora-3a,4a-
diaza-s-indacene tetramethylrhodamine (BODIPY TMR) channel (ex: 543 nm, em: 569 nm)
with a flatbed scanner. Equal protein loading was assessed via silver stain.
Intact mammalian cell labeling with JCP174-BT
Cells were seeded at a density of 300,000 cells per well into a 6-well plate and allowed to adhere
overnight. Intact adherent cells were pre-treated with different concentrations of JCP174 or
palmostatin B for 1 hour at 37˚C. Media was removed, cells were washed with PBS, and media
containing 1 μM (unless otherwise indicated) of JCP174-BT was added to cells for an addi-
tional 1 hour at 37˚C. Cells were washed with PBS and lysates were prepared by incubating pel-
lets in TBS with 0.5% NP40 for 30 minutes on ice. Lysates were clarified via centrifugation at
13,000 rpm for 30 minutes, and total protein concentration was measured via BCA assay.
Lysates were diluted to 20 μg total protein in a volume of 15 μL in TBS with 0.5% NP40, sam-
ples were prepared with 1× SDS-PAGE loading buffer (4× stock), boiled for 5 minutes, and
resolved by SDS-PAGE. Fluorescent signal was visualized in the BODIPY TMR channel (ex:
543 nm, em: 569 nm) with a flatbed scanner. Where applicable, western blot analyses were per-
formed with anti-HsAPT1 antibody (ProteinTech, rabbit polyclonal 16055-1-AP) (1:1000) and
anti-GAPDH (Millipore, mouse monoclonal MAB374) (1:300).
Intact parasite labeling with JCP174-BT
Intact extracellular T. gondii tachyzoites harvested by syringe lysis of heavily infected host-cells
were pre-treated with different concentrations of JCP174 or palmostatin B for 15 minutes at
37˚C. Parasites were washed and labeled with 1 μM (unless otherwise indicated) of JCP174-BT
for 1 hour at 37˚C. Parasites were washed with PBS and lysates were prepared by incubating
pellets in Tris-buffered saline (TBS, 50 mM TRIS, 150 mM NaCl, pH 7.4) with 0.5% NP40 and
0.1% SDS for 30 minutes on ice. Lysates were clarified via centrifugation in a benchtop micro-
centrifuge at 13,000 rpm for 30 minutes, and total protein concentration was measured via
BCA assay. Lysates were diluted to 20 μg total protein in a volume of 15 μL in TBS buffer with
0.5% NP40, samples were prepared with 1× SDS-PAGE loading buffer (4× stock), boiled for 5
minutes, and resolved by SDS-PAGE. Fluorescent signal was visualized in the BODIPY TMR
channel (ex: 543 nm, em: 569 nm) with a flatbed Typhoon scanner.
HsAPT1 IP
Intact mammalian U-2 OS cells were labeled with 1 μM JCP174-BT for 1 hour at 37˚C. Cells
were washed with PBS and lysates were prepared by incubating pellets in TBS buffer with 0.5%
NP40 for 30 minutes on ice. Lysates were clarified via centrifugation at 13,000 rpm for 30 min-
utes, and total protein concentration was measured via BCA assay. To immunoprecipitate
HsAPT1, 100 μg total protein was incubated with anti-HsAPT1 antibody (ProteinTech) in
250 μL of IP buffer (50 mM TRIS, 150 mM NaCl, pH 7.4, 0.5% NP40) for 15 minutes on ice,
then immobilized on Protein G resin (40 μL of 50:50 slurry) with agitation overnight at 4˚C. The
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Activity-based probe for acyl-protein thioesterases
reaction was washed 2 × 500 μL of IP buffer, 1 × 500 μL of IP buffer without NP40, aspirated dry
and eluted by boiling with SDS-PAGE loading buffer. Input (10 μg), supernatant, and elution
samples were resolved by SDS-PAGE. Fluorescent signal was visualized in the BODIPY TMR
channel (ex: 543 nm, em: 569 nm) with a flatbed scanner before performing western blot analysis
for HsAPT1. For western blot analysis, anti-HsAPT1 antibody (ProteinTech, rabbit polyclonal)
(1:1000) primary and protein G HRP (Life technologies) (1:5000) secondary were used.
General synthetic methods
All reagents were HPLC grade and used without further purification. The LC-MS data were
acquired using an Agilent HPLC in tandem with an API 150 mass spectrometer (AppliedBio-
systems/SCIEX) equipped with an electrospray interface. The general synthesis of the chloroi-
socoumarin scaffold has been described previously [12,29], and was followed with minor
modifications.
2-(2-(but-3-yn-1-yloxy)-2-oxoethyl)-5-nitrobenzoic acid
A suspension of 4-nitrohomophthalic acid [30] (.53 g, 2.4 mmol) and 3-butyn-1-ol (5 equiv.) in
toluene (2 mL/mmol) was heated to 70˚ C. A catalytic amount (~0.1 equiv.) of pTSOH was
added and the reaction was stirred overnight until TLC indicated consumption of the starting
material. The reaction was quenched with diluted sodium bicarbonate and extracted twice with
ethyl acetate. The aqueous layer was acidified with 1M HCl and extracted twice with ethyl ace-
tate. The combined organic layers were combined and dried with MgSO4 and concentrated in
vacuo to afford the monoesterified product in 27% yield. 1H NMR (400 MHz, Chloroform-d) δ
8.98 (d, J = 2.5 Hz, 1H), 8.39 (dd, J = 8.4, 2.5 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 4.24 (t, J = 6.7 Hz,
2H), 4.21 (s, 2H), 2.54 (td, J = 6.7, 2.7 Hz, 2H), 2.01 (t, J = 2.7 Hz, 1H); 13C NMR (100 MHz,
Chloroform-d) δ 170.1, 170.0, 147.4, 143.5, 133.9, 130.1, 127.7, 127.0, 79.9, 70.2, 63.1, 40.6, 19.0.
3-(3-Butynoxy)-4-chloro-7-nitro-isocoumarin
PCl5 (3 equiv.) was added to a solution of the monoesterified product (.15 g, .54 mmol) in toluene
(7 mL) and solution was stirred overnight at 70˚ C. TLC (20% EtOAc/Hexanes) indicated com-
plete consumption of the starting material. The reaction mixture was cooled and diluted with
ethyl acetate and extracted with sodium bicarbonate. The aqueous layer was extracted 2× with
ethyl acetate. The combined organic layers were washed with sodium bicarbonate and brine and
subsequently dried with MgSO4 and concentrated in vacuo. The product was purified using silica
column chromatography (5–20% EtOAc/Hexanes) to afford a yellow solid (30% yield). 1H NMR
(400 MHz, Chloroform-d) δ 9.04 (d, J = 2.4 Hz, 1H), 8.53 (dd, J = 9.0, 2.3 Hz, 1H), 7.84 (d, J = 9.0
Hz, 1H), 4.56 (t, J = 6.8 Hz, 2H), 2.75 (td, J = 6.8, 2.7 Hz, 2H), 2.05 (t, J = 2.6 Hz, 1H).
3-(3-Butynoxy)-4-chloro-7-amino-isocoumarin (JCP174)
The nitro-isocoumarin product (.055 g, .19 mmol) was suspended in 4:1 EtOH:H2O (5 mL)
and Fe (s) was added (10 equiv.) followed by 8 μL concentrated HCl. The mixture was refluxed
at 80˚ C until disappearance of the starting material by TLC, approximately 1 hour. The mix-
ture was cooled, diluted with EtOAc, and filtered through a pad of celite. The filtrate was
washed with brine, dried with MgSO4 and concentrated in vacuo. The crude mixture was puri-
fied by reversed-phase HPLC (20% ACN/H2O +0.1% TFA to 43% ACN/H2O +0.1% TFA) to
afford a yellow solid (54% yield after HPLC). 1H NMR (400 MHz, DMSO-d6) δ 7.44 (d, J = 8.6
Hz, 1H), 7.31 (d, J = 2.3 Hz, 1H), 7.19 (dd, J = 8.6, 2.4 Hz, 1H), 4.32 (t, J = 6.4 Hz, 2H), 2.90 (t,
J = 2.6 Hz, 1H), 2.66 (td, J = 6.4, 2.6 Hz, 2H).
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Activity-based probe for acyl-protein thioesterases
JCP174-BODIPY TMR
JCP174-alkyne (9.0 μmol) was solubilized in 100 μL degassed MeOH. An equivalent volume of
BODIPY-TMR-azide dissolved in dioxane was added, followed by a catalytic amount (~0.1
equiv) of TBTA and tetrakis(acetonitrile)copper(I) hexafluorophosphate. The mixture was stir-
red at room temperature for 3 h until complete consumption of the starting material was
observed by TLC (5% MeOH/DCM, 3 drops of triethylamine). The reaction was diluted with
DCM and MeOH and concentrated in vacuo. The compound was purified by silica column
chromatography (1% MeOH/DCM) and further purified by reversed-phase HPLC to yield 0.6
mg JCP174-BODIPY TMR (9% yield). 1H NMR (600 MHz, Acetonitrile-d3) δ 7.82 (d, J = 8.9
Hz, 2H), 7.57 (s, 1H), 7.46 (d, J = 8.6 Hz, 1H), 7.33 (d, J = 2.0, 1H), 7.33 (s, 1H), 7.15 (dd, J = 8.6,
2.5 Hz, 1H), 7.03 (d, J = 4.0 Hz, 1H), 6.98 (d, J = 8.9 Hz, 2H), 6.58 (d, J = 3.8 Hz, 1H), 6.40 (t,
J = 4.0 Hz 1H), 4.48 (t, J = 6.5 Hz, 2H), 4.15 (t, J = 7.1 Hz, 2H), 3.83 (s, 3H), 3.10–3.06 (m, 4H),
2.70 (t, J = 7.2 Hz, 2H), 2.48 (s, 3H), 2.27 (t, J = 7.3 Hz, 2H), 2.22 (s, 3H), 1.88 (m, 2H).
Supporting information
S1 Fig. Purification of HsAPT1 and HsAPT2. Overloaded Coomassie stained SDS-PAGE gel
of purified recombinant HsAPT1 and HsAPT2 protein.
(DOCX)
S2 Fig. Synthesis of JCP174-BT. Click reaction between JCP174-alk and BODIPY-TMR azide
to generate the ABP JCP174-BT.
(DOCX)
S3 Fig. 4-NPO activity assay with JCP174-BT. Dose-response curves for HsAPT1 and
HsAPT2 recombinant enzyme with JCP174-BT in the 4-NPO esterase activity assay.
(DOCX)
S4 Fig. FP-rho labeling of matched cancer cell lines. Mammalian cell lysates were labeled
with FP-rho and resolved via SDS-PAGE. Pairs of oncogenic cell lines from derived from three
tissue types (carcinomas of the breast, ovary and prostate) were chosen to contrast low meta-
static/aggressive potential (MCF7, OVCAR-3, and LNCaP) versus high metastatic potential
(MDA-MB-231, SKOV-3, and PC-3) [25]. For each oncogenic pair, wedges indicate low to
high metastatic potential. FP-rho fluorescent signal was visualized with a flatbed scanner (top
panel). Total HsAPT1 protein level was visualized by western blot (middle panel). GAPDH
was used as a loading control (lower panel).
(DOCX)
S1 Table. IC50 values of JCP174 and palmostatin B over time in the 4-NPO esterase assay.
(DOCX)
S2 Table. RMA-normalized mRNA expression data of HsAPT1 and HsAPT2 for cancer
cell lines from the Cancer Cell Line Encyclopedia (CCLE)[26].
(DOCX)
Acknowledgments
We would like to thank C. Contag and S. Rogalla for providing the cancer cell lines used in
this manuscript; B. Martin for providing the APT1 and APT2 inhibitors used in this study;
and members of the Bogyo lab for helpful discussions.
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Activity-based probe for acyl-protein thioesterases
Author Contributions
Conceptualization: Megan Garland, Christopher J. Schulze, Matthew A. Child, Matthew
Bogyo.
Data curation: Megan Garland, Ian T. Foe, Matthew A. Child, Matthew Bogyo.
Formal analysis: Megan Garland, Christopher J. Schulze, Matthew A. Child, Matthew Bogyo.
Funding acquisition: Matthew Bogyo.
Investigation: Megan Garland, Christopher J. Schulze, Ian T. Foe, Wouter A. van der Linden.
Methodology: Megan Garland, Wouter A. van der Linden, Matthew A. Child, Matthew
Bogyo.
Project administration: Megan Garland, Matthew Bogyo.
Supervision: Matthew A. Child, Matthew Bogyo.
Validation: Megan Garland, Ian T. Foe, Wouter A. van der Linden, Matthew A. Child.
Visualization: Matthew A. Child, Matthew Bogyo.
Writing – original draft: Megan Garland, Matthew A. Child, Matthew Bogyo.
Writing – review & editing: Megan Garland, Matthew A. Child, Matthew Bogyo.
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