Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S-Transferases

Article abstract of DOI:10.1021/jacs.7b07378

Glutathione S-transferases (GSTs) comprise a diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione (GSH) to endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured, the isoform-specific contribution to the metabolism of xenobiotics in complex biological samples has not been possible. We have developed two activity-based probes (ABPs) that characterize active GSTs in mammalian tissues. The GST active site is composed of a GSH binding "G site" and a substrate binding "H site". Therefore, we developed (1) a GSH-based photoaffinity probe (GSTABP-G) to target the "G site", and (2) an ABP designed to mimic a substrate molecule and have "H site" activity (GSTABP-H). The GSTABP-G features a photoreactive moiety for UV-induced covalent binding to GSTs and GSH-binding enzymes. The GSTABP-H is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and "G" and "H" site specificity was carried out using a series of competition experiments in the liver. Herein, we present robust tools for the characterization of enzyme- and active site-specific GST activity in mammalian model systems.

Full text of DOI:10.1021/jacs.7b07378

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Communication
Activity-Based Probes for Isoenzyme- and Site-Specific
Functional Characterization of Glutathione S-transferases
Ethan G. Stoddard, Bryan Killinger, Reji N. Nair, Natalie Sadler, Regan F Volk,
Samuel O Purvine, Anil K. Shukla, Jordan Ned Smith, and Aaron T. Wright
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b07378 • Publication Date (Web): 25 Oct 2017
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Ethan G. Stoddard, Bryan J. Killinger, Reji N. Nair, Natalie C. Sadler, Regan F. Volk, Samuel O.
Purvine, Anil K. Shukla, Jordan N. Smith, and Aaron T. Wright
Biological Sciences Division, Pacific Northwest National Laboratory, 902 Battelle Blvd, Richland, Washington 99352, Unitꢀ
ed States
Supporting Information Placeholder
ABSTRACT: Glutathione Sꢀtransferases (GSTs) comprise a
diverse family of phase II drug metabolizing enzymes whose
shared function is the conjugation of reduced glutathione (GSH)
to endoꢀ and xenobiotics. Although the conglomerate activity of
these enzymes can be measured, the individual contribution from
specific isoforms and their contribution to the metabolism of xeꢀ
nobiotics in complex biological samples has not been possible.
We have developed two activityꢀbased probes (ABPs) that characꢀ
terize active GSTs in mammalian tissues. The GST active site is
comprised of a GSH binding “G site” and a substrate binding “H
site”. Therefore, we developed (1) a GSHꢀbased photoaffinity
probe (GSTABPꢀG) to target the “G site”, and (2) an ABP deꢀ
signed to mimic a substrate molecule and have “H site” activity
(GSTABPꢀH). The GSTABPꢀG features a photoreactive moiety
for UVꢀinduced covalent binding to GSTs and GSHꢀbinding enꢀ
zymes. The GSTABPꢀH is a derivative of a known mechanismꢀ
based GST inhibitor that binds within the active site and inhibits
GST activity. Validation of probe targets and “G” and “H” site
specificity was carried out using a series of competition experiꢀ
ments in the liver. Herein, we present robust tools for the characꢀ
terization of enzymeꢀ and active siteꢀspecific GST activity in
mammalian model systems.
Glutathione Sꢀtransferases (GSTs) are a diverse group of
phase II drug metabolizing enzymes whose shared function is the
conjugation of glutathione (GSH) to various electrophilic endoꢀ
Figure 1. Activityꢀ and affinityꢀbased probes designed to target active
GSTs. (A) Function of active GSTs; the conjugation of reduced glutathiꢀ
one (GSH) to xenobiotic (X) stabilized by “G” and “H” site binding by
each substrate, respectively. (B) Structure of affinityꢀbased GSTABP-G
designed to target GST “G” sites (C) Structure of activityꢀbased
GSTABP-H designed to target GST “H” sites. GSTABP-G and
GSTABP-H probe dependent labeling of 1 µM recombinant human
GSTM1 (D) and mouse liver cytosol (E). BP – benzophenone.
1
and xenobiotics. GSTs have been implicated in the conjugation
of endogenously produced oxidized metabolites including
propenal, 4ꢀhydroxynonenals, organic hydroperoxides, phosphoꢀ
2
ꢀ5
lipids, and fatty acid peroxides. Reactive species generated by
P450 monoxygenation of exogenous polyaromatic hydrocarbons
9ꢀ12
ing agents and are inactivated via GSH conjugation.
Due to
(PAHs) are also conjugated to GSH, highlighting the importance
their function in the detoxification of endogenous and exogenous
electrophiles, an exploration of enzymeꢀspecific response to perꢀ
turbation is critical for understanding mammalian metabolism of
xenoꢀ and endobiotics.
of GSTs in the protection of macromolecules such as DNA or
6ꢀ8
proteins from modification by P450ꢀactivated PAH metabolites.
Additionally, many chemotherapeutic drugs, such as 1,3ꢀbisꢀ(2ꢀ
chloroethyl)ꢀ1ꢀnitrosourea and chlorambucil, function as alkylatꢀ
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Figure 2. LCꢀMS/MS chemoproteomics results of probe enriched and probe competed mouse liver cytosol (n=3). All samples were incubated with 10 µM
probe or an equal concentration of vehicle control. Enrichment was calculated as AMT tag abundances from probeꢀenriched samples divided by a no UV
(for GSTABP-G) or DMSO only (for GSTABP-H) control. Significance was determined using a paired tꢀtest with a twoꢀtailed distribution. (A) Volcano
plot of GSTABP-G enrichment. In black: all GSTs that did not demonstrate competitive inhibition of probe labeling. In green: GSTs whose probe labeling
was competitively inhibited by 2.5× excess 2,3ꢀdichloroꢀ1,4ꢀnapthoquinone (Dichlon) over probe. In blue: GSTs whose probe labeling was competitively
inhibited by 2.5× excess Dichlon and 2.5× excess Sꢀhexylglutathione. (B) Volcano plot of GSTABP-H enrichment. In black: all GSTs whose probe labeling
was not competitively inhibited. In blue: GSTs whose probe labeling was competitively inhibited by 20× Sꢀhexylglutathione. In red: GSTs whose probe
labeling was competitively inhibited by 10× Nꢀethylmaleimide.
GSTs are categorized by their subcellular location into cytoꢀ
solic, mitochondrial, and microsomal superfamilies and are furꢀ
ther divided into classes based on sequence homology. GSTs
contain a GSH binding “G” site and a substrateꢀbinding “H” site.
While the G site is highly conserved, the H site is variable beꢀ
tween classes, contributing to interꢀclass diversity in substrate
We developed two probes to enable activityꢀbased protein
profiling of GSTs at the GSHꢀbinding G site and the substrate
binding H site (Figure 1A), and validated their ability to target
and measure the activity of GSTs in mammalian model systems.
The first probe (GSTABP-G) was designed to target the GSHꢀ
binding G site of GSTs by mimicking GSH (Figure 1B). A photoꢀ
reactive benzophenone and a terminal alkyne were appended to
the thiol of GSH. This allows for irreversible binding of the probe
to GST targets when UV irradiated, and the subsequent enrichꢀ
ment of targeted proteins following click chemistry. GST enꢀ
zymes recognize and bind the peptidic structure of GSH, whereas
the GSH thiol functions as a nucleophile for GSTꢀmediated atꢀ
tachment to xenobiotics. Thereby, to target the G site we modified
the thiol with the alkyne and benzophenone containing linker. To
complement the G site targeting photoaffinity probe, we develꢀ
oped a mechanismꢀbased probe designed to characterize H site
activity (Figure 1C). The second probe (GSTABP-H) is derived
from Dichlon (2,3ꢀdichloroꢀ1,4ꢀnaphthoquinone), a broad specꢀ
trum irreversible inhibitor of human and rodent GST
1
specificity. Expression of GSTs is often nonꢀindicative of the
propensity of these enzymes for their detoxifying GSH transferase
activity. This discrepancy between expression and activity can be
attributed to known postꢀtranslational modifications, alternative
enzymeꢀspecific nonꢀtransferase activities, and activity altering
1
3ꢀ16
proteinꢀprotein interactions.
Measurement of GST activity
using commercial activity assays provides information regarding
the total GSH conjugating ability of a system, but fail to reveal
enzymeꢀspecific activity. Due to incongruence of GST expression
and activity, in combination with a limited toolkit for studying
GST activity, robust tools to measure enzymeꢀspecific GST acꢀ
tivity are needed.
17ꢀ18
isoenzymes
. We hypothesized that GSTABP-H would bind H
sites due to Dichlon’s hydrophobicity and electrophilicity, which
are properties of H site binding molecules. In addition, evidence
suggests the Dichlon moiety may also have capacity to bind conꢀ
served catalytic tyrosines within GST H sites. For example, a
GST mu isoenzyme has previously been found to be irreversibly
bound by 2ꢀ(Sꢀglutathionyl)ꢀ3,5,6ꢀtrichloroꢀ1,4ꢀbenzoquinone.
The proposed binding mechanism involves an interaction between
the catalytic tyrosines and the chlorinated quinone moiety shared
by 2ꢀ(Sꢀglutathionyl)ꢀ3,5,6ꢀtrichloroꢀ1,4ꢀbenzoquinone and Diꢀ
19
chlon. Thus, we posited an alkyneꢀappended Dichlon derivative
would make an effective H site targeting activity based probe.
To validate irreversible labeling of active GSTs by the ABPs,
we first evaluated concentration dependent labeling of a recombiꢀ
nant GST (Figure 1D). GSTABP-G and GSTABP-H were apꢀ
plied with increasing concentrations to the recombinant human
GSTM1, a GST isoenzyme that is highly expressed in mammalian
liver. After 30 min of incubation, a fluorescent rhodamine reporter
was added by click chemistry, and SDSꢀPAGE revealed both
probes exhibit concentrationꢀdependent GSTM1 (Figure 1D).
Both probes were then applied with increasing concentrations to
the cytosolic fraction of mouse liver lysate to further test probe
targeting (Figure 1E). Both probes strongly label two distinct
Figure 3. Competitive inhibition of GSTABP-G and GSTABP-H labelꢀ
ing of mouse liver cytosol to delineate G and H site binding (n=3). 10
µM probe was used in all samples. Increasing concentrations of Sꢀ
hexylglutathione (GSꢀhexyl) or ethacrynic acid (EA) was incubated with
mouse liver cytosol for 30 min before probe labeling for 30 min. Afterꢀ
wards, rhodamine was attached to the probeꢀprotein complexes via click
chemistry. Proteins were resolved via SDSꢀPAGE followed by fluoresꢀ
cence gel imaging and quantification (arbitrary units) of probe labeled
GST bands. All replicates were normalized to a % labeled protein value
(probed samples with no inhibitor = 100%). Normalized values were
plotted and EC50 values were determined with a best fit curve.
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bands at 24 kDa. The lower band is likely GSTP1, a 23.6 kDa
protein highly abundant in the liver. The upper band shows probꢀ
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able GST targets from the mu and alpha classes, the molecular
weights of which are ~26 kDa. In addition to these proteins, the
GSTABP-H shows concentrationꢀdependent labeling of various
higher molecular weight proteins. While the GSTABP-G demonꢀ
strates high selectivity, the GSTABP-H seems more susceptible
to offꢀtarget labeling, likely due to its strong electrophilic nature
(Figure 1E). To determine the specific GST isoforms targeted by
each probe, we performed LCꢀMS based proteomics analyses of
mouse liver labeling, and labeling in the presence of specific inꢀ
hibitors (Figure 2). Proteomics revealed that the GSTABP-G
shows high specificity for GSTs (Figure 2A). 27 probeꢀtargeted
proteins were determined to be statistically significant targets of
GSTABP-G at a foldꢀchange of 3 over no UV exposure controls.
Of these 27 proteins, eight are GSTs and include members of mu,
alpha, pi, theta, kappa, and zeta classes. The remaining proteins
include five proteins with known GSH binding, one with known
antioxidant and drug binding activity, and five proteins highly
abundant in liver with no known GSH binding activity (SI Table
S1). The GSTABP-H also facilitated the enrichment of 12 memꢀ
bers of the GST mu, alpha, pi, theta, omega, kappa, and zeta clasꢀ
ses (Figure 2B; SI Table S2). However, as with the gel studies,
proteomics results for GSTABP-H indicate considerably more off
target labeling than GSTABP-G. An evaluation of the offꢀtargets
reveals that many of these proteins have known reactive thiols,
akin to the GSTs. Within GSTs, GSTABP-G shows selectivity
for GSTM2, M1, A3, P1, M4, T1, K1, MAAI (Z) and GSTABP-
H for GSTT2, T1, and Z isoenzymes. These results validate the
effectiveness of both probes for isoenzymeꢀspecific targeting of
many members of cytosolic GST classes. To demonstrate the
selectivity of the probes, and the value of an ABP approach over
global abundance profiling, we compared ABP labeling results to
global proteomics analysis of liver lysate. Only six GSTs were
identified by global analysis, but ABPꢀlabeling resulted in the
detection of 12 GSTs, including all of the GSTs detected in unenꢀ
riched lysate (SI Table S3). The ABPs enable characterization of
active and low abundance GSTs.
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Figure 4. (A) Liver and lung GSTABP-G and GSTABP-H targets (from
probe enrichment and LCMSꢀMS analysis) depicted as a Venn diagram.
(B) Relative abundances of GSTABP-G and GSTABP-H GST targets in
hepatic cytosol from untreated versus corn oil treated mice. Proteins with
* denotes significant alteration in protein abundance from SD to HFD
mice.
GSTABP-H selectively targets GST H sites (Figure 3). We also
competed GSTABP-G labeling with ethacrynic acid and found it
to inhibit GSTABP-G labeling at 0.44 µM (to 10 µM probe).
Thus, ethacrynic acid may block both GSTABP-G and
GSTABP-H access to the G site (Figure 3). It is also possible that
the presence of ethacrynic acid in the H site interferes with the
alkyne and benzophenone moieties of GSTABP-G that likely
reside in the H site.
To determine specific labeling of GSTs and GSHꢀbinding proꢀ
teins, we performed proteomic analysis of Sꢀhexylglutathione
competition in liver cytosol. GSTABP-H and GSTABP-G labelꢀ
ing of GSTA3, M1, and P1 was significantly inhibited by Sꢀ
hexylglutathione (fold change>3.0, p≤0.05), and were the top
three of all inhibited targets when sorted by foldꢀchange represꢀ
sion, indicating that both probes are labeling the active site of
GSTs (Figure 2; SI Table S4; SI Table S5).
To distinguish between activityꢀspecific binding and general
reactivity of the probes, and to characterize their selectivity for G
or H site specificity, we competed GSTABP-H and GSTABP-G
labeling of mouse liver lysate against relevant inhibitors. To
evaluate G site labeling by GSTABP-G, GSH was added in exꢀ
cess. Gel and LCꢀMS analysis showed no significant inhibition
The subsite complementarity of the probes provide a unique
approach to examine each probe’s enzyme specificity. We postuꢀ
lated Dichlon could effectively inhibit GSTABP-G labeling by
physically competing the alkyne and benzophenone moieties that
likely extend into the H site, as with Sꢀhexylglutathione. Competiꢀ
tion with Dichlon selectively inhibits labeling of a 24 kDa protein,
the approximate weight of several GSTs (Figure S1). We propose
this inhibition is likely due to competition between Dichlon and
the thiolꢀappended alkyne/benzophenone moiety for H site occuꢀ
pancy. Therefore, not only do these results suggest that the
GSTABP-G is binding within the GST active site, they also proꢀ
vide further evidence that Dichlon, the GSTABP-H parent moleꢀ
cule, binds within the GST active site. LCꢀMS was performed on
Dichlon (25 µM) competed GSTABP-G labeled mouse liver
cytosol (Figure 2A). GSTABP-G labeling of GSTP1, M4, A3,
M1, K1, and M2 was significantly inhibited when competed with
Dichlon (FC>2, p≤0.05) (Figure 2A). This indicates that the
GSTABP-H parent molecule selectively inhibits a variety of GST
classes near the same binding region as the GSTABP-G, further
confirming that the GSTABP-H is binding to the active site of
GSTs. To investigate the impact of the alkyne on the reactivity of
the GSTABP-H’s Dichlon moiety, we competed GSTABP-H
labeling of liver cytosol with Dichlon (Figure S1). Inhibition reꢀ
sulted in decreases in all measurable fluorescent bands, indicating
that the alkyne does not significantly affect reactivity.
(data not shown). We hypothesize that this lack of inhibition is
attributable to the formation of glutathione disulfides (GSꢀSG) in
our in vitro proteome sample. To circumvent the requisite addiꢀ
tion of potentially activityꢀmodifying reducing agents to prevent
GSꢀSG formation, we competed probe labeling with Sꢀ
hexylglutathione, a GSH conjugate incapable of forming disulfide
bonds. Sꢀhexylglutathione inhibits GSTABP-G labeling of GSTs
in a concentration dependent manner, with an effective concentraꢀ
tion of 1.77 µM (to 10 µM probe), suggestive of the GSTABP-G
specificity for the G site (Figure 3).
To investigate GSTABP-H targeting of GST H sites, we
compared competition of GSTABP-H inhibition by the G site
inhibitor Sꢀhexylglutathione to competition by ethacrynic acid, a
20ꢀ23
known H site inhibitor.
Sꢀhexylglutathione shows significant
inhibition of GSTABP-H labeling with an EC50 of 41.5 µM (Figꢀ
ure 3). It is plausible this competition is due to a known interacꢀ
tion between Sꢀhexylglutathione’s conjugated hexyl moiety and
2
4
the GST H site. Although our G site inhibitor decreases
GSTABP-H binding, we expected the H site inhibitor, ethacrynic
acid, to be much more effective. We found ethacrynic acid to be
nearly 12× more potent in inhibiting GSTABP-H enzyme labelꢀ
ing compared to Sꢀhexylglutathione, indicating that the
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Finally, to differentiate between the general reactivity of the
Synthetic schemes and protocol; SDSꢀPAGE analyses showing
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GSTABP-H versus its activityꢀspecific labeling, we performed
competitive probe labeling using Nꢀethylmaleimide (NEM), a
cysteine alkylating agent. Proteomic analysis of 100 µM NEM
competed GSTABP-H labeling failed to compete for binding of
most GST isoenzymes, indicating cysteine binding may not be the
primary amino acid targeted by GSTABP-H (Figure 2B; SI Table
S5). In combination with previous studies suggesting tyrosine
binding by chlorinated quinones , these results are suggestive
that tyrosine is the primary residue irreversibly bound by
GSTABP-H.
concentration dependent labeling and competition studies with
corresponding determination of EC50 values. The Supporting Inꢀ
formation is available free of charge on the ACS Publications
website.
AUTHOR INFORMATION
Corresponding Author
19
*aaron.wright@pnnl.gov
Notes
The authors declare no competing financial interests. The mass
spectrometry proteomics data have been deposited to the Proteoꢀ
meXchange Consortium via the PRIDE partner repository with
the dataset identifier PXD006920 and 10.6019/PXD006920.
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Following our investigation into the labeling mechanisms of
the ABPs, we evaluated changes in siteꢀspecific GST activity of
organs relevant to xenobiotic metabolism including liver, lung,
2
5
kidney, intestine, spleen, and heart lysates. We compared GST
activity determined via probe labeling and fluorescence gel imagꢀ
ing with a colorimetric GST activity assay that measures the total
conglomerate of GST activity. Fluorescence intensity of ABP
labeling was determined by quantification of the fluorescence
signal. As anticipated, liver GST activity was highest by ABP
labeling, followed by lung and kidney. These measurements
closely correspond with total GST activity determined by the
assay (Figure S2). We then conducted proteomics studies on lung
lysate. 11 GST isoenzymes were enriched by GSTABP-H, and 3
GST enzymes were enriched by GSTABP-G (Figure 4A; SI Taꢀ
ble S6). Both probes showed high enrichment of lung GSTA4, a
protein not significantly enriched by either probe in liver lysate, as
ACKNOWLEDGMENT
This research was supported by the National Institutes of Health
National Institute of Environmental Health Sciences (P42
ES016465), and employed proteomics capabilities supported by
the NIH NIGMS Research Resource for Integrative Biology (P41
GM103493). A portion of the research was performed using
EMSL, a DOE Office of Science User Facility sponsored by the
Office of Biological and Environmental Research. PNNL is a
multiprogram laboratory operated by Battelle for US DOE Conꢀ
tract DEꢀAC06ꢀ76RL01830.
25
anticipated from prior studies . While GSTABP-H and
REFERENCES
GSTABP-G show enrichment of GSTM4 in the liver, no activity
was detected in lung lysate. GSTABP-H also enriches GSTM6 in
liver lysate, but not lung; in agreement with mRNA expression
(1) Armstrong, R.N. Compr. Tox. 2010, 295.
(2) Danielson, U. H.; Esterbauer, H.; Mannervik, B. Biochem. J. 1987,
25
247, 707.
analyses . This data demonstrates the effectiveness of these comꢀ
plementary probes to examine the tissueꢀspecific contribution of
GSTs in xenobiotic metabolism.
(3) Berhane, K.; Widersten, M.; Engström, A.; Kozarich, J. W.;
Mannervik, B. PNAS 1994, 91, 1480.
(4) Singhal, S. S.; Singh, S. P.; Singhal, P.; Horne, D.; Singhal, J.;
Awasthi, S. Toxicol. Appl. Pharmacol. 2015, 289, 361.
Finally, to validate that the probes detect physiologically releꢀ
vant alterations to GST activity, we compared ABPꢀdetermined
GST activity in intestines of mice fed standard (10% fat) or high
fat, obesogenic, chow (60% fat) over a 20 week period. Statistiꢀ
cally significant alterations in several GST isoenzymes were seen
in response to diet induced obesity, including members of mu and
pi GST classes (Figure 4B; Table S7). The activity increases in
intestinal GSTs in obese mice is likely a response to oxidative
(5) Singhal, S. S.; Zimniak, P.; Awasthi, S.; Piper, J. T.; He, N. G.;
Teng, J. I.; Petersen, D. R.; Awasthi, Y. C. Arch. Biochem. Biophys. 1994,
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Townsend, A. J. Chem.-Biol. Interact. 2009, 179, 240.
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stress resulting from obesity.
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82.
9) Morrow, C. S.; Smitherman, P. K.; Diah, S. K.; Schneider, E.;
We anticipate that successful targeting of GSTs in mammalian
model systems by ABPs will enable a much improved understandꢀ
ing of the role of GSTs in human drug metabolism and xenobiotic
exposure. The GSHꢀconjugating activity of GSTs is affected by
various factors, including PTMs, making transcriptomics and
global proteomics poor indicators of activity. The ability to anaꢀ
lyze the enzymeꢀspecific activity of GSTs is required to advance
investigations into the pathways involved in GSH conjugation. Of
the 19 cytosolic mammalian GSTs encoded in the genome, the
probes successfully label 13; demonstrating that the ABPs can
broadly profile individual GST activities. The ABPs also facilitate
enrichment of members of all GST classes. Probe labeling and
MS analysis of other tissues in which the remaining GSTs show
higher activity may reveal comprehensive cytosolic GST targetꢀ
ing. We assert that this chemoproteomics approach, using these
complementary activityꢀ and affinityꢀbased probes, provides a
required advance for understanding of human drug metabolism
and response to exposure.
(
Townsend, A. J. J. Biol. Chem. 1998, 273, 20114.
(10) Meyer, D. J.; Gilmore, K. S.; Harris, J. M.; Hartley, J. A.; Ketterer,
B. Br. J. Cancer 1992, 66, 433.
(11) Larsson, A.ꢀK.; Shokeer, A.; Mannervik, B. Arch. Biochem.
Biophys. 2010, 497, 28.
(12) AliꢀOsman, F.; Caughlan, J.; Gray, G. S. Cancer Res. 1989, 49,
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(13) Townsend, D. M.; Manevich, Y.; He, L.; Hutchens, S.; Pazoles, C.
J.; Tew, K. D. J. Biol. Chem. 2009, 284, 436.
(14) Board, P. G.; Coggan, M.; Chelvanayagam, G.; Easteal, S.;
Jermiin, L. S.; Schulte, G. K.; Danley, D. E.; Hoth, L. R.; Griffor, M. C.;
Kamath, A. V.; Rosner, M. H.; Chrunyk, B. A.; Perregaux, D. E.; Gabel,
C. A.; Geoghegan, K. F.; Pandit, J. J. Biol. Chem. 2000, 275, 24798.
(15) Theodoratos, A.; Blackburn, A. C.; Coggan, M.; Cappello, J.;
Larter, C. Z.; Matthaei, K. I.; Board, P. G. Int. J. Obes. 2012, 36, 1366.
(16) Singh, S. Cancer Chemother. Pharmacol. 2015, 75, 1.
(
17) van Ommen, B.; Ploemen, J. H.; Bogaards, J. J.; Monks, T. J.;
Gau, S. S.; van Bladeren, P. J. Biochem. J. 1991, 276, 661.
18) Vos, R. M. E.; Van Ommen, B.; Hoekstein, M. S. J.; De Goede, J.
(
H. M.; Van Bladeren, P. J. Chem.-Biol. Interact. 1989, 71, 381.
(19) Ploemen, J. H.; Johnson, W. W.; Jespersen, S.; Vanderwall, D.;
van Ommen, B.; van der Greef, J.; van Bladeren, P. J.; Armstrong, R. N. J.
Biol. Chem. 1994, 269, 26890.
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(20) Ploemen, J. H.; van Ommen, B.; Bogaards, J. J.; van Bladeren, P.
, S.; Klaassen, C. D. Toxicol. Sci. 2007, 100, 513.
J. Xenobiotica 1993, 23, 913.
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Pharmacol. 1990, 40, 1631.
22) Cameron, A. D.; Sinning, I.; L'Hermite, G.; Olin, B.; Board, P. G.;
Mannervik, B.; Jones, T. A. Structure 1995, 3, 717.
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Federici, G.; Parker, M. W. Biochemistry 1997, 36, 576.
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M.; Federici, G.; Parker, M. W. J. Mol. Biol. 1992, 227, 214.
(26) Marseglia, L.; Manti, S.; D’Angelo, G.; Nicotera, A.; Parisi, E.; Di
Rosa, G.; Gitto, E.; Arrigo, T. Int. J. Mol. Sci. 2015, 16, 378.
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Figure 1. Activityꢀ and affinityꢀbased probes designed to target active GSTs. (A) Function of active GSTs; the
conjugation of reduced glutathione (GSH) to xenobiotic (X) stabilized by “G” and “H” site binding by each
substrate, respectively. (B) Structure of affinityꢀbased GSTABPꢀG designed to target GST “G” sites (C)
Structure of activityꢀbased GSTABPꢀH designed to target GST “H” sites. GSTABPꢀG and GSTABPꢀH probe
dependent labeling of 1 µM recombinant human GSTM1 (D) and mouse liver cytosol (E). BP –
benzophenone.
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Figure 2. LCꢀMS/MS chemoproteomics results of probe enriched and probe competed mouse liver cytosol
(n=3). All samples were incubated with 10 µM probe or an equal concentration of vehicle control.
Enrichment was calculated as AMT tag abundances from probeꢀenriched samples divided by a no UV (for
GSTABPꢀG) or DMSO only (for GSTABPꢀH) control. Significance was determined using a paired tꢀtest with a
twoꢀtailed distribution. (A) Volcano plot of GSTABPꢀG enrichment. In black: all GSTs that did not
demonstrate competitive inhibition of probe labeling. In green: GSTs whose probe labeling was
competitively inhibited by 2.5× excess 2,3ꢀdichloroꢀ1,4ꢀnapthoquinone (Dichlon) over probe. In blue: GSTs
whose probe labeling was competitively inhibited by 2.5× excess Dichlon and 2.5× excess Sꢀ
hexylglutathione. (B) Volcano plot of GSTABPꢀH enrichment. In black: all GSTs whose probe labeling was not
competitively inhibited. In blue: GSTs whose probe labeling was competitively inhibited by 20× Sꢀ
hexylglutathione. In red: GSTs whose probe labeling was competitively inhibited by 10× Nꢀethylmaleimide.
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Figure 3. Competitive inhibition of GSTABPꢀG and GSTABPꢀH labeling of mouse liver cytosol to delineate G
and H site binding (n=3). 10 µM probe was used in all samples. Increasing concentrations of Sꢀ
hexylglutathione (GSꢀhexyl) or ethacrynic acid (EA) was incubated with mouse liver cytosol for 30 min
before probe labeling for 30 min. Afterwards, rhodamine was attached to the probeꢀprotein complexes via
click chemistry. Proteins were resolved via SDSꢀPAGE followed by fluorescence gel imaging and
quantification (arbitrary units) of probe labeled GST bands. All replicates were normalized to a % labeled
protein value (probed samples with no inhibitor = 100%). Normalized values were plotted and EC50 values
were determined with a best fit curve.
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Figure 4. (A) Liver and lung GSTABP-G and GSTABP-H targets (from probe enrichment and LCMS-MS
analysis) depicted as a Venn diagram. (B) Relative abundances of GSTABP-G and GSTABP-H GST targets in
hepatic cytosol from untreated versus corn oil treated mice. Proteins with * denotes significant alteration in
protein abundance from SD to HFD mice.
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