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INTRODUCTION |
Interleukin (IL)1-1 is a
proinflammatory mediator produced in abundance by lipopolysaccharide
(LPS)-activated monocytes and macrophages (1). Unlike most other
cytokines generated by these cells, IL-1 is not constitutively
released. Rather, efficient IL-1 export requires that the
cytokine-producing cell encounter an effector that initiates an unusual
posttranslational processing mechanism leading to cytokine release
(2-4). A separate secretion stimulus is needed because the initial
IL-1 translation product lacks a signal sequence (5, 6), a recognition
motif that directs nascent membrane and secretory polypeptides to the
endoplasmic reticulum (7). From this location, polypeptides destined
for secretion typically proceed to the cell surface via a common
secretory apparatus involving the Golgi complex and small secretory
vesicles (8). In the absence of the recognition marker for entry to the
endoplasmic reticulum, newly synthesized IL-1 accumulates within the
cytoplasmic compartment (9, 10). Proteins retained in this compartment
generally are not thought to function as extracellular mediators.
Separate but related genes encode the procytokine forms of IL-1
and
IL-1
(5, 6). In response to proper cellular stimuli, the 31-kDa
translation products of both genes are proteolytically processed to the
mature 17-kDa cytokines. In the case of IL-1
, the mature and
procytokine species both are capable of binding to the IL-1 receptor
complex and initiating cytokine signaling (11). In the case of IL-1
,
proteolytic maturation is required to generate a receptor-competent
ligand (11). Pro-IL-1
is cleaved by caspase-1 (12, 13), one of a
family of intracellular cysteine proteases that initiate and/or execute
apoptotic responses (14). Stimuli that promote efficient IL-1
posttranslational processing in vitro include extracellular
ATP (2, 15, 16), the potassium ionophore nigericin (3, 17), bacterial
toxins such as Escherichia coli hemolysin (18), cytolytic
T-cells (2, 19), and protegrins (20). All these agents induce membrane
depolarization and cell death, and K+ efflux from the
target cell appears to be a requirement for the cytokine response (15,
21). K+ efflux increasingly has been reported to be an
important element of many apoptotic responses (22-24).
ATP-induced IL-1
posttranslational processing proceeds via
activation of the P2X7 receptor, a ligand-gated ion
channel. Immediately following ATP binding, the P2X7
receptor acts as a non-selective cation channel (25-27). However,
prolonged ligation causes the channel to transition to a "porelike"
conductance state (28, 29). Receptor mutagenesis has suggested that the
intracellular carboxyl terminus of the receptor polypeptide is
necessary for pore formation (25), but whether the pore forms as a
result of a change in the receptor itself or as a result of an
association with other polypeptides remains to be determined (30, 31). Changes attendant to P2X7 receptor activation include
activation of various stress kinases (32, 33), transcription factors (34) and phospholipases (35, 36), and membrane depolarization (37). In
some cell systems, P2X7 receptor activation does not result
in immediate cell death (33). On the other hand, sustained P2X7 receptor activation in cells such as monocytes,
macrophages, and microglia initiates a rapid and dramatic death
response (15, 38, 39). Factors that determine whether a
P2X7 receptor-responding cell follows a fatal course remain
to be elucidated.
IL-1 is a key contributor to many inflammatory disease processes, and
both preclinical animal model as well as human clinical studies
employing various inhibitors of IL-1 have provided evidence of
therapeutic utility (40-43). Thus, great interest exists in finding
novel mechanisms for regulating IL-1 activity. We recently identified a
series of cytokine release inhibitory drugs (CRIDs) that potently and
selectively disrupt stimulus-induced IL-1
posttranslational processing (44). These agents do not directly inhibit caspase-1, and
they do not act as antagonists of the P2X7 receptor; their mechanism of action remains to be established. This paper describes studies aimed at identifying their molecular target. Our results indicate that GST Omega 1-1, an atypical member of the glutathione S-transferase superfamily previously associated with
stress-induced apoptosis, is a CRID-binding protein. Therefore, GST
Omega 1-1 may perform an important, although currently unidentified,
role in ATP-induced IL-1
posttranslational processing.
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EXPERIMENTAL PROCEDURES |
Reagents--
1-(4-Chloro-2,6-diisopropylphenyl)-3-[2-fluoro-5-oxiranyl
benzenesulfonyl]urea (CP-452,759; CRID 1),
[14C]CRID 1, 1-(1,2,3,5,6,7- hexahydro-s-indacen-4-yl)-3-[2-fluoro-5-oxiranylbenzenesulfonyl]urea (CP-470,947; CRID 2), and [14C]CRID 2 were prepared as
detailed previously (45). CP-456,773 (CRID 3) was prepared by treatment
of 4-(1-hydroxy-1-methylethyl)furan-2-sulfonic acid amide (46) with
sodium hydride and
4-isocyanato-1,2,3,5,6,7-hexahydro-s-indacene in
tetrahydrofuran (47). CRID 3 was isolated as the sodium salt by
precipitation with pentane and filtration, and was purified by
recrystallization from isopropyl alcohol (m.p. of free acid = 153-154 °C). 1,2-Epoxy-3-(4-nitrophenoxy)propane was purchased from
Ryan Scientific (Isle of Palms, SC).
1-(1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)-3-[2-fluoro-5-(2-amino-4-[1-(carboxymethylcarbamoyl)-2-(2-hydroxyethylsulfanyl)ethylcarbamoyl]butyric acid)benzenesulfonyl]urea trisodium salt (CRID 2-SG) was prepared by
reaction of glutathione with CRID 2 in the presence of 0.1 M sodium methoxide in methanol and was purified by
trituration from tetrahydrofuran. Human carbonic anhydrase (type II)
and porcine liver carboxyl esterase were purchased from Sigma. A
plasmid encoding human glyoxalase I was obtained from Dr. Bengt
Mannervik (Uppsala University, Uppsala, Sweden) and expressed in
E. coli; the recombinant polypeptide was purified and
assayed as previously described (48).
Protein Sequencing--
Sequence analysis by automated Edman
degradation was performed on an Applied Biosystems model 494 Procise
sequencing system.
In-gel Digestions and LC-MS--
Proteins isolated in the form
of Coomassie Blue-stained gel bands were reduced with DTT,
S-alkylated with iodoacetamide, and digested with trypsin
(Promega chemically modified, sequencing grade) or Glu-C (Roche Applied
Science, sequencing grade) by the method of Stone and Williams (49).
Peptide mapping of the digests by LC-MS was conducted using a Vydac C18
column (type 218TP51) operating at 0.1 ml/min with a gradient of
acetonitrile in 0.1% trifluoroacetic acid. A Hewlett-Packard model
1090 chromatograph was used in tandem with a Thermo Finnigan LCQ
ion-trap mass spectrometer using a standard Finnigan electrospray
interface and controlled by Xcalibur software. For experiments to
identify the site of affinity labeling, the chromatograph was an
Agilent 1100 operating at 5 µl/min with a Vydac type 218MS5.510
column (0.5 × 100 mm). The HPLC solvents were: A, 0.02%
trifluoroacetic acid; B, 0.02% trifluoroacetic acid in
CH3CN. From initial conditions of 1.6% B (0-2 min), the
gradient consisted of steps from 1.6 to 35% B (2-98 min) and from 35 to 80%B (98-108 min). The standard Finnigan electrospray interface
was modified by replacing the sample needle with a coated fused silica
tip (TaperTip; part no. TT150-50-50-CE-5, New Objective Inc., Woburn,
MA). Data-dependent MS/MS was performed on MS peaks
exceeding a preset threshold (50), and proteins were identified by data
base searching using a version of the SEQUEST program (51) licensed
from Finnigan. When necessary, the spectrometer was programmed to
select specific peaks for multistage MS using standard programming
features of Xcalibur.
Labeling of Intact Cells with an Epoxide CRID--
Human
mononuclear cells were isolated from normal volunteers as previously
detailed (15). 2.5 × 107 mononuclear cells were
seeded into each well of a 60-mm dish, and monocytes were allowed to
attach for 2 h at 37 °C. Media and nonattached cells were then
removed, and 5 ml of fresh serum-free medium (Invitrogen; catalog no.
12065-074) containing 0.1% penicillin/streptomycin and 100 ng/ml macrophage colony stimulating factor was added to each
dish. These cultures were incubated overnight at 37 °C in a 5%
CO2 environment after which FBS and LPS (Sigma; E. coli serotype 055:B5) were introduced to achieve final
concentrations of 5% and 10 ng/ml, respectively. Following an
additional 3-h incubation, media were removed, the cells were washed
several times with RPMI medium devoid of FBS, and then 3 ml of RPMI
1640 medium containing 20 mM Hepes, pH 6.9, and the
indicated concentration of either [14C]CRID 1 or
[14C]CRID 2 was added, and the cultures were incubated at
37 °C for 60 min. The cells subsequently were washed repeatedly with
RPMI 1640 medium containing 25 mM Hepes, 5 mM
sodium bicarbonate, pH 6.9, 1% FBS, 2 mM glutamine, 1%
penicillin/streptomycin (Chase Medium). At this point, labeled cells in
some experiments were subjected to ATP activation; 3 ml of fresh Chase
Medium containing 2 mM ATP was added to each dish and the
cultures were incubated for 60 min. Following this treatment, the
dishes were placed on ice and media were removed for IL-1
analysis
(by ELISA). Cells, with or without ATP treatment, were extracted with 1 ml of 25 mM Hepes, pH 7.0, 150 mM NaCl, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 0.1 mM PMSF, and
0.5% saponin for 30 min on ice. These extracts subsequently were
clarified by centrifugation (45,000 rpm in a Beckman TLA.4 rotor).
Resulting supernatants were recovered as the soluble fraction and were
concentrated by Centricon YM-30 (Millipore, Bedford, MA)
centrifugation. The final concentrates were lyophilized overnight, then
resuspended in SDS sample buffer. The saponin-insoluble microsomal
pellets were solubilized directly into SDS sample buffer. All
SDS-containing samples subsequently were heated at 56 °C for 3 min;
heating to higher temperatures was avoided because of breakdown of the
radiolabel. The disaggregated samples then were fractionated by
SDS-polyacrylamide gel electrophoresis, and the resulting dried gel was
profiled by autoradiography and/or phosphorimager analysis.
IL-1 Assays--
To allow visualization of the mature cytokine
species, a metabolic labeling approach was employed to study IL-1
posttranslational processing. For this approach, 1 × 107 mononuclear cells were added to each well of six-well
multi-dishes in 2 ml of RPMI 1640 containing 25 mM Hepes,
5% FBS, 2 mM glutamine, 1% penicillin/streptomycin, pH
7.2 (Maintenance Medium). Monocytes were allowed to adhere for 2 h, after which the supernatants were discarded and the attached cells
were rinsed twice and then incubated in Maintenance Medium overnight at
37 °C in a 5% CO2 environment. The cultured monocytes
then were incubated with 10 ng/ml LPS for 2 h and labeled for 60 min in 1 ml of methionine-free RPMI 1640, containing 1% dialyzed FBS,
25 mM Hepes, pH 7.2, and 83 µCi/ml [35S]methionine (Amersham Biosciences; 1000 Ci/mmol). The
pulse medium subsequently was discarded, the radiolabeled cells were
rinsed once with 2 ml of Chase Medium, and then 1 ml of Chase Medium, with or without a test agent, was added to each well. Where indicated, ATP was added (from a 100 mM stock solution, pH 7.0) to
achieve a final concentration of 2 mM. Radiolabeled
monocytes were treated with ATP at 37 °C for 3 h after which
the medium was recovered and clarified by centrifugation; the resulting
supernatants were harvested and adjusted to 1% in Triton X-100, 0.1 mM PMSF, 1 mM iodoacetic acid, 1 µg/ml
pepstatin, and 1 µg/ml leupeptin by addition of concentrated stock
solutions of these reagents. Adherent monocytes were solubilized by
addition of 1 ml of an extraction buffer composed of 25 mM
Hepes, 1% Triton X-100, 150 mM NaCl, 0.1 mM
PMSF, 1 mM iodoacetic acid, 1 µg/ml pepstatin, 1 µg/ml
leupeptin, and 1 mg/ml ovalbumin, pH 7.0; 50 µl of this extraction
buffer also was added to the pellets obtained after clarification of
the media supernatants, and these samples were combined with their
corresponding cell extracts. After a 30-min incubation on ice, both the
media and cell extracts were clarified by centrifugation at 45,000 rpm
for 30 min.
For the ELISA-based assay, 2 × 105 mononuclear cells
were seeded into each well of 96-well plates in a total volume of 0.1 ml. Monocytes were allowed to adhere for 2 h, after which the supernatants were discarded and the attached cells were rinsed twice
and then incubated in Maintenance Medium overnight at 37 °C in a 5%
CO2 environment. These cultured monocytes were activated with 10 ng/ml LPS for 2 h after which the activation medium was removed, the cells were rinsed twice with 0.1 ml of Chase Medium, and
then 0.1 ml of Chase Medium containing a test agent was added and the
plate was incubated for 30 min; each test agent concentration was
evaluated in triplicate wells. ATP then was introduced (from a 100 mM stock solution, pH 7.0) to achieve a final concentration of 2 mM, and the plate was incubated at 37 °C for an
additional 3 h. Media were harvested and clarified by
centrifugation, and their IL-1
content was determined by ELISA (R&D
Systems, Minneapolis, MN).
Isolation of CRID-labeled THP-1 Cell Polypeptides--
THP-1
cells were obtained from American Type Culture Collection (Rockville,
MD) and activated with 200 ng/ml LPS for 2 h. These activated
cells (5 × 108 cells) then were incubated with 10 µM [14C]CRID 1 in 100 ml of hypotonic
medium (27 mM NaCl, 0.9 mM CaCl2, 0.5 mM MgCl2, 0.53 mM KCl, 0.29 mM KH2PO4, 20 mM Hepes,
5 mM glucose, 5 mM NaHCO3, 10 ng/ml
LPS, pH 7.0) as a suspension culture for 60 min at 37 °C, after
which the cells were harvested by centrifugation, washed with
phosphate-buffered saline, and suspended in 50 ml of 25 mM
Hepes, 30 mM KCl, 30 mM sodium gluconate, 0.9 mM CaCl2, 0.5 mM MgCl2,
1 µg/ml leupeptin, 1 µg/ml pepstatin, pH 7.0. This suspension was
subjected to nitrogen cavitation (650 p.s.i. for 15 min), and the
resulting lysate was clarified by ultracentrifugation. The supernatant
was recovered and applied to an anion exchange resin (10-ml HiTrap Q
column; Amersham Biosciences) equilibrated in 20 mM Tris,
20 mM NaCl, 1 mM MgCl2, 1 mM DTT, 1 µg/ml pepstatin, and 1 µg/ml leupeptin, pH
8.0, and bound proteins were eluted with a linear 200-ml gradient from
20 to 250 mM NaCl. An aliquot of each fraction was
monitored by liquid scintillation counting, and peak fractions were
pooled and concentrated; their polypeptide content subsequently was
assessed by SDS-PAGE and autoradiography. Under the conditions used,
the 31-kDa radiolabeled polypeptide failed to bind to the anion
exchanger and was recovered in the flow-through (Fig. 3). In contrast,
the 32- and 56-kDa species bound to the column and were eluted at
distinct regions of the NaCl gradient (Fig. 3). The radiolabeled 32-kDa
polypeptide co-migrated with a discrete Coomassie Blue staining
polypeptide and was excised from the gel and analyzed. The 31-kDa
polypeptide, however, required additional purification. Material that
ran through the anion exchange column was fractionated on a Superose 12 HR 10/30 column, and the 31-kDa polypeptide was eluted from this sizing
column as a single peak. When analyzed by two-dimensional gel
electrophoresis and autoradiography, this peak was found to contain a
single radiolabeled polypeptide that corresponded to a distinct
Coomassie Blue staining polypeptide (data not shown). This was excised
from the dried gel and analyzed. The 56-kDa enriched peak from the
anion exchange column was dialyzed overnight against 20 mM
sodium phosphate, 20 mM NaCl, 1 mM
MgCl2, 1 mM CaCl2, 1 µg/ml
pepstatin/leupeptin, pH 6.0, and then fractionated on a HiTrap SP
column (5 ml). A linear gradient of NaCl (from 20 to 500 mM) was applied to the column, and a single radioactive
peak was eluted. Fractions comprising this peak were pooled and
concentrated by Centricon YM-30 centrifugation. This concentrate then
was applied to the Superose 12 column. Again, a single peak of
radioactivity was observed. Fractions within this peak were pooled,
concentrated by Centricon YM-30 centrifugation, and fractionated by
SDS-PAGE. Following autoradiography, the Coomassie Blue staining
polypeptide corresponding to the radiolabeled 56-kDa species was
excised for sequence analysis by digestion and LC-MS.
CRID Affinity Chromatography--
Thiopropyl-Sepharose (7 g;
Amersham Biosciences) initially was suspended in buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.0) containing 50 mM DTT to generate free sulfhydryl
groups. The activated resin then was washed to remove DTT and suspended in 70 ml of 10 mM Tris, 100 mM NaCl, 10 mM EDTA, pH 9.3, containing 120 mg of CRID 2; to allow
success of the coupling to be monitored, a small amount of
[14C]CRID 2 was added to the reaction mixture. After a
16-h incubation at 4 °C, the resin was recovered and washed
extensively with 10 mM Tris, 100 mM NaCl, 1 mM EDTA, 50 mM DTT, pH 7.0. Next, the resin was
washed free of DTT and treated with 14 mM
N-ethylmaleimide in 0.1 M sodium bicarbonate,
100 mM NaCl, pH 8.0, for 30 min at room temperature to cap
unreacted free sulfhydryl groups. Following this step, the resin was
washed extensively with 50 mM Tris, 5 mM DTT,
pH 7.0. A 10-ml column of the resin was employed for the purification
of THP-1 cell polypeptides; the resin was equilibrated in 25 mM Hepes, 100 mM NaCl, 5 mM DTT,
0.3% Triton X-100, 1 µg/ml pepstatin, 1 µg/ml leupeptin, pH
7.0.
4.5 × 107 THP-1 cells were harvested by
centrifugation and suspended in 20 ml of methionine-free RPMI medium
containing 1% dialyzed FBS and 1 mCi of [35S]methionine
(1000 Ci/mmol). The cells were labeled for 1 h after which they
were harvested by centrifugation, washed with cold PBS to remove free
[35S]methionine, and then mixed with 2.3 × 108 unlabeled THP-1 cells. The combined cell pellet was
suspended in 20 ml of hypotonic buffer (25 mM Hepes, 25 mM NaCl, 5 mM DTT, pH 7.0, containing
CompleteTM protease inhibitors (Roche Applied Science,
Mannheim, Germany) and subjected to nitrogen cavitation (650 p.s.i. for
15 min). The resulting lysate was clarified by low speed centrifugation to remove unbroken cells and cell debris; the supernatant subsequently was subjected to high speed centrifugation to yield a cytosolic (soluble) fraction. This fraction was applied to the CRID affinity column; individual 2-ml fractions were collected and monitored for radioactivity.
Labeling of Recombinant GST Omega--
Bacterially expressed
protein was isolated as described (52). 1 µg of the recombinant
protein was incubated in 0.02 ml of PBS, 1 mM DTT
containing the indicated concentrations of [14C]CRID 2 in
the presence or absence of potential effector molecules. After a 30-min
incubation at 4 °C, 6 µl of 6× SDS sample buffer were added, and
the mixtures were heated at 56 °C for 3 min and analyzed by
SDS-polyacrylamide gel electrophoresis. Resulting dried gels were
profiled by autoradiography and/or phosphorimager analysis.
For LC-MS analysis of the protein adduct, GST Omega 1-1 (900 µg in 1 ml of 50 mM Tris, pH 7.6, 100 mM NaCl, 5 mM DTT) was incubated with 35 µM CRID 2 (final Me2SO concentration was 0.01%) for 10 min at room
temperature. The reaction mixture was applied to a Sephadex G25 column
equilibrated in 50 mM Tris, pH 7.2, 100 mM
NaCl, 5 mM DTT to remove excess CRID 2, desalted by
reversed-phase HPLC as described (53), and dried in a centrifugal
concentrator. The dried proteins were redissolved in 8 M
urea, reduced with DTT, S-carbamoylmethylated by
treatment with iodoacetamide, and digested with trypsin.
 |
RESULTS |
CRID Affinity Labeling Studies--
Two novel
diarylsulfonylurea-based cytokine release inhibitory drugs were
synthesized containing an epoxide group appended to the pharmacophore
(Fig. 1A); synthesis of these
compounds was described earlier (45). The selection of an epoxide as
the reactive moiety was based on the expectation that this group reacts
selectively to form covalent adducts with free sulfhydryl groups on
proteins, and on the earlier demonstration that ATP-induced IL-1
posttranslational processing is disrupted by non-selective
alkylating agents such as N-ethylmaleimide (38). Both of the
epoxide-containing agents, CRID 1 and CRID 2, demonstrated
dose-dependent inhibition of ATP-induced IL-1
posttranslational processing (Fig. 1B). CRID 1 and CRID 2 yielded IC50 values in the monocyte-based assay of 350 and
250 nM, respectively (Fig. 1B). Importantly,
equivalent concentrations of an epoxide-bearing non-diarylsulfonylurea
test agent, 1,2-epoxy-3-(4-nitrophenoxy)propane (Fig. 1A),
did not impair ATP- induced IL-1
production (Fig. 1B).
Therefore, when they were tested at similar concentrations, the
pharmacodynamic activity demonstrated by the two epoxide-bearing diarylsulfonylurea-based compounds was not shared with a structurally distinct epoxide-containing agent.

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Fig. 1.
CRIDs inhibit IL-1
posttranslational processing. A, structures of
pharmacological agents employed in this study. B,
LPS-activated human monocytes were treated with the indicated
concentration of effector for 30 min and then ATP was introduced to the
medium to initiate IL-1 posttranslational processing. After a 3-h
incubation, media were harvested and their IL-1 content was
determined by ELISA. The amount of cytokine is expressed as a
percentage of that released from control cells in the absence of an
effector and indicated as a function of test agent concentration. Each
data point is the average of triplicate determinations.
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To identify proteins for which function may be disrupted by CRID
binding, radiolabeled derivatives of both CRID 1 and CRID 2 (45) were
applied to cultured human monocytes. Monocytes treated with
[14C]CRID 1 incorporated radioactivity into a limited
number of discrete cytosolic proteins (Fig.
2A). At concentrations
0.3
µM, [14C]CRID 1 labeled a single species
with an apparent molecular mass of 31 kDa (Fig. 2A). As the
concentration of this test agent was increased, polypeptides with
apparent molecular masses of 32 and 56 kDa also became evident. In
contrast, the membrane-enriched fraction derived from the CRID-treated
monocytes contained no obvious radiolabeled proteins as detected by
SDS-PAGE and autoradiography (data not shown).

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Fig. 2.
[14C]CRIDs covalently bind to a
limited number of cellular polypeptides. A,
LPS-activated human monocytes were treated with the indicated
concentration of [14C]CRID 1 for 60 min, after which a
cytosolic fraction was prepared and analyzed by SDS-PAGE and
autoradiography. B, LPS activated human monocytes were
treated with the indicated concentration of [14C]CRID 2 for 60 min, after which a cytosolic fraction was analyzed by SDS-PAGE
and autoradiography. C, prior to harvesting the CRID-labeled
cells in panel A, the cultures were washed free
of non-bound test agent, and then treated with 2 mM ATP to
engage IL-1 posttranslational processing. After a 60-min incubation,
media supernatants were harvested and analyzed for IL-1 content by
ELISA; the amount of extracellular cytokine is expressed as a
percentage of that recovered from control cells in the absence of the
CRID and indicated as a function of test agent concentration. For
comparison, radioactivity incorporated into the three polypeptides
shown in panel A was determined by phosphorimager
analysis. The maximum level of incorporation for each polypeptide was
set to 100%; the relative level of incorporation at each test agent
concentration then was determined (as a percentage of the maximum) and
is indicated. This type of correlation experiment was performed three
and two times, respectively, with CRID 1 and CRID 2. The apparent
molecular masses of the radiolabeled species are indicated as 56, 32, and 31 kDa.
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A similar experiment was performed with [14C]CRID 2, and
an identical set of cytosolic polypeptides incorporated the
radiolabeled pharmacophore (Fig. 2B). Concentrations of
[14C]CRID 2
1 µM resulted in the
labeling of a single 31-kDa radiolabeled polypeptide. Increasing the
test agent concentration to 3 and 10 µM enhanced
incorporation into the 32- and 56-kDa species, whereas incorporation
into the 31-kDa species appeared to reach saturation (Fig.
2B). As with cells treated with [14C]CRID 1, incorporation of CRID 2-derived radioactivity into membrane proteins
was minimal (as detected by SDS-PAGE and autoradiography; data not shown).
Prior to harvesting [14C]CRID 1-treated cells in the
above experiment, the test agent was removed from the medium, and the
cells were washed to remove non-bound pharmacophore. They then were treated with ATP to engage IL-1 posttranslational processing. Media
recovered from the ATP-treated cultures subsequently were assessed for
IL-1
content by ELISA. Relative to monocytes treated with the
nucleoside triphosphate without prior test agent exposure, pharmacophore-treated cells demonstrated a dose-dependent
inhibition in cytokine production (Fig. 2C). In an attempt
to correlate the extent of radiolabeling of the three individual
cytosolic polypeptides with the observed pharmacological response,
radioactivity incorporated into each polypeptide (expressed as a
percentage of that observed at the highest tested
[14C]CRID 1 concentration) also is indicated in Fig.
2C. Viewed in this manner, labeling of the 31-kDa
polypeptide correlated with the extent of IL-1 inhibition, whereas
labeling of the 32- and 56-kDa polypeptides occurred at test agent
concentrations exhibiting maximal inhibition of the cytokine response
(Fig. 2C).
Identification of Binding Polypeptides--
Following treatment of
THP-1 cells with [14C]CRID 1, radiolabeled polypeptides
possessing apparent molecular masses of 31, 32, and 56 kDa were
detected (Fig. 3A). As in the
monocyte system, the 31-kDa polypeptide labeled at lower concentrations
of the pharmacophore than did the 32- and 56-kDa species (Fig.
3A). At the highest test agent concentration (10 µM), the relative abundance of the 32-kDa radiolabeled
species appeared higher in extracts derived from THP-1 cells than in
those recovered from monocytes (compare Fig. 3A to Fig.
2A), but the labeling patterns for the two cell types were
similar overall. Therefore, THP-1 cells were employed as a starting
source for isolation of the binding proteins. A procedure for
generating enriched preparations of the three radiolabeled polypeptides
is detailed under "Experimental Procedures." A key step in this
isolation was fractionation of the [14C]CRID 1-labeled
cytosol preparation on an anion exchange column that separated the
three radiolabeled polypeptides (Fig. 3B). The 31-kDa
protein did not bind to the anion exchange resin and was recovered in
the flow-through fractions (Fig. 3B). In contrast, the 32- and 56-kDa species bound and were eluted with increasing salt; the
56-kDa species eluted earlier (peak C, Fig.
3B) than the 32-kDa species (peak D,
Fig. 3B). The three proteins were further purified as
described under "Experimental Procedures."

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Fig. 3.
Isolation of radiolabeled polypeptides from
THP-1 cells. A, THP-1 cells (pretreated with 50 nM phorbol 12-myristate 13-acetate for 2 days to promote
attachment) were treated with the indicated concentration of
[14C]CRID 1 after which a cytosolic extract was prepared
and analyzed by SDS-PAGE. B, a cytosol fraction prepared
from THP-1 cells previously treated with 10 µM
[14C]CRID 1 was fractionated by anion exchange
chromatography. Individual fractions were collected, and aliquots of
each were analyzed by liquid scintillation counting. The flow-through
peak (FT) as well as fractions corresponding to peaks and
shoulders (A-G) were pooled, and a portion of each was
subjected to SDS-PAGE and autoradiography. An autoradiogram is shown;
migration positions of the 56-, 32-, and 31-kDa species are
indicated.
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NH2-terminal sequence analysis of the radiolabeled 31-kDa
polypeptide blotted to polyvinylidene difluoride from a two-dimensional gel indicated that the protein possessed a blocked amino terminus. Following tryptic digestion and HPLC fractionation of the digest, a
number of peak fractions were subjected to automated Edman sequencing and matrix-assisted laser desorption/ionization time-of-flight MS.
Several peptide sequences were obtained that matched predicted tryptic
peptides encoded by a deposited human cDNA (accession no. U90313),
which also has a murine ortholog (accession no. U80819). The murine
ortholog originally was identified as a resistance factor to
radiation-induced lymphocyte apoptosis (54). The human protein, GST
Omega 1-1, recently was identified as a novel member of the GST
superfamily (52).
NH2-terminal sequence analysis of the 32-kDa polypeptide
blotted to polyvinylidene difluoride from an SDS gel indicated that this protein was also blocked. Tryptic and Glu-C digests were prepared
and fractionated by HPLC. Sequencing of multiple peptides led to
identification of the 32-kDa polypeptide as CLIC1 (accession no.
U93205), a protein belonging to a family of unusual ion channels (55,
56). A relationship between GST Omega 1-1 and CLIC1 previously was
described based on sequence homology (57). Alignment of the amino acid
sequences indicates that these two proteins share an absolute identity
of 15% (Fig. 4); a region near the amino
terminus (residues 24-38 of CLIC1 and 32-46 of GST Omega 1-1) is
particularly well conserved. This is noteworthy, as Cys32
of GST Omega 1-1 resides at its putative active center (52).

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Fig. 4.
Sequence alignment of CLIC-1 and GST
Omega 1-1. Underlined residues correspond to those
identified by Edman sequence analysis and/or mass spectrometry.
Residues conserved between the two polypeptides are boxed.
Sequence alignment was generated using MegAlign (DNASTAR, Inc.).
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Trypsin digestion of the 56-kDa polypeptide excised from an SDS gel
yielded peptides matching those present in human carboxyl esterase as
demonstrated by LC-MS and data base searching. The mass of carboxyl
esterase, 60 kDa (58), is in close agreement with the apparent mass of
the 56-kDa polypeptide.
Identification of CRID-binding Proteins by Affinity
Chromatography--
A CRID affinity matrix was generated by coupling
CRID 2 to Thiopropyl-Sepharose, and soluble extracts derived from
[35S]methionine-labeled THP-1 cells were applied to a
column containing this matrix. Following sample application, a large
peak of radioactivity was recovered in the flow-through fractions (Fig.
5A). The column subsequently
was washed to achieve a constant base-line level of eluting
radioactivity, after which p-nitrophenol (100 µM) was added to the buffer in an attempt to disrupt weak
interactions. This resulted in a minimal increase in the amount of
radioactivity eluted from the resin (Fig. 5A). Subsequent
addition of CRID 3 (5 mM) to the elution buffer
consistently eluted a small peak of radioactivity; allowing the column
to sit overnight in the presence of CRID 3 eluted additional
radioactivity (Fig. 5A, inset). In total, CRID 3 eluted 0.1-0.4% of the radioactivity applied to the column. Finally,
the column was washed with 0.1% SDS; this denaturing detergent eluted
a peak of radioactivity accounting for 14-16% of the total applied
radioactivity (Fig. 5A). Overall, radioactivity recovered
from the column ranged from 89 to 98% of the total applied.

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Fig. 5.
CRID affinity chromatography.
A, THP-1 cytosol (derived from 1.3 × 109
cells) was applied to a 10-ml column of CRID 2-Thiopropyl-Sepharose.
The column was washed first with buffer and then sequentially with 100 µM p-nitrophenol, 5 mM CRID 3, and
0.1% SDS (all in buffer); arrows indicate where these
agents were applied. Following application of CRID 3, the elution was
allowed to proceed up through fraction 105, after which the flow was
stopped and the column was allowed to sit overnight before flow was
resumed (inset). Aliquots of the individual fractions were
subjected to liquid scintillation counting, and the distribution of
radioactivity is indicated. B, individual fractions were
pooled corresponding to the following: FT, flow-through
(fractions 1-13); NP, p-nitrophenol (fractions
74-79); 1, fractions 96-99; 2, fractions
100-105; 3, fractions 106-110; 4; fractions
111-115; and SDS, fractions 130-135. A sample of the
starting cell extract (L) also was analyzed. The apparent
molecular masses of the three predominant polypeptides recovered in the
CRID 3 elution are indicated as 31, 28, and 22 kDa. Standards of known
molecular mass (Std) are included on the
left.
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Fractions corresponding to the various peaks of radioactivity were
pooled and their polypeptide content analyzed by SDS-PAGE and
autoradiography. Relative to the polypeptide pattern loaded onto the
column, the flow-through peak contained a similar composition of
radiolabeled polypeptides (Fig. 5B). The
p-nitrophenol eluate contained one major polypeptide of 28 kDa (Fig. 5B). The CRID 3 eluate reproducibly contained two
or three major polypeptide species with molecular masses of 22, 28, and
31 kDa (Fig. 5B). The 22-kDa species was not detected in one
out of three experiments. Finally, the SDS eluate contained a large
number of polypeptides (Fig. 5B); for the most part, these
polypeptides were distinct from those eluted by CRID 3 and from those
recovered in the flow-through (Fig. 5B).
Following SDS-PAGE, the three major radiolabeled proteins recovered in
the CRID 3 eluate were identified by LC-MS and data base searching
following trypsin digestion. Results of this analysis identified the
22-, 28-, and 31-kDa polypeptides as glyoxalase I, carbonic anhydrase
(type II), and GST Omega 1-1, respectively. Based on Coomassie
staining, carbonic anhydrase was the most abundant polypeptide in the
CRID 3 eluate. However, as noted above, elution of this polypeptide
from the affinity column also occurred in the presence of
p-nitrophenol, and prolonged washing of the column with
buffer alone led to its elution (data not shown).
Recombinant GST Omega 1-1 Incorporates [14C]CRID
2--
Incubation of human recombinant GST Omega 1-1 with
[14C]CRID 2 resulted in a dose-dependent
incorporation of radioactivity into the polypeptide (Fig.
6A). In view of the apparent
conservation of an active site cysteine between GST Omega 1-1 and CLIC1
(Fig. 4) and the expectation that [14C]CRID 2 reacts with
free sulfhydryl groups to mediate its covalent attachment to proteins,
we reasoned that Cys32 in GST Omega 1-1 may be the site of
attachment. Therefore, this site was mutated to an alanine residue, and
the altered protein was treated with [14C]CRID 2. Concentrations of the radioactive pharmacophore resulting in robust
incorporation of radioactivity into wild type GST Omega 1-1 yielded no
discernible incorporation into the alanine-containing mutant protein
(Fig. 6B). Thus, Cys32 appeared to be the site
of covalent attachment.

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Fig. 6.
Recombinant GST Omega 1-1 is labeled by
[14C]CRID 2. A, human recombinant GST
Omega 1-1 was incubated with the indicated concentration of
[14C]CRID 2 for 30 min at 4 °C, after which the
reaction mixtures were analyzed by SDS-PAGE. B, recombinant
GST Omega 1-1 or its C32A mutant counterpart were incubated with the
indicated concentration of [14C]CRID 2 for 30 min at
4 °C before being analyzed by SDS-PAGE and autoradiography.
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The site of modification was studied directly by capillary LC-MS
peptide mapping of tryptic digests of modified and control GST Omega
1-1. A single modification was confirmed by mass spectrometry of the
intact modified protein (data not shown). A control sample, not exposed
to CRID 2, was otherwise treated in the same way. LC-MS of the control
digest, including data-dependent MS/MS and combined with
post-run data base searching using the SEQUEST algorithm, demonstrated
the presence of the presumed active-site peptide FCPFAER. This peptide,
with its cysteinyl residue as the S-carbamoylmethyl derivative, was eluted as a distinct peak near 42 min (marked with
asterisk in Fig.
7A). Sequence coverage of the
polypeptide was near 90%.

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Fig. 7.
CRID 2 modified Cys32 of GST
Omega 1-1. A, HPLC chromatogram of tryptic digest of
control protein; the peak marked with an asterisk was shown
by on-line MS/MS to be the S-carbamoylmethylated derivative
of the active-site peptide FCPFAER (data not shown). B, HPLC
chromatogram of tryptic digest of GST Omega 1-1 singly modified with
CRID 2. The peaks marked with asterisks were not present in
the control digest; note also the disappearance of the active-site
peptide peak from the control. C, on-line MS/MS of
MH22+ (monoisotopic
m/z = 643.1) from the first-eluted new peak
from modified protein. Peaks are interpreted according to modification
of the Cys residue in FCPFAER by the 416.4-Da CRID 2. Inset,
hypothetical intermediate for neutral loss of a 173-Da aminodiindane
moiety from the peptide. D, MS3 of the
m/z = 556.6 ion generated in MS/MS. Peaks
are interpreted as classical b and y sequence ions of FCPFAER modified
at Cys with the 243.4-Da residue of CRID 2 remaining after neutral loss
of 173 Da.
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Analysis of CRID 2-modified GST Omega-1 revealed only a trace of the
active-site peptide with S-carbamoylmethylcysteine (note disappearance of the 42-min peak in Fig. 7B), but
demonstrated the presence of a peptide with mass 416 Da higher than
that of FCPFAER, consistent with modification of Cys32 by
the 416-Da epoxide CRID 2. Three peaks with the mass properties of this
product were eluted in the region near 92-93 min (marked with
asterisks in Fig. 7B).
MS/MS of this product (Fig. 7C) gave weak peaks
corresponding to classical sequence ions for the predicted structure,
but was dominated by peaks interpreted as being caused by neutral loss
of the 172-Da aminodiindanyl moiety plus an additional proton to
preserve neutrality (173 Da in all). The intermediate stage of a
hypothetical mechanism for this neutral loss is depicted in the
inset to Fig. 7C. Thus, the major peak in the
MS/MS spectrum at m/z = 556.6 was
interpreted as arising from neutral loss of 173 Da from
MH22+ of 643.4, leading to a doubly charged ion
with m/z = 556.6. The classical
b2 ion was detected at m/z = 667.0, but neutral loss of 173 Da from this fragment yielded a more
intense ion at m/z = 493.9. The
y5 ion at m/z = 619.3 was the
major simple sequence ion detected, consistent with the anticipated
relatively high lability of the peptide bond located to the
NH2-terminal side of Pro.
To pursue more complete confirmation of the sequence, the doubly
charged ion at m/z = 556.6 was subjected to
MS3 using the programmable capacities of the LCQ ion trap
spectrometer. The result (Fig. 7D) was interpreted in terms
of classical sequence fragmentation of FCPFAER, with the Cys residue
now having mass of 346.5 Da after loss of the 173-Da aminodiindane.
Continuous sets of congruent sequence ions from y1 to
y6 and from b2 to b6 furnished
conclusive evidence that Cys32 in the active site peptide
FCPFAER was the site of modification by CRID 2.
Material with the mass of CRID 2-modified active-site peptide occurred
in three unequal peaks with retention time near 92-93 min (Fig.
7B). All showed very similar behavior in multistage MS. The
multiplicity of peaks was believed to originate with two factors, these
being (i) the presence of two enantiomers of CRID 2 in the reagent and
(ii) the potential for the Cys32 nucleophile to be
alkylated by either of the epoxide carbon atoms.
The crystal structure of GST Omega 1-1 indicates that Cys32
is located near the boundary of the two sites in the polypeptide corresponding to the glutathione and electrophilic substrate binding pockets of prototype GST family members (52). Therefore, we asked if
S-alkylglutathione analogs disrupt binding of GST Omega 1-1 to [14C]-CRID 2; a number of GSTs, including porcine GST
Omega 1-1 (59), bind these glutathione adducts tightly because they
mimic enzyme reaction products (60). Glutathione sulfonate (2 mM) produced little effect on the incorporation (Fig.
8A). Likewise,
S-methylglutathione was inactive at all tested
concentrations (up to 200 µM). As the chain length of the
alkyl group increased, however, progressively greater inhibitory
potencies were observed (Fig. 8A). Thus,
S-ethyl-, S-butyl-, S-hexyl-, and
S-octylglutathione yielded IC50 values of
350, 18, 3.0, and 0.9 µM, respectively.
Further elongation of the alkyl group to 10 carbon atoms did not lead
to an additional increase in potency; the IC50 for
S-decylglutathione was 1.2 µM (Fig.
8A). The presence of a large aliphatic chain was not
required for effective inhibitory activity as
S-(p-nitrobenzyl)glutathione and
S-(1-adamantyl)glutathione yielded IC50 values
of 1.8 and 3.0 µM, respectively (Fig. 8A).
Several other S-substituted glutathione adducts demonstrated
modest IC50 values including oxidized glutathione (350 µM),
S-(p-azidophenylacyl)glutathione (100 µM),
S-(p-chlorophenacyl)glutathione (95 µM), and S-D-lactoylglutathione
(200 µM). Results of these competition experiments,
therefore, suggest that GST Omega 1-1 can bind to
S-substituted glutathione adducts and that this binding prevents access of Cys32 to [14C]CRID 2.

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Fig. 8.
S-Substituted glutathione adducts
block [14C]CRID incorporation. A, GST
Omega 1-1 was preincubated with the indicated concentration of each
individual glutathione analog and then 0.5 µM
[14C]CRID 2 was introduced. Reaction mixtures were
incubated at 4 °C for 30 min and then analyzed by SDS-PAGE. The
amount of radioactivity incorporated into GST Omega 1-1 was determined
by phosphorimager analysis (expressed as a percentage relative to the
quantity incorporated in the absence of an inhibitor) and is indicated
as a function of test agent concentration. Compounds and their
estimated IC5O values (in parentheses) are: A,
S-lactoylglutathione (200 µM); B,
glutathione sulfonate (>1 mM); C, oxidized
glutathione (350 µM); D,
S-hexylglutathione (3 µM); E,
S-methylglutathione (>100 µM); F,
S-(p-nitrobenzyl)glutathione (1.8 µM); G,
S-(p-azidophenylacyl)glutathione (100 µM); H, S-butylglutathione (18 µM); I, S-ethylglutathione (350 µM); J, S-octylglutathione (630 nM); K,
S-(p-chlorophenacyl)glutathione (95 µM); L, S-decylglutathione (1 µM); M, S-(1-adamantyl)glutathione
(3 µM). B, recombinant GST Omega 1-1 was
incubated for 30 min at 4 °C with the indicated concentration of
CRID 2-SG adduct and then 2 µM [14C]CRID 2 was introduced. Following a 30-min incubation, the reaction mixtures
were analyzed by SDS-PAGE and autoradiography.
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One additional glutathione adduct was synthesized and assessed in the
competition assay. CRID 2 was conjugated to reduced glutathione via the
epoxide group of the pharmacophore to generate an adduct of the
CRID with glutathione (CRID 2-SG). This adduct blocked incorporation of
[14C]CRID 2 into GST Omega 1-1 (Fig. 8B);
based on phosphorimager analysis of the gel, the adduct yielded an
IC50 of 25 µM.
CRID 2-SG Blocks ATP-induced IL-1
Processing--
If GST Omega
1-1 is the target responsible for the pharmacological action of CRIDs,
then other agents that bind to this polypeptide may act as inhibitors
of ATP-induced IL-1
posttranslational processing. To explore this
possibility, several S-alkylglutathione derivatives (S-methyl-, S-hexyl-, S-octyl-, and
S-decylglutathione) were tested individually in the monocyte
cytokine production assay; all were inactive as inhibitors of
ATP-induced IL-1
posttranslational processing (Fig.
9A). On the other hand,
monocytes treated with CRID 2-SG demonstrated a
dose-dependent inhibition in cytokine production; the
IC50 in this assay was 18 µM (Fig.
9C). CRID 2 on its own demonstrated a
dose-dependent response, yielding an IC50 value
of 70 nM (Fig. 9C). Therefore, attachment of the
glutathione tripeptide lowered CRID potency, but did not eliminate
activity. Confirmation of this inhibitory effect was achieved using a
metabolic assay format.
LPS-activated/[35S]methionine-labeled human monocytes
were treated with ATP in the absence or presence of the adduct after
which IL-1
released to the medium was recovered by
immunoprecipitation. In the absence of the adduct, large amounts of the
17-kDa mature cytokine species were released to the medium (Fig.
9D). Inclusion of the CRID 2-SG adduct in the culture medium
dose-dependently inhibited release of the 17-kDa species
(Fig. 9D). Moreover, as observed with cells treated with a
CRID (42), adduct-arrested cells did not compensate by releasing
pro-IL-1
to the medium (Fig. 9D). S-Hexylglutathione (1.25 mM) did not inhibit release of 17-kDa IL-1
,
confirming its inactivity observed in the ELISA format (Fig.
9A).

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Fig. 9.
CRID 2-SG inhibits ATP-induced
IL-1 posttranslational processing.
A, LPS-activated human monocytes were treated with 2 mM ATP in the presence of the indicated concentration of
S-substituted glutathione adduct. After 3 h of
treatment, media were harvested and assessed for IL-1 content by
ELISA. The amount of cytokine is indicated (as a percentage of that
recovered in the absence of an effector) as a function of test agent
concentration. B, LPS-activated human monocytes were
incubated with 1 µM [14C]CRID 2 in the
absence or presence of a potential competitor (where present, the
competitor was added 30 min prior to the radiolabeled probe). Following
a 60-min incubation at 37 °C, the cells were harvested,
disaggregated, and subjected to SDS-PAGE and autoradiography. The
region of the autoradiogram corresponding to 31-kDa GST Omega 1-1 (arrow) is indicated. C, LPS-activated human
monocytes were preincubated with the indicated effector and then
treated (in the continued presence of the effector) with 2 mM ATP. Following a 3-h incubation, media were harvested
and assessed for IL-1 content by ELISA. Cytokine levels (expressed
as a percentage of that generated by cells in the absence of an
effector) are indicated as a function of test agent concentration.
D, LPS-activated/[35S]methionine-labeled human
monocytes were preincubated for 15 min with the indicated concentration
of either CRID 2-SG or S-hexylglutathione and then treated
with 2 mM ATP (in the continued presence of the two test
agents) for 3 h. Media were recovered, and IL-1 was isolated by
immunoprecipitation. The immunoprecipitates were fractionated by
SDS-PAGE; an autoradiogram of the gel is shown. Each condition was
performed in duplicate, and migration positions of the 31-kDa proform
and 17-kDa mature forms of IL-1 are indicated.
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The inability of S-alkylglutathione analogs to inhibit the
ATP response may indicate that these adducts did not access GST Omega
1-1 within intact cells; nonesterified glutathione analogs generally
are not membrane-permeant (61). To assess this, monocytes were treated
with [14C]CRID 2 in the absence and presence of an excess
of S-hexylglutathione. In the absence of a potential
effector, monocytes treated with [14C]CRID 2 readily
incorporated radioactivity into 31-kDa GST Omega 1-1 (Fig.
9B). Co-treatment with 1 or 5 mM
S-hexylglutathione did not reduce this incorporation (Fig.
9B). On the other hand, monocytes treated with
[14C]CRID 2 in the presence of the CRID 2-SG adduct
displayed reduced incorporation of the radiolabel into GST Omega 1-1;
concentrations >1 mM were effective (Fig. 9B).
Thus, whereas both S-hexylglutathione and CRID 2-SG blocked
incorporation of [14C]CRID 2 into rGST Omega 1-1, only
the latter blocked [14C]CRID 2 incorporation into
cell-associated GST Omega 1-1.
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DISCUSSION |
Based on the unusual requirement for a separate secretory stimulus
to promote efficient mature IL-1
export from LPS-activated human
monocytes, we previously set out to identify inhibitors of this process
using an intact cell assay format (44). This search led to discovery of
diarylsulfonylureas as a potent and selective class of CRIDs. These
agents block mature cytokine production independent of the initiating
stimulus, and they arrest the process in a manner that allows the
activated cells to maintain plasma membrane latency (44). This
fingerprint shares features with several non-selective anion transport
inhibitors, such as tenidap and ethacrynic acid, which also arrest
stimulus-induced IL-1
production (62); the molecular mechanism by
which these agents disrupt the cytokine process is not known. In an
attempt to elucidate the mechanism by which diarylsulfonylureas disrupt
the cellular process, we employed a multifaceted approach. Results of
our search identify GST Omega 1-1 as a diarylsulfonylurea-binding
protein and suggest that this interaction is responsible for inhibition of ATP-induced IL-1
posttranslational processing. Evidence
supporting these conclusions include demonstrations that: 1)
14C-labeled epoxide-containing diarylsulfonylureas bind
irreversibly to GST Omega 1-1 and this binding correlates with
inhibition of cytokine production, 2) GST Omega 1-1 binds reversibly to
a diarylsulfonylurea affinity column, and 3) a CRID-glutathione adduct
inhibits ATP-induced IL-1
posttranslational processing and interacts
with GST Omega 1-1 within intact monocytes.
The radiolabeled epoxide-containing diarylsulfonylureas CRID 1 and CRID
2 both covalently labeled three monocyte-soluble polypeptides corresponding to GST Omega 1-1 (31 kDa), CLIC1 (32 kDa), and
carboxylesterase (56 kDa). Alveolar macrophages are known to express
and to secrete carboxylesterase (58); the exact role of this enzyme in
the macrophage is not clear. Relative to concentrations of the test agents required to irreversibly block ATP-induced IL-1
processing, carboxylesterase labeling was observed only at concentrations in excess
of those required to inhibit the cytokine response. Moreover, when
tested as a direct inhibitor of purified carboxylesterase, 50 µM CRID 2 yielded a modest inhibitory effect (55%; data
not shown). Therefore, carboxylesterase is not considered a good
candidate to account for the CRID pharmacodynamic effect.
GST Omega 1-1 and CLIC1 are related polypeptides sharing 15% sequence
identity. Moreover, the crystal structure of GST Omega 1-1 predicts the
presence of an active site cysteine (Cys32) (52), and this
residue is conserved in CLIC1. Importantly, substitution of alanine for
cysteine32 in GST Omega 1-1 dramatically reduced the
ability of the recombinant protein to incorporate
[14C]CRID 2, and LC-MS peptide analysis indicated that
this cysteine residue is responsible for nucleophilic attack of the
pharmacophore-associated epoxide group. Incorporation into GST Omega
1-1 occurred at lower concentrations of the radiolabeled pharmacophores
than did labeling of CLIC1, suggesting that the former has a higher
binding affinity for the diarylsulfonylureas or that CLIC1 has an
inherently lower nucleophilic capacity. Recent resolution of the x-ray
crystal structure of CLIC1 indicates that this polypeptide not only
shares sequence similarity to GST Omega 1-1 but also structural
attributes (63). Therefore, the ability of [14C]CRID 2 to
label both of these polypeptides may reflect conservation of structure
and/or function.
Concentrations of [14C]CRID 2 required to label
cell-associated GST Omega 1-1 correlated with the extent of inhibition
of IL-1
posttranslational processing. It is noteworthy that
cell-associated GST Omega 1-1 incorporated the epoxide-bearing
pharmacophores. In the previous x-ray crystallography study,
Cys32 of recombinant GST-Omega was found to readily form a
mixed disulfide bond with glutathione (52). Although intracellular
concentrations of reduced glutathione are quite high (64),
concentrations of the oxidized species generally are much lower and
this latter species is likely necessary for formation of the mixed
disulfide with GST Omega 1-1. Thus, in the context of a typical
intracellular reducing environment, cell-associated GST Omega 1-1 appears to exist with the sulfhydryl side chain of Cys32 unmodified.
A concern with the affinity labeling approach is that incorporation of
the epoxide-bearing pharmacophore by a protein may occur simply as a
result of a highly nucleophilic sulfhydryl group. In view of
nucleophilic cysteines being associated with many cellular proteins,
however, this explanation does not appear consistent with the observed
selectivity of the labeling profile. As an alternative approach, an
affinity matrix was generated in which the epoxide group of CRID 2 was
conjugated to an insoluble support, making it unavailable to react with
protein-associated nucleophiles. Using this resin, glyoxalase I,
carbonic anhydrase, and GST Omega 1-1 demonstrated
CRID-dependent binding interactions. Enzymatic activity of
recombinant glyoxalase I was inhibited by 45% in the presence of 50 µM CRID 2 (data not shown), a level not considered sufficient to account for the submicromolar CRID-like effect of this
compound. Carbonic anhydrase (type II) also bound to the CRID affinity
column, but elution of this enzyme was not dependent on a competing
diarylsulfonylurea. Rather, bound carbonic anhydrase was eluted in the
absence of a soluble competitor, although the presence of
p-nitrophenol or CRID 3 enhanced its rate of elution. In an
enzyme activity assay using commercial human carbonic anhydrase type
II, 50 µM CRID 2 neither activated nor inhibited
activity. Moreover, a known inhibitor of carbonic anhydrase,
chlorothiazide, did not block ATP-induced IL-1
posttranslational
processing (data not shown). Thus, the interaction of CRIDs with
carbonic anhydrase is not considered responsible for the CRID
pharmacological effect.
Of the three polypeptides recovered from the affinity column, only GST
Omega 1-1 also was identified by affinity labeling. As noted above,
binding to the affinity matrix is not dependent on formation of a
covalent bond; the epoxide group of the affinity ligand tethers the
pharmacophore to the resin and thus is not available to interact with
Cys32 on GST Omega 1-1. Therefore, GST Omega 1-1 recognizes
the diarylsulfonylurea pharmacophore independent of the presence of the epoxide.
As noted earlier, the x-ray crystal structure of GST Omega 1-1 predicts
the presence of a glutathione-binding site similar to that found in
prototypical GST family members (52). Based on this, a number of
S-substituted glutathione adducts were assessed as
inhibitors of [14C]CRID 2 incorporation into recombinant
GST Omega 1-1. Structurally distinct adducts demonstrated
dose-dependent reductions in radiolabel incorporation, with
S-octylglutathione being the most potent inhibitor tested.
This inhibition is consistent with [14C]CRID 2 binding to
Cys32, which is situated adjacent to the glutathione
binding pocket of the enzyme (52). On the assumption that binding to
GST Omega 1-1 is responsible for the CRID effect, we asked whether the
S-substituted glutathione adducts blocked ATP-induced
IL-1
posttranslational processing. Several tested adducts did not
inhibit the ATP response, and they did not block incorporation of
[14C]CRID 2 into cell-associated GST Omega 1-1. Because
the glutathione adducts block incorporation into recombinant but not
cell-associated protein, we assume that the plasma membrane poses a
barrier that does not allow their access to GST Omega 1-1. Significantly, however, the CRID 2-SG adduct did inhibit ATP-induced
IL-1
posttranslational processing and did reduce incorporation of
[14C]CRID 2 into monocyte-associated GST Omega 1-1;
attributes that selectively allow this adduct to penetrate the plasma
membrane are unknown. The ability of the CRID 2-SG adduct to retain
CRID activity despite a large structural change in the pharmacophore attendant to the presence of the tripeptide is remarkable and completely consistent with GST Omega 1-1 being involved in the cellular
response mechanism. In this regard, ethacrynic acid, a compound
structurally distinct from diarylsulfonylureas, also effectively
inhibits ATP-induced IL-1 posttranslational processing (62). In
addition to inhibiting ion transport processes, ethacrynic acid is
known to inhibit members of the GST superfamily (65), and its ability
to disrupt the IL-1 response further suggests a GST involvement in the
cellular process.
GST Omega 1-1 is distinguished from other GST family members by the
presence of an extended amino terminus and an active center cysteine
residue; most GSTs possess an active center tyrosine or threonine
residue (60, 66). Absence of a side-chain hydroxyl group results in
loss of prototypical glutathione conjugating activity (67); this group
has been shown by x-ray crystallography to stabilize the thiolate anion
of glutathione (60, 66). The active center of GST Omega appears more
comparable with that expected for a glutaredoxin-type of activity than
for a GST (52, 68). Indeed, human GST Omega 1-1 demonstrates modest
glutathione-dependent thiol transferase activity (52).
Other activities have been reported for GST Omega 1-1. For example, the
rat enzyme is reported to be a dehydroascorbate reductase (69), the
human enzyme is reported to modulate activity of the ryanodine receptor
(57), and the murine enzyme (p28) is reported to be a
glutathione-binding stress protein (54). The murine protein originally
was identified on the basis that it was up-regulated in a population of
T-cells resistant to radiation-induced apoptosis (54). Interestingly, this protein changed its subcellular localization in response to heat
stress (54). In line with the stress connection, human GST Omega 1-1 demonstrates stronger sequence homologies with a number of plant and
lower organism GSTs than to prototype mammalian GSTs, and many of these
enzymes are induced in response to stress stimuli such as salt and
oxygen (70-72). The actual enzymatic function of GST Omega 1-1 remains
to be elucidated.
Is GST Omega 1-1 likely to function in the context of stimulus-coupled
IL-1
posttranslational processing? Based on reported activities of
GST Omega-1, two types of functional contributions can be envisioned.
The first is based on its similarity to CLIC1. Despite our recovery of
CLIC1 from the soluble fraction of THP-1 cells, this polypeptide is
reported to serve as a chloride channel both in its recombinant state
after incorporation into lipid vesicles and in its native state within
cells (55, 56). GST Omega-1 itself has not been reported to possess ion
channel activity, but CLIC1 and GST Omega-1 are related polypeptides;
they share sequence homology, possess similar x-ray crystal structures,
and are labeled by epoxide-bearing CRIDs. We assume, therefore, that these two polypeptides share functional attributes. ATP-induced IL-1
posttranslational processing is accompanied by dramatic changes in
ionic homeostasis, and yield of extracellular cytokine is greatly affected by changes to the ionic composition of the medium. For example, increasing extracellular K+ (15), removal of
extracellular Na+ (73), or replacement of extracellular
Cl
with chaotropic anions inhibits the cellular response
(74). Thus, CLIC1 and GST Omega-1 may function to facilitate an
important ionic flux. In this regard, E. coli contains a
glutathione-gated potassium efflux system that operates in response to
stress and redox state (75, 76) and perhaps GST Omega-1 is a component of this type of mechanism.
An alternate function for GST Omega-1 is suggested based on similarity
between its active center and that of glutaredoxin (52). Glutaredoxin
possesses both dehydroascorbate reductase and thioltransferase
activities (77, 78). Effectors that induce monocyte/macrophage IL-1
processing in vitro promote dramatic changes to the
intracellular environment and ultimately cause the responding cell to
die. The best-studied effector, ATP, works via the P2X7
receptor. When ligated, this receptor can initiate activation of
caspases, phospholipases, and stress kinases (32-36); all of these
activities signal that the responding cell is engaged in a stress
response. Moreover, a recent study using rat microglial cells noted
that P2X7 receptor activation promotes a burst of H2O2 generation (79), suggesting that receptor
ligation also alters intracellular redox state. During conditions of
oxidative stress, increased levels of oxidized glutathione and
glutathionylation of select cellular polypeptides can be observed
(80-84). This reversible posttranslational modification can lead to
altered protein/enzyme function, suggesting a new type of regulatory
mechanism. For example, tyrosine hydroxylase becomes glutathionylated
and inactivated when PC12 cells are treated with the prooxidant diamide
(85). Removal of the glutathione moiety restores enzyme activity, and glutaredoxin catalyzes this thioltransferase type of reaction (85, 86).
Caspases, as a family of cysteine proteases, represent prime targets
for glutathionylation at their active center thiols. Indeed, purified
caspase-3 forms a mixed disulfide and is inactivated following
treatment with oxidized glutathione (87). Interestingly, activation of
caspase-8 via the death-inducing signaling complex in
FAS-induced lymphocytes is dependent on the presence of
glutathione (88). The glutathione-dependent step has not
been determined, but appears to occur after assembly of the protein
complex (88). The mechanism of procaspase-1 activation is not well
understood, but like caspase-8, appears to involve a large protein
assembly termed the inflammasome (89). Perhaps an ATP-induced change in
the intracellular redox state promotes formation of glutathione-protein mixed disulfides leading to inactivation of cellular polypeptides such
as caspase-1 or a member of the inflammasome, function of which is
critical to the cellular response. To maintain activity of critical
sulfhydryl-containing enzymes, GST Omega-1 may act in concert with
glutaredoxin to reduce glutathione-protein mixed disulfides and
preserve catalytic function. Although recombinant GST Omega-1
demonstrates very low activity as a thioltransferase when assessed with
the artificial substrate hydroxyethyl disulfide (52), the possibility
that this type of activity could be achieved with a protein-linked
disulfide remains to be tested. As noted above, the Omega class of GSTs
is evolutionarily conserved in plants. The Arabidopsis
thaliana genome, for example, contains at least five separate
genes encoding GST family members possessing the conserved CPF active
center motif (90). Although biological function of the plant enzymes
also remains to be elucidated, conservation within both the plant and
animal kingdoms suggests that this family of enzymes serves an
important function worthy of further investigation.