From the Department of Immunology, Lerner Research
Institute, Cleveland Clinic, Cleveland, Ohio 44195
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ABSTRACT |
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Cytokine-inducible nitric oxide synthase (iNOS)
is a homodimeric enzyme that generates nitric oxide (NO) and
L-citrulline from L-arginine
(L-Arg) and O2. The N-terminal oxygenase domain (amino acids 1-498; iNOSox) in each subunit binds heme,
L-Arg, and tetrahydrobiopterin (H4B), is the
site of NO synthesis, and is responsible for the dimeric interaction,
which must occur to synthesize NO. In both cells and purified systems,
iNOS dimer assembly is promoted by H4B, L-Arg,
and L-Arg analogs. We examined the ability of imidazole and
N-substituted imidazoles to promote or inhibit dimerization
of heme-containing iNOSox monomers, or to affect iNOS dimerization in
cells. Imidazole, 1-phenylimidazole, clotrimazole, and miconazole all
bound to the iNOSox monomer heme iron. Imidazole and
1-phenylimidazole promoted iNOSox dimerization, whereas
clotrimazole (30 µM) and miconazole (15 µM)
did not, and instead inhibited dimerization normally promoted by
L-Arg and H4B. Clotrimazole also bound to
iNOSox dimers in the absence of L-Arg and H4B
and caused their dissociation. When added to cells expressing iNOS,
clotrimazole (50 µM) had no effect on iNOS protein expression but almost completely inhibited its dimerization and consequent NO synthesis over an 8-h culture period, without affecting calmodulin interaction with iNOS. Thus, imidazoles can promote or
inhibit dimerization of iNOS both in vitro and in cells,
depending on their structure. Bulky imidazoles like clotrimazole block
NO synthesis by inhibiting assembly of the iNOS dimer, revealing a new
means to control cellular NO synthesis.
Nitric oxide (NO)1 is
synthesized from L-arginine (L-Arg) in animals
by the NO synthases (NOSs, for reviews, see Refs. 1-3). Three NOS
isoforms have been characterized which differ in primary sequence, gene
chromosomal location, and activation by Ca2+ (4-6). A
neuronal NOS isoform (nNOS) that is present in brain and skeletal
muscle (7, 8), and an endothelial NOS isoform (eNOS) expressed in the
vasculature or brain (9, 10) are dependent on calmodulin (CaM) binding
for activity, which is reversible and occurs in response to elevated
intracellular Ca2+. In contrast, a continuously active NOS
isoform (iNOS) is expressed in cells exposed to inflammatory cytokines
or bacterial products (11), and is neither stimulated by
Ca2+ nor blocked by CaM antagonists due to its containing
tightly-bound CaM (12). Numerous pathologies are attributed to excess
NO production by iNOS (13-16) and have led to a quest for specific
inhibitors of this isoform. Work has focused on a broad range of
molecules including substrate analogs, guanidine derivatives,
thioureas, and heterocycles, with some specific inhibitors beginning to
emerge (17-27).
Although the NOS isoforms differ regarding their primary sequence and
mode of expression, they all are bi-domain enzymes comprised of a
C-terminal reductase domain that contains binding sites for NADPH, FAD,
FMN, and CaM, and an N-terminal oxygenase domain that contains binding
sites for heme, tetrahydrobiopterin (H4B), and L-Arg (5, 28-31). The NOS heme shares characteristics with
the heme in cytochromes P-450 in that it coordinates to the protein through a cysteine thiolate (30-32), can bind O2 as a
sixth ligand (33), and may directly participate in oxygen activation
and product formation (33-37). Thus, as with the cytochromes P-450, the NOS heme represents a potential target for enzyme inhibition. In
fact, compounds that bind directly to the NOS heme iron such as CO, NO,
CN, imidazole, and N-phenyl imidazoles all inhibit NO
synthesis (22, 34, 38-40).
The NOSs are only active as homodimers (41-43). For iNOS (29) and
possibly nNOS and eNOS (44-46), only the oxygenase domains of two
subunits interact to form the dimer, with the reductase domains
attached as extensions that may destabilize the dimer (47). Both the
oxygenase and reductase domain of each NOS isoform can fold and
function independent of one another (45-47). For example, the iNOS
oxygenase domain (iNOSox, amino acids 1-498) is expressed in
Escherichia coli as a dimer, exhibits normal affinity toward L-Arg and H4B, and catalyzes NO synthesis from
the reaction intermediate N-hydroxy-L-Arg either
in a H2O2-supported reaction or when supplied with NADPH and its reductase domain (48-50). A crystal structure of
dimeric iNOSox with L-Arg and H4B bound has
been published (32).
Full-length NOS monomers isolated from mammalian cells are devoid of
H4B and heme, but contain bound FAD and FMN, functional NADPH and CaM binding sites, and can catalyze electron transfer to
artificial acceptors such as cytochrome c at rates that
match their respective dimeric forms (41, 42). Work with full-length iNOS and nNOS monomers indicates that their dimerization minimally requires that heme be inserted into the protein during the dimerization reaction (41, 42, 51). The heme-containing NOS monomer may be an
intermediate on the path toward forming a stable dimer (52), and
although it does not accumulate in mammalian cells containing H4B and L-Arg (41), it can be generated
in vitro by dissociating purified iNOS dimers with urea (47,
53). Dimerization of iNOS monomers is also stimulated by
H4B and L-Arg (41, 47, 53) and is inhibited in
cells by NO, which interferes with heme insertion into the monomers
(54). Dimerization activates iNOS in part by enabling electron transfer
between enzyme flavin and heme prosthetic groups (55), and by
facilitating productive binding of substrate and H4B (41,
49).
Because dimer assembly is critical for NO synthesis, it is a
potential target for therapeutic intervention. A study of iNOS dimerization as promoted by L-Arg showed that its effect
was stereospecific but not unique, because several L-Arg
and guanidine analogs that bind to the iNOS dimer also promoted dimer
assembly (41, 56). Two substrate analogs that exhibit greater affinity
toward the iNOS dimer than L-Arg,
N On the basis of these considerations, we tested if imidazoles might
positively or negatively influence iNOS dimer assembly. Because
imidazoles bind to the NOS heme iron, their binding should not depend
on NOS dimeric structure, but simply on whether heme is incorporated
into the protein. In fact, the crystal structure of a heme-containing
iNOSox monomer shows that two molecules of imidazole can bind within
the monomer's distal heme pocket (57), one coordinating to the heme
iron and the other binding to the carboxylate of Glu-371, which also
binds the guanidino nitrogens of L-Arg (32, 48). We
therefore examined the ability of imidazole and
N1-substituted imidazoles to promote or
antagonize the dimerization of full-length iNOS and iNOSox monomers,
and compared results to those obtained with L-Arg,
H4B, or 7-nitroindazole, which are all known to stabilize
the NOS dimer or promote its assembly without binding to the heme iron
(56, 58). We found that imidazoles can promote or inhibit iNOS
dimerization depending on their structure, and act through a mechanism
that involves binding to the heme iron. On this basis, we characterize
an antifungal imidazole as a potent inhibitor of iNOS dimer assembly
both in a purified system and in cultured cells.
Materials--
Interferon- Cell Culture and Protein Preparation--
RAW 264.7 macrophage
cell cultures (500 ml) were grown in spinner flasks and induced to
express iNOS by adding 10 units/ml interferon-
The oxygenase domain of iNOS (amino acids 1-498, iNOSox) containing a
6-hisitidine C terminus was expressed in E. coli and purified in the absence of L-Arg and H4B
essentially as detailed in Siddhanta et al. (60), while
full-length iNOS containing a 6-histidine tag at its N terminus was
expressed in E. coli and purified in the absence of
L-Arg and H4B as described in Wu et al. (61). The purified iNOSox and full-length iNOS proteins were
70-80% dimeric as isolated and were dissociated into monomers with
urea according to previous methods (47, 60). Briefly, iNOSox was
diluted to 3 µM in 40 mM HEPES, pH 7.5, containing 10% glycerol, 3 mM DTT, and 5 M
urea at 4 °C. After 1 h, this solution (~1 ml) was
sequentially dialyzed at 4 °C against 200 ml of the same buffer
containing 5 M urea for 90 min, against 200 ml of the same
buffer containing 2 M urea for 4 h, and against 200 ml
the same buffer containing 0.1 M urea overnight. The same procedure was used for full-length iNOS except 2.5 M urea
was used to dissociate the dimer and intermediate dialysis with 2 M urea was omitted. These procedures resulted in
preparations that contained 60-80% monomer and 20-40% dimer
(percentages reflect distribution of iNOS protein mass) as determined
by gel filtration chromatography. The urea-generated iNOS monomers
contained heme but no H4B (47, 53) and were used within 1 day of their preparation.
NO Synthesis Activity--
The NO synthesis activity of
activated cell cultures or of soluble cell supernatants was estimated
using the colorimetric Griess assay for nitrite as described previously
(41). Culture fluid was analyzed directly for nitrite. NO synthesis
activity of cell supernatants or column fractions was determined in
100-µl incubations containing aliquots of cell supernatants or column fractions and 40 mM Tris buffer, pH 7.8, 1 mM
NADPH, 2 mM L-Arg, 3 mM DTT,
protease inhibitors, and 4 µM each of FAD, FMN, and H4B as described previously (41). Incubations were run for
60 min at 37 °C prior to analyzing for nitrite. NO synthesis
activity of purified full-length iNOS was determined using the
spectrophotometric oxyhemoglobin assay. Cuvette samples (350 µl)
contained iNOS, 5 µM oxyhemoglobin, 0.3 mM
DTT, 1 mM L-Arg, 0.5 mg/ml bovine serum albumin, 1300 units/ml catalase, and 150 units/ml superoxide dismutase in 40 mM EPPS, pH 7.6. Reactions were initiated by adding
100 µM NADPH and the rate of NO synthesis was measured at
401 nm, using an extinction coefficient of 38 mM Spectroscopy--
Optical spectra were recorded at room
temperature on a Hitachi U-2110 spectrophotometer as detailed in
Sennequier and Stuehr (56). Spectra were collected and processed using
SpectraCalc software (Galactic Industries Corp., Salem, NH). Titrations
of N-substituted imidazoles were performed by adding 3-µl
aliquots of concentrated stock solutions (giving a final concentration range of 1-50 µM and 1-20 µM,
respectively, for clotrimazole and miconazole) to cuvettes containing 1 ml of Bis-Tris buffer, pH 7.6, 1 mM DTT, iNOSox, and
L-Arg, or H4B as stated in the text. Spectra
were collected after each addition. Binding constants were derived from
double-reciprocal plots of the absorbance difference (peak to trough in
the difference spectra) versus the additive concentration.
Dimer/Monomer Detection and Formation--
iNOS dimer and
monomer in supernatants of lysed cells or purified iNOSox samples were
estimated by fractionating 100 µl of sample on a Superdex 200 gel
filtration column at 4 °C. The column was run at 0.5 ml/min with 40 mM Bis-Tris propane buffer, pH 7.4, containing 2 mM DTT, 10% glycerol, and 200 mM NaCl. Under
these conditions, dimer does not dissociate into monomer nor do
monomers form dimers.2 For
samples containing iNOSox, eluted protein was monitored at 280 nm and
the peaks were assigned to be dimer or monomer based on the elution
volumes of protein standards and authentic iNOSox monomer and dimer
(47). The dimer-monomer distribution in iNOSox samples was estimated
based on relative peak heights. For cell supernatants, aliquots of each
column fraction were subject to SDS-PAGE, proteins were transferred
onto a Nytran membrane, and iNOS was detected using an anti-iNOS
polyclonal antibody as detailed in Albakri and Stuehr (54). The
SDS-PAGE used a gradient of 5-20% acrylamide which allowed us to
probe the membrane for CaM (17 kDa) as well as for iNOS (130 kDa).
After iNOS bands were visualized and quantitated by scanning
densitometry, the membrane was stripped of the antibody directed
against iNOS and reprobed using a mouse antibody directed against
bovine CaM by a standard procedure (12).
Dimerization of iNOSox or full-length iNOS monomers was performed by
incubating the protein (1 µM) at room temperature in 40 mM HEPES buffer, pH 7.5, containing 3 mM DTT,
0.5 mg/ml bovine serum albumin and L-Arg, H4B,
and/or compounds as noted in the text. The iNOSox dimer-monomer
distribution was estimated by fractionating 100 µl of the incubate at
various times on the Superdex gel filtration column as noted above. To
monitor dimerization of iNOSox spectroscopically, the reaction was
carried out in a 300-µl cuvette and optical spectra were recorded
between 250 and 700 nm every 10 min. Dimerization of full-length iNOS
was followed by assaying NO synthesis activity of aliquots removed at
indicated times.
The structures of the substituted imidazoles and indazole used in
this study are depicted in Fig. 1. The
ability of imidazole, phenylimidazoles, and 7-nitroindazole to bind to
a heme-containing iNOSox monomer and affect dimerization was
investigated by incubating monomers with each compound and monitoring
their binding and dimerization over a 40-min period with UV/visible
spectroscopy and gel filtration chromatography. The iNOSox preparation
used here was initially ~70% monomeric based on gel filtration (data
not shown) and in the presence of DTT exhibited a split Soret
absorbance peak at 378 and 459 nm (Fig.
2A), indicative of a DTT
thiolate coordinating as a sixth heme ligand to the ferric iNOSox
monomer and residual dimer (53, 59). Adding imidazole to the iNOS
monomer preparation caused immediate conversion to a species exhibiting
a single Soret band with absorbance maximum at 429 nm (Fig.
2A), indicating that imidazole quickly displaced DTT as a
sixth ligand to the heme iron in the iNOSox monomer and residual dimer.
The spectrum of imidazole-bound iNOSox did not change over the
subsequent 40-min incubation period. In contrast, an iNOSox monomer
incubated with L-Arg and H4B for 40 min
underwent gradual spectral changes to generate a species with Soret
maxima at 398 nm (Fig. 2A, inset), which is the typical
spectral change observed during dimerization of iNOSox monomers
promoted by L-Arg and H4B (47), and indicates gradual displacement of DTT from the ferric heme as dimerization progresses. Incubation of iNOSox monomers with 7-nitroindazole also
caused a gradual spectral change that indicated displacement of DTT,
and a high degree of dimerization (data not shown).
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-amino-L-Arg and
L-thiocitrulline (21, 24), did not promote dimer assembly,
suggesting they might function as antagonists. However, they were
incapable of blocking dimer assembly in the presence of excess
L-Arg (56), consistent with their sharing a common
binding site but being generally unable to bind to iNOS monomers (32,
48, 49). This suggests that substrate analogs have little inherent
capacity to block iNOS dimerization.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
was a gift from Genentech, South
San Francisco, CA. Antibodies against iNOS and CaM were from
Transduction Laboratories (Lexington, KY) and Upstate Biotechnology
Inc. (Lake Placid, NY). The enhanced chemiluminescence (ECL) kit for
immunodetection was from Amersham International PLC (Little Chalfon,
United Kingdom). Bacterial culture materials were purchased from Difco
Labs (Detroit, MI) and Becton Dickinson Microbiology Systems
(Cockeysville, MD). Clotrimazole and miconazole were purchased from
Sigma, while 7-nitroindazole, 1-phenylimidazole, and 2-phenylimidazole
were purchased from Aldrich. Mammalian cell culture materials were
purchased from Life Technologies Inc. (Gaithersburg, MD), ampicillin
was from Apothecon (Princeton, NJ). Pefabloc and lysozyme were from
Boehringer-Mannheim GmBH (Germany). Ni2+-nitriloacetate
resin was from Novagen Inc. (Madison, WI). Gel filtration protein
standards were from Bio-Rad. Clotrimazole and miconazole stock
solutions were prepared in Me2SO, while all other imidazoles were dissolved in buffer.
and 1 µg/ml
E. coli lipopolysaccharide as previously detailed (41).
Clotrimazole was added as a 2-ml Me2SO solution to
each culture as described below. In some cases, iNOS monomers and
dimers were purified from the soluble RAW 264.7 cell fraction by
sequential chromatography on 2',5'-ADP Sepharose and Mono-Q anion
exchange columns using fast protein liquid chromatography (41).
1 cm
1.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (18K):
[in a new window]
Fig. 1.
Structures of imidazoles and the
indazole used in this study.
View larger version (18K):
[in a new window]
Fig. 2.
Comparative ability of imidazole and
L-Arg/H4B to promote spectral changes and
dimerization of iNOSox monomers. Urea-generated iNOSox monomers
(1-2 µM) were incubated in Hepes buffer containing 3 mM DTT alone, DTT plus 3 mM imidazole, or DTT
plus 1 mM L-Arg and 10 µM
H4B. Spectra were recorded every 10 min up to 1 h.
Panel A, spectra obtained before
( ) and immediately after adding 3 mM imidazole (- - -). Inset, consecutive
spectra collected at 0, 10, 20, 30, 40, and 60 min after adding
L-Arg and H4B. Arrows show the
direction of changes with time. Panel B, gel filtration
profiles (A280) of the three iNOSox samples
after their 40-min incubation. On the basis of protein standards, the
protein peak eluting at 10.5 ml is iNOSox dimer, and the peak at 13 ml
is the iNOSox monomer.
A significant portion of imidazole-bound iNOSox monomer assembled into a dimer over the 40-min incubation period (Fig. 2B). The amount of dimer present in the sample incubated with imidazole exceeded the amount of dimer present in the sample incubated with DTT alone,3 and was comparable to that obtained for monomers incubated with L-Arg and H4B (Fig. 2B). Spectral and dimerization results obtained using 1-phenylimidazole were identical to those obtained with imidazole (data not shown), indicating that a phenyl substituent at the N1 position has no adverse effect on either heme binding or dimerization. In contrast, incubating iNOSox monomers with 2-phenylimidazole did not result in spectral change or dimerization (data not shown), consistent with 2-phenylimidazole binding poorly to heme proteins due to a steric interaction between the phenyl ring and porphyrin (22).
The rate of iNOS dimerization in the presence of imidazole was investigated using urea-generated heme-containing monomers of full-length iNOS and assaying recovery of NO synthesis, which is a dimer-specific activity used to measure iNOS dimerization (41, 47, 53). As shown in Fig. 3, there was an increase in NO synthesis activity within the first 20 min of incubation followed by a more gradual increase. This time course is similar to that observed for iNOS dimerization in response to L-Arg and H4B (47, 49, 53), and suggests that dimerization follows binding of imidazole to the heme iron, which occurs within seconds after mixing. Recovery of NO synthesis was also associated with an increase in the proportion of dimer from 30 to 70% over the course of the imidazole incubation as determined by gel filtration chromatography of the 0 and 180-min samples (data not shown). We conclude that imidazole and small N1-substituted imidazoles can promote dimer assembly after binding to the heme iron. This differs from L-Arg, H4B, or 7-nitroindazole, which promote dimer assembly without binding to the heme iron.
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We then investigated if larger N1-substituted imidazoles would bind to iNOSox monomer and affect dimer assembly. Addition of clotrimazole (30 µM) or miconazole (15 µM) led to an immediate spectral change identical to that obtained when imidazole was added (data not shown), indicating that these bulky imidazoles bind rapidly to the monomer heme iron. The affinity of the iNOSox monomer heme for clotrimazole and miconazole was determined by perturbation difference spectrophotometry (shown for clotrimazole in Fig. 4A). The binding constants derived from double-reciprocal plots of spectral change versus wavelength were 7.3 ± 1.0 µM for clotrimazole (Fig. 4B) and 12 ± 0.4 µM for miconazole (data not shown). The constant for clotrimazole binding to iNOSox is in agreement with its inhibitory constant for NO production by stimulated hepatocytes (13 µM) (62). We conclude the iNOSox monomer heme can bind large N1-substituted imidazoles with high affinity, consistent with the monomer containing a relatively open distal heme pocket (57).
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Gel filtration analysis of iNOSox monomers after incubation with either clotrimazole or miconazole for 40 min showed that it remained monomeric in both cases (Fig. 5, top horizontal row), indicating that these bulky imidazoles did not promote dimer assembly. We therefore examined their ability to antagonize L-Arg- and H4B-promoted dimerization. Monomers were preincubated for 10 min either with buffer alone or containing various concentrations of clotrimazole or miconazole, and then exposed to 1 mM L-Arg and 10 µM H4B to promote dimerization, which was assayed after an additional 10 and 40 min incubation. In the absence of clotrimazole and miconazole, significant dimer assembly occurred within 10 min of incubation with L-Arg and H4B and generated ~90% dimer by 40 min (Fig. 5, first vertical row). Clotrimazole or miconazole inhibited dimer assembly as promoted by L-Arg/H4B in a concentration-dependent manner (Fig. 5), with miconazole being somewhat more effective than clotrimazole. In cases where little or no dimerization occurred (30 µM clotrimazole or 15 µM miconazole), spectral change characteristic of L-Arg- and H4B-promoted dimerization (see Fig. 2A, inset) was not observed, whereas in cases of partial dimerization (for example, at 10 or 20 µM clotrimazole) we observed a time-dependent increase in absorbance centered at 396 nm, consistent with some H4B- and L-Arg-promoted dimerization and displacement of clotrimazole (Fig. 6).
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We next investigated whether clotrimazole or miconazole could dissociate an iNOSox dimer4 that had or had not been preincubated with L-Arg plus H4B. Dimer not exposed to L-Arg and H4B bound clotrimazole (15 µM) or miconazole (30 µM) rapidly, as judged by an immediate spectral change from the split Soret peak to a single peak centered at 429 nm (data not shown; identical to that observed for imidazole in Fig. 2A). Their binding was associated with subsequent conversion of dimer to monomer over a 40-min incubation as determined by gel filtration (Fig. 7, traces A-C). In contrast, iNOSox dimer that had been preincubated with L-Arg and H4B was unable to bind clotrimazole or miconazole, as judged by a lack of spectral change (data not shown), and also did not significantly dissociate into monomer over a 40-min incubation (Fig. 7, trace D). Thus, clotrimazole and miconazole only bound to (and dissociated) dimeric iNOSox in the absence of L-Arg plus H4B.
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On the basis of the above data, we tested if clotrimazole would block
assembly of dimeric iNOS in the RAW 264.7 macrophage cell line. In a
typical experiment, a 500-ml spinning cell culture was made 50 µM in clotrimazole, incubated for 30 min, then induced to
express iNOS by adding lipopolysaccharide and interferon- and
incubated for an additional 8 h before harvesting and cell lysis.
Control cultures received Me2SO alone but were otherwise identically activated and processed. The nitrite concentration in the
control culture increased from 2 to 30 µM over the 8-h induction period (Fig. 8), consistent
with induced expression of active dimeric iNOS, but the nitrite level
did not increase in the culture given clotrimazole. Cell supernatants
prepared from representative matched control and clotrimazole-treated
cultures (500 ml each) contained 28 and 23 mg of soluble protein,
respectively, indicating that clotrimazole caused little or no cell
lysis under these conditions. Supernatant prepared from the
clotrimazole-treated culture displayed a NO synthesis specific activity
that was 6-fold lower than the control supernatant (0.38 versus 2.3 nmol of nitrite/min/mg of protein, respectively),
but contained a similar amount of iNOS protein as the control as judged
by Western analysis (data not shown), consistent with corresponding
observations in activated hepatocytes (62). Thus, the
clotrimazole-treated culture contained iNOS in a predominantly inactive
form.
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Inactive iNOS could arise from clotrimazole inhibiting dimer assembly, or alternatively, by clotrimazole inhibiting CaM binding to iNOS (63, 64), which would likely prevent its proper folding (61). We differentiated between these two possibilities by determining iNOS dimer-monomer ratios in the cell lysates and checking whether CaM was associated to iNOS. The control activated supernatant (Fig. 9, panel A) contained both dimeric and monomeric iNOS in relatively equal proportion, consistent with previous reports (41, 54), whereas the clotrimazole-treated supernatant (Fig. 9, panel B) predominantly contained monomeric iNOS. Scanning densitometry estimated the dimer-monomer ratios to be 45:55 and 15:85 in each case, respectively. Reprobing the Western blots with an antibody directed against CaM showed that CaM was present in the same fractions that contained the iNOS dimer and monomer, with the majority running at an apparent molecular mass of 17 kDa (shown in Fig. 9, panel C, for the cell lysate from clotrimazole-treated cells), as is typically observed for iNOS in denaturing gels (12, 61). Measurement of NO synthesis activity in each gel filtration fraction (Fig. 9, panel D) confirmed that much less iNOS dimer was present in the supernatant from clotrimazole-treated cells. We also checked for iNOS-associated CaM by determining the cytochrome c reductase activity of iNOS monomer purified from clotrimazole-treated cells. Monomer reductase activity in the absence or presence of added Ca2+/CaM was 2.0 ± 0.3 and 3.1 ± 0.5 mol of cytochrome c reduced/min/mmol of iNOS, respectively, similar to the value reported for iNOS monomers purified from conventional cultures (2.5 ± 0.4 mol of cytochrome c reduced/min/mmol; Ref. 41). We conclude that clotrimazole inhibited iNOS dimer assembly in the cells without significantly preventing CaM binding to iNOS.
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DISCUSSION |
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Our data show that imidazoles can positively or negatively regulate iNOS dimer assembly. Small molecules like imidazole and 1-phenylimidazole promoted iNOS dimerization, whereas bulky imidazoles like clotrimazole and miconazole did not, and instead inhibited iNOS dimerization both in a purified system and in cultured cells. Their ability to block iNOS dimer assembly was unexpected and may suggest new strategies to control NO synthesis in cells.
How small molecules influence NOS dimer assembly is not clear. Outside of a few cell culture studies (54, 65, 66), our current understanding is based on work with purified NOS proteins or subcellular preparations. Some general features have emerged. For example, various derivatives of L-Arg and guanidine, various pteridines, ethylisothiourea, and 7-nitroindazole all promote NOS subunit dimerization (41, 47, 51, 56, 67) or stabilize the dimer (42, 43, 58). This suggests structural constraints are minimal, and the promoting effect is not limited to molecules acting as substrates or cofactors for NO synthesis. The active molecules noted above all bind to dimeric NOS isoforms with moderate to good affinity, but appear to bind poorly or not at all to NOS monomers, suggesting their binding sites are incompletely formed in the monomer. In addition, while most of these molecules seem to bind near the distal heme pocket of NOS, they do not ligate directly to the heme iron, and instead bind through interactions with the protein. When molecules of this type are added to heme-containing iNOS monomers, they cause a gradual shift to five-coordinate high-spin heme that proceeds according to the tempo of iNOS dimerization, as determined independently by monitoring protein fluorescence and catalytic changes associated with dimerization (47, 58). Together, these features support a mechanism in which NOS subunits first interact reversibly to form a loose dimer before the promoter molecules bind.5 Subsequent promoter molecule binding then stabilizes the dimeric structure and drives the monomer-dimer equilibrium toward stable dimer (42, 52).
Our current results suggest that imidazoles may not function by this mechanism, but instead affect dimerization by binding directly to the iNOS monomer heme iron. This possibility is supported by the immediate and complete binding of imidazole when it was added to iNOS monomers, the rate at which dimerization occurred when imidazole was present at a concentration approximately 100 times Kd, and the fact that imidazole remained bound to the heme after the dimer had formed. Its binding to the monomer heme appears critical, because dimerization is not promoted by imidazoles that cannot bind to heme (i.e. 2-phenylimidazole). Thus, two means for promoting iNOS dimerization appear possible: one involves subunit dimeric interaction as an initial step followed by promoter binding to stabilize the dimeric structure (for example, H4B and L-Arg); while the other may involve promoter binding as an initial event followed by productive subunit interaction (imidazole). Both events may cause a common change in iNOS structure that stabilizes the dimeric form.
Clotrimazole bound immediately to an iNOSox monomer or to a L-Arg- and H4B-free dimer, but it was unable to bind to a H4B- and L-Arg-saturated dimer. This provides further evidence that the distal heme pockets of an iNOS monomer or H4B- and L-Arg-free dimer are similar in being exposed to the solvent and able to accommodate large heme iron ligands like clotrimazole or DTT. Bound H4B and L-Arg excludes these bulky ligands. Because clotrimazole could only dissociate iNOSox dimers with which it formed a heme-iron complex, it likely affects iNOS quaternary structure after binding to the heme iron, like the smaller imidazoles.
A scheme that incorporates clotrimazole (and miconazole) inhibition
into a current model for iNOS dimer assembly (52) is illustrated in
Fig. 10. Heme-free iNOS monomers
(iNOSM) must incorporate heme (Fe) in order to dimerize
(41, 42). Heme-containing iNOS partitions in a monomer-loose dimer
equilibrium (iNOSMFe iNOSDFe). H4B and L-Arg, if present, can bind to the
dimer and drive formation of stable, active iNOS dimers
(iNOSDFe(Arg, H4B)). Clotrimazole reversibly
binds to the heme iron of a monomer and prevents its dimerization; it
can also reversibly bind to the heme in a loose dimer and cause it to
dissociate. Both processes lead to monomer accumulation. Occupancy of
the distal heme pocket by clotrimazole versus
H4B plus L-Arg appears to be mutually
exclusive. This equilibrium binding model is consistent with
clotrimazole slowing iNOSox dimerization in response to
L-Arg and H4B (Fig. 5), rather than making it
completely impossible.
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How do bulky imidazoles like clotrimazole prevent iNOS dimerization at
the molecular level? That clotrimazole binds to iNOSox monomer is
consistent with crystal structures that reveal an open heme pocket in
the monomer (57). Computer modeling of a clotrimazole molecule bound to
the heme of an iNOSox monomer or dimer predicts extensive collisions
between clotrimazole's phenyl moieties and iNOSox -strands 8b and
9b, which are located above the heme. Modeling also shows that the
phenyl groups do not extend far enough out of the pocket to directly
contact the dimer interface
region.6 Thus, distortion of
iNOS
-strands 8b and 9b is likely to occur upon binding
clotrimazole, which may perturb nearby structural elements that
participate in forming the dimer interface.
The function we propose for clotrimazole and miconazole in Fig. 10
differs from their other known effects. For example, clotrimazole and
miconazole inhibit cytochrome P450 catalysis (69), notably in the
course of oxidation of
N-hydroxy-L-Arg (70). This occurs
because clotrimazole and miconazole are hydrophobic enough to enter the
P450 active site, which allows their imidazole nitrogen to bind to the
heme iron and prevent substrate access and O2 binding. A
similar mechanism cannot operate in an active iNOS dimer, which by
virtue of its bound H4B and L-Arg excludes
bulky imidazoles from the active site. Clotrimazole and miconazole are
also known to inhibit CaM binding to receptor proteins (63, 64) and to
nNOS at higher concentrations (22). However, our dimerization
experiments utilized iNOSox, which does not have a CaM-binding site.
Significantly, clotrimazole also inhibited dimerization of full-length iNOS in cells that were actively engaged in iNOS protein synthesis, leading to accumulation of iNOS monomers in the clotrimazole-treated cells and a lack of NO synthesis. The effect of clotrimazole on cellular iNOS dimer assembly is striking if one considers that blocking NO synthesis during iNOS expression normally increases the proportion of dimer from 50 to 90% of the total iNOS (54). Our data argue for a heme-based mode of action for clotrimazole in the cells. For example, the clotrimazole-treated cells grew normally and expressed a normal amount of protein, confirming it is not toxic at the concentration used (62, 71). Immunochemical and catalytic data indicated that iNOS monomers from clotrimazole-treated cells still contained a normal amount of bound CaM. This is consistent with clotrimazole being unable to dissociate CaM from purified iNOS dimer or inhibit its NO synthesis when added to the assay system (12, 72). The high affinity of iNOS toward CaM (12) likely constrains clotrimazole to interact primarily in a heme-dependent manner. Our findings are in apparent conflict with Bogle and Vallance (71), who report to have purified CaM-free iNOS from cells treated with econazole, a structural relative of miconazole. Whether structural differences allow econazole to function through a CaM-based mechanism deserves further study.
Although clotrimazole appears to inhibit cellular iNOS assembly through a heme-based mechanism, the exact point where inhibition occurs is still unclear. For example, clotrimazole could possibly affect events upstream from dimerization that control heme availability or insertion into iNOS monomers. Indeed, iNOS monomers which accumulated in the clotrimazole-treated cells were heme-free, suggesting clotrimazole affects heme incorporation into iNOS. Heme-free iNOS monomers also accumulate in cells actively generating NO, because NO apparently blocks heme insertion (54). In any case, our current data indicate that cellular assembly of active iNOS is remarkably sensitive to clotrimazole, and in principle may be sensitive to any bulky imidazole that can bind to the monomer heme iron.
Clotrimazole will likely inhibit iNOS dimerization in animal cells only
when it is present during active iNOS protein synthesis and dimer
assembly. This is because clotrimazole cannot bind to the heme iron of
an assembled iNOS dimer that has already incorporated H4B
and L-Arg, and almost all animal cells and cell lines
synthesize H4B and take in L-Arg (73).
Nevertheless, cytokine induction of iNOS is long lasting (74), tissues
like lung epithelium continuously express iNOS (75), and cellular
assembly of dimer is sufficiently slow (66) to make iNOS susceptible to
clotrimazole inhibition. It remains to be seen whether cellular
assembly of constitutive NOS isoforms is also blocked by bulky
imidazoles, or if structural analogs with specificity toward a
particular isoform can be developed.
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ACKNOWLEDGEMENTS |
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We thank Pam Clark for excellent technical assistance, and Drs. Brian Crane, John Tainer, John Parkinson, and Serpil Erzurum for manuscript review and stimulating discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA53914 and by Berlex Biosciences.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a research grant from Rhône-Poulenc, France. Current address: Ecole Nationale Supérieure des Mines de Paris, 60 boulevard Saint-Michel, 75272 Paris Cedex 06, France.
¶ Established Investigator of the American Heart Association. To whom correspondence should be addressed: Immunology NN-1, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-6950; Fax: 216-444-9329; E-mail: stuehrd{at}cesmtp.ccf.org.
The abbreviations used are:
NO, nitric oxide; L-Arg, L-arginine; BSA, bovine serum albumin; CaM, calmodulin; clotrimazole, 1-(o-chloro-,
-diphenylbenzyl)imidazole; DTT, dithiothreitol; EPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic
acid; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; H4B, (6R)-5,6,7,8-tetrahydro-L-biopterin; miconazole, 1-[2,4-dichloro-
-[(2,4-dichlorobenyloxy]phenylethyl]-imidazole; NADPH,
-nicotinamide adenine dinucleotide, reduced; iNOS, nitric
oxide synthase; iNOSox, iNOS oxygenase domain; PAGE, polyacrylamide gel
electrophoresis; Bis-Tris, 2-[bis)2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
2 N. Sennequier, D. K. Ghosh, and D. J. Stuehr, unpublished results.
3 Molecules which coordinate to the iNOS heme iron do not necessarily promote dimer assembly (for example, DTT).
4 When isolated from E. coli in the absence of L-Arg or H4B, the iNOSox is approximately 60-80% dimeric (49).
5 Although loose dimers have not been observed in mammalian cells, all NOS form a loose heme-containing dimer when overexpressed in E. coli in the absence of known dimer-promoting molecules (43, 61, 68).
6 B. R. Crane and J. T. Tainer, personal communication.
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REFERENCES |
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