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Antifungal Imidazoles Block Assembly of Inducible NO Synthase into an Active Dimer

1999; Elsevier BV; Volume: 274; Issue: 2 Linguagem: Inglês

10.1074/jbc.274.2.930

ISSN

1083-351X

Autores

Nicolas Sennequier, Dennis W. Wolan, Dennis J. Stuehr,

Tópico(s)

Oxidative Organic Chemistry Reactions

Resumo

Cytokine-inducible nitric oxide synthase (iNOS) is a homodimeric enzyme that generates nitric oxide (NO) andl-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 andN-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 byl-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. Cytokine-inducible nitric oxide synthase (iNOS) is a homodimeric enzyme that generates nitric oxide (NO) andl-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 andN-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 byl-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) 1The abbreviations used are: NO, nitric oxide; l-Arg, l-arginine; BSA, bovine serum albumin; CaM, calmodulin; clotrimazole, 1-(o-chloro-α,α-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-β-[(2,4-dichlorobenyloxy]phenylethyl]-imidazole; NADPH, β-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. is synthesized from l-arginine (l-Arg) in animals by the NO synthases (NOSs, for reviews, see Refs. 1Marletta M.A. Cell. 1994; 78: 927-930Abstract Full Text PDF PubMed Scopus (815) Google Scholar, 2Nathan C. Xie Q. J. Biol. Chem. 1994; 269: 13725-13728Abstract Full Text PDF PubMed Google Scholar, 3Griffith O.W. Stuehr D.J. 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Abu-Soud H.M. Stuehr D.J. Biochemistry. 1996; 35: 1444-1449Crossref PubMed Scopus (64) Google Scholar). For example, the iNOS oxygenase domain (iNOSox, amino acids 1–498) is expressed inEscherichia coli as a dimer, exhibits normal affinity towardl-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 (48Gachhui R. Ghosh D.K. Wu C. Parkinson J. Crane B.R. Stuehr D.J. Biochemistry. 1997; 36: 5097-5103Crossref PubMed Scopus (72) Google Scholar, 49Ghosh D.K. Wu C. Pitters E. Moloney M. Werner E.R. Mayer B. Stuehr D.J. Biochemistry. 1997; 36: 10609-10619Crossref PubMed Scopus (154) Google Scholar, 50Ghosh D.K. Abu-Soud H.M. Stuehr D.J. Biochemistry. 1995; 34: 11316-11320Crossref PubMed Scopus (50) Google Scholar). A crystal structure of dimeric iNOSox with l-Arg and H4B bound has been published (32Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (626) Google Scholar). 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 (41Baek K.J. Thiel B.A. Lucas S. Stuehr D.J. J. Biol. Chem. 1993; 268: 21120-21129Abstract Full Text PDF PubMed Google Scholar, 42Klatt P. Pfieffer S. List B.M. Lehner D. Glatter O. Bachinger H.P. Werner E.R. Schmidt K. Mayer B. J. Biol. Chem. 1996; 271: 7336-7342Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). 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 (41Baek K.J. Thiel B.A. Lucas S. Stuehr D.J. J. Biol. Chem. 1993; 268: 21120-21129Abstract Full Text PDF PubMed Google Scholar, 42Klatt P. Pfieffer S. List B.M. Lehner D. Glatter O. Bachinger H.P. Werner E.R. Schmidt K. Mayer B. J. Biol. Chem. 1996; 271: 7336-7342Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 51Riveros-Morino V. Heffernan B. Torres B. Chubb A. Charles I. Moncada S. Eur. J. Biochem. 1995; 230: 52-57Crossref PubMed Scopus (52) Google Scholar). The heme-containing NOS monomer may be an intermediate on the path toward forming a stable dimer (52Stuehr D.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 339-359Crossref PubMed Scopus (452) Google Scholar), and although it does not accumulate in mammalian cells containing H4B and l-Arg (41Baek K.J. Thiel B.A. Lucas S. Stuehr D.J. J. Biol. Chem. 1993; 268: 21120-21129Abstract Full Text PDF PubMed Google Scholar), it can be generatedin vitro by dissociating purified iNOS dimers with urea (47Ghosh D.K. Abu-Soud H.M. Stuehr D.J. Biochemistry. 1996; 35: 1444-1449Crossref PubMed Scopus (64) Google Scholar,53Abu-Soud H.M. Loftus M. Stuehr D.J. Biochemistry. 1995; 34: 11167-11175Crossref PubMed Scopus (97) Google Scholar). Dimerization of iNOS monomers is also stimulated by H4B and l-Arg (41Baek K.J. Thiel B.A. Lucas S. Stuehr D.J. J. Biol. Chem. 1993; 268: 21120-21129Abstract Full Text PDF PubMed Google Scholar, 47Ghosh D.K. Abu-Soud H.M. Stuehr D.J. Biochemistry. 1996; 35: 1444-1449Crossref PubMed Scopus (64) Google Scholar, 53Abu-Soud H.M. Loftus M. Stuehr D.J. Biochemistry. 1995; 34: 11167-11175Crossref PubMed Scopus (97) Google Scholar) and is inhibited in cells by NO, which interferes with heme insertion into the monomers (54Albakri Q.A. Stuehr D.J. J. Biol. Chem. 1996; 271: 5414-5421Abstract Full Text PDF PubMed Scopus (145) Google Scholar). Dimerization activates iNOS in part by enabling electron transfer between enzyme flavin and heme prosthetic groups (55Siddhanta U. Presta A. Fan B. Wolan D. Rousseau D.L. Stuehr D.J. J. Biol. Chem. 1998; 273: 18950-18958Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), and by facilitating productive binding of substrate and H4B (41Baek K.J. Thiel B.A. Lucas S. Stuehr D.J. J. Biol. Chem. 1993; 268: 21120-21129Abstract Full Text PDF PubMed Google Scholar,49Ghosh D.K. Wu C. Pitters E. Moloney M. Werner E.R. Mayer B. Stuehr D.J. Biochemistry. 1997; 36: 10609-10619Crossref PubMed Scopus (154) Google Scholar). 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 (41Baek K.J. Thiel B.A. Lucas S. Stuehr D.J. J. Biol. Chem. 1993; 268: 21120-21129Abstract Full Text PDF PubMed Google Scholar, 56Sennequier N. Stuehr D.J. Biochemistry. 1996; 35: 5883-5892Crossref PubMed Scopus (61) Google Scholar). Two substrate analogs that exhibit greater affinity toward the iNOS dimer than l-Arg,N ω-amino-l-Arg andl-thiocitrulline (21Narayanan K. Spack L. McMillan K. Kilbourn R.G. Hayward M.A. Masters B.S.S. Griffith O.W. J. Biol. Chem. 1995; 270: 11103-11110Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 24Wolff D.J. Lubeski A. Umansky S. Arch. Biochem. Biophys. 1994; 314: 360-366Crossref PubMed Scopus (72) Google Scholar), did not promote dimer assembly, suggesting they might function as antagonists. However, they were incapable of blocking dimer assembly in the presence of excessl-Arg (56Sennequier N. Stuehr D.J. Biochemistry. 1996; 35: 5883-5892Crossref PubMed Scopus (61) Google Scholar), consistent with their sharing a common binding site but being generally unable to bind to iNOS monomers (32Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (626) Google Scholar,48Gachhui R. Ghosh D.K. Wu C. Parkinson J. Crane B.R. Stuehr D.J. Biochemistry. 1997; 36: 5097-5103Crossref PubMed Scopus (72) Google Scholar, 49Ghosh D.K. Wu C. Pitters E. Moloney M. Werner E.R. Mayer B. Stuehr D.J. Biochemistry. 1997; 36: 10609-10619Crossref PubMed Scopus (154) Google Scholar). This suggests that substrate analogs have little inherent capacity to block iNOS dimerization. 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 (57Crane B.R. Arvai A.S. Gachhui R. Wu C. Ghosh D.K. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1997; 278: 425-431Crossref PubMed Scopus (337) Google Scholar), 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 (32Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (626) Google Scholar, 48Gachhui R. Ghosh D.K. Wu C. Parkinson J. Crane B.R. Stuehr D.J. Biochemistry. 1997; 36: 5097-5103Crossref PubMed Scopus (72) Google Scholar). We therefore examined the ability of imidazole andN 1-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 (56Sennequier N. Stuehr D.J. Biochemistry. 1996; 35: 5883-5892Crossref PubMed Scopus (61) Google Scholar, 58Klatt P. Schmidt K. Lehner D. Glatter O. Bachinger H.P. Mayer B. EMBO J. 1995; 14: 3687-3695Crossref PubMed Scopus (266) Google Scholar). 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. Interferon-γ 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. RAW 264.7 macrophage cell cultures (500 ml) were grown in spinner flasks and induced to express iNOS by adding 10 units/ml interferon-γ and 1 μg/mlE. coli lipopolysaccharide as previously detailed (41Baek K.J. Thiel B.A. Lucas S. Stuehr D.J. J. Biol. Chem. 1993; 268: 21120-21129Abstract Full Text PDF PubMed Google Scholar). 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 (41Baek K.J. Thiel B.A. Lucas S. Stuehr D.J. J. Biol. Chem. 1993; 268: 21120-21129Abstract Full Text PDF PubMed Google Scholar). 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. (60Siddhanta U. Wu C. Abu-Soud H.M. Zhang J. Ghosh D.K. Stuehr D.J. J. Biol. Chem. 1996; 271: 7309-7312Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), while full-length iNOS containing a 6-histidine tag at its N terminus was expressed in E. coli and purified in the absence ofl-Arg and H4B as described in Wu et al. (61Wu C. Zhang J. Abu-Soud H. Ghosh D.K. Stuehr D.J. Biochem. Biophys. Res. Commun. 1996; 222: 439-444Crossref PubMed Scopus (89) Google Scholar). 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 (47Ghosh D.K. Abu-Soud H.M. Stuehr D.J. Biochemistry. 1996; 35: 1444-1449Crossref PubMed Scopus (64) Google Scholar, 60Siddhanta U. Wu C. Abu-Soud H.M. Zhang J. Ghosh D.K. Stuehr D.J. J. Biol. Chem. 1996; 271: 7309-7312Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Briefly, iNOSox was diluted to 3 μm in 40 mm HEPES, pH 7.5, containing 10% glycerol, 3 mm DTT, and 5 murea 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 2m 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 (47Ghosh D.K. Abu-Soud H.M. Stuehr D.J. Biochemistry. 1996; 35: 1444-1449Crossref PubMed Scopus (64) Google Scholar, 53Abu-Soud H.M. Loftus M. Stuehr D.J. Biochemistry. 1995; 34: 11167-11175Crossref PubMed Scopus (97) Google Scholar) and were used within 1 day of their preparation. 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 (41Baek K.J. Thiel B.A. Lucas S. Stuehr D.J. J. Biol. Chem. 1993; 268: 21120-21129Abstract Full Text PDF PubMed Google Scholar). 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 mmNADPH, 2 mml-Arg, 3 mm DTT, protease inhibitors, and 4 μm each of FAD, FMN, and H4B as described previously (41Baek K.J. Thiel B.A. Lucas S. Stuehr D.J. J. Biol. Chem. 1993; 268: 21120-21129Abstract Full Text PDF PubMed Google Scholar). 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 mmDTT, 1 mml-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−1 cm−1. Optical spectra were recorded at room temperature on a Hitachi U-2110 spectrophotometer as detailed in Sennequier and Stuehr (56Sennequier N. Stuehr D.J. Biochemistry. 1996; 35: 5883-5892Crossref PubMed Scopus (61) Google Scholar). 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, andl-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. 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. 2N. Sennequier, D. K. Ghosh, and D. J. Stuehr, unpublished results. 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 (47Ghosh D.K. Abu-Soud H.M. Stuehr D.J. Biochemistry. 1996; 35: 1444-1449Crossref PubMed Scopus (64) Google Scholar). 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 (54Albakri Q.A. Stuehr D.J. J. Biol. Chem. 1996; 271: 5414-5421Abstract Full Text PDF PubMed Scopus (145) Google Scholar). 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 (12Cho H.J. Xie Q.-w. Calaycay J. Mumford R.

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