Free Radical Biology & Medicine 51 (2011) 1116–1125
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Free Radical Biology & Medicine
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Original Contribution
Nox2 B-loop peptide, Nox2ds, specifically inhibits the NADPH oxidase Nox2
Gábor Csányi a,1, Eugenia Cifuentes-Pagano a,1, Imad Al Ghouleh a, Daniel J. Ranayhossaini a, Loreto Egaña a,Lucia R. Lopes b, Heather M. Jackson c, Eric E. Kelley a,d, Patrick J. Pagano a,⁎a Vascular Medicine Institute, Department of Pharmacology & Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15260, USAb Department of Pharmacology, Biomedical Sciences Institute, University of São Paulo, 05508 900 São Paulo, Brazilc Department of Pathology and Experimental Medicine, Emory University School of Medicine, Atlanta, GA 30322, USAd Department of Anesthesiology, University of Pittsburgh, Pittsburgh, PA 15260, USA
In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1,Nox2, and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibitionof these isoforms is an area of intense current investigation. In this study, we postulated that Nox2ds, apeptidic inhibitor that mimics a sequence in the cytosolic B-loop of Nox2, would inhibit ROS production by theNox2-, but not the Nox1- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2dswas assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1-,or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibitedsuperoxide (O2
•−) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electronparamagnetic resonance confirmed that Nox2ds inhibits O2
•− production by Nox2 oxidase. In contrast, Nox2dsdid not inhibit ROS production by either Nox1- or Nox4-oxidase. These findings demonstrate that Nox2ds is aselective inhibitor of Nox2-oxidase and support its utility to elucidate the role of Nox2 in organpathophysiology and its potential as a therapeutic agent.
Reactive oxygen species (ROS) play an important role in thepathogenesis of cardiovascular disorders, including systemic andpulmonary hypertension, atherosclerosis, stroke, and restenosis [1,2].ROS are often considered highly reactive toxic by-products of oxygenmetabolism. However, it is also known that ROS contribute to a widerange of physiological processes, including regulation of vascular tone,cellular signaling, gene expression, angiogenesis, cellular senescence,and cell growth [3–6]. NADPH oxidases (Nox) are the major source ofROS in the cardiovascular system [7,8] and strong evidence suggeststhat Nox proteins contribute to oxidative damage in response to awide variety of stimuli, including cytokines, hormones, metabolicfactors, and mechanical injury [1]. Therefore, the selective blockade ofthe undesirable actions of Nox-derived ROS is expected to be an
important therapeutic strategy for treating oxidative stress-relatedcardiovascular pathologies.
The mechanism of production of the prototypical ROS superoxideanion (O2
•−) from NADPH oxidase has been extensively studied inphagocytes [9]. The phagocytic NADPH oxidase is an enzyme complexcomposed of two transmembrane “anchoring” subunits, Nox2 (akagp91phox) and p22phox; three cytosolic components (p47phox, p67phox,and p40phox); and the small GTPase Rac2 [10]. Upon activation, theregulatory subunit p47phox is phosphorylated and translocates to themembrane with the activator subunit p67phox and p40phox. Preventionof this translocation/assembly in phagocytes formed the basis forpeptidic inhibitor development by our group [11,12]. The assembledphagocyte NADPH oxidase catalyzes the transfer of one electron fromNADPH through FAD and two heme groups to molecular oxygen toform O2
•−, a key determinant of nitric oxide bioavailability and theforerunner of multiple other biological ROS.
It is now known that the Nox family consists of seven members,namely Nox1, Nox2, Nox3, Nox4, Nox5, dual oxidase (DUOX) 1, andDUOX2, which differ in tissue distribution, subcellular localization,regulation, activity, and pathophysiological function [13], but retaintheir ability to transfer electrons from NADPH to oxygen to formeither O2
•− or its dismuted metabolite hydrogen peroxide (H2O2) [10].In rodents, the major family members are Nox1, Nox2, and Nox4.Nox1 is localized to caveolae in the plasmamembrane and endosomes[13], and Nox4 has been identified in focal adhesions [14], the nucleus[5], and the endoplasmic reticulum [15]. Nox1 is constitutively active
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and requires the respective p47phox and p67phox homologues NOXO1(Nox organizer subunit 1) and NOXA1 (Nox activator subunit 1) foractivation [16,17]. Nevertheless, previous data in smooth muscle cellsseem to suggest that p47phox and p67phox might supplant NOXO1 andNOXA1 in the Nox1-oxidase system [18]. Takeya et al. [17] showedthat COS cells transfected with Nox1/NOXO1/NOXA1 constitutivelygenerate O2
•− that can be further stimulated by phorbol myristateacetate (PMA). Moreover, O2
•− production was significantly higher inNox1/NOXO1/NOXA1- than in Nox1/NOXO1/p67phox- or Nox1/p47phox/NOXA1-transfected COS cells, whereas Nox1/p47phox/p67phox-transfected cells were inactive [17].
In contrast, Nox4 does not require the conventional subunitsp47phox and p67phox or their homologues for activation and thepossibility of a role for other cytosolic modulators is the focus of activeresearch [19]. In addition, multiple reports suggest that Nox4 differsfrom other Nox isoforms in producing primarily H2O2 [20].
Because excessive Nox-derived ROS contribute to the progressionof a wide spectrum of diseases, the Nox family of oxidases is a highlysought after therapeutic target, and the selective blockade ofindividual Nox isoforms is an area of intense investigation [21]. Todate, several potential inhibitors have been identified, yet most ofthem seem to exhibit low selectivity, potency, and bioavailability, andnone to our knowledge selectively inhibits Nox2 [21].
Our laboratorywas the first to rationally design a peptidic inhibitorin its original cell-permeative chimeric form (Nox2 docking se-quence–tat; Nox2ds-tat, aka gp91ds-tat) targeting the assembly ofNox2; in that study, Nox2ds-tat attenuated angiotensin II (AngII)-induced vascular O2
•− production and blood pressure elevation inmice[12]. Numerous studies demonstrated the effectiveness of thischimeric peptide inhibitor to attenuate or abolish ROS levels innormal or diseased tissue, consistent with the expression of Nox2[12,22–25]. However, the specificity of the B-loop nonchimericpeptide Nox2ds for Nox2 has not been demonstrated to ourknowledge. Recent studies suggest that certain amino acid sequencesin the B-loop of Nox2 bind to the dehydrogenase (DH) domain in theC-terminal tail of Nox2 and Nox4 and that this binding is required foractivity [26]. This, along with significant homology in the B-loopsamong isoforms raised concern for non-isoform-specific inhibition ofdifferent Nox's by Nox2ds. As the major vascular isoforms of NADPHoxidase are Nox1, Nox2, and Nox4, and because ROS derived fromthese isoforms have been implicated in many cardiovascularpathologies, we set out to test the inhibitory effect of Nox2ds onthese isoforms and postulated that Nox2ds is specific for Nox2, but notNox1 or Nox4, inhibition and its attendant ROS production.
Materials and methods
Materials
Cytochrome c, superoxide dismutase (SOD), lithium dodecylsulfate (LiDS), catalase, diphenyleneiodonium chloride (DPI), horse-radish peroxidase (HRP),Nω-nitro-L-argininemethyl ester (L-NAME),rotenone, and phenylmethanesulfonyl fluoride (PMSF) were pur-chased from Sigma–Aldrich (St. Louis, MO, USA). L-012was purchasedfrom Wako Chemicals USA (Richmond, VA, USA). Febuxostat waspurchased from Axon Medchem (Groningen, The Netherlands).Amplex red was purchased from Invitrogen/Molecular Probes(Eugene, OR, USA). Protease inhibitor cocktail was purchased fromRoche Diagnostics GmbH (Mannheim, Germany). Nox2ds and ScrmbNox2ds were synthesized by the Tufts University Core Facility(Boston, MA, USA). The sequence of Nox2ds in human Nox2 is asfollows: NH3-CSTRVRRQL-CONH2. The Scrmb Nox2ds sequence is asfollows: NH3-CLRVTRQSR-CONH2. In both cases, as has been the casesince the original publication on these peptides, the NH3 grouprepresents the amino end and NH2 represents the amide of thecarboxy terminus, a consequence of the synthetic procedure. The
purity of Nox2ds and Scrmb Nox2ds was 97.7% and 92.4%, respec-tively. Because these studies were carried out in cell-free systems, it isimportant to note that the peptides used in this study did not require achimeric design containing the tat peptide for cell permeation [12,27].
Cell lines
All cell culture reagents were obtained from Invitrogen, unlessindicated otherwise. COS-22 (COS-7 cells stably expressing humanp22phox) and COS-Nox2 (aka COS-phox) cells (COS-7 cells stablyexpressing human p22phox, Nox2, p47phox, and p67phox) were kindlyprovided by Dr. Mary C. Dinauer (Indiana University School ofMedicine, Bloomington, IN, USA). Superoxide production in intactCOS-Nox2 cells was characterized elsewhere [28]. COS-22 cells weremaintained in Dulbecco'smodified Eaglemedium (DMEM)with 4.5 g/L glucose, L-glutamine, and sodium pyruvate, containing 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, and100 μg/ml streptomycin (complete medium) supplemented with1.8 mg/ml G418 (Calbiochem/EMB Bioscience, Gibbstown, NJ, USA).COS-Nox2 cells were maintained in complete medium supplementedwith 1.8 mg/ml G418, 1 μg/ml puromycin (Sigma), and 0.2 mg/mlhygromycin B (Invitrogen, Carlsbad, CA, USA).
Plasmid preparation, amplification, and purification
Plasmids encoding full-length human cDNAs for Nox1(pcDNA3.1-hNox1), NOXO1 (pcDNA3.1-hNOXO1), NOXA1(pCMVsport 6-hNOXA1), p47phox (pCMV-Tag4A-hp47), and Nox4(pcDNA3-hNox4) were kindly provided by Dr. David Lambeth(Emory University, Atlanta, GA, USA) [29,30]. Plasmids encodingNox1, NOXO1, or NOXA1 were transformed and amplified intoEscherichia coli strain TOP10 (Invitrogen). Plasmids were purifiedusing a QIAfilter plasmid purification kit (Qiagen, Valencia, CA, USA).For human Nox4 expression, the BglII/NotI restriction fragment fromthe pcDNA3-hNox4 was subcloned into the plasmid pcDNA3.1/Hygro(−) (Invitrogen) to generate pcDNA3.1/Hygro-hNox4. Thefragment sequence, in-frame insertion, and orientation werevalidated by DNA sequencing after PCR amplification. pcDNA3.1/Hygro-hNox4 was amplified into E. coli strain TOP10 and purifiedwith a QIAfilter plasmid purification kit.
Transfection
Cell transfection was carried out using Lipofectamine LTX and Plusreagent (Invitrogen), according to the manufacturer's instructions.COS-22 cells were transiently cotransfected with pcDNA 3.1-hNox1,pCMVsport 6-hNOXA1, and pcDNA3.1-hNoxO1 (COS-Nox1/NOXO1/NOXA1 cells). Cells were used 24 h after transfection. For stabletransfection of Nox4, COS-22 cells were transfected with pcDNA3.1/Hygro-hNox4 (COS-Nox4 cells). COS-Nox4 cells were selected incomplete medium supplemented with 0.2 mg/ml hygromycin B and1.8 mg/ml G418. Stable transfectants were maintained in cultureunder the same conditions. Adherent cells were harvested byincubating with 0.05% trypsin/EDTA for 5 min at 37 °C. After additionof DMEM/10% FBS to neutralize the trypsin, the cells were pelletedby centrifugation at 1000 g for 5 min at 4 °C and used for theexperiments.
Superoxide-generating activity in COS cells
Superoxide production was measured in intact COS-Nox1/NOXO1/NOXA1 and COS-22 cells by L-012 chemiluminescence. Superoxideproduction in COS-Nox1/NOXO1/NOXA1 and COS-Nox2 cell-freesystems was measured by both SOD-inhibitable cytochrome creduction and electron paramagnetic resonance (EPR).
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L-012 chemiluminescenceCOS-Nox1/NOXO1/NOXA1 and COS-22 cells were replated into 96-
well white microplates (Greiner-Bio One, Germany) at a density of5×104 cells/well. The cells were incubated at 37 °C in phosphate-buffered saline (PBS) containing 400 μM luminol derivative L-012 for30 min. Luminescence was quantified over time using a BioTekSynergy 4 hybridmultimodemicroplate reader (BioTek,Winooski, VT,USA). The specificity of L-012 for O2
•−was confirmed by the addition ofSOD (150 U/ml).
Cytochrome c assayCOS-Nox2, COS-Nox1/NOXO1/NOXA1, and COS-22 cells were
suspended to a concentration of 5×107 cells/ml in ice-cold disruptionbuffer (8 mM potassium, sodium phosphate buffer, pH 7.0, 131 mMNaCl, 340 mM sucrose, 2 mM NaN3, 5 mM MgCl2, 1 mM EGTA, 1 mMEDTA, and protease inhibitor cocktail) [31]. The cells were lysed by fivefreeze/thaw cycles and passed through a 30-gauge needle five times tofurther lyse the cells. Cell disruption was confirmed by phase-contrastmicroscopy. The cell lysatewas centrifuged at 1000 g for 10 min at 4 °Cto remove unbroken cells, nuclei, and debris. Throughout all theseprocedures, extreme care was taken to maintain the lysate at atemperature close to 0 °C. Superoxide production was calculated fromthe initial linear rate (over 10 min) of SOD-inhibitable cytochrome creduction quantified at 550 nm using the extinction coefficient of 21.1mM−1 cm−1 (Biotek Synergy 4 hybridmultimodemicroplate reader).The oxidase assay buffer consisted of 65 mM sodium phosphate buffer(pH 7.0), 1 mM EGTA, 10 μM FAD, 1 mM MgCl2, 2 mM NaN3, and0.2 mM cytochrome c [31]. The components of the cell-free systemwere added in the following order: oxidase assay buffer, cell lysate(5×105 cell equivalents/well), and Nox2ds/Scrmb Nox2ds peptides ata final concentration of 0.1, 0.3, 1.0, 3.0, and 10 μM. The plates wereplaced on an orbital shaker to mix contents for 5 min at 120movements/min at room temperature. LiDS, an established lipidactivator of phagocyte cell-free systems, was added at a concentrationof 130 μM, and O2
•− productionwas initiated by the addition of 180 μMNADPH. The concentration of Nox2ds peptide that caused 50%inhibition of O2
•− production (IC50) in COS-Nox2 cell lysates wascalculated by Prism 5 (GraphPad Software, La Jolla, CA, USA).
To test the effect of Nox2ds on the hybrid Nox1 cell-free system,COS-22 cells were separately transfected with Nox1, NOXO1, p47phox,NOXA1, or p67phox and the Nox1 membrane component, and NOXO1,p47phox, NOXA1, and p67phox cytosolic extracts were individuallyprepared. Cells were lysed as described above and cell lysates werecentrifuged at 160,000 g for 60 min at 4 °C to separate the membranefrom the cytosol. The organizer subunit NOXO1 or p47phox waspreincubated with 10 μM Nox2ds for 10 min, and then NOX1 andNOXA1 or p67phox were added consecutively. After the preincubationperiod, LiDS (130 μM) was added to induce the assembly of theoxidase and O2
•− production was initiated by 180 μM NADPH.Superoxide production was measured using cytochrome c asdescribed above.
Electron paramagnetic resonanceThe spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetra-
methylpyrrolidine hydrochloride (CMH; Alexis Corp., San Diego, CA,USA) was used to examine O2
•− production in COS-Nox2 cell lysatesusing a Bruker eScan table-top EPR spectrometer (Bruker Biospin,USA). Superoxide production was measured in oxidase assay buffer(65 mM sodium phosphate buffer, pH 7.0, 1 mM EGTA, 10 μM FAD,1 mM MgCl2, 2 mM NaN3) supplemented with 50 μM CMH. Celllysates (5×105 cell equivalents) were incubated with Nox2ds andScrmb Nox2ds peptides for 5 min at room temperature. After thepreincubation period, LiDS was added at a concentration of 130 μMand O2
•− production was initiated by the addition of 180 μM NADPH.Analyses of the EPR spectra peak heights were used to quantify theamount of O2
•− produced by the lysates and were compared with
buffer-only control spectra or spectra in the presence of 10 μMNox2ds, 10 μM Scrmb Nox2ds, or 150 U/ml SOD. The effects of Nox2dsand Scrmb Nox2ds were expressed as SOD-inhibitable formation ofCM• radical. To minimize the deleterious effects of contaminatingmetals, the buffers were treated with Chelex resin and contained25 μM deferoxamine (Noxygen Science Transfer, Germany).
In separate experiments the O2•−-scavenging activity of Nox2ds
was determined by EPR using the xanthine/xanthine oxidase O2•−-
generating system. Assay mixtures contained PBS, 5 mU/ml xanthineoxidase, 50 μM CMH, and 25 μM deferoxamine in a final volume of100 μl. The reference samples contained 100 U/ml SOD. After additionof 25 μMxanthine, CM• radical formationwasmonitored for 10 min inthe absence and presence of 10 μM Nox2ds or Scrmb Nox2ds.
Hydrogen peroxide-generating activity
H2O2-producing activity was quantified in intact COS-Nox4 cellsusing Amplex red, according to the previously described methods[32]. H2O2 production was quantified in the COS-Nox4 cell-freesystem as described previously [32]. Briefly, COS-Nox4 cells (5×107
cells/ml) were disrupted in ice-cold disruption buffer (PBS containing0.1 mM EDTA, 10% glycerol, protease inhibitor cocktail, and 0.1 mMPMSF) by freeze/thaw cycles as we described above. Incubation ofCOS-Nox4 cell lysate with Nox2ds was performed in assay buffer(25 mM Hepes, pH 7.4, containing 0.12 M NaCl, 3 mM KCl, 1 mMMgCl2, 0.1 mM Amplex red, and 0.32 U/ml HRP) for 5 min at roomtemperature on an orbital shaker (120 movements/min), before theaddition of 36 μM NADPH, to initiate H2O2 production. This concen-tration of NADPH was used because it was found that higherconcentrations interferedwith Amplex red fluorescence. Fluorescencemeasurementsweremade using a Biotek Synergy 4 hybridmultimodemicroplate reader with a 530/25-excitation and a 590/35-emissionfilter. A standard curve of known H2O2 concentrations was developedusing the Amplex red assay (as per the manufacturer's instructions)and was used to quantify H2O2 production in the COS-Nox4 cell-freesystem. Nox4 activity was obtained by subtracting nontransfectedCOS-22 cell lysate activity from COS-Nox4 cell lysate activity. Thereaction was monitored at room temperature for 10 min, and theemission increase was linear during this interval.
Superoxide-generating activity in HEK 298 cells
To test the effect of Nox2ds on inducible Nox1 activity we usedNox1/NOXO1/NOXA1-transfected HEK 298 cells (hereafter referred toas HEK-Nox1) as it was shown previously that O2
•− production in HEK-Nox1 cells was significantly stimulated by PMA treatment [17]. HEK-Nox1 and nontransfected HEK 298 cells were replated into 96-wellwhite microplates at a density of 5×104 cells/well, and O2
•− wasmeasured in the absence and presence of PMA (1 μM) using L-012(400 μM). The effect of Nox2ds on inducible Nox1 activity was testedon LiDS-stimulated O2
•− in the HEK-Nox1 cell-free system. HEK 298cells were separately transfected with Nox1, NOXO1, or NOXA1.Nox1-containing membranes and NOXO1 and NOXA1 cytosolicextracts from each preparation were prepared as described above.The Nox1 organizer subunit NOXO1 was preincubated with 10 μMNox2ds for 10 min, and then NOX1 and NOXA1 were addedconsecutively. After the preincubation period, LiDS (130 μM) wasadded to induce the assembly of the oxidase and O2
•− production wasinitiated by 180 μM NADPH. Superoxide production was measuredusing cytochrome c.
Enzyme-linked immunosorbent assay (ELISA)
ELISA experiments were performed to test the mechanism bywhich Nox2ds could inhibit O2
•− production in the COS-Nox2 and notin the COS-Nox1 cell-free system. Neutravidin-coated plates (Thermo
Fig. 1. Nox2ds dose-dependently inhibits O2•− production from Nox2. COS-Nox2 cell
lysate was preincubated with various concentrations of Nox2ds (from 0.1 to 10 μM) andScrmb Nox2ds (from 0.1 to 10 μM) for 5 min at 25 °C. After the addition of 130 μM LiDS,O2•− production was initiated by the addition of 180 μM NADPH and measured by the
initial linear rate of SOD-inhibitable cytochrome c reduction. Superoxide production isexpressed as nmol O2
•−/min/107 cell equivalents. Data represent the means±SEM of 7–16 experiments. For comparison, O2
•− production in nontransfected COS-22 cell lysate isshown. *pb0.05 indicates significant difference in O2
•− production between Nox2ds andScrmb Nox2ds treatment. †pb0.05 indicates significant difference between COS-Nox2and COS-22 cell lysate activity.
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Scientific, Rockford, IL, USA)were incubated with biotinylated Nox2ds(biotin-Nox2ds, 6 μM) or biotinylated Scrmb Nox2ds (biotin-Scrmb,6 μM) (Tufts University Core Facility) for 2 h at room temperature. Theplates were washed three times using wash buffer (25 mM Tris,150 mM NaCl, 0.1% bovine serum albumin, 0.05% Tween 20, pH 7.2).After 1 h incubation at room temperature with COS-22, COS-22-p47phox (COS-22 cells transfected with p47phox), or COS-22-NOXO1(COS-22 cells transfected with NOXO1) cytosolic fraction, rabbitpolyclonal p47phox (1:500; Santa Cruz Biotechnology, Santa Cruz, CA,USA) or rabbit NOXO1 (1:500; Rockland Immunochemicals, Gilberts-ville, PA, USA) antibodies were used to detect p47phox or NOXO1bound to Nox2ds or its scrambled control, respectively. After 1 hincubation and extensive washing, bound primary antibodies weredetected by the addition of FITC-labeled goat anti-rabbit IgG antibody(1:500; Sigma–Aldrich). The fluorescence of each well was measuredusing a Biotek Synergy 4 hybrid multimode microplate reader(excitation 488 nM, emission 518 nM).
Fluorescence polarization
The Nox4 dehydrogenase domain (amino acids 304–578) recom-binant protein fused to glutathione S-transferase (GST) was expressedand purified as described previously [26] from E. coli usingGlutathione Sepharose 4B (GE Healthcare, Waukesha, WI, USA). Theability of the Nox2ds or Scrmb Nox2ds to compete off Nox4 B-loopfrom the Nox4 DH domain was measured by competition assaysmonitored by fluorescence polarization with a Synergy 2 multimodemicroplate reader and Gen5 software package (BioTek). FITC-labeledand unlabeled Nox4 B-loop peptides were synthesized and purified bythe Emory University Microchemical Facility. Unlabeled Nox4 B-loop(amino acids 77–106), Nox2ds, or Scrmb Nox2ds peptides weretitrated against a constant amount of Nox4 DH protein (60 nM) andFITC-labeled Nox4 B-loop peptide (31 nM) in assay buffer containing10 mM Hepes (pH 7.0), 75 mM NaCl, 1 μM FAD, and 0.05% Tween 20.Data were fit to a one-site competitionmodel to obtain LogEC50 valuesand 95% confidence intervals.
Western blot
Western blot experiments were preformed to validate theexpression of the catalytic membrane subunits (Nox1, Nox2, andNox4), as well as the organizer (p47phox and NOXO1) and activator(p67phox and NOXA1) subunits, in each reconstituted Nox system(Supplementary Fig. 1). Cell lysates were subjected to SDS–PAGE.Blots were probed with rabbit polyclonal Nox2 (1:250; Upstate CellSignaling Solutions, Temecula, CA, USA), rabbit polyclonal Nox1(1:500; Santa Cruz Biotechnology), rabbit polyclonal Nox4 (1:500;Novus Biologicals, Littleton, CO, USA), rabbit polyclonal NOXO1(1:500; Rockland Immunochemicals), rabbit polyclonal p47phox
(1:500; Santa Cruz Biotechnology), mouse polyclonal NOXA1(1: 500, Abcam, Cambridge, MA, USA), or mouse monoclonalp67phox 81.1 (kindly provided by Dr. Mark T. Quinn) antibodies andthen incubated with their respective secondary antibodies (1:10000,IRDye antibodies; Li-Cor Biotechnology, Lincoln, NE, USA). Loading ofequal amounts of proteins was confirmed by reprobing the mem-branes with a mouse monoclonal β-actin (1:500, Santa CruzBiotechnology) antibody. Blots were scanned using the Odysseyinfrared imaging system (Li-Cor Biotechnology).
Statistical analysis
All results are expressed as means±SEM. Comparisons betweenindividual concentrations of Nox2ds and ScrmbNox2dswere assessedby two-way ANOVA, followed by a Bonferroni post hoc t test. pb0.05was considered statistically significant.
Results
Nox2ds dose-dependently inhibits O2•− production from Nox2
Cytochrome c assayTo confirm that Nox2 is the major source of O2
•− in the COS-Nox2cell-free system, COS-Nox2 cell lysate was pretreated with the nitricoxide synthase inhibitor L-NAME (100 μM), the mitochondrialelectron transport inhibitor rotenone (50 μM), or the xanthineoxidase inhibitor febuxostat (100 nM). Preincubation with rotenone,L-NAME, or febuxostat did not decrease O2
•− production significantlyin the COS-Nox2 cell-free system (nmol O2
•−/min/107 cell equivalents:1.69±0.15, 1.95±0.21, 1.64±0.13, and 1.32±0.1 for vehicle-, L-NAME-, rotenone-, and febuxostat-treated COS-Nox2 lysates, respec-tively, n=3). These results suggest that the major source of O2
•− in theCOS-Nox2 cell-free system is the Nox2 NADPH oxidase.
To investigate whether B-loop peptide Nox2ds inhibits O2•−
production in the Nox2 system, SOD-inhibitable cytochrome creduction was measured in Nox2ds-pretreated COS-Nox2 cell lysates.Addition of LiDS to lysates derived fromCOS-Nox2 cells stimulatedO2
•−
production in a reaction that was dependent on the presence ofNADPH (1.33±0.1 and 0.40±0.1 nmol O2
•−/min/107 COS-Nox2 lysatecell equivalents for LiDS- and vehicle-treated COS-Nox2 lysates,respectively, pb0.05). Superoxide production in nontransfected COS-22 cell lysates (control) was 0.16±0.1 nmol O2
•−/min/107 COS-22lysate cell equivalents (Fig. 1). As demonstrated in Fig. 1, preincuba-tion of COS-Nox2 cell lysates with Nox2ds concentration-dependentlyinhibited O2
•− production, displaying an IC50 of 0.74 μM. In contrast,preincubation of COS-Nox2 cell lysates with Scrmb Nox2ds did notinhibit Nox2.
EPR spectroscopy confirms inhibition of Nox2 oxidase by Nox2dsThe inhibitory effect of Nox2ds on O2
•− production was confirmedby EPR. The reaction of the hydroxylamine spin probe CMH with O2
•−
results in formation of a nitroxide radical (CM•), yielding acharacteristic three-line spectrum (Fig. 2A). Superoxide formationwas assayed as NADPH-dependent, SOD-inhibitable formation of CM•
radical. Superoxide production in Nox2 lysates was stimulated by LiDSin the presence of NADPH and this was inhibited by SOD (150 U/ml;Fig. 2A). Preincubation of COS-Nox2 cell lysates with Nox2ds (10 μM),but not with Scrmb Nox2ds (10 μM), significantly inhibited CM•
radical formation (Figs. 2B and C). Fig. 2C shows cumulative and
Fig. 2.Nox2ds inhibits O2•− production from Nox2 as measured by EPR. (A) COS-Nox2 cell lysate was incubated with 130 μM LiDS, and O2
•− production was initiated by the addition of180 μM NADPH in the absence and presence of 150 U/ml SOD. CM• radical formation was measured for 10 min at 25 °C. *pb0.05 indicates significant difference in O2
•− productionbetween LiDS- and vehicle-treated and LiDS- and LiDS+SOD-treated COS-Nox2 lysates. (B) O2
•− production in COS-Nox2 cell lysate was assayed as LiDS-stimulated (130 μM),NADPH-dependent (180 μM), SOD-inhibitable (150 U/ml) formation of CM• radical. COS-Nox2 cell lysate was preincubated with Nox2ds (10 μM) and Scrmb Nox2ds (10 μM)peptides (before LiDS treatment) and assayed for O2
•− production (representative scans). (C) Cumulative and averaged SOD-inhibitable CM• radical formation in vehicle-, 10 μMNox2ds-, and 10 μMScrmbNox2ds-treated COS-Nox2 lysates. Data represent themeans±SEM of four or five experiments. *pb0.05 indicates significant difference in O2
•− productionbetween Nox2ds and vehicle treatment.
Fig. 3. Nox2ds does not inhibit O2•− production from Nox1. COS-Nox1 cell lysate was
preincubated with various concentrations of Nox2ds (from 0.1 to 10 μM) for 5 min at25 °C. O2
•− production was initiated by the addition of 180 μMNADPH and measured bythe initial linear rate of SOD-inhibitable cytochrome c reduction. O2
•− production isexpressed as nmol O2
•−/min/107 cell equivalents. Data represent the means±SEM ofthree experiments.
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averaged SOD-inhibitable CM• radical intensities in vehicle-, 10 μMNox2ds-, and 10 μM Scrmb Nox2ds-treated COS-Nox2 lysates.
Nox2ds does not inhibit O2•− production from Nox1
L-012 chemiluminescence of transiently transfected Nox1To evaluate the activity of the transiently transfected canonical
Nox1-oxidase system (comprising Nox1, NOXO1, and NOXA1), wholeCOS-Nox1/NOXO1/NOXA1 cell activity was assessed by L-012 chemi-luminescence. We confirmed previous results showing that the Nox1/NOXO1/NOXA1 reconstituted system produces O2
•− in the absence ofPMA [relative light units (RLUs) were 52,977±1921 and 8734±158for COS-Nox1 and COS-22 control cells, respectively, pb0.05, n=3,[17]. SOD (150 U/ml) abolished the chemiluminescent signal (177±9and 108±16 RLUs for SOD-treated COS-Nox1/NOXO1/NOXA1 andCOS-22 cells, respectively, pb0.05, n=3). PMA (1 μM) did notstimulate O2
•− in COS-Nox1/NOXO1/NOXA1 cells (56,537±2741 and5903±541 RLUs for PMA-treated COS-Nox1/NOXO1/NOXA1 andCOS-22 cells, respectively, n=3). These data suggested that thecanonical Nox1 system in COS cells is constitutively active and cannotbe further stimulated by PMA.
However, a previous study showed that O2•− in HEK 298 cells
transfected with Nox1/NOXO1/NOXA1 was significantly enhanced by
PMA stimulation [17].We transfectedHEK 298 cells withNox1/NOXO1/NOXA1 and measured O2
•− in the absence and presence of PMA (1 μM).PMA significantly stimulated O2
COS-22 cells were separately transfected with Nox1, NOXO1, p47phox, NOXA1, orp67phox and lysed and the Nox1 membrane component and the NOXO1, p47phox,NOXA1, and p67phox cytosolic extracts were separately prepared. The respectiveorganizer subunit (NOXO1 or p47phox) was preincubated with vehicle (control) or10 μM Nox2ds for 10 min, and then Nox1-containing membrane and NOXA1/p67phox
were added consecutively. After the preincubation period, LiDS (130 μM) was added toinduce the assembly of the oxidase, and O2
•− production was initiated by 180 μMNADPH. O2
•− was measured by the initial linear rate of SOD-inhibitable cytochrome creduction. O2
•− production is expressed as nmol O2•−/min/107 cell equivalents. Data
represent the means±SEM of three to six experiments.* pb0.05, significant difference in O2
•− production between Nox1 and nontransfectedCOS-22 cell lysates.
# pb0.05, significant difference between vehicle (control) and LiDS treatment.
1121G. Csányi et al. / Free Radical Biology & Medicine 51 (2011) 1116–1125
(RLUs were 12,946±199 and 8560±337 for PMA-stimulated and-unstimulated HEK-Nox1/NOXO1/NOXA1 cells, respectively, n=4,pb0.05). RLUs were 912±40.5 and 755±65.3 for PMA-stimulatedand -unstimulated nontransfected HEK 298 cells, respectively (n=4).
Cytochrome c assay of COS-Nox1 cell-free systemIn contrast to the COS-Nox2 cell-free system and in line with the
constitutive activity of COS-Nox1/NOXO1/NOXA1 cells, the cyto-chrome c assay of the COS-Nox1/NOXO1/NOXA1 cell-free systemrevealed that the canonical COS-Nox1 does not require LiDS foractivation (nmol O2
•−/min/107 cell equivalents were 1.30±0.3 and0.16±0.1 for COS-Nox1 and COS-22 cell lysates, respectively,pb0.05). Preincubation of COS-Nox1 cell lysates with variousconcentrations of Nox2ds did not inhibit O2
•− production (Fig. 3).In separate experiments, we tested whether the lack of an
inhibitory effect of Nox2ds on the Nox1 system is due to the stableinteraction of NOXO1 and NOXA1 with Nox1. The Nox1 organizersubunit NOXO1 was preincubated with vehicle (control) or 10 μMNox2ds for 10 min, and then NOX1 and NOXA1 were addedconsecutively. After the preincubation period, LiDS was added andO2
•− production was measured. Our results demonstrate thatpreincubation of NOXO1 with Nox2ds did not inhibit the canonicalNox1 (Table 1). Previous studies reported that Nox1 can be activatednot only by NOXO1 and NOXA1 but also by p47phox and p67 phox [33].We therefore tested the effect of Nox2ds on O2
•− production on allpossible permutations of the Nox1-oxidase system. As shown inTable 1, Nox2ds did not significantly inhibit O2
•− production in eitherthe canonical or the hybrid Nox1 system. This was particularly evidentin the hybrid purported to exist in large-vessel smooth muscle cells(Nox1/p47/NOXA1). Moreover, 1 μM Nox2ds demonstrated no effect
Table 2Effect of Nox2ds on inducible O2
•− production (O2•−/min/107 cell equivalents) in the
HEK-Nox1 cell-free system.
Nox system Control (no LiDS) LiDS 10 μM Nox2ds+LiDS
Nox1/NOXO1/NOXA1 0.53±0.01 0.98±0.2* 1.2±0.2
HEK 298 cells were separately transfected with Nox1, NOXO1, or NOXA1 and lysed andthe Nox1 membrane component and the NOXO1 and NOXA1 cytosolic extracts wereseparately prepared. NOXO1 was preincubated with vehicle (control) or 10 μMNox2dsfor 10 min, and then Nox1-containing membrane and NOXA1 were addedconsecutively. After the preincubation period, LiDS (130 μM) was added to inducethe assembly of the oxidase and O2
•− production was initiated by 180 μM NADPH. O2•−
was measured by the initial linear rate of SOD-inhibitable cytochrome c reduction. O2•−
production is expressed as nmol O2•−/min/107 cell equivalents. Data represent the
means±SEM of six experiments.* pb0.05, significant difference in O2
•− production between vehicle (control) andLiDS treatment.
on LiDS-stimulated O2•− production in the COS-Nox1/p47phox/p67phox
cell-free system (1.52±0.18 vs 1.59±0.10 nmol O2−/min/107 cell
equivalents for LiDS vs 1 μM Nox2ds+LiDS, respectively).
Cytochrome c assay of HEK-Nox1 cell-free systemIn contrast to the COS-Nox1/NOXO1/NOXA1 cell-free system, LiDS
treatment significantly increased O2•− production in the HEK-Nox1/
NOXO1/NOXA1 cell-free system (Table 2). The effect of Nox2ds wastested on LiDS-stimulated O2
•− in the HEK-Nox1/NOXO1/NOXA1 cell-free system. Our results demonstrate that preincubation of NOXO1with 10 μMNox2ds did not inhibit LiDS-stimulated O2
•− production inthe HEK-Nox1/NOXO1/NOXA1 cell-free system (Table 2).
Nox2ds does not inhibit H2O2 production from Nox4
Previous studies reported that Nox4 produces mainly H2O2 [20];however, it is possible that Nox4 could produce O2
•− that is rapidlyconverted to H2O2 by SOD. Before cell lysis, the ability of intact COS-Nox4 cells to produce H2O2 was confirmed by Amplex red fluorescence.COS-Nox4 cells showed approximately twofold higher H2O2-generatingactivity than nontransfected COS-22 cells [4818±19 vs 2577±6.5relative fluorescence units (RFU) for COS-Nox4 vs COS-22 cells,respectively; pb0.05]. The Nox4-dependent activity was inhibited by82% with DPI (5 μM; 2977±227 RFU for DPI-treated COS-Nox4 cells).
To test whether these cells produce O2•−, L-012 chemilumines-
cence was performed. Our data demonstrated that there was nodifference in O2
•− production between COS-Nox4 and nontransfectedCOS-22 cells (RLUs were 1766.7±41.3 and 1701.0±115.8 for COS-Nox4 and COS-22 cells, respectively). Based on the results of theseexperiments the effect of Nox2ds was tested on H2O2 production inCOS-Nox4 lysates using Amplex red. Nox4-derived H2O2 productionwas calculated by subtracting nontransfected COS-22 cell lysateactivity from COS-Nox4 cell lysate activity. Preincubation of COS-Nox4cell lysates with Nox2ds did not inhibit H2O2 production (Fig. 4).
p47phox, but not NOXO1, binds to Nox2ds
Nox2ds was designed to selectively inhibit the interactionbetween the cytosolic B-loop of Nox2 and p47phox by binding top47phox and preventing its translocation to the membrane. To confirmthis binding and to test for possible binding of Nox2ds to NOXO1,ELISA experiments were performed on lysates from p47phox- orNOXO1-transfected COS-22 cells incubated in ELISA plates withneutravidin-immobilized biotinylated Nox2ds or its scrambled con-trol. As shown in Fig. 5A, using p47phox antibody to detect binding
Fig. 4. Nox2ds does not inhibit H2O2 production from Nox4. COS-Nox4 cell lysates werepreincubated with various concentrations of Nox2ds (from 0.1 to 10 μM) for 5 min at25 °C and H2O2 production was initiated by the addition of 36 μM NADPH. Thefluorescence of Amplex red was measured for 10 min at 25 °C. Nox4 activity wastabulated by subtracting nontransfected COS-22 cell lysate activity from COS-Nox4 celllysate activity. H2O2 production is expressed as nmol H2O2/min/107 cell equivalents.Data represent the means±SEM of 6–10 experiments.
1122 G. Csányi et al. / Free Radical Biology & Medicine 51 (2011) 1116–1125
(followed by fluorescently-tagged secondary antibody), fluorescenceintensity in biotinylated Nox2ds-bound wells was significantly higherwhen cytosolic fractions of COS-22-p47phox vs COS-22 were added.These data indicate that p47phox binds to Nox2ds. Binding of p47phox
was sequence specific as p47phox binding to the scrambled sequenceproduced significantly less fluorescence (Fig. 5A). In contrast, therewas no difference in fluorescence intensity when cytosolic fractions ofCOS-22-NOXO1 vs COS-22 were added to plates bound with Nox2ds,followed by incubation with NOXO1 antibody (Fig. 5B).
Fig. 6. Nox2ds does not compete with the Nox4 DH domain for B-loop binding. Bindingbetween GST-fused recombinant Nox4 DH domain and Nox2ds or Scrmb Nox2ds wasmeasured by the ability of each peptide to compete with FITC-conjugated Nox4 B-looppeptide for Nox4 DH protein. Fluorescence polarization detected binding betweenfluorescently labeled Nox4 B-loop peptide and GST-Nox4 DH protein. Data were fit toone-site competition curves using GraphPad Prism to obtain LogEC50 values. Reportedare the mean LogEC50 of three experiments with SEM; unlabeled WT Nox4 B-loop,LogEC50 3.0±0.08 nM; Nox2ds, LogEC50 5.5±0.5 nM; Scrmb, LogEC50 5.4±0.3 nM.Difference between LogEC50 of unlabeled WT Nox4 B-loop and that of Nox2ds, pb0.03;WT Nox4 B-loop and Scrmb, pb0.01; no statistically significant difference betweenNox2ds and Scrmb.
Nox2ds does not interact with the Nox4 DH domain
Binding between Nox4 B-loop peptide and the DH domain of Nox4was previously shown and implicated in Nox4 activity [26]. Usingfluorescence polarization, we tested whether Nox2ds or ScrmbNox2ds competes with the Nox4 B-loop for binding to the Nox4 DHdomain. As shown in Fig. 6, the unlabeled Nox4 B-loop effectivelycompeted off the FITC-Nox4 B-loop in the low-micromolar range;however, both Nox2ds (LogEC50 5.5±0.5 nM) and Scrmb Nox2ds(LogEC50 5.4±0.3 nM) were not effective at concentrations below0.1 mM.
Fig. 5. p47phox, but not NOXO1, binds to Nox2ds. ELISA experiments were performed totest whether (A) p47phox and (B) NOXO1 bind to Nox2ds and/or Scrmb Nox2ds.Biotinylated Nox2ds and scrambled peptides were first bound to neutravidin-coatedplates and then incubated with COS-22, COS-22-p47phox (COS-22 cells transfected withp47phox), or COS-22-NOXO1 (COS-22 cells transfected with NOXO1) cytosolic extractsfor 1 h at room temperature. Rabbit polyclonal p47phox or rabbit NOXO1 antibodieswere used to detect Nox2ds-bound p47phox or NOXO1, respectively. After 1 h incubationand extensive washing, bound primary antibodies were detected by FITC-labeled goatanti-rabbit IgG antibody. The fluorescence of each well was measured using a BiotekSynergy 4 hybrid multimode microplate reader (excitation 488 nM, emission 518 nM).Data represent the means±SEM of three or four experiments. *pb0.05 between COS-22 and COS-22-p47phox cytosolic extracts in Nox2ds wells. #pb0.05 between Nox2dsand Scrmb Nox2ds wells incubated with COS-22-p47phox extracts.
Nox2ds does not inhibit xanthine oxidase or directly scavenge O2•−
To test for the possible nonspecific inhibitory effect of Nox2ds onanother major source of mammalian O2
•−, xanthine oxidase, as well asthe possibility that Nox2ds could be acting as an O2
•− levels produced by the classicalxanthine oxidase (XO)/xanthine (X) O2
•−-generating system. Pre-incubation of Nox2ds or Scrmb Nox2ds (10 μM for 10 min) did notalter XO-generated O2
•− as assessed by EPR (100±2.4, 92.5±1.8, and92.4±1.8% of radical intensities for XO/X control, 10 μM Nox2ds+X/XO, and 10 μM Scrmb Nox2ds+X/XO, respectively; Fig. 7). XO aloneproduced only a small signal (2.9±0.4%) and XO+X in the presenceof 100 U/ml SOD yielded 15.7±0.3% radical intensity (Fig. 7).
Discussion
Previously, our laboratory and many others demonstrated theeffectiveness of the chimeric peptide Nox2ds-tat (Nox2ds linked to asmall portion of HIV coat named tat to allow cell permeation) inreducing or abolishing ROS levels in normal or diseased tissue known
Fig. 7. Nox2ds does not inhibit xanthine oxidase (XO) or directly scavenge XO-derivedO2•−. The O2
•−-scavenging activity of Nox2ds was determined by EPR using the xanthine/xanthine oxidase O2
•−-generating system. Assay mixtures contained PBS, 5 mU/ml XO,50 μMCMH, and 25 μMdeferoxamine in a final volume of 100 μl. The reference samplescontained 100 U/ml SOD. After addition of 25 μM xanthine (X), CM• radical formationwas monitored for 10 min in the absence and presence of 10 μM Nox2ds or ScrmbNox2ds. Data represent the means±SEM of six experiments. *pb0.05 indicatessignificant difference in CM• radical intensities between XO+X and XO and betweenXO+X and 100 U/ml SOD+XO+X.
1123G. Csányi et al. / Free Radical Biology & Medicine 51 (2011) 1116–1125
to contain Nox [11,12,25] and in attenuating the progression ofcardiovascular disease processes involving increased Nox activity[12,22–24]. However, until now the specificity of inhibition of Nox2ds(nonchimeric B-loop peptide) among vascular Nox isoforms has notbeen investigated. To test the specificity of Nox2ds, we examined itspotential inhibitory activity in cell-free assays using reconstitutedsystems of the major murine vascular Nox isoforms, Nox1, Nox2, andNox4. Our data show that Nox2ds concentration-dependentlyinhibited O2
•− production in a COS-Nox2 cell-free system and thatNox2ds is a potent and efficacious inhibitor of NADPH oxidase Nox2,with an IC50 of 0.74 μM. Furthermore, our results demonstrate thatNox2ds does not inhibit ROS production in either the COS-Nox1 or theCOS-Nox4 oxidase system. The results of this study demonstrateselectivity of Nox2ds peptide in differentiating the contribution ofNox2 vs Nox1 and Nox4 to Nox-derived ROS production. Thesefindings have broad implications for distinguishing the role of Nox2 ina wide range of disease processes and support its potential use as aNox2-targeted therapeutic agent.
There is a large body of evidence that Nox2 is amajor source of ROSin vascular and cardiac tissues [7,8] and that Nox2-derived ROS areinvolved in many cardiovascular disease processes, including athero-sclerosis [34], hypertension [35], vascular injury [36], ischemic stroke[37], and diabetic vasculopathy [38]. Moreover, Nox2 has beenimplicated in a broad spectrum of other diseases [22,24,39,40].Thus, there is great interest in designing selective Nox2 inhibitors toassess the individual contribution of this enzyme to overall ROSproduction in cardiovascular pathologies as well as to directlymodulate ROS levels and reduce oxidative stress in patients withcardiovascular and other disorders. To date, a variety of antioxidantsand small-molecule Nox inhibitors have been developed, yet none ofthem to our knowledge selectively inhibit Nox2 [21]. Previously, thepeptide-based inhibitor PR-39, which binds to the SH3 domains ofp47phox, was shown to prevent association of p47phox with Nox2[41,42]. Nonetheless, as PR-39 binds to SH3 domains in general, it isnot an isoform-specific inhibitor and is likely to have multiple effectsin other enzyme systems that possess this domain [41]. Importantly,rationally-targeted, sequence-specific peptide-based inhibitors arelikely to yield the greatest specificity among peptidic inhibitorsbecause of their potential for being Nox-isoform specific. It thusbecomes evident that, in designing isoform-specific peptidic Noxinhibitors, it is important to take advantage of differences that existbetween the sequences of these isoforms. Indeed, Nox1 shares ~60%amino acid identity with Nox2, whereas Nox4 shares only ~39%identity with Nox2 [43], allowing for selective targeting of theseisoforms with peptide sequences unique between them. Furthermore,because of their ability to precisely model intrinsic peptide sequencesin the holoenzyme and thereby compete with the proteins’ in-teractions and activities directly, unique peptide sequences and theirpeptidomimetics have the potential to be among the most effectiveinhibitors of Nox. This, of course, does not take into account thelimitations on the use of peptidic inhibitors in chronic diseaseprevention, which include the limited bioavailability of orallyadministered peptides. However, currently under intense investiga-tion are alternative means for delivery of peptides, includingnanoparticle technologies, which have the potential of supersedingthese pharmacokinetic barriers.
Our laboratory developed a sequence-specific peptidic inhibitor,Nox2ds, which was designed to selectively target the interactionbetween the cytosolic B-loop of Nox2 and p47phox, preventing theassembly of Nox2-based enzyme complex [12]. Indeed, the results ofthe present study demonstrate the selectivity of Nox2ds in thatNox2ds binds to p47phox but not to NOXO1, thus providing anexplanation for why Nox2ds inhibits O2
•− production in COS-Nox2, butnot in the COS-Nox1/NOXO1/NOXA1 system. Importantly, the bindingof p47phox to Nox2ds was sequence specific as p47phox did not bind toScrmb Nox2ds and, interestingly, Nox2ds did not inhibit either COS-
Nox1/p47phox/NOXA1 or COS-Nox1/p47phox/p67phox oxidase, suggest-ing that the interaction between the cytosolic B-loop of Nox1 andp47phox is not important for Nox1 activity. Perhaps more importantly,the data presented in this study demonstrate that Nox2ds does notinhibit inducible Nox1 activity. Moreover, because a previous studyshowed binding between Nox4 B-loop peptide and the DH domain ofNox4 (implicated in Nox4 activity) [26], the possibility that Nox2ds orScrmb Nox2ds could compete off the Nox4 B-loop from the Nox4 DHdomain was tested using fluorescence polarization. The data demon-strate that the Nox4 B-loop competed off labeled Nox4 B-loop bindingto the Nox4 DH domain quite effectively, in the low-micromolarrange. In contrast, Nox2ds and ScrmbNox2ds demonstrated equal andpoor competition with the Nox4 B-loop for binding to the Nox4 DHdomain (millimolar concentrations). The very high pharmacologicalconcentrations as well as the fact that Nox2ds and Scrmb Nox2ds hadequal effects suggest that Nox2ds does not interfere with the Nox4 DHdomain and thus Nox4 activation.
The active sequence of Nox2ds was identified previously byrandom-sequence peptide phage display library analysis and hadbeen shown to inhibit NADPH oxidase activity in a neutrophil cell-freesystem, which expresses Nox2 [11]. Moreover, Touyz et al. demon-strated that Nox2ds-tat inhibited AngII-stimulated oxidase activity inhuman microvascular endothelial cells, which are known to expressNox2 [18]. On the other hand, Griendling et al. tested the ability ofNox2ds-tat to inhibit NADPH oxidase activity in nondiseased rat aorticsmooth muscle cells, which do not contain Nox2, and found noinhibitory effect (unpublished data). Consistent with these findings,multiple studies successfully applied Nox2ds-tat to attenuate ROSlevels in normal and diseased tissues, implicating Nox2 in a variety ofmodels [12,22–25]. Data from our laboratory demonstrated cellpermeation and in vivo effectiveness of Nox2ds-tat in an AngII-induced hypertension model in which co-infusion of the peptidesignificantly decreased vascular ROS production, reduced intercellularadhesion molecule-1 expression, inhibited leukocyte infiltration, andattenuated medial hypertrophy [12,44]. Moreover, Jacobson et al.reported that Nox2ds-tat prevented balloon angioplasty-induced O2
•−
production and neointimal hyperplasia of the rat carotid artery [27].Furthermore, application of Nox2ds to adventitial fibroblasts, whichshow abundant expression of the Nox2 system [45], by adenoviraltechniques attenuated AngII-induced carotid artery medial hypertro-phy and lipid peroxidation by-product 4-hydroxynonenal depositionin vivo [46]. Until the current study, however, specificity of Nox2ds forNox2 oxidase was not known.
Identification of isoform-specific Nox inhibitors has been a majorchallenge in the Nox field. Indeed, in many studies, whether Noxenzymes are the source of the ROS could not be definitivelydetermined because of the lack of specific inhibitors. For a longtime, DPI has been used to determine Nox involvement even though itis well established that DPI inhibits a multitude of flavin-containingenzymes, including nitric oxide synthase, xanthine oxidase, andNADH dehydrogenase (mitochondrial complex I) [41,47]. This isfurther complicated by the fact that putative Nox inhibitors couldattenuate Nox-derived ROS by a number of ways other than specificinhibition of Nox activity. These include (a) acting on upstreamsignaling molecules, (b) interfering with activation pathways thataffect the assembly of the enzyme, (c) decreasing cytosolic levels ofNADPH, (d) upregulating or activating endogenous antioxidantenzymes causing a rise in cellular ROS scavenging activity, and (e)affecting expression of mRNA and/or protein level of different NADPHoxidase components. Although our studies have not addressed all ofthese potentially indirect influences of Nox2ds, the use of reconsti-tuted cell-free Nox systems is a major step in testing for its specificity.Indeed, in this study, Nox2ds was shown to selectively and potentlyinhibit the Nox2 system, yielding an IC50 of 0.74 μM. This is in linewith a previous study in phagocytes, in which the same peptidesequence inhibited O2
•− level with an IC50 of 1 μM [48]. The inhibitory
1124 G. Csányi et al. / Free Radical Biology & Medicine 51 (2011) 1116–1125
effect of Nox2ds was sequence specific as evident in the lack ofinhibition by the scrambled sequence. Moreover, Nox2ds was highlyefficacious in its inhibition of Nox2 oxidase, causing approximately85% inhibition of activity at 10 μM. Most importantly, Nox2dsdisplayed no ability to inhibit either Nox1 or Nox4 oxidase. Thisclear selectivity of Nox2ds for inhibition of Nox2 and not Nox1 orNox4 suggests that Nox2ds is indeed sequence specific and thatNox2ds or its chimeras would not inhibit other Nox's or other moredistinct enzyme systems that are expected to be more diverse in theirsequences. Nevertheless, studies are under way to further interrogatethe ability of Nox2ds to inhibit other Nox isoforms as well as topossess off-target effects.
Because the assays currently used to detect ROS have exhibited thepotential for artifacts, we used EPR spectroscopy to confirm ourresults. In support of our initial findings with the cytochrome c assay,EPR experiments demonstrated that preincubation of COS-Nox2 celllysates with Nox2ds significantly inhibited O2
•− production. Thedegree of inhibition was lower with EPR than the cytochrome cassay (approximately 50% vs 85% with EPR and cytochrome c,respectively). We do not have a definitive explanation for thisdiscrepancy but we expect this to be related to the differences inthe sensitivity as well as differences in background signal between thetwo assays. Importantly, the ineffectiveness of the scrambled controlsequence of Nox2ds at inhibiting O2
•− detection by EPR argues in favorof an isoform-specific effect of Nox2ds.
We tested the effect of Nox2ds on ROS production in Nox1 andNox4 reconstituted systems and showed that preincubation of COS-Nox1 and COS-Nox4 lysates with Nox2ds failed to inhibit ROSproduction. In addition, we tested whether Nox2ds could directlyalter O2
•− levels generated by the classical xanthine oxidase/xanthinesystem. Our results demonstrated that preincubation of Nox2ds withxanthine oxidase and xanthine did not alter xanthine oxidase-mediated O2
•− levels, suggesting that Nox2ds is unable to directlyinhibit this enzyme nor directly scavenge O2
•− derived from theenzyme.
To our knowledge, no single available Nox2-specific inhibitor isready for use in clinical trials. Ideal Nox inhibitors show specificity,absence of toxicity, lack of off-target effects, desirable pharmacoki-netic profiles, and in vivo efficacy in well-established animal modelsof disease. Relevant to this point, there are no reports of toxicity or anynonspecific actions of Nox2ds or its chimera Nox2ds-tat, whichsupports its specificity for inhibiting the unique protein–proteininteraction it was designed to target in Nox2. In addition, there havebeen no reports to date of immunosuppressive or immunogenicresponses to either Nox2ds-tat or scrambled control sequences afteracute administration or chronic treatment of the drug [41]. This mayseem surprising because inhibition of Nox2 oxidase in phagocytescould be expected to lead to reduced microbe killing and infection.However, there are two potential reasons for this. First, our originalreport on the peptide chimera displayed a small effect on macrophageROS production [12]. As proposed in that report, it is plausible thatproteases on the surface of leukocytes degrade the peptide, therebyreducing its effect on these cells. Second, Kume and Dinauer havereported that even very small amounts of O2
•− production inphagocytes from patients with chronic granulomatous disease aresufficient for microbicidal activity [49]. Thus the levels of inhibitionrendered by Nox2ds in phagocytes may avert dysfunction in thesecells. Nevertheless, further investigations are clearly required beforetoxicity, off-target effects, and pharmacokinetic profiles of Nox2ds areestablished.
Notwithstanding the broadly demonstrated effectiveness ofNox2ds-tat by parenteral, peritoneal, subcutaneous, and directapplication to blood vessels using gene therapy [46,50], thelimitations in the use of peptides as “druggable” therapeutics areobvious. These include a very limited oral bioavailability due topeptide degradation in the gut. As mentioned earlier, however, these
issues are being circumvented by novel technologies, including theuse of nanotechnologies and the aerosolization of Nox2ds forapplication directly into lungs. Indeed preliminary studies by ourgroup show that nebulization of Nox2ds-tat into mouse lungssubstantially reduces right ventricular hypertrophy in pulmonaryhypertension (unpublished findings).
Overall, our findings demonstrate for the first time that Nox2ds is aselective inhibitor of Nox2 and support its utility in differentiating thecontribution of Nox2 vs Nox1 (canonical or hybrid) and Nox4 NADPHoxidases to Nox-derived ROS production. These findings have broadimplications ranging from the use of this inhibitor as a means todelineate the role of Nox2 in cardiovascular and other pathologies tosupport for its potential use as a therapeutic agent.
Supplementarymaterials related to this article can be found onlineat doi:10.1016/j.freeradbiomed.2011.04.025.
Acknowledgments
This work was supported by National Institutes of Health GrantsHL079207 and HL55425. We thank Sheila Frizzell for her criticalreview of the manuscript. P.J.P. is an Established Investigator of theAmerican Heart Association. P.J.P. receives research support from theVascular Medicine Institute, the Institute for Transfusion Medicine,and the Hemophilia Center of Western Pennsylvania. G.C. is arecipient of an American Heart Association Postdoctoral Fellowship.The authors wish to sincerely thank Dr. Edgar Pick for his criticalreview of our data as well as methodological advice.
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