Free Radical Biology & Medicine xxx (2008) xxx–xxx

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Original Contribution

Persistent susceptibility of cathepsin B to irreversible inhibition by nitroxyl (HNO) inthe presence of endogenous nitric oxide

Antti J. Väänänen a,b, Pertteli Salmenperä a, Mika Hukkanen c, Katrina M. Miranda d, Ari Harjula e,Pekka Rauhala a, Esko Kankuri a,e,⁎a Department of Pharmacology, Institute of Biomedicine, Biomedicum Helsinki, P.O. Box 63, FIN-00014 University of Helsinki, Finlandb The Brain laboratory, Department of Biological and Environmental Sciences, P.O. Box 65, FIN-00014 University of Helsinki, Finlandc Department of Anatomy, Institute of Biomedicine, Biomedicum Helsinki, P.O. Box 63, FIN-00014 University of Helsinki, Finlandd Department of Chemistry, University of Arizona, P.O. Box 210041, Tucson, AZ 85721-0041, USAe Cell Therapy Research Consortium, Helsinki University Central Hospital, 3rd Department of Surgery, P.O. Box 340, FIN-00029 HUS, Finland

Abbreviations: DEA/NO, diethylamine nonoate; DTTIFN-γ, gamma-interferon; L-NAME, L-NG-nitroarginine mcharide; NO, nitric oxide; NOS, nitric oxide synthase, SDSpolyacrylamide gel electrophoresis.⁎ Corresponding author. Fax: +358 9 191 25364.

E-mail address: ESKO.KANKURI@HELSINKI.FI (E. Kan

0891-5849/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.freeradbiomed.2008.05.025

Please cite this article as: Väänänen, A. J.;presence of endogenous nitric oxide, Free R

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Article history:

Nitrosation of enzyme regu Received 19 November 2007Revised 2 April 2008Accepted 21 May 2008Available online xxxx

latory cysteines is one of the key posttranslational modification mechanisms ofenzyme function. Frequently such modifications are readily reversible; however, cysteine proteases, such ascathepsin B, have been shown to be covalently and permanently inactivated by nitroxyl (HNO), the one-electron reduction product of NO. Owing to the high reactivity of HNO with NO, endogenous NO productioncould provide direct protection for the less reactive protein cysteines by scavenging HNO. Additionally,endogenous cellular production of NO could rescue enzyme function by protective nitrosation of cysteinesprior to exposure to HNO. Thus, we studied the effect of endogenous NO production, induced by LPS or IFN-γ,on inhibition of cysteine protease cathepsin B in RAW macrophages. Both LPS and IFN-γ induce iNOS withgeneration of nitrate up to 9 μM in the media after a 24-h stimulation, while native RAW 264.7 macrophagesneither express iNOS nor generate nitrate. After the 24-h stimulation, the HNO-releasing Angeli's salt (0–316 μM) caused dose-dependent and DTT-irreversible loss of cathepsin B activity, and induction of iNOSactivity did not protect the enzyme. The lack of protection was also verified in an in vitro setup, wherepapain, a close structural analogue of cathepsin B, was inhibited by Angeli's salt (2.7 μM) in the presence ofthe NO donor DEA/NO (0–316 μM). This clearly showed that a high molar excess of DEA/NO (EC50 406 μM) isneeded to protect papain from the DTT-irreversible covalent modification by HNO. Our results provide firstevidence on a cellular level for the remarkably high sensitivity of active-site cysteines in cysteine proteasesfor modification by HNO.

© 2008 Elsevier Inc. All rights reserved.

Introduction

Inflammatory insults stimulate immunocompetent cells, such asmacrophages, to generate nitric oxide (NO) in high amounts via theCa-independent inducible form of NO synthase (iNOS) [1]. The manyfates of NO within an inflammatory focus have been intenselyinvestigated, and this highly reactive small molecule has beenattributed with immunomodulatory as well as cytotoxic properties[2,3]. Until recent years the biochemical interest on nitrogen oxidesmainly focused on NO and its autoxidation products or oxidants, suchas peroxynitrite, generated on reaction of NO with O2

- [4,5]. Increased

, dithiotreitol; HNO, nitroxyl;ethyl ester; LPS, lipopolysac-

-PAGE, sodium dodecyl sulfate-

kuri).

l rights reserved.

et al., Persistent susceptibilitadic. Biol. Med. (2008), doi:1

knowledge on NO biochemistry as well as enhanced methodology,however, has enabled us to unravel the biological properties of theone-electron reduction product of NO, nitroxyl, which under physio-logical conditions is present as protonated species HNO [6]. Thefundamental chemistry and biochemical properties of HNO have beensubject to extensive reviews recently [7,8].

At the site of inflammation with induced focal production of NO,not only the environment but also the NO-producing cells themselvesbecome exposed to high concentrations of nitrogen oxides. Intracel-lularly, NO and its autoxidation products readily react with thiols andthus regulate, for example, cysteine-dependent enzymes throughnitrosation of their key active-site cysteine residues [3,9]. In additionto regulatory functions, such nitrosation of cysteines may also protectcysteines from oxidation by such species as peroxynitrite or HNO [10].We recently discovered that the lysosomal cysteine protease cathe-psin B is a highly susceptible intracellular target for HNO [11]. Inmonocyte/macrophages, we found HNO to irreversibly inhibitcathepsin B activity independently of several-fold induction of the

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intracellular protective thiol, glutathione (GSH) [11]. Interestingly,modification of cathepsin B active-site cysteine by HNO is irreversible,and the site cannot be regenerated by addition of excess DTT in vitronor by intracellular thiols, such as GSH, in vivo [7,10,12,13].Furthermore, such HNO modification of cysteines has been proposedto be unique among the nitrogen oxides, and may present afingerprint of HNO-induced cysteine modification [14].

Our previous work in vitro suggested that prior modification bynitrosation or mixed disulfide generation of the active-site cysteine incysteine proteases could protect enzyme activity against HNO-induced abrogation [10]. In order for cysteine protease protection tooccur by nitrosation in the intracellular milieu, a prior reaction of theactive-site cysteine with the autoxidation products of NO would berequired. Furthermore, the reported reactivity of HNO with NO(5.8×106 M−1 s−1) [6] is an order of a magnitude higher than that ofHNO with L-cysteine (5×105 M−1 s−1) [15]. Thus, in addition to such“prenitrosation” of cysteines, NO could scavenge HNO from cellular orin vitro systems (reactions (1)–(3) [6]), diminishing the effects of HNOon the less reactive protein cysteines.

HNOþ NO⇄HN2O2 ð1ÞHN2O2 þ NO⇄HN3O3 ð2Þ

HN3O3→N2Oþ HNO2 ð3Þ

Despite the numerous reported pathways potentially leading togeneration of HNO in vivo, to date no confirming evidence for itsgeneration has been shown. These difficulties associated with the, yethypothetical, generation of HNO in vivo can be circumvented by usingAngeli's salt [16,17]. It releases HNO in a pH and temperature-dependent fashion with a half-life of 2.5 min at physiologicalconditions [18,19,20].

As a direct continuation of our previous work [10,11], we nowinvestigated the ability of endogenous NO, as derived from iNOS inRAW macrophages, to protect intracellular cathepsin B againstirreversible inhibition by HNO. Moreover, the interference ofconcomitant NO generation was also investigated in a controlledaerobic in vitro system using papain, a close structural analoque ofcathepsin B [21].

Materials and methods

Chemicals and reagents

Angeli's salt and DEA/NO were used as HNO and NO donors,respectively, and purchaced from Cayman Chemicals (Ann Arbor, MI).The donors were stored in -80°C. For the experiments the donors weredissolved in ice-cold 10 mM NaOH to produce a 100-fold stocksolution which was added to either cell-culture media or papainsolution to initiate the release of nitrogen oxides. Unless otherwisespecified, all other reagents were obtained from Sigma (St. Louis, MO).

Cell-line and culture conditions

Murine RAW 264.7 cells (ATCC number TIB-71) were maintainedunder standard cell culture conditions (+37°C, 5% CO2) in D-MEMmedium, supplemented with 10% fetal calf serum (Gibco BRL, GrandIsland, NY) and antibiotics (penicillin G 100 U/ml, amphotericin B250 ng/ml, and streptomycin 100 μg/ml, Gibco BRL).

The cells were stimulated in the growth medium for 0–24 h witheither Escherichia coli lipopolysaccharide (LPS; O111:B55, Sigma;500 ng/ml), mouse recombinant IFN-γ (0-100 IU/ml, PeproTech Inc.,Rocky Hill, NJ), or TNF-α (0–10 ng/ml, PeproTech Inc.). Whereindicated, L-NG-nitroarginine methyl ester (L-NAME) was used at aconcentration of 500 μM to inhibit NOS activity. Thereafter the mediawere frozen at−80°C for the measurement of nitrite and nitrate whilethe cells were washed twice in serum and antibiotic-free basal D-

Please cite this article as: Väänänen, A. J.; et al., Persistent susceptibilitpresence of endogenous nitric oxide, Free Radic. Biol. Med. (2008), doi:1

MEM and used for the nitrogen oxide donor experiments orhomogenized for the Western blot analysis.

For the nitrogen oxide donor tests, 5 μl of Angeli's salt stocksolution or vehicle only was added into 500 μl of D-MEM covering thecells and the cultures were incubated for 1 h at +37°C prior tomeasurement of cathepsin B activity.

Cathepsin B activity assay

The cathepsin B activity assay was based on fluorescent assaydescribed for cathepsin L by Barret and Kirschke [22]. The modifiedassay is based on DTT reactivation of cathepsin B in the cell lysate andsubsequent detection of AMC release from the cathepsin B substrateZ-Arg-Arg-7-(4-methyl)coumarylamide (Calbiochem) using the in-house protocol which has been previously described in detail [11]. Thereleased AMC was quantitated by spectrofluorometer (Wallac Victor2,1420 Multilabel counter) using excitation and emission wavelengthsof 355 and 460 nm, respectively. The reagent blankwas deducted fromall readings, which were subsequently referenced to the proteincontent and expressed as percentage of indicated control.

Nitrite and nitrate assay

The combined nitrite and nitrate content of the cell culture mediawas measured using a commercial assay kit (Nitrate/Nitrite Fluoro-metric Assay Kit, Cayman Chemicals). The nitrite detection is based onfluorophore formation on reaction of nitrite with 2,3-diamino-naphthalene, which was quantitated by spectrofluorometery (WallacVictor2, 1420 Multilabel counter) using excitation and emissionwavelengths of 355 and 430 nm, respectively, as previously described[11].

Western blot analysis

The cells were scraped and sonicated into 70 μl of homogenizationbuffer (50mM sodium acetate, 2.5mMDTT, 2.5mMEDTA,1% Triton X-100; pH 5.5) [11]. Following centrifugation (10,000 g for 10 min), a 30-μl aliquot was boiled for 5 min with an equal volume of standardLaemmli buffer (4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.004%bromophenol blue, 125 mM Tris-HCl) and stored at −20°C for amaximum of 2 weeks.

The samples were resolved by 4–20% gradient SDS-PAGE andtransferred to nitrocellulose membranes, which were blocked withnonfat powdered milk (2.5% in Tris-buffered saline). Immunoreactiveproteins were visualized with antibodies to cathepsin B (R and Dsystems, Minneapolis, MN), iNOS (Santa Cruz Biotechnology, SantaCruz, CA), and beta-actin (Labvision Inc. Fremont, CA), followed bysecondary horeradish peroxidase-coupled anti-rabbit IgG (Santa CruzBiotechnology), or anti-mouse IgG+IgM (Jackson Immunoresearch).The ECL reaction (Pierce, Rockford, IL) was detected and quantifiedusing ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA).

Immunocytochemistry and laser scanning confocal microscopy

Indirect immunofluorescence was used for the localization andassessment of relative expression level of iNOS and cathepsin B in LPS-(500 ng/ml) or IFN-γ-(100 U/ml) stimulated RAW 264.7 macrophages.The cells were fixed in−20°C methanol and incubated with 1:100normal donkey serum prior to application of mouse anti-mouse iNOSIgG (BD Transduction Laboratories, Cat. No. N39120) or goat anti-mouse cathepsin B IgG (R and D Systems, Cat. No. AF965) for 1 h. AlexaFluor donkey anti-mouse 488 and donkey anti-goat 568 conjugateswere applied at a concentration of 0.4 μg/ml for 1 h (Invitrogen/Molecular Probes). Cell nuclei were visualized with 1 μM monomericcyanine nucleic acid stain TO-PRO-3 (Invitrogen/Molecular Probes).Confocal microscopy was carried out using a Leica TCS SP2 AOBS

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system (Leica Microsystems AG, Mannheim, Germany) with HCX PLAPO CS 40/ 1.25 NA objective, and 488, 568, and 633 nm laserexcitation for Alexa Fluor 488 and 568 conjugates, and TO-PRO-3,respectively. Stacks including nine images each were acquired with1024×1024 lateral resolution and 160 nm z-sampling frequency. Theimages were deconvolved with 10 iterations using theoretical point-spread function and iterative maximum-likelihood estimation algo-rithms of Huygens Essential (Scientific Volume Imaging BV, Hilver-sum, the Netherlands).

Protein content

Protein concentration was assayed by the method of Bradford [23]using commercially available reagent and protocol (Sigma) from the10,000 g supernatants after dilution of the samples by 1:50 withdistilled water, and referenced to a BSA standard curve (0–50 μg/ml).

Cell viability

The release of LDH from cells was measured by a commerciallyavailable assay kit (Cytotoxicity Detection Kit, Roche, Mannheim,Germany) according to the manufacturer's instructions. For themeasurement, a small aliquot of the supernatant from the Angeli'ssalt-(316 μM) or vehicle-(0.1 mM NaOH, final) treated cells was takenafter the 1-h incubation and diluted to 1:100 with Milli-Q-filteredwater. The LDH activity of this sample was then referenced to a fullylysed control sample (homogenized by sonication in the presence of1% Triton-X 100), which was taken to represent themaximal release ofLDH from the studied cell load.

Papain activity assay

The commercial papain batch (Sigma) was reduced as previouslydescribed by 5 mM DTT for 1 h at room temperature and dialyzedagainst PBS containing 50 μMDETAPACwith 6 buffer changes [10]. Thepapain sample was then diluted to 0.1 mg/ml for the experimentsbased on A280 (0.1%) ε=2.47 cm−1 [24].

The papain samples (500 μl) were incubated with DEA/NO (0–316 μM) or GSH (0–316 μM) in the presence or absence of Angeli's salt(2.7 μM). After a 1-h incubation at +37°C a 100-μl aliquot was taken foractivity measurement, carried out as previously described using L-BAPNA as the substrate [10]. The rest of the papain sample wasincubated for a further 1 h with 10 mM DTT (final) prior to activitymeasures.

The papain activity was referenced to vehicle-treated controlsample, which was also incubated with DTT with the samples.

Statistical analysis

All experiments were carried out in triplicate, and statisticalanalysis and IC50 calculations were performed using Graph Pad Prism4.0 (GraphPad Software Inc., San Diego, CA).

Results

Effect of LPS or IFN-γ stimulation on iNOS and cathepsin B expressionand activity

Stimulation of RAW 264.7 macrophages with LPS resulted in atime-dependent increase in iNOS expression (Fig. 1A). Similarupregulation of iNOS was seen after a 24-h stimulation with IFN-γ(100 IU/ml), but not after stimulation with 10 ng/ml TNF-α (Fig. 1B).Thus for the subsequent studies, a 24-h stimulation with LPS or IFN-γwas chosen.

The endogenous NO production was manipulated by using L-NAME at 500 μMduring the stimulation, which subsequently caused a

Please cite this article as: Väänänen, A. J.; et al., Persistent susceptibilitpresence of endogenous nitric oxide, Free Radic. Biol. Med. (2008), doi:1

marked upregulation of iNOS expression during the IFN-γ (100 IU/ml)stimulation (Fig.1B), suggesting activity of a negative feedback loop byNO to iNOS transcription as has been previously demonstrated [25,26].

Native RAW 264.7 cells did not express detectable iNOS (Figs. 1A,1B, and 1F), or produce detectable quantities of NO autoxidationproducts in themedium (Figs.1C and 1D). Stimulationwith LPS or IFN-γ caused marked accumulation of nitrite/nitrate in the medium (Figs.1C and 1D) consistent with the upregulation iNOS expression (Figs.1A,1B, and 1F). After a 24-h induction period, LPS-and IFN-γ-(100 IU/ml)stimulated macrophages produced 8.0 and 9.1 μM nitrite/nitrateconcentration into medium, respectively. Accumulation of NO oxida-tion products was reduced by 66±0.8 and 81±3.8% subsequent toaddition L-NAME (500 μM) to the growth medium during the LPS orIFN-γ (100 IU/ml) stimulation, respectively.

LPS or IFN-γ stimulations did not cause marked differences in theexpression level of cathepsin B in RAW 264.7 macrophages (Figs. 1Aand 1B). However, the cellular location and basal activity werechanged subsequent to LPS stimulation, with cathepsin B havingrather a cytosolic than lysosomal location (Fig. 1F) and 55±4.4% loweractivity compared to nonstimulated control cells. Addition of L-NAME(500 μM) for 24 h during the LPS stimulation abolished the inhibitoryeffect of LPS stimulation on the cathepsin B activity (Fig.1E). Leupeptin(100 μM), as the positive control, completely inhibited cathepsin Bactivity.

Inhibition of cathepsin B in RAW 264.7 macrophages by acute HNO insultusing Angeli's salt

Angeli's salt dose dependently inhibited cathepsin B activity innonstimulated as well as LPS-stimulated and IFN-γ-stimulated cells.After a 1-h exposure to Angeli's salt (316 μM), cathepsin B activity was53±2.6, 25±2.2, and 57±2.6% in nonstimulated, LPS-, and IFN-γ-(100 IU/ml) treated cells, respectively (Figs. 2A and 2B). When thesestimulations were carried out in the presence of L-NAME (500 μM), therespective activities in Angeli's salt-(316 μM) treated cells werereduced to 36±4.9, 42±3.0, and 68±6.0% in nonstimulated, LPS-,and IFN-γ-(100 IU/ml) stimulated cells, respectively (Figs. 2A and 2B).Thus L-NAME augmented Angeli's salt-induced cathepsin B inhibitionin native RAW 264.7 macrophages, but protected cathepsin B activityin stimulated RAW 264.7 cells.

Angeli's salt at 316 μM did not induce acute necrotic cell death incontrol or stimulated RAW cells, LPS, or IFN-γ-stimulated cells asmeasured by LDH release from the cells. The LDH release was 2.4±0.2,3.3±0.4, and 3.5±0.5% of total LDH load in control, LPS, and IFN-γ-stimulated cells, respectively, after 1-h Angeli's salt (316 μM)exposure. LDH release from respective NaOH-(0.1 mM final) treatedvehicle controls for Angeli's salt was in a similar range, indicating thatAngeli's salt in the concentration used in this study did not cause acutecytotoxicity.

Effect of DEA/NO and GSH on inhibition of papain in vitro

To study the inhibition of the papain-family cysteine proteases byAngeli's salt or HNO in the presence of NO, papainwas used as a targetmolecule. In mammals, cathepsin B is a close structural analogue ofpapain [21], whereas papain shows higher stability in solution thatenables in vitro DTT reactivation experiments.

The NO-donor DEA/NO dose dependently inhibited papain activityunder aerobic in vitro conditions after a 1-h incubation reaching a53±0.4% inhibition at 316 μM concentration. This effect was com-pletely reversed by DTT (Fig. 3A) as previously described for PAPA/NO-inhibited papain [10].

In the papain preparation used for the studies, 2.7 μM Angeli's saltcaused a 98±0.9% inhibition. Thus papain was treated with a 2.7 μMAngeli's salt and the indicated concentrations of DEA/NO (0-316 μM)(Fig. 3B), resulting in a modest rescue of papain activity reaching 15±

y of cathepsin B to irreversible inhibition by nitroxyl (HNO) in the0.1016/j.freeradbiomed.2008.05.025

Fig. 1. The effect of TNF-α, IFN-γ, and LPS on iNOS expression, NO production, and cathepsin B activity in RAW 264.7 macrophages. (A) Representative immunoblots for cathepsin B(Cath B), iNOS, and beta-actin (Actin) are shown following activation of RAW 264.7 macrophages by LPS (500 ng/ml) for the indicated time in the absence or presence of L-NAME(1 mM). (B) Dose response of iNOS induction by TNFα (ng/ml) or IFN-γ in the absence or presence of L-NAME (1 mM). (C) The effect of LPS (500 ng/ml) on cumulative production ofnitrite and nitrate in the media in the absence (solid bars) or in the presence of L-NAME (1 mM, open bars) is shown at the indicated time points. (D) Cumulative production of nitriteand nitrate in the media following 24-h activation with indicated concentration of IFN-γ (IU/ml). Solid bars without L-NAME, open bar with L-NAME (1 mM). (E) The effect of a 24-hstimulation with IFN-γ (50–100 IU/ml) or LPS (500 ng/ml) on cathepsin B activity as a percentage of vehicle control following indicated stimulation in the absence (solid bars) or inthe presence of L-NAME (1 mM, open bars). Leupeptin (LEU; 100 μM) was used as a control inhibitor. (F) Immunocytochemical demonstration of cathepsin B (red) and iNOS (green)expression in control (CTRL), LPS-(500 ng/ml), and IFN–γ (100 IU/ml) stimulated RAW 264.7 macrophages after a 24-h stimulation. The cell nuclei have been counterstained withTO-PRO-3 (cyan). ⁎Pb0.05 compared to all other groups. All quantitative data are expressed as mean±SE from triplicate samples. (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of this article.)

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1.0% of vehicle-treated control samples activity at 316 μM DEA/NOconcentration. Since NO autoxidation-inactivated papain is reacti-vated by DTT (Fig. 3A) [10], the extent of papain reactivation by DTTtreatment was studied in the Angeli's salt-and DEA/NO-treated

Fig. 2. The effect of LPS (500 ng/ml) or IFNγ (0–100 IU/ml) on inhibition of cathepsin B by Anthe indicated agents for 24 h prior to 1-h incubationwith AS (0–316 μM) followed bymeasure(0–316 μM) in control and LPS- and LPS+L-NAME-stimulated RAW 264.7 cells. Bars indicate cbars). ⁎⁎Pb0.01 compared to respective AS-induced inhibition in controls without LPS or L-Ncontrol (IFNγ) and IFNγ-stimulated (IFNγ 50 or 100 IU/ml)±L-NAME (1 mM) RAW 267.4 macontrol (100%; relative activities of controls shown in Fig. 1E). PN0.05 for all samples compatriplicate samples.

Please cite this article as: Väänänen, A. J.; et al., Persistent susceptibilitpresence of endogenous nitric oxide, Free Radic. Biol. Med. (2008), doi:1

samples. The activity of papain samples was increased compared topre-DTT-treated samples at higher than 31.6 μM DEA/NO concentra-tions, reaching 64±0.9% of vehicle and DTT-treated control samplesafter the DTT reactivation of 316 μM DEA/NO concentration (Fig. 3B).

geli's salt (AS) in RAW 264.7 macrophages. RAW 264.7 macrophages were stimulated byment of cathepsin B activity. (A) The activity of cathepsin B after 1-h incubationwith ASathepsin B activity as a percentage of respective non-AS-treated control samples (openAME treatment. (B) The effect of AS (316 μM) on cathepsin B activity in nonstimulated

crophages. Solid bars show the effect of AS 316 μM referenced to respective stimulationred to nonstimulated cells after AS 316 μM exposure. All data shown as mean±SE from

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Fig. 3. The effect of HNO-scavenging NO donor DEA/NO or GSH on inhibition of cysteine protease papain in vitro by Angeli's salt. (A) Inhibition of papain (0.1 mg/ml) after 1-hincubation period with the indicated concentration of DEA/NO (0–316 μM) is shown (open squares). Following the initial incubation the samples were further incubated with 10 mM(final) DTT and the activity was measured (solid squares). The data are expressed as a percentage of vehicle-treated papain (open squares) or DTT-treated control samples (solidsquares). (B) Similar experiment as in A, but all incubations were carried out in the presence of Angeli's salt (2.7 μM). Open squares show papain activity after a 1-h incubation periodwith Angeli's salt (2.7 μM) and DEA/NO (0–316 μM), while solid squares show papain activity following a 1-h posttreatment with 10 mM DTT. (C) Papain was incubated with 2.7 μMAngelis's salt and GSH (0–316 μM) and the activity referenced to vehicle-treated control (open squares). Thereafter the samples were further incubated for 1 h with 10mMDTT (final)and the activity was referenced to DTT-treated vehicle control. All data expressed as mean±SE (if larger than symbol size) from n=3–6 replicates.

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Since the reactivity of the active cysteine site in papain canoutcompete the reaction of HNO with NO, the effect of GSH againstinhibition of papain by HNO was tested by adding GSH (0-316 μM)with 2.7 μM Angeli's salt to papain. In line with well-characterizedhigh reactivity of HNO with GSH [15,27], addition of GSH provided adose-dependent protection against inhibition of papain (Fig. 3C; IC50

19 μM). DTT posttreatment did not affect the inhibition of papain (Fig.3C; IC50 22 μM).

Discussion

This investigation was set to elucidate the potency of intracellularNO production to protect susceptible critical protein cysteines frommodification by HNO. NO can either directly scavenge HNO [6](reactions (1)-(3) and Fig. 4, pathway 5) or cause, on autoxidation,nitrosation of cysteines [2,3] (Fig. 4, reaction (1)) providing resistanceto subsequent modification by HNO [10]. Our results show thatcellular NO production, as induced by LPS or IFN-γ, does not protectthe cysteine protease cathepsin B in RAW 264.7 murine macrophagesagainst HNO. These data further stress the susceptibility andvulnerability of cathepsin B to HNO.

We observed a similar rate of inhibition of cathepsin B by HNO ashad been previously shown for the human THP-1 monocyte/macro-phage cell line [10]. However, since the THP-1 cells do not readilyexpress iNOS or produce NO [11] they do not facilitate theexamination of the effects of endogenous NO production on theactions of HNO. Based on their ability to produce high concentrationsof NO on stimulation, the RAW 264.7 macrophages provide a cellularmodel to study the effect of NO on inhibition of cysteine proteases,such as cathepsin B, by HNO. Interestingly, basal cathepsin B activitywas greatly inhibited after LPS stimulation. This inhibitionmay be dueto peroxynitrite formation in the LPS-stimulated cells [28] since thecathepsin B assay involved DTT reactivation that does not activateperoxynitrite-inhibited cysteine proteases [10,29] and L-NAME, aninhibitor of NOS activity, reversed the LPS-induced inhibition. More-over, at 24 h of stimulation LPS markedly altered cellular morphologythat could alter cathepsin B susceptibility to reactive nitrogen species.It must be borne in mind that also HNO, if hypothetically produced bythe LPS-stimulated macrophages, would show a similar effect on

Please cite this article as: Väänänen, A. J.; et al., Persistent susceptibilitpresence of endogenous nitric oxide, Free Radic. Biol. Med. (2008), doi:1

cathepsin B activity. More experimentation is needed to demonstratethis hypothesis.

The dose-dependent HNO-induced inhibition of cathepsin B inRAW 264.7 macrophages was not attenuated by induction of iNOSeither with LPS or IFN-γ stimulation. In fact, in LPS-stimulated cellsthe relative loss of cathepsin B activity was slightly higher followingexposure to Angeli's salt, while in IFN-γ-stimulated cells induction ofiNOS did not affect the inhibition caused by Angeli's salt. This stronglysuggests that endogenous NO production does not protect cathepsin Bfrom irreversible inhibition by HNO. Furthermore, while L-NAMEcaused a marked reduction in NO production it did not aggravate thecathepsin B inhibition; rather it protected against the inhibitioncaused by Angeli's salt. Thus it is apparent that even at these highconcentrations as produced by the macrophages, endogenous NOproduction fails to protect cathepsin B activity from HNO-inducedabrogation.

Since the reactivity of HNOwith NO exceeds by order of magnitudethe reactivity of HNO with L-cysteine, the observed lack of protectionof cathepsin B by NO is surprising, and emphasizes the remarkablesensitivity of cathepsin B to irreversible inhibition by HNO. Thecellular NO concentrations, after maximal stimulation, are in themicromolar range, and thus could be overrun by the HNO fromAngeli's salt. However, it is noteworthy that the concentration ofcathepsin B is in a much lower range, and thus NO should present amarkedly more attractive target for HNO. Furthermore, the cytosol isrich in GSH which has been considered one of the main targets forHNO owing to its millimolar intracellular concentration and 100-foldhigher reactivity compared to NO [7].

It has been proposed that the functional mechanism of someproteins, including cysteine proteases, may make them sensitivetargets for HNO due to deprotonation of the active-site cysteine,caused by adjacent nucleophilic amino acids [7,8,12]. The reported pKa

value for the cysteine forming the thiolate-imidazolium ion pair at thecatalytic center of cathepsin B and papain is 5.5 [21,30]. Thus, thelower pKa which has been proposed to sensitize yeast glyceraldehydedehydrogenase (GAPDH) to inhibition by HNO compared to depletionof the cellular GSH pool [12] may also apply to cysteine proteases.Since the reactivity of HNO with GSH exceeds that of HNO with NO,such effects support the lack of NO-induced protection againstinhibition of cathepsin B activity by HNO.

y of cathepsin B to irreversible inhibition by nitroxyl (HNO) in the0.1016/j.freeradbiomed.2008.05.025

Fig. 4. A schematic presentation on the potential reactions in a reaction system consisting of cysteine-harboring protein (PROT-SH), Angeli's salt-released HNO, and NO or GSH. Onnitrosation (1) by autoxidation product of NO a DTT-reversible S-nitrosoprotein is formed, while on reaction with HNO a transient N-hydroxysulfenamide species is formed (2). TheN-hydroxysulfenamide can subsequently react with either GSH (3) to form a DTT-reversible mixed disulfide between GSH and protein or undergo rearrangement into DTT-irreversible sulfinamide species (4). Additionally either GSH or NO can directly scavenge HNO (5).

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To gain a better understanding on the relative reactivity of cysteineprotease active-site cysteine with HNO in the presence of either NO orGSH, we used an in vitro setup to study the effect of NO donor DEA/NOand GSH, respectively, on the inhibition of papain activity by HNO.Under aerobic conditions DEA/NO inhibited papain at a somewhatlower potency than PAPA/NO in the same system [10]. This could bedue to a shorter degradation half-life of DEA/NO compared to PAPA/NO, which may not be optimal for the cysteine-modifying NOautoxidation product generation. DEA/NO has a similar half-lifecompared to Angeli's salt [19] and is thus an optimal NO donor forstudying the modulatory effects of concomitant NO generation on theeffects of Angeli's salt. Surprisingly, in the presence of higherconcentrations of DEA/NO there was some residual activity (Fig. 3B).This is likely due to the lower stability of nitrosated papain, since asignificant portion of the protease is apparently inactivated by theDTT-reversible nitrosation of the active site (Fig. 3B, Fig. 4, reaction (1)). Therelative potency of DEA/NO (EC50 406 μM) to protect papain from DTT-irreversible inactivation by HNO is surprisingly low, particularly sinceDEA/NO releases twice as much NO compared to HNO release fromAngeli's salt [19].

In the utilized in vitro setup the potential consumption pathwaysfor HNO released from the degradation of Angeli's salt are dimeriza-tion to nitrous oxide (8×106 M-1 s-1 [6]), reaction with oxygen(3×103 M-1 s-1 [15]), or the active-site cysteine of papain. Angeli's saltinactivates papain in a 1:1 stoichiometric fashion when papain istitrated against fixed low Angeli's salt concentration (3.16 μM). Thuswhen Angeli's salt concentration is kept low, other consumptivepathways are not dominant [15]. It has been previously reported thatunder 5 μM Angeli's salt in aerobic buffer causesN99% of the HNO toreact with O2, which can be stoichiometrically inhibited by thiols suchas GSH and N-acetylcysteine [15]. It is noteworthy that in the cellculture studies when higher concentrations of Angeli's salt are utilizedthe reactions with oxygen and autoxidation become more likely, thuslimiting the flux of HNO. The eightfold higher concentration of GSHneeded for protecting half of papain from inactivation by HNOsuggests that reaction of the active-site cysteine (Fig. 4, reaction (2)) issuperior to the reaction of GSH with HNO (Fig. 4, reaction (5)). When4.2 μM (0.1 mg/ml) papain containing 3.2 μM titrable cysteine isexposed to 3.16 μM Angeli's salt with IC50 concentration of GSH(31.5 μM), the potential reaction of HNOwith the thiols takes place at a1:1 rate. Thus,

kGSH HNO½ � GSH½ � ¼ kpapain HNO½ � papain½ �kpapain ¼ kGSH GSH½ � papain½ �−1:

Please cite this article as: Väänänen, A. J.; et al., Persistent susceptibilitpresence of endogenous nitric oxide, Free Radic. Biol. Med. (2008), doi:1

The kGSH is known as 2×106 M-1 s-1 [15]; thus kpapain can beapproximated as 2×107 M-1 s-1.

The reaction of papain cysteine with HNO initiates a multistepreaction, where the initial step inactivates the enzyme dependent oncysteine at its catalytic site. On reaction of cysteine with HNO, anunstabile N-hydroxysulfenamide is formed (Fig. 4, reaction (2)[31,32]), which could subsequently react with GSH causing ageneration of mixed disulfide between papain and GSH (Fig. 4,reaction (3)), that could be readily reactivated by DTT. However,papain inactivated by HNO in the presence of a significant excess ofGSH is not reactivated by DTT (Fig. 3C). Thus, the active site of papain ismore prone to react with HNO than GSH, and the N-hydroxysulfena-mide form of papain then readily undergoes rearrangement into DTT-irreversible sulfinamide (Fig. 4, reaction (4)) prior to reaction of N-hydroxysulfenamide intermediate with GSH. The mixed disulfidegeneration would require that GSH can access the catalytic site of theenzyme; this could be hindered by sterical factors. However, papaincan be readily inhibited by GSNO [10,33] and such inhibition ispronounced in the presence of metal-chelating agent DETAPAC [10],which increases the stability of S-nitrosothiols. Finally, GSNO andother S-nitrosothiols have been shown to generate mixed disulfideswith the active-site cysteine of papain [33] and therefore the presenceof significant sterical obstacles for GSH seems unlikely.

In conclusion, the results from this study show that NO is unable toprotect cysteine proteases cathepsin B or papain from inhibition byHNO, likely due to significantly greater reactivity of HNO with theactive-site cysteine in these proteases. Thus HNO can affect cellularproteins, such as cysteine proteases, even under conditions where NOproduction is induced.

Acknowledgments

The authors thank Lahja Eurajoki for expert assistancewith the cellculture experiments.

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