(Received for publication, July 17, 1996, and in revised form, February 18, 1997)
From the Julius Friedrich Cohnheim-Minerva Center for
Phagocyte Research, Department of Human Microbiology, Sackler School of
Medicine, Tel Aviv University, Tel Aviv 69978, Israel and
§ Pentapharm Ltd., Engelgasse 109, CH-4002, Basel, Switzerland
The elicitation of an oxidative burst
in phagocytes rests on the assembly of a multicomponental complex
(NADPH oxidase) consisting of a membrane-associated flavocytochrome
(cytochrome b559), representing the redox
element responsible for the NADPH-dependent reduction of
oxygen to superoxide (O2), two cytosolic components
(p47phox, p67phox), and the small GTPase Rac (1 or 2).
We found that 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), an
irreversible serine protease inhibitor, prevented the elicitation of
O
2 production in intact macrophages and the amphiphile-dependent activation of NADPH oxidase in a
cell-free system, consisting of solubilized membrane or purified
cytochrome b559 combined with total cytosol or
a mixture of recombinant p47phox, p67phox, and Rac1.
AEBSF acted at the activation step and did not interfere with the
ensuing electron flow. It did not scavenge oxygen radicals and did not
affect assay reagents. Five other serine protease inhibitors (three
irreversible and two reversible) were found to lack an inhibitory
effect on cell-free activation of NADPH oxidase. A structure-function
study of AEBSF analogues demonstrated that the presence of a sulfonyl
fluoride group was essential for inhibitory activity and that compounds
containing an aminoalkylbenzene moiety were more active than
amidinobenzene derivatives. Exposure of the membrane fraction or of
purified cytochrome b559, but not of cytosol or
recombinant cytosolic components, to AEBSF, in the presence of a
critical concentration of the activating amphiphile lithium dodecyl
sulfate, resulted in a marked impairment of their ability to support
cell-free NADPH oxidase activation upon complementation with untreated
cytosol or cytosolic components. Kinetic analysis of the effect of
varying the concentration of each of the three cytosolic components on
the inhibitory potency of AEBSF indicated that this was inversely
related to the concentrations of p47phox and, to a lesser
degree, p67phox. AEBSF also prevented the amphiphile-elicited
translocation of p47phox and p67phox to the membrane.
These results are interpreted as indicating that AEBSF interferes with
the binding of p47phox and/or p67phox to cytochrome
b559, probably by a direct effect on cytochrome b559.
The production of reactive oxygen radicals represents the major microbicidal mechanism of phagocytes (1). Oxygen radicals are also generated, in lesser amounts, by some nonphagocytic cells, sharing the enzymatic machinery characteristic of phagocytes (2), and, under certain conditions, by plant cells (3). Interest in reactive oxygen species has also been stimulated by accumulating evidence for their involvement in the pathogenesis of diseases, ranging from respiratory distress syndrome to ischemia-reperfusion injury in several organs (4).
The primordial oxygen radical produced by phagocytes is superoxide
(O2).1 It is generated, in
response to appropriate stimuli, by NADPH-derived one-electron
reduction of molecular oxygen, a reaction catalyzed by a membrane-bound
heterodimeric flavocytochrome (cytochrome b559)
(reviewed in Refs. 5-7). Cytochrome b559
contains two redox centers, FAD and heme, and electron flow from NADPH
to oxygen in initiated by the interaction of cytochrome with two
cytosolic proteins, p47phox and p67phox, and the small
GTPase, Rac1 or Rac2 (reviewed in Ref. 8). In the intact cell, this
interaction is made possible by the stimulus-dependent translocation of the cytosolic components to the plasma membrane, leading to the assembly of what is known as the NADPH oxidase complex.
Activation of NADPH oxidase, resulting in O
2 generation, can
be reproduced in vitro by a cell-free system consisting of either phagocyte membranes and cytosol (9, 10) or of a mixture of
purified or recombinant components, exposed to a critical amount of an
anionic amphiphile (arachidonate or SDS) (11, 12).
A number of inhibitors of the O2 generating NADPH oxidase have
been described (reviewed in Ref. 13). The search for such inhibitors is
propelled by two incentives: (a) to serve as tools for
understanding the structure and mechanism of activation of the NADPH
oxidase, and (b) their potential use as therapeutic agents
in diseases associated with production of oxygen radicals at an
inappropriate site or time. In most studies, inhibitors were tested for
an effect on intact phagocytes. The main drawback of such an approach
is that it does not permit a distinction between an effect on membrane
receptors and signal transduction and a direct effect on the components
of the NADPH oxidase complex. Alternative strategies rest on examining
the effect of inhibitors on subcellular fractions originating from
stimulated cells or on NADPH oxidase activation in the cell-free
system. These latter approaches should allow the selection of
inhibitors with a direct effect on the assembled complex or on
individual components of the NADPH oxidase complex. So far, the only
compound having gained wide acceptance as a relatively specific direct
inhibitor of NADPH oxidase is diphenylene iodonium (14).
In the present paper we report that
4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF; also known as
Pefabloc SC), originally developed as an irreversible serine protease
inhibitor, prevents the activation of the O2 generating NADPH
oxidase in both intact stimulated macrophages and in cell-free systems.
The effect is shared by some structurally related compounds, indicating
that we are dealing with a new class of NADPH oxidase inhibitors.
Evidence is presented in favor of the proposal that AEBSF interferes
with the interaction of p47phox and/or p67phox with
cytochrome b559, probably by chemical
modification of cytochrome b559.
Chemicals and Reagents
AEBSF hydrochloride (>99% pure), lithium dodecyl sulfate
(LiDS), and common laboratory chemicals (at the highest purity
available) were from Merck. 4-(2-Aminoethyl)-benzenesulfonamide
(AEBSNH2, 99% pure) was purchased from Aldrich.
4-(2-Aminoethyl)-benzenesulfonic acid hydrochloride (AEBSAc),
4-(2-N-methylaminoethyl)-benzenesulfonyl fluoride
hydrochloride (MAEBSF), and 4-(amidino)-benzenesulfonyl fluoride
hydrochloride (pABSF) were synthesized by Pentapharm Ltd., Basle,
Switzerland. n-Octyl--D-glucopyranoside
(octyl glucoside) was a product of Pfanstiehl Laboratories. Aprotinin
was obtained from Fluka. Guanosine 5
-3-O-(thio)triphosphate
(GTP
S) was purchased from Boehringer. The following chemicals were
obtained from Sigma: NADPH (tetrasodium salt, 95% pure),
ferricytochrome c (from horse heart, 95% pure), superoxide
dismutase (from bovine blood), xanthine, xanthine oxidase (from
buttermilk), phorbol 12-myristate 13-acetate (PMA),
N-formyl-Met-Leu-Phe (fMLP), calcium ionophore A23187, phenylmethanesulfonyl fluoride (PMSF),
4-(amidino)-phenylmethanesulfonyl fluoride hydrochloride (pAPMSF),
benzamidine hydrochloride hydrate, leupeptin hemisulfate salt,
n-tosyl-L-phenylalanine chloromethyl ketone
(TPCK), and 3,4-dichloroisocoumarin (DCIC).
Protease inhibitors were dissolved in water at a concentration at least 50-fold higher than the highest concentration used in NADPH oxidase inhibition assays, witih the exception of PMSF, TPCK, and DCIC, which were dissolved in ethanol, methanol, and dimethyl sulfoxide, respectively.
Preparation of Macrophages and Subcellular Fractions
Macrophages were obtained from the peritoneal cavity of guinea pigs injected with mineral oil 5-6 days before cell harvest. The cells were washed and made erythrocyte-free, as described previously (10), and suspended in Earle's balanced salt solution (2 × 108 cells/ml), for assays involving intact cells. The membrane and cytosol fractions were obtained from cells disrupted by sonication, as described before (15), with some modifications (16).
Solubilization of Membranes and Purification of Cytochrome b559
Membranes were washed in 1 M KCl in cell homogenization buffer (15, 16) and solubilized in 40 mM octyl glucoside, as described (17). Before use in the cell-free assay, solubilized membrane was diluted in detergent-free solubilization buffer, to bring the concentration of octyl glucoside to 4-5 mM. Cytochrome b559 was purified from the original solubilized membrane and relipidated by a modification (18) of our original method (19). Spectral analysis of cytochrome b559 preparations was performed as described in Ref. 19.
Preparation of Recombinant Cytosolic NADPH Oxidase Components
Baculoviruses carrying cDNAs for human p47phox and
p67phox were kind gifts of Dr. Thomas L. Leto (National
Institutes of Health, Bethesda, MD). Suspension cultures of Sf9 cells
(2 × 106 cells/ml), grown in serum-free medium
(SF-900 II SFM, Life Technologies) were infected with the recombinant
baculoviruses and harvested 72 h after infection. The recombinant
proteins were prepared as described by Leto et al. (20).
Briefly, the cells were disrupted by sonication using three 30-s
pulses, in a 250 watt apparatus (Sonics and Materials), centrifuged at
12,000 × g for 10 min, and the supernatant fractions
recentrifuged at 100,000 × g for 1 h. The
recombinant proteins were purified by ion exchange chromatography of
the high speed supernatants, referred to as "crude extracts." Thus,
crude extract, containing p47phox, was applied to a SP
Sepharose column (HiLoad 16/10, Pharmacia Biotech), equilibrated with
50 mM sodium phosphate buffer, pH 7.0, supplemented with 1 mM dithioerythritol, and was eluted with a 0-0.3
M NaCl gradient in the same buffer (210 ml, at a flow rate
of 1.5 ml/min). Crude extract, containing p67phox, was applied
to a Q-Sepharose column (HiLoad 16/10, Pharmacia Biotech Inc.)
equilibrated with 20 mM Tris-HCl, pH 7.5, supplemented with
1 mM dithioerythritol, and eluted with a 0-0.5
M NaCl gradient in the same buffer (210 ml, at a flow rate
of 1.5 ml/min). Purified recombinant proteins were analyzed by
SDS-polyacrylamide gel electrophoresis; p47phox was 99% pure,
whereas p67phox was 90% pure. Recombinant Rac1 was isolated
from Escherichia coli transformed with Rac1 cDNA
subcloned into the bacterial expression vector pGEX2T (a kind gift of
Dr. Thomas L. Leto, National Institutes of Health, Bethesda, MD), as
described by Kwong et al. (21). Briefly, the pellet of
bacterial culture overexpressing the glutathione S-transferase-Rac1 fusion protein was disrupted by
sonication, by three pulses of 30 s, and the homogenate was
clarified by centrifugation at 12,000 × g for 5 min.
The fusion protein was absorbed from the supernatant on
glutathione-agarose (Sigma) and Rac1 was cleaved from the resin-bound
fusion protein by thrombin. Thrombin was removed by absorption on
benzamidine-Sepharose 6B (Pharmacia Biotech Inc.). Purified Rac1 was
loaded with GTPS, as described before (22).
O2 Production by Intact Macrophages
Macrophages (107 cells), in a volume of 50 µl,
were added to 940 µl of Earle's balanced salt solution (containing
1.8 mM CaCl2), supplemented with 80 µM ferricytochrome c, in disposable
spectrophotometer cuvettes. Inducers of an oxidative burst were added
in a volume of 10 µl and the cuvettes placed in the thermostatted
(37 °C) cuvette holder of a Uvikon 860 spectrophotometer (Kontron
Instruments). Stimulus-dependent O2 production
was assayed by the continuous recording of superoxide dismutase
inhibitable ferricytochrome c reduction, measured at 550 nm,
and the maximal rate was calculated from the linear sector of the
change in absorbance curve (23).
Assays for Cell-free Activation of NADPH Oxidase
OAnionic amphiphile (LiDS)-activated
O2 production in vitro was assayed by
ferricytochrome c reduction in mixtures consisting of
solubilized membrane and either total cytosol or a combination of
p47phox, p67phox, and Rac1-GTP
S, as described (10,
16). In some experiments, the membrane was replaced by purified
relipidated cytochrome b559. Cell-free assays
were performed either by the "one-step" method, in which activation
by LiDS and NADPH-dependent O
2 production occur
simultaneously, or by the "two-step" method (24) permitting, at
least partial, separation of the activation and catalytic stages of the
reaction.
LiDS-dependent NADPH oxidation
by cell-free mixtures, of a composition identical to those utilized in
the O2 production assays, was measured as described in Ref.
25.
LiDS- and NADPH-dependent oxygen consumption by cell-free mixtures, consisting of membrane and recombinant cytosolic components, was assayed with the aid of a Clark oxygen electrode (Yellow Springs Instruments), as described (26).
Translocation of p47phox and p67phox to the Membrane
The translocation of p47phox and p67phox
to the membrane fraction of macrophages was studied in the
LiDS-activated cell free system. Macrophage membranes (equivalent to
1.6 × 107 cells) were mixed with total macrophage
cytosol (equivalent to 1.5 × 107 cells) in a total
volume of 1 ml of NADPH oxidase assay buffer (10), not containing
ferricytochrome c, and incubated for 5 min at 24 °C, in
the absence or presence of 185 µM LiDS (a concentration found to induce maximal O2 production under these
conditions). The mixture was centrifuged at 15,800 × g
for 30 min at 4 °C and, after careful removal of the supernatant,
the membrane pellet was resuspended in 1 ml of NADPH oxidase assay
buffer and recentrifuged at 15,800 × g for 15 min.
After removal of the supernatant, the sedimented membranes were
resuspended in 30 µl of electrophoresis sample buffer containing 2%
SDS, heated at 95 °C for 5 min, and subjected to SDS-polyacrylamide
gel electrophoresis and immunoblotting, as described (19). The blots
were probed with a mixture of goat polyclonal anti-recombinant
p47phox and anti-recombinant p67phox antibodies
(20) (kind gifts of Dr. Thomas L. Leto, National Institutes of
Health, Bethesda, MD), followed by peroxidase-conjugated rabbit
anti-goat IgG (Jackson ImmunoResearch). The reactive bands were
detected by enhanced chemiluminescence, using reagents manufactured by Amersham International, and exposure to Rx medical x-ray film (Fuji).
O2 production can be elicited in guinea pig
peritoneal macrophages by a variety of stimulants (27). We chose three
stimulants (PMA, fMLP, and the calcium ionophore, A23187), known to
activate NADPH oxidase in intact cells by distinct transductional
mechanisms, and used these at concentrations found to elicit maximal
O
2 generation in cells in suspension at 37 °C. In the
presence of AEBSF, a dose-dependent inhibition of
O
2 production elicited by 2 µM PMA was observed (Fig. 1A). When AEBSF was added just before
the addition of PMA, the IC50 was close to 1 mM
and inhibition was almost complete at 5 mM AEBSF. At the
latter concentration, AEBSF also caused a total suppression of
O
2 production elicited by 1 µM fMLP or 10 µM A23187 (results not shown). This result was compatible
with three interpretations: (a) AEBSF acts as a scavenger of
O
2; (b) it acts on a signal transduction path
shared by all three stimulants; and (c) it has a direct
effect on the NADPH oxidase complex.
AEBSF Does Not Scavenge O
We examined the effect of
AEBSF on the ability to measure O2 generation by the
xanthine-xanthine oxidase system, using the ferricytochrome
c reduction assay. AEBSF (0.5-5 mM) did not
interfere with the detection of O
2 generated by xanthine (0.2 mM) and xanthine oxidase (0.018 units/ml); the difference
between O
2 detection in the absence or presence of AEBSF (up
to 5 mM) was less than 10% of the control value.
NADPH oxidase can be activated in a cell-free system by
certain anionic amphiphiles, a mechanism which circumvents the
transductional pathways active in the intact phagocyte (9, 10). We,
therefore, examined the effect of AEBSF on O2 production in
several forms of cell-free activation systems. The simplest one
consisted of solubilized macrophage membranes and unfractionated
cytosol, both derived from unstimulated cells. AEBSF was found to
exhibit a concentration-related, inhibitory influence on LiDS-induced
O
2 production, with an IC50 of 0.206 ± 0.014 mM (n = 8) (Fig. 1B). We next
investigated the effect of AEBSF on a semi-recombinant cell-free system
(11, 12), consisting of either solubilized macrophage membrane or
purified cytochrome b559, incorporated in
phospholipid liposomes, combined with recombinant p47phox,
p67phox, and Rac1-GTP
S. AEBSF was found to act as an
inhibitor of O
2 production in this system, too, composed of
four purified NADPH oxidase components in the virtual absence of any
other protein (Fig. 1C). In the course of the latter
experiments we became aware of an unexpected variability in the
inhibitory potency of AEBSF which was not found in the cell-free system
consisting of solubilized membrane and unfractionated cytosol. Analysis
of this phenomenon revealed that, in the presence of a constant amount
of membrane or purified cytochrome b559, the
inhibitory effect of AEBSF was inversely related to the concentration
of p47phox. A more detailed exploration of the mechanism of
this correlation is provided in the penultimate section.
To further ascertain that inhibition by AEBSF of O2 production
in the cell-free system is due to a direct effect on NADPH oxidase, we
examined the influence of AEBSF on two additional indicators of NADPH
oxidase activation. These were LiDS-elicited NADPH oxidation (25) and
LiDS- and NADPH-dependent oxygen consumption (26), by
mixtures of membrane and cytosol or membrane and recombinant cytosolic
components. We found that AEBSF suppressed NADPH oxidation in a
concentration-related manner; when cell-free mixtures were analyzed in
parallel by ferricytochrome c reduction, the
IC50 of AEBSF values were identical in the two assays.
Also, at 5 mM, AEBSF inhibited oxygen consumption by 95%.
We concluded that AEBSF acts by interacting with one or more of the
components of the NADPH oxidase complex.
The O2 generating NADPH oxidase
has a Km for NADPH of 20-40 µM (6)
and the enzyme activated by amphiphile under cell-free conditions
exhibits the same kinetic characteristics (9, 10). The effect of AEBSF
(2 mM) on NADPH oxidase activation in the cell-free system
was examined at concentrations of NADPH varying from 10 to 400 µM and analyzed by Michaelis-Menten plotting. This
demonstrated that inhibition of AEBSF was noncompetitive with NADPH.
Membrane solubilization and purification of cytochrome b559 results in a partial loss of FAD from the
cytochrome (11, 19, 28), which explains the dependence of NADPH oxidase
activation, in the cell-free system, on exogenous FAD (9). We,
therefore, investigated whether AEBSF interfered with the reflavination
of cytochrome b559, by assessing the effect of
varying the concentration of exogenous FAD on the inhibitory effect of
AEBSF. We found that the degree of inhibition by AEBSF
(IC50) was independent of the concentration of exogenous
FAD (from 10 to 100 µM). Finally, we explored the
possibility that AEBSF competes with the activating amphiphile for
interaction with a component of the NADPH oxidase complex. We found
that increasing the concentration of LiDS, from the optimal activating
range of 120-140 µM to up to 180 µM, did not reverse or reduce the inhibitory effect of AEBSF (see also Fig.
4).
AEBSF Interferes with Activation of NADPH Oxidase but Not with Electron Flow
Activation of NADPH oxidase, in intact cells and in
broken cell preparations, is considered to be the result of the
assembly of the individual components of the enzyme into a
membrane-localized complex (reviewed in Ref. 8), this being followed by
the induction of electron flow within the flavocytochrome
b559 dimer. The "activation" and "electron
flow" stages can be separated in a two-step cell-free activation
assay (24). This consists of exposing a mixture of NADPH oxidase
components to an optimal concentration of activating amphiphile in a
small volume (100 µl) for a fixed time interval, in the absence of
substrate NADPH, followed by 10-fold dilution and the initiation of
electron flow by addition of NADPH. The 10-fold dilution results in a
reduction in the concentration of amphiphile to a subactivating level
and causes the virtual interruption of further assembly of the enzyme
complex. We utilized this approach to identify the event affected by
the inhibitor, by adding AEBSF either to the activation mixture at time
0 or after the completion of activation. As apparent in Fig.
2, AEBSF was a much more effective inhibitor when
present during the assembly of the NADPH oxidase complex than when
added at the completion of assembly. The finding that AEBSF was not
totally inactive when added at the end of step one is probably due to
the fact that some enzyme assembly continues taking place in the course
of the second step. The ability of AEBSF to suppress O2
production when added to the components of the NADPH oxidase complex,
in the absence of electron flow, also indicates that its mechanism of
action is distinct from that of diphenylene iodonium, which acted as an
inhibitor only under reducing conditions, such as those generated by
NADPH oxidase turnover (29).
Reversibility of Inhibition by AEBSF
Use was made of the
two-step cell-free assay for determining whether inhibition of NADPH
oxidase activation by AEBSF was reversible. For this purpose,
activation mixtures were prepared in the absence and presence of a
single concentration of AEBSF (2 mM) and incubated with an
optimal concentration of LiDS for 5 min. Following this, the reaction
mixtures were diluted in assay buffer to reduce the amphiphile
concentration, and re-exposed or not to an activating concentration of
LiDS for 3 min. The results illustrated in Fig. 3
demonstrate that re-exposure to LiDS of mixtures treated with 2 mM AEBSF partially reverses the inhibition. Thus,
O2 production by activation mixtures treated with AEBSF was
22.67 ± 1.12% of that of control preparations, whereas
re-exposure of the AEBSF-treated preparations to LiDS led to resurgence
of O
2 production, reaching 55.55 ± 1.99% of that
measured in untreated preparations.
Specificity of AEBSF-mediated Inhibition
AEBSF was developed originally as an irreversible serine protease inhibitor (30, 31). It was, therefore, essential to establish whether its inhibitory effect on NADPH oxidase activation was related to its anti-protease activity. Hence, we examined the ability of five additional serine protease inhibitors to affect NADPH oxidase activation in the cell-free system. These were the irreversible inhibitors PMSF (0.1-2 mM) and TPCK (10-200 µM), the "mechanism based" irreversible inhibitor, DCIC (0.25 mM), and the reversible inhibitors aprotinin (0.3 µM) and leupeptin (20 µM). None of these exhibited an inhibitory effect on NADPH oxidase activation. We next explored the possibility that inhibition might be related to the concentration of activating amphiphile. As apparent in Fig. 4, PMSF, TPCK, and DCIC did not possess an inhibitory effect at any LiDS concentration whereas AEBSF was a potent inhibitor over the whole range of amphiphile concentrations. On the contrary, PMSF, TPCK, and DCIC had an enhancing effect on NADPH oxidase activation, at concentrations of LiDS below those required for maximal activation.
Structural Requirements for Inhibitory ActionWe investigated
the capacity of several analogues of AEBSF to interfere with the
activation of NADPH oxidase. One group consisted of aminoethylbenzene
derivatives and included: MAEBSF, identical to AEBSF except for the
presence of an amino-linked methyl group; AEBSAc, a product of alkaline
hydrolysis of AEBSF, found to be inactive as a protease inhibitor (31);
and AEBSNH2, in which an amide group replaces the fluoride
found in AEBSF, also reported to lack protease inhibitory activity
(32). As apparent in Fig. 5A, AEBSAc and
AEBSNH2 were totally inactive, whereas MAEBSF exhibited an
inhibitory potency identical to that of AEBSF (IC50 = 0.285 ± 0.005 mM; n = 3). The second
group consisted of amidinobenzene derivatives. Among these, pAPMSF was
reported to be a potent irreversible serine protease inhibitor (33),
and benzamidine acts as a peptidase inhibitor (34). As seen in Fig.
5B, only pABSF was a moderate inhibitor of NADPH oxidase
activation; its IC50 (1.49 ± 0.05; n = 3) was 7.5-fold higher than that of AEBSF. We also examined the
influence of an excess of the two inactive analogues, AEBSAc and
AEBSNH2, sharing the aminoethylbenzene structure with
AEBSF, on inhibition of NADPH oxidase activation by AEBSF. Neither of the two analogues, at 5 mM, was capable of reducing the
inhibitory effect of AEBSF, assayed at concentrations ranging from 0.1 to 5 mM. These results demonstrate that a sulfonyl fluoride
group, adjacent to the benzene ring, is essential for inhibitory
activity and that aminoalkylbenzene derivatives are more potent
inhibitors than amidinobenzene compounds.
Identification of the Molecular Target of AEBSF
We next
investigated whether interaction between AEBSF and a specific NADPH
oxidase component can be demonstrated. The basic design of these
experiments was to expose subcellular fractions or individual NADPH
oxidase components to AEBSF for a fixed time interval in a small
volume, followed by 10-fold dilution into assay buffer containing the
untreated complementary fraction or a mixture of the other, untreated
NADPH oxidase components, activating LiDS and NADPH. Dilution resulted
in a reduction in AEBSF concentration to subinhibitory levels, limiting
its effect to the component initially exposed to the compound. We
examined the effect of AEBSF on total membrane, total cytosol,
relipidated cytochrome b559, and recombinant
p47phox, p67phox, and Rac1-GTPS. Each of these was
exposed or not to AEBSF in the absence or presence of LiDS, the maximal
concentration of amphiphile being chosen not to exceed that permitting
the recovery of NADPH oxidase activity, upon complementation with
untreated components.
As apparent in Table I, exposure of total solubilized
membrane or purified and relipidated cytochrome
b559 to AEBSF, in the presence but not in the
absence of LiDS, resulted in marked inhibition of their capacity to
support O2 production upon complementation with cytosol and
activation by LiDS. Total cytosol as well as p47phox and Rac1
were insensitive to treatment by AEBSF; a minor effect of AEBSF on
p67phox was evident but this result was difficult to interpret
because of considerable interexperimental variability and because,
among all NADPH oxidase components, p67phox was the most
sensitive to denaturation by LiDS.
|
The ability of AEBSF to inactivate cytochrome
b559 was investigated in more detail by
assessing the dose-dependence of the inactivation of solubilized
membrane by AEBSF, in the presence and absence of LiDS. As seen in Fig.
6, AEBSF inhibited the capacity of the membrane to
support O2 production only in the presence of LiDS, with an
IC50 of 2.18 mM.
AEBSF Interferes with the Interaction between Cytochrome b559 and p47phox and/or p67phox
This series of experiments was initiated as
an extension of the preliminary finding that, in the presence of a
constant amount of cytochrome b559, the
inhibitory effect of AEBSF could be overcome by increasing the
concentration of cytosolic component p47phox. The consensus
opinion is that an essential event in the assembly of the NADPH oxidase
complex is the binding of p47phox to cytochrome
b559 (reviewed in Ref. 8). In light of the
results described in the preceding section, it seemed likely that AEBSF competes with p47phox for interaction with cytochrome
b559. We, therefore, investigated the influence
of varying the concentration of p47phox on the inhibition of
NADPH oxidase activation by AEBSF, in the presence of a constant amount
of membrane and saturating concentrations of p67phox and
Rac1-GTPS (Fig. 7A). In additional
experiments, we varied the concentrations p67phox (Fig.
7B) and Rac1-GTP
S (Fig. 7C), in the presence
of saturating concentrations of the other two cytosolic components. In
all experiments, three concentrations of cytosolic component were
chosen, two of which were on the ascending slope of previously
established dose-response curves, whereas the third one represented a
saturating concentration. The data were analyzed by plotting the
"concentration of AEBSF" (on a logarithmic scale) against
"inhibition of NADPH oxidase activation" and IC50 of
AEBSF values, for each concentration of cytosolic component, were
calculated. As seen in Fig. 7A, when p47phox was
present in subsaturating concentrations, sigmoid curves were generated
and IC50 values for AEBSF decreased with decreasing concentrations of p47phox. IC50 of AEBSF values
were moderately reduced by lowering the concentration of
p67phox (Fig. 7B) and not significantly affected by
varying the concentration of Rac1-GTP
S (Fig. 7C). These
results are compatible with the hypothesis that AEBSF interferes with
the binding of p47phox to cytochrome
b559 but we cannot rule out the possibility that AEBSF also inhibits a direct interaction between p67phox and
cytochrome b559 (see "Discussion").
The hypothesis that AEBSF acts as a competitive inhibitor of
p47phox was further tested by performing a Michaelis-Menten
analysis of the effect of AEBSF on the activation of NADPH oxidase at
four concentrations of p47phox (14, 21, 28, and 35 nM), all within the ascending slope of the concentration-response curve. As seen in the Lineweaver-Burk plot in
Fig. 8, AEBSF, in the concentration range of 0.5 to 2 mM, behaves as a competitive inhibitor with respect to
p47phox. At concentrations of AEBSF exceeding 2 mM,
a noncompetitive or mixed type of inhibition pattern was obtained.
Similar kinetic analysis of the effect of AEBSF on NADPH oxidase
activation at varying concentrations of either p67phox or
Rac1-GTPS revealed noncompetitive or mixed type inhibition patterns.
AEBSF Prevents Amphiphile-dependent Translocation of p47phox and p67phox to the Membrane
Activation of NADPH oxidase in intact cells is
accompanied by translocation of fractions of p47phox and
p67phox to the plasma membrane (35). The anchoring of both
cytosolic components to the membrane is dependent on the presence of
cytochrome b559 (36). Also, translocation of
p67phox to the membrane occurs only in the presence of
p47phox (36), suggesting that at least one of the functions of
p47phox is to serve as an escort protein for p67phox.
It was, therefore, of interest to investigate the effect of AEBSF on
the translocation of cytosolic components to the membrane in the
cell-free system (37). As seen in Fig. 9, the addition of AEBSF to mixtures of macrophage membranes and cytosol, activated by
a concentration of LiDS to result in maximal
NADPH-dependent O2 production, markedly inhibited
the translocation of both p47phox and p67phox to the
membrane. Parallel testing of the sedimented membranes, derived from
cell-free mixtures activated in the absence and presence of 4.5 mM AEBSF, for O
2 production, showed that lack
of translocation was accompanied by an 82% inhibition of NADPH oxidase
activation. As apparent in Fig. 9B, translocation of
p47phox and p67phox (and its inhibition by AEBSF) was
strictly dependent on the presence of membranes and was not the result
of nonspecific precipitation of cytosolic components by LiDS (35).
We found that AEBSF, originally developed as a water-soluble
irreversible serine protease inhibitor of relatively low toxicity, interferes with the activation of the O2 generating NADPH
oxidase both in intact macrophages and in cell-free systems in a
concentration-dependent manner. A remarkable feature of
this inhibition is that it affects all forms of cell-free activation,
from broken cell systems, consisting of total membrane and
unfractionated cytosol, to mixtures of purified cytochrome
b559 and three recombinant cytosolic components.
While this paper was in preparation, we became aware of the report by Remold-O'Donnell and Parent (38), describing the inhibitory effect of
2 mM AEBSF on O
2 production by human neutrophils
in response to PMA and opsonized zymosan. AEBSF (0.1-0.2
mM) was recently found to block priming of human monocytes
for enhanced O
2 production in response to PMA and fMLP
but was reported not to affect the response of unprimed cells, in
the above concentration range (32).
There is a large body of literature linking the elicitation of an
oxidative burst to the activation of cellular proteases (summarized in
Ref. 39). Some of the evidence for such a connection is based on the
effect of synthetic or natural protease inhibitors, particularly serine
protease inhibitors, on oxygen radical production by intact phagocytes.
Results of such experiments have to be interpreted with caution since
it became evident that the effects of protease inhibitors on
O2 production were frequently unrelated to their antiproteolytic activity (39-42). Our results indicate, indeed, that
AEBSF affects NADPH oxidase activation, at least in cell-free preparations, by a mechanism unrelated to its protease inhibitory activity. This claim is supported by the following arguments: (a) five other serine protease inhibitors (PMSF, TPCK, DCIC,
leupeptin, and aprotinin) were found to be inactive when tested under
identical conditions (it is of interest that DCIC which, like AEBSF,
was found to block O
2 production by intact neutrophils (38),
did not inhibit NADPH oxidase activation in the cell-free system); (b) among protease inhibitors, structurally related to AEBSF
(PMSF and pAPMSF), there was no relation between NADPH oxidase
activation inhibitory and antiproteolytic activities, and
(c) AEBSF was inhibitory in a cell-free system composed
exclusively of purified native and recombinant proteins, and,
therefore, free of contaminating proteases.
The inhibitory effect of AEBSF on NADPH oxidase activation exhibited a pronounced structural specificity. A number of derivatives of benzylamine and benzamidine, to which categories AEBSF is related, were shown to function as competitive serine protease inhibitors (43). The presence of a reactive sulfonyl fluoride group on the aromatic ring generates compounds capable of forming a covalent bond with their target enzymes (30, 44). We found that, among four compounds sharing an aminoethylbenzene moiety (AEBSF, AEBSAc, AEBSNH2, and MAEBSF), only AEBSF and MAEBSF, which possess a reactive sulfonyl fluoride group, were inhibitory. Among three amidinobenzene derivatives (benzamidine, pAPMSF, and pABSF) only pABSF, possessing a sulfonyl fluoride group adjacent to the benzene ring exhibited moderate NADPH oxidase inhibitory activity. PMSF and TPCK, two serine protease inhibitors bearing limited structural similarity to AEBSF, but lacking the basic amino group present in AEBSF, were found to be inactive in the cell-free system. We conclude that two structural elements are the minimal prerequisites for the possession of NADPH oxidase inhibitory property: a positively charged aminoalkyl moiety and a reactive sulfonyl fluoride group.
The search for the molecular target of AEBSF led to the conclusion that this is, most likely, cytochrome b559. This proposal is based principally on the finding that treatment of either total membrane fraction or purified and relipidated cytochrome b559 with AEBSF, in the presence of a critical concentration of LiDS, resulted in a dose-dependent reduction in their ability to cooperate with untreated components in the assembly of NADPH oxidase. Neither total cytosol nor any of the three cytosolic components were sensitive to AEBSF to a comparable degree. A remarkable feature of this effect is its absolute dependence on the simultaneous presence of an anionic amphiphile. The amphiphile may make the liposome-enclosed cytochrome b559 more accessible to AEBSF or may induce a conformational change in cytochrome b559 itself, normally associated with the process of activation, but also leading to exposure of an AEBSF-binding domain. Evidence for a direct effect of anionic amphiphiles, at NADPH oxidase activating concentrations, on cytochrome b559 was recently presented by the demonstration that arachidonate and SDS enhance interaction of cytochrome b559 with the NADPH oxidase inhibitory heme ligand, butyl isocyanide (45).
The results of systematic screening of the effect of varying the concentration of each of the three cytosolic components on the IC50 of AEBSF support the proposal that AEBSF prevents the binding of p47phox to cytochrome b559 and, consequently, interferes with the assembly of a functionally active NADPH oxidase complex. Such a mechanism is also in agreement with the Michaelis-Menten analysis of the inhibition of NADPH oxidase activation by AEBSF at various concentrations of p47phox. There is extensive evidence demonstrating interaction between p47phox and the small (46, 47) and large (48-50) subunits of cytochrome b559. Several groups also reported that, in p47phox, a region extending from residues 315 to 347, or part of it, represents a site of interaction with cytochrome b559 (51-53). This region is rich in basic residues, especially arginines, and it is possible that it forms electrostatic bonds with acidic residues on one of the two subunits of cytochrome b559.
However, the IC50 of AEBSF is also affected, albeit to a lesser degree, by the concentration of p67phox. This effect can be explained by assuming that p67phox binds to cytochrome b559 and that AEBSF interferes with this binding. There is, indeed, recent functional evidence for a direct interaction between p67phox and cytochrome b559 (54, 55). An alternative explanation could be provided by an indirect effect of varying the concentration of p67phox on the binding of p47phox to cytochrome b559. Thus, based on kinetic evidence, an enhancement of p47phox binding to cytochrome b559 by p67phox was demonstrated (56), and a marked reduction in the translocation of p47phox to the plasma membrane in neutrophils of p67phox-deficient patients, was recently reported (cited in Ref. 57). Our finding that AEBSF inhibits the activation-related translocation of both p47phox and p67phox to the membrane can be explained by either distinct effects on the binding of each of the two cytosolic components to cytochrome b559 or by an effect on the binding of one component affecting the binding of the other component.
When working as a protease inhibitor, AEBSF acts as a bifunctional reagent (30). The charged aminoethyl moiety acts as a substrate analogue, capable of forming ionic complexes with trypsin and related proteases because of its similarity to basic amino acids, whereas the chemically reactive sulfonyl fluoride group endows the inhibitor with the capacity of forming a stable covalent bond with the enzyme (30, 43, 44). We propose that AEBSF also acts as a bifunctional inhibitor of NADPH oxidase assembly, binding by the intermediary of its aminoethyl end, to an acidic domain on one of the subunits of cytochrome b559. This, essentially reversible step is followed by the formation of an irreversible bond, mediated by the sulfonyl fluoride group. The finding that, in the 0.5-2 mM range, AEBSF behaved as a competitive inhibitor, with respect to p47phox, but that this behavior was replaced by a noncompetitive pattern, at higher concentrations, suggests that the establishment of stable bonds becomes increasingly dominant with increasing concentrations of AEBSF. While this manuscript was under review, we became aware of a recent communication2 that AEBSF, under certain conditions (such as, at high concentrations), forms covalent adducts with proteins, other than serine proteases. These interactions appear to involve tyrosine and lysine residues and the free amino terminus.
Further exploration of the mechanism of inhibition of NADPH oxidase by AEBSF in particular and by aminoalkylbenzenesulfonyl fluorides in general is of both theoretical and practical relevance. Thus, it should lead to a better understanding of the molecular basis of the assembly of the NADPH oxidase complex and to the design of drugs to be used in circumstances associated with the undesired production of oxygen radicals. This latter aim, however, is dependent on the clarification of the relevance of effects found in the cell-free system to the situation in intact cells. Nevertheless, the fact that AEBSF is a stable, water-soluble and relatively nontoxic compound, possessing both NADPH oxidase suppressory and anti-proteolytic properties, makes it an interesting model compound for the design of antiinflammatory drugs.
We thank Dr. T. L. Leto (National Institutes of Health, Bethesda, MD) for providing baculoviruses carrying cDNAs for p47phox and p67phox and Rac1 plasmid in E. coli and S. Hanft for word processing.