Modulation of SCFβ-TrCP-dependent IκBα Ubiquitination by Hydrogen Peroxide*

Reactive oxygen species are known to participate in the regulation of intracellular signaling pathways, including activation of NF-κB. Recent studies have indicated that increases in intracellular concentrations of hydrogen peroxide (H2O2) have anti-inflammatory effects in neutrophils, including inhibition of the degradation of IκBα after TLR4 engagement. In the present experiments, we found that culture of lipopolysaccharide-stimulated neutrophils and HEK 293 cells with H2O2 resulted in diminished ubiquitination of IκBα and decreased SCFβ-TrCP ubiquitin ligase activity. Exposure of neutrophils or HEK 293 cells to H2O2 was associated with reduced binding between phosphorylated IκBα and SCFβ-TrCP but no change in the composition of the SCFβ-TrCP complex. Lipopolysaccharide-induced SCFβ-TrCP ubiquitin ligase activity as well as binding of β-TrCP to phosphorylated IκBα was decreased in the lungs of acatalasemic mice and mice treated with the catalase inhibitor aminotriazole, situations in which intracellular concentrations of H2O2 are increased. Exposure to H2O2 resulted in oxidative modification of cysteine residues in β-TrCP. Cysteine 308 in Blade 1 of the β-TrCP β-propeller region was found to be required for maximal binding between β-TrCP and phosphorylated IκBα. These findings suggest that the anti-inflammatory effects of H2O2 may result from its ability to decrease ubiquitination as well as subsequent degradation of IκBα through inhibiting the association between IκBα and SCFβ-TrCP.

chronic obstructive pulmonary disease, sepsis, and ischemiareperfusion injury, that are characterized by activation of neutrophils, macrophages, and other cell populations to release cytokines and other proinflammatory mediators, many of which are under the regulatory control of the transcription factor NF-B (2)(3)(4)(5). Although initial reports indicated that ROS, such as superoxide and hydrogen peroxide (H 2 O 2 ), exerted proinflammatory effects through activation of NF-B, more recent studies have shown that ROS are not only responsible for inducing inflammation but also can have potent anti-inflammatory properties. In particular, increases in intracellular concentrations of H 2 O 2 have been demonstrated to diminish TLR4 (Toll-like receptor 4)-induced activation of NF-B and production of proinflammatory cytokines in neutrophils, epithelial cells, and other cell populations (6 -8).
NF-B p50/p65 heterodimers are retained in the cytoplasm by binding to the inhibitory molecule IB␣ (inhibitor of NF-B) (9). However, in response to external stimuli, such as engagement of TLR4, IB␣ is degraded by a three-step process involving phosphorylation by IKK kinases, polyubiquitination by the SCF ␤-TrCP (Skp1-cullin-F-box/␤-transducin repeat-containing protein) complex, and degradation of ubiquitinated IB␣ by the 26 S proteasome, thereby exposing the nuclear localization sequence in NF-B and permitting translocation of NF-B to the nucleus (10 -12). Previous studies from our laboratory showed that exposure of LPS-stimulated neutrophils to H 2 O 2 was associated with diminished degradation of IB␣ and decreased translocation of NF-B to the nucleus, providing a potential mechanism for the anti-inflammatory properties of H 2 O 2 . Increased intracellular concentrations of H 2 O 2 did not affect LPS-induced activation of IKK or phosphorylation of IB␣ (13). Although H 2 O 2 has been shown to diminish 26 S proteasome activity, at least in part through oxidative modification and S-glutathionylation of the Rpn2 regulatory particle (14), the extent of stabilization of IB␣ in LPS-stimulated neutrophils cultured with H 2 O 2 was greater than that produced by treatment with MG132, a specific inhibitor of the 26 S proteasome (6). Such results suggest that H 2 O 2 may affect additional processes, such as the ubiquitination of IB␣, which are involved in targeting IB␣ for degradation by the 26 S proteasome and which contribute to the activation of NF-B (15)(16)(17).
In the present experiments, we examined the ability of H 2 O 2 to modulate IB␣ ubiquitination and SCF ␤-TrCP activity after TLR4 engagement. We found that exposure to H 2 O 2 or inhibition of catalase, a situation associated with increased intracellular concentrations of H 2 O 2 (6,13), resulted in diminished association of SCF ␤-TrCP with IB␣ as well as decreased ubiquitination of IB␣.

EXPERIMENTAL PROCEDURES
Mice-Male C57BL/6, C3HeB/FeJ, or acatalasemic C3Ga. Cg-Cat B/J mice, 8 -12 weeks of age, were purchased from Jackson Laboratory (Bar Harbor, ME). The mice were kept on a 12-h/12-h light/dark cycle with free access to food and water. All experiments were conducted in accordance with institutional review board-approved protocols (University of Alabama at Birmingham Institutional Animal Care and Use Committee).
Neutrophil Isolation and Culture-Bone marrow neutrophils were isolated as described previously (6,25,26). Neutrophil purity was consistently Ͼ97%, as determined by Wright-Giemsa-stained cytospin preparations. Neutrophils were cultured in RPMI 1640 medium containing 0.5% fetal bovine serum and treated as indicated in the figure legends. Neutrophil viability as determined by trypan blue staining was consistently Ͼ95%.
Acute Lung Injury Model-Acute lung injury was induced by intratracheal administration of 1 mg/kg LPS in 50 l of phosphate-buffered saline as described previously (8,13,27,28). Briefly, mice were anesthetized with isoflurane and then suspended by their upper incisors on a 60°incline board. The tongue was then gently extended, and LPS solution was deposited into the pharynx (8,25,29). Mice were pretreated with saline or ATZ (500 mg/kg body weight dissolved in 0.9% saline) intraperitoneally, and 4 h later, LPS (1 mg/kg) was administered intratracheally. Lungs were harvested 24 h after LPS administration.
Construction of Expression Plasmids and Recombinant Protein Expression-A full-length human ␤-TrCP cDNA was purchased from Open Biosystems and cloned into 3ϫFLAG-CMV10 (Sigma) for mammalian expression. Four FLAGtagged point mutant constructs of ␤-TrCP-C308A (MB1), C348A (MB2), C471A (MB5), and C511A (MB6) were generated using PCR mutagenesis. An IKK␤ cDNA containing N-terminal amino acids 1-420 was obtained from Open Biosystems and cloned into 3ϫFLAG-CMV10. Full-length human Roc1, Skp1, and UBCH3/Cdc34 (Open Biosystems) were cloned into pcDNA-Myc vector for mammalian expression as Myc-tagged proteins. A full-length cDNA for ␤-catenin was purchased from Open Biosystems. The IB␣ construct in pET15b was kindly provided by Dr. Gourisankar Ghosh (University of California, San Diego, La Jolla, CA). IB␣ and ␤-catenin were cloned into pGEX vector (GE Healthcare) for bacterial expression as N-terminal GST fusion proteins. GST-tagged recombinant proteins were purified using glutathione-Sepharose (GE Healthcare).
In Vitro Phosphorylation of IB␣ and ␤-Catenin-Phosphorylation of IB␣ or ␤-catenin was performed using 2 g of GSTtagged substrate protein, 50 ng of IKK␤ (Cell Signaling, Danvers, MA), or GSK3␤ (SignalChem, Richmond, Canada), in 50 l of 1ϫ kinase buffer (Cell Signaling) and 2 mM ATP for 1 h at room temperature. The phosphorylated products were stored at Ϫ80°C until used.
In Vitro Ubiquitination Assay-Cultured cells and neutrophils were lysed, or lungs of mice were homogenized in lysis buffer consisting of 50 mM Tris, pH 8.0, 5 mM EDTA, 150 mM NaCl, 10 mM NaF, 2 mM Na 3 VO 4 , protease inhibitor mixture (1:100, v/v) (Sigma), and 0.5% Nonidet P-40. Protein concentrations were determined using Bradford's reagent (Bio-Rad). To immunoprecipitate SCF ␤-TrCP , 1 mg of cell lysates or lung homogenates was incubated with anti-cullin-1 mouse monoclonal antibody in 1 ml of lysis buffer containing 5% glycerol for 2 h, followed by the addition of 30 l of Protein A-Sepharose beads (Sigma) and incubation overnight at 4°C with continuous stirring. The beads were then washed three times with lysis buffer and twice with ligase buffer (50 mM Tris, pH 7.5, 25 mM MgCl 2 , 2 mM Na 3 VO 4 , 10 mM NaF). Ubiquitination was performed with the washed beads resuspended in 30 l of solution containing 100 ng of E1-GST (Boston Biochem, Cambridge, MA), 500 ng of His-tagged UbcH3 (Boston Biochem), 1 g of ubiquitin-FLAG (BioMol, Plymouth Meeting, PA), Energy Regeneration Solution (Boston Biochem) (a mixture that contains MgCl 2 , ATP, and ATP-regenerating enzymes to recycle hydrolyzed ATP (i.e. AMP and ADP to ATP)), 100 M ATP, and 200 ng of phosphorylated substrate for 1 h at 30°C. The reaction was then stopped by adding SDS-PAGE loading buffer, followed by boiling the samples for 15 min, and resolution on 6% SDS-PAGE. Ubiquitination of substrates was detected by either anti-ubiquitin antibodies or antibodies to IB␣ or ␤-catenin.
GST Pull-down of ␤-TrCP Using Phospho-GST-IB␣ or Phospho-GST-␤-Catenin-Phosphorylated GST-IB␣ or phosphorylated GST-␤-catenin (500 ng) was added to 1 mg of protein obtained from cell lysates or lung extracts in 1 ml of lysis buffer containing 5% glycerol. Glutathione-Sepharose beads were added, and the mixture was incubated at 4°C with continuous stirring for 1 h. The beads were washed three times with lysis buffer, and IB␣-or ␤-catenin-bound ␤-TrCP was analyzed by immunoblotting with specific antibodies to FLAG or ␤-TrCP.
Cytokine Enzyme-linked Immunosorbent Assay-Levels of tumor necrosis factor-␣ from neutrophils into culture media were determined using commercially available enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN), according to the manufacturer's instructions and as previously described (7,8,13,34).
Labeling of ␤-TrCP Free Cysteine Thiols-The extent of free (unoxidized) cysteine residues within ␤-TrCP was determined using the biotinylated iodoacetyl ethylenediamine (BIAM)-labeling assay (14, 39 -42). Briefly, cell lysates (0.5 mg/ml) obtained from HEK 293 cells that transiently expressed ␤-TrCP-FLAG were incubated with BIAM (100 M) for 30 min at room temperature, and then excess BIAM was removed by passing the extracts through Bio-Gel P10. Next, BIAM-protein conjugates were precipitated with streptavidin-agarose for 1 h at 4°C. Samples were washed four times with lysis buffer containing 0.05% SDS and then subjected to reducing SDS-PAGE and Western blot analysis with antibodies to ␤-TrCP or FLAG peptide. Cells were also directly incubated with BIAM (200 M) for 20 min, and then cell lysates were passed through Bio-Gel P10 to remove excess BIAM. The amount of BIAM-␤-TrCP adduct formation was determined using streptavidin-agarose pull-down followed by Western blot analysis with antibodies specific to ␤-TrCP or FLAG peptide.
Statistical Analyses-For each experiment, neutrophils were isolated and pooled from groups of mice (n ϭ 3-4), and all conditions were studied at the same time. Data are presented as means Ϯ S.D. for each experimental group. One-way analysis of variance, the Tukey-Kramer multiple comparison test (for multiple groups), or Student's t test (for comparisons between two groups) were used. p Ͻ 0.05 was considered significant.

Inhibitory Effects of Hydrogen Peroxide on TLR4-induced IB␣ Degradation-
In recent studies, we demonstrated that increased intracellular levels of H 2 O 2 were associated with diminished degradation of IB␣ and reduced 26 S proteasomal activity in LPS-stimulated neutrophils (6,8). As shown in Fig. 1, A-C, whereas exposure of LPS-stimulated neutrophils or 293-hTLR4/MD2-CD14 cells to H 2 O 2 resulted in diminished degradation of IB␣, the extent of IB␣ stabilization in H 2 O 2treated cells was greater than that found after blockade of 26 S proteasomal function with MG132 in the absence of H 2 O 2 . These results suggested that the mechanisms by which H 2 O 2 prevents IB␣ degradation may extend beyond proteasomal inhibition.
Because phosphorylation of IB␣ is required for its polyubiquitination and subsequent degradation by the 26 S proteasome, we hypothesized that a mechanism by which H 2 O 2 might diminish TLR4-induced degradation of IB␣ was through inhibiting IB␣ phosphorylation. To examine this possibility, 293-hTLR4/MD2-CD14 cells were stimulated with LPS in the presence or absence of H 2 O 2 . As shown in Fig. 1C, H 2 O 2 exposure appeared to have no effect on LPS-induced phosphorylation of IB␣. Although exposure to LPS or LPS and H 2 O 2 both resulted in enhanced phosphorylation of IB␣, the amount of total IB␣ decreased in LPS-treated cells but not in those treated with both LPS and H 2 O 2 (Fig. 1D). Next, we examined the effects of H 2 O 2 and MG132 on 26 S proteasomal activity. As shown in Fig. 1E, incubation of HEK 293 cells with MG132 resulted in inhibition of trypsin-like and chymotrypsin-like proteasomal activity; such inhibition of 26 S proteasomal function was more pronounced in cells treated with MG132 as compared with that found in cells treated with H 2 O 2 alone (Fig. 1E). As shown in Fig. 1F, preincubation with H 2 O 2 or MG-132 had inhibitory effects on the release of tumor necrosis factor-␣ from LPS-stimulated neutrophils.
The experiments shown in Fig. 1, A-D, indicate that although H 2 O 2 appeared to be a less potent inhibitor of 26 S proteasomal activity than was MG132, exposure of LPS-treated cells to H 2 O 2 appeared to stabilize IB␣ levels to a greater extent than did MG132. These results suggest that in addition to its inhibitory actions on the 26 S proteasome, H 2 O 2 also may affect upstream events that result in IB␣ stabilization, such as through modulating IB␣ ubiquitination.
Effects of H 2 O 2 on IB␣ Ubiquitination by SCF ␤-TrCP -Ubiquitination of IB␣ occurs after TLR4 engagement (43) and is required for the processing and degradation of IB␣ by the 26 S proteasome. As shown in Fig. 2, A and B, exposure of neutrophils or 293-hTLR4/MD2-CD14 cells to LPS resulted in increased ubiquitination of IB␣, an effect that was enhanced by blockade of 26 S proteasomal activity following the addition of the proteasomal inhibitor MG132 to the cultures. In contrast, LPS-induced ubiquitination of IB␣ was diminished in a concentration-dependent manner after exposure of cells to The E3 ubiquitin ligase SCF ␤-TrCP is responsible for the ubiquitination of IB␣ (15,44,45). Therefore, given the diminished ubiquitination of IB␣ in H 2 O 2 -exposed cells, it seemed possible that H 2 O 2 might affect SCF ␤-TrCP activity. As shown in Fig.  2, C and D, SCF ␤-TrCP isolated from neutrophils or 293-hTLR4/ MD2-CD14 cells cultured with H 2 O 2 was less able to ubiquitinate IB␣ than was SCF ␤-TrCP purified from cells that had not been exposed to H 2 O 2 .
In initial experiments to determine the mechanism for the inhibitory actions of H 2 O 2 on SCF ␤-TrCP ubiquitin ligase activity, we examined the effect of H 2 O 2 on cullin-1 neddylation, a process that has been shown to result in enhanced SCF ␤-TrCP activity (45). Despite the reduction of SCF ␤-TrCP activity in H 2 O 2 -exposed cells, incubation of HEK 293 cells with H 2 O 2 resulted in increased cullin-1 neddylation (Fig. 3A). Such results indicate that the ability of H 2 O 2 to diminish SCF ␤-TrCP activity was not due to inhibition of cullin-1 neddylation.
Because the H 2 O 2 -induced decrease in SCF ␤-TrCP ubiquitin ligase activity might be due to alterations in the composition of the SCF ␤-TrCP complex, we determined binding of ␤-TrCP to Skp1, cullin-1 to Skp1, and cullin-1 to Roc1 in HEK 293 cells that were transfected with ␤-TrCP-FLAG and Skp1-Myc, cullin-1-FLAG and Skp1-Myc, or cullin-1-Flag and Roc 1-Myc and then exposed or not to increasing concentrations of H 2 O 2 . As shown in Fig. 3, B, C, and D, H 2 O 2 did not appear to affect binding of Skp1 to cullin-1, ␤-TrCP to Skp1, or cullin-1 to Roc1. These data suggest that alterations in the composition of SCF ␤-TrCP are not responsible for the ability of H 2 O 2 to inhibit its ubiquitin ligase activity.
Because H 2 O 2 does not affect SCF ␤-TrCP complex formation, we next examined the effect of H 2 O 2 on the binding affinity of SCF ␤-TrCP to phosphorylated IB␣. Within the SCF ␤-TrCP complex, the F-box protein ␤-TrCP is responsible for substrate acquisition, including binding to phosphorylated IB␣ and ␤-catenin (18,24). For these experiments, we transfected 293-hTLR4/MD2-CD14 cells with IB␣-Myc, ␤-TrCP-FLAG, and IKK␤-FLAG constructs and then treated the cells with increasing doses of H 2 O 2 followed by immunoprecipitation of IB␣.
Enhanced Intracellular H 2 O 2 Concentrations in Vivo Are Associated with Diminished SCF ␤-TrCP Activity and Association with IB␣-Intracellular concentrations of H 2 O 2 are increased in acatalasemic mice and in mice treated with ATZ, a specific inhibitor of catalase (13). These conditions therefore permit exploration of the effects of increased intracellular H 2 O 2 on SCF ␤-TrCP activity under in vivo conditions. As shown in Figs. 5 and 6, SCF ␤-TrCP isolated from the lungs of acatalasemic mice or mice treated with ATZ is less able to ubiquitinate IB␣ and also demonstrates diminished binding to phosphorylated IB␣ as compared with SCF ␤-TrCP from control mice (Figs. 5B and 6D). In addition, intratracheal administration of LPS resulted in enhanced ubiquitination of IB␣ in the lungs of control mice but not in the lungs of mice treated with ATZ before LPS exposure (Fig. 6A). Although SCF ␤-TrCP isolated from the lungs of LPS-exposed mice showed increased ability to ubiquitinate either phosphorylated IB␣ or phosphorylated ␤-catenin, there was no increase in ubiquitin ligase activity of SCF ␤-TrCP that had been purified from mice treated with ATZ before LPS exposure (Fig. 6, B and C). These results are consistent with the ability of increased intracellular H 2 O 2 levels to inhibit SCF ␤-TrCP activity and decrease IB␣ turnover even under basal conditions (Figs. 5A and 6D).
Cysteine Thiols of ␤-TrCP Undergo Oxidative Modification after Exposure to H 2 O 2 -Reactive oxygen species (and particularly H 2 O 2 ) can affect intracellular signaling through posttranslational protein modification and particularly through oxidation of cysteine thiols (1,42,46,47). To determine if cellular exposure to H 2 O 2 produces modification of cysteines in ␤-TrCP, we labeled free, unmodified cysteine residues by the addition of BIAM to cells incubated with increasing concentrations of H 2 O 2 . As shown Fig. 7A, the number of unaltered cysteines in ␤-TrCP available for BIAM-cysteine adduct formation was decreased in a dose-dependent manner by cellular incubation with H 2 O 2 , a situation associated with increased intracellular concentrations of H 2 O 2 and other reactive oxygen species (supplemental Fig. 1). Similarly, incubation of cell extracts with H 2 O 2 resulted in diminished BIAM association with ␤-TrCP, consistent with H 2 O 2 -dependent oxidative modification of cysteine residues in ␤-TrCP (Fig. 7B).

Cysteine 308 in the Blade 1 ␤-TrCP ␤-Propeller Region Is Required for Binding between ␤-TrCP and Phosphorylated
IB␣-␤-TrCP contains a total of 25 cysteine residues. The binding site of ␤-TrCP to phosphorylated substrates, including phosphorylated IB␣, has been localized to the top face of the WD40 domain of ␤-TrCP (19, 48 -50). There are a total of 12 cysteines in the seven blades of the ␤-TrCP WD40 ␤-propeller region that are likely to interact with phosphorylated substrates, including phosphorylated IB␣; of these 12 cysteines, only four are likely to exist as thiolate anions as a result of being close to positively charged amino acids (i.e. Cys-Leu in Blade 1, Leu-Cys-Leu in Blade 2, Arg-Cys-Leu in Blade 5, and Arg-Cys-X-Arg in Blade 6) (Fig. 8A). These four cysteines in Blades 1, 2, 5, and 6 are therefore potentially more vulnerable to oxidation by H 2 O 2 and were targets for site-directed mutagenesis in ␤-TrCP. In these experiments, Cys 3 Ala point mutations in ␤-TrCP were generated in the targeted cysteines in Blades 1, 2, 5, and 6, and then the FLAG-tagged mutants or wild type ␤-TrCP were expressed in HEK 293 cells.
As shown in Fig. 8B, mutation of cysteine 308 in Blade 1 of ␤-TrCP to alanine reduces binding of phosphorylated IB␣ with ␤-TrCP, whereas mutation of cysteine 348 in Blade 2, cysteine 471 in Blade 5, or cysteine 511 in Blade 6 in the WD40 domain of ␤-TrCP did not affect binding between ␤-TrCP and phosphorylated IB␣. These results demonstrate that cysteine 308 in Blade 1 of ␤-TrCP participates in binding between ␤-TrCP and phosphorylated IB␣. Of note, whereas exposure of cells to H 2 O 2 increased intracellular oxidation of DCF-DA, an indicator of intracellular ROS formation, transient expression or ␤-TrCP or exposure to LPS had no effects on DCF-DA oxidation (supplemental Fig. 1).

DISCUSSION
Previous studies have shown that increased intracellular concentrations of H 2 O 2 exert anti-inflammatory effects on TLR4 induced neutrophil activation (51,52). Exposure of neutrophils to H 2 O 2 stabilizes cytoplasmic concentrations of IB␣, both in resting cells and after TLR4 engagement, and also diminishes LPS-induced nuclear translocation of NF-B (6 -8, 13). Although initial experiments with HeLa cells suggested that exposure to H 2 O 2 enhanced nuclear translocation of NF-B, subsequent studies (51,(53)(54)(55)(56) in other cell populations found the opposite effect, with H 2 O 2 inhibiting NF-B activation. Such disparate findings suggested that the role of H 2 O 2 in affecting pathways relating to the activation of NF-B is likely to be cell type-specific. Several potential mechanisms for the ability of H 2 O 2 to inhibit degradation of IB␣ and to diminish activation of NF-B have been proposed (6,13,(57)(58)(59). Phosphorylation of serine 32 and 36 in IB␣ by the IKK complex is required to initiate the subsequent ubiquitination and degradation of IB␣ in the 26 S proteasome (60 -62). Although previous studies in C10 and aortic smooth muscle cells demonstrated that H 2 O 2 treatment resulted in inhibition of IKK␤ (52), we did not find any effects of H 2 O 2 exposure on IKK activity in neutrophils (7,51). Similarly, the present studies did not demonstrate any alterations in the phosphorylation of IB␣ after incubation of 293-hTLR4/MD2-CD14 cells with H 2 O 2 . Our laboratory and others have demonstrated that increased intracellular levels of H 2 O 2 result in inhibition of 26 S proteasomal function in neutrophils or other cell populations (6,63). If inhibition of the 26 S proteasome was the primary mechanism leading to stabilization of intracellular levels of IB␣ in H 2 O 2treated cells, then one would also expect to find increased concentrations of polyubiquitinated IB␣ after H 2 O 2 exposure. However, as shown in the present experiments, incubation of LPS-stimulated neutrophils or 293-hTLR4/MD2-CD14 cells with H 2 O 2 resulted in diminished ubiquitination of IB␣, consistent with inhibition of ubiquitin ligase activity. Our studies also found that exposure of neutrophils or HEK 293 cells to H 2 O 2 resulted in inhibition of the activity of SCF ␤-TrCP , the specific E3 ubiquitin ligase responsible for ubiquitination of IB␣, thereby providing a mechanism that may explain the ability of increased intracellular concentrations of H 2 O 2 to prevent degradation of IB␣, activation of NF-B, and expression of NF-B proinflammatory genes, such as tumor necrosis factor-␣, after TLR4 engagement (Fig. 9).
Ubiquitination of target proteins is a multistep process. After activation by an E1 enzyme, ubiquitin is transferred to an active cystine of a ubiquitin-conjugating enzyme (E2). A ubiquitin ligase (E3) then transfers ubiquitin from the E2 ubiquitin-conjugating enzyme to the target protein either by forming an E3-ubiquitin thioester intermediate in the case of HECT E3 ubiquitin ligases or by facilitating the transfer of ubiquitin directly from the E2 to the substrate for RING finger E3 ubiquitin ligases (60,61). SCF ␤-TrCP , a RING finger E3, appears to be specific for ubiquitination of phosphorylated IB␣ as well as for phosphorylated ␤-catenin (11,24,64,65). SCF ␤-TrCP includes several structural and functional components: ␤-TrCP, a F-box protein that binds to phosphorylated IB␣ and ␤-catenin; the adapter protein SKP1, C and D, SCF ␤-TrCP complex was immunoprecipitated (IP) from lung homogenates of control or acatalasemic mice using anti-cullin-1 antibodies followed by measurement of IB␣ ubiquitination in vitro. Representative Western blots using anti-phospho-IB␣ antibodies show the amounts of Ub n -IB␣ (C). In C, lung extracts from control and acatalasemic mice were incubated with recombinant phospho-IB␣-GST followed by pull-down with glutathione-Sepharose and Western blotting with anti-␤-TrCP antibodies. The amounts of ␤-TrCP in the lung extracts as well as recombinant phospho-IB␣-GST added to lung extracts (input) are shown. Input indicates the levels of ␤-TrCP in lung homogenates obtained from control or acatalasemic mice. Similar results were obtained from three independent experiments. FIGURE 6. SCF ␤-TrCP activity is diminished in the lungs of mice treated with aminotriazole. A-E, C57BL/6 mice were treated intraperitoneally with saline or the catalase inhibitor ATZ and then 4 h later were given LPS (1 mg/kg) intratracheally. Lung homogenates were obtained 2 h after LPS administration. In A, lung homogenates were subjected to immunoprecipitation (IP) with anti-Ub antibodies, and Ub n -IB␣ conjugates were determined using Western blots (WB) developed with anti-phospho-IB␣ antibodies. B and C, SCF ␤-TrCP complexes were immunoprecipitated from lung homogenates of control or ATZ-treated mice with anti-cullin-1 antibodies followed by determination of the ability of the precipitated SCF ␤-TrCP to ubiquitinate phospho-GST-IB␣ or phospho-␤-catenin in vitro. D, representative Western blots are shown as well as mean optical band density of the levels of IB␣ obtained from lung homogenates of control and mice treated with ATZ (means Ϯ S.D., n ϭ 3 per each group of mice, **p Ͻ 0.01). E, lung homogenates were incubated with recombinant phospho-IB␣-GST and then precipitated with glutathione-Sepharose. The amounts of ␤-TrCP in the lung extracts as well as recombinant phospho-IB␣-GST added to lung extracts (input) are shown. Representative Western blots are shown. Two additional experiments provided similar results.
In the present experiments, we found that exposure of 293-hTLR4/MD2-CD14 cells to H 2 O 2 was associated with diminished activity of SCF ␤-TrCP . Because phosphorylated IB␣ was used in ubiquitination assays containing purified E1 and E2, the decreased ubiquitination of IB␣ found with SCF ␤-TrCP immu-noprecipitated from H 2 O 2 -treated cells must be due to decreased ubiquitin ligase activity of SCF ␤-TrCP and not to diminished phosphorylation of the substrate, as might occur through oxidation-induced changes in IB␣ or inhibition of IKK or to inactivation of the E1 or E2 enzymes through modification of cysteine or other amino acids by H 2 O 2 .
Neddylation of cullin-1 is involved in SCF ␤-TrCP complex assembly and enhances its ubiquitin ligase activity (45,66). A previous study showed that H 2 O 2 can diminish SCF ␤-TrCP ubiquitin ligase activity by inhibiting the neddylation of cullin-1 (67). Contrary to these previous findings, we found that cullin-1 neddylation is not affected in H 2 O 2 -treated cells although such conditions result in decreased activity of SCF ␤-TrCP . Such results indicate that the inhibitory effects of H 2 O 2 on SCF ␤-TrCP activity are not due to modulation of cullin-1 neddylation.
A potential mechanism for the inhibitory effect of H 2 O 2 on IB␣ ubiquitination may be through alteration in SCF ␤-TrCP complex formation. The results of the present experiments indicate that H 2 O 2 exposure does not affect SCF ␤-TrCP complex composition but does result in diminished binding between SCF ␤-TrCP and phosphorylated IB␣. Because binding of phosphorylated IB␣ to ␤-TrCP is required for ubiquitination of IB␣ by the SCF ␤-TrCP complex, the H 2 O 2 -induced inhibition of interaction between IB␣ and SCF ␤-TrCP provides a potential mechanism for the ability of H 2 O 2 to inhibit IB␣ ubiquitination. Because cysteine thiol modification is a common mechanism by which H 2 O 2 modulates protein function (46,68), it is possible that cysteines in ␤-TrCP that are critical for binding IB␣ are oxidized in H 2 O 2exposed cells. In these studies, exposure of ␤-TrCP to H 2 O 2 resulted in diminished BIAM adduct formation, consistent with oxidative modification of cysteine residues in ␤-TrCP.
Using site-directed mutagenesis of specific cysteines in the ␤-TrCP WD40 region that are likely to interact with phosphorylated IB␣, we found that Cys-308 in Blade 1 of the ␤-TrCP ␤-propeller region is required for optimal binding between phosphorylated IB␣ and ␤-TrCP and is likely to be involved in  the H 2 O 2 -induced reduction in binding between SCF ␤-TrCP and phosphorylated IB␣. These results are consistent with the previously reported findings that alkylation of cysteine thiols of ␤-TrCP by N-ethylmaleimide significantly diminished ubiquitination of IB␣, whereas alkylation of cullin-1 and Roc1 with N-ethylmaleimide had no effect on IB␣ ubiquitination (20).
Exposure of the lungs to LPS during Gram-negative pneumonia, during systemic Gram-negative infections, or through environmental factors produces acute inflammatory changes associated with neutrophil migration into the pulmonary parenchyma and airways, release of reactive oxygen species and proinflammatory cytokines, and the development of lung injury (69 -71). Catalase facilitates the conversion of H 2 O 2 to H 2 O and O 2 , and intracellular H 2 O 2 concentrations are increased in neutrophils from acatalasemic mice or mice treated with aminotriazole, an inhibitor of catalase, reflecting the importance of catalase in regulating oxidant balance. There is diminished severity of LPS-induced pulmonary inflammation and lung injury in mice that are acatalasemic or treated with aminotriazole (13). The present experiments, showing diminished SCF ␤-TrCP ubiquitin ligase activity in the lungs of aminotriazole-treated mice and of acatalasemic mice, provide a potential mechanism for the protective effects of catalase inhibition or its absence on LPS-associated lung injury. In particular, similar to the findings in H 2 O 2 -treated HEK 293 cells, there was decreased binding between phosphorylated IB␣ and SCF ␤-TrCP immunoprecipitated from the lungs of aminotriazole-treated and acatalasemic mice as well as diminished ability of SCF ␤-TrCP to ubiquitinate phosphorylated IB␣ or a second substrate, phosphorylated ␤-catenin. These results show that the inhibitory effects of H 2 O 2 on SCF ␤-TrCP ubiquitin ligase activity are not specific for IB␣ but rather reflect a generic decrease of its ability to associate with and ubiquitinate its substrates.
The present findings, showing that H 2 O 2 inhibits SCF ␤-TrCP ubiquitin ligase activity through diminishing association with its substrates, including phosphorylated IB␣, provide a novel mechanism for the anti-inflammatory actions of H 2 O 2 ( Fig. 9) Although our results indicate that H 2 O 2 can diminish association between phosphorylated IB␣ and ␤-TrCP, it is also possible that H 2 O 2 affects additional functions of the SCF ␤-TrCP complex, such as coordination of Cdc34-dependent transfer of ubiquitin to IB␣. Although oxidation of cysteine thiols by H 2 O 2 has been shown to participate in regulating the activity of kinases, such as IKK and Akt, as well as transcriptional factors, such as the p50 subunit of NF-B, that are involved in inflammatory processes, there were no previous data demonstrating that H 2 O 2 or other ROS could affect ubiquitination, an important regulatory pathway in physiologic and pathophysiologic processes. Although our experiments focused on interactions between H 2 O 2 , IB␣, and SCF ␤-TrCP , it is possible that H 2 O 2 may also participate in affecting the function of other ubiquitin ligases, providing an expanded role for ROS as participants in modulating cellular regulation and activation.
Acknowledgment-We thank Dr. Jack Lancaster, Jr. for helpful advice. FIGURE 9. Proposed mechanism for the ability of H 2 O 2 to inhibit LPS-induced IB␣ degradation and NF-B activation. 1, in unstimulated cells, IB␣ is bound to the p50/p65 NF-B heterodimeric complex, thereby preventing nuclear translocation of NF-B. 2, engagement of TLR4 by LPS leads to IKK-dependent phosphorylation of IB␣ that then associates with and is ubiquitinated by the SCF ␤-TrCP complex. Ubiquitinated IB␣ is targeted for degradation by the 26 S proteasome (3), followed by nuclear translocation of NF-B and enhanced transcription of NF-B-dependent genes (4). Increases in the intracellular level of H 2 O 2 suppress ubiquitination of phosphorylated IB␣ through inhibiting association with ␤-TrCP and the SCF ␤-TrCP complex (2) and also inhibits degradation of ubiquitinated IB␣ by diminishing activity of the 26 S proteasome (3). Inhibition of IB␣ ubiquitination and proteasomal degradation results in maintenance of cytoplasmic levels of IB␣ and prevention of nuclear translocation of NF-B.