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J Biol Chem, Vol. 274, Issue 34, 23787-23793, August 20, 1999


4-Hydroxy-2-nonenal-mediated Impairment of Intracellular Proteolysis during Oxidative Stress
IDENTIFICATION OF PROTEASOMES AS TARGET MOLECULES*

Kunihiko Okada, Chantima Wangpoengtrakul, Toshihiko Osawa, Shinya ToyokuniDagger , Keiji Tanaka§, and Koji Uchida

From the Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, the Dagger  Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, and the § Tokyo Metropolitan Institute of Medical Science, 18-22, Honkomagome 3-chome, Bunkyo-ku, Tokyo 113, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oxidative stress is associated with important pathophysiological events in a variety of diseases. It has been postulated that free radicals and lipid peroxidation products generated during the process may be responsible for these effects because of their ability to damage cellular components such as membranes, proteins, and DNA. In the present study, we provide evidence that oxidative stress causes a transient impairment of intracellular proteolysis via covalent binding of 4-hydroxy-2-nonenal (HNE), a major end product of lipid peroxidation, to proteasomes. A single intraperitoneal treatment with the renal carcinogen, ferric nitrilotriacetate, caused oxidative stress, as monitored by accumulation of lipid peroxidation products and 8-hydroxy-2'-deoxyguanosine, in the kidney of mice. In addition, transient accumulation of HNE-modified proteins in the kidney was also found by competitive enzyme-linked immunosorbent assay and immunohistochemical analyses. This and the observation that the HNE-modified proteins were significantly ubiquitinated suggested a crucial role of proteasomes in the metabolism of HNE-modified proteins. In vitro incubation of the kidney homogenates with HNE indeed resulted in a transient accumulation of HNE-modified proteins, whereas the proteasome inhibitor significantly suppressed the time-dependent elimination of HNE-modified proteins. We found that, among three proteolytic activities (trypsin, chymotrypsin, and peptidylglutamyl peptide hydrolase activities) of proteasomes, both trypsin and peptidylglutamyl peptide hydrolase activities in the kidney were transiently diminished in accordance with the accumulation of HNE-modified proteins during oxidative stress. The loss of proteasome activities was partially ascribed to the direct attachment of HNE to the protein, based on the detection of HNE-proteasome conjugates by an immunoprecipitation technique. These results suggest that HNE may contribute to the enhanced accumulation of oxidatively modified proteins via an impairment of ubiquitin/proteasome-dependent intracellular proteolysis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Several lines of evidence indicate that oxidative stress may play an important role in various pathological states including cancer, neurodegeneration, atherosclerosis, diabetes, cancer, and rheumatoid arthritis, as well as in drug-associated toxicity, post-ischemic reoxygenation injury, and aging (1). Oxidative stress is one of the mechanisms that contribute to structural changes or misfolding of proteins. It has been proposed that the reactive oxygen species and lipid peroxidation products resulting from episodes of oxidative stress promote the modification of cellular proteins (2). The extent of accumulation of oxidatively modified proteins depends on both the rate of production and the efficiency of removal of the modified proteins (3). It is known that, in most cells, intracellular proteolytic enzymes selectively degrade the oxidatively modified proteins (4-6). Proteolytic capabilities are therefore considered to be secondary defense systems that can avert or delay the accumulation of altered proteins (3, 7, 8).

There is increasing evidence that aldehydes produced during lipid peroxidation reactions are causally involved in many of the pathophysiological effects associated with oxidative stress in cells and tissues. Aldehydes are often considered to be end products in lipid peroxidation; however, they are still reactive with various biomolecules, such as proteins and phospholipids, generating stable products at the end of a series of lipid peroxidation reactions (9). Among the aldehydes that originate from the peroxidation of cellular membrane lipids, 4-hydroxy-2-nonenal (HNE)1 is believed to be largely responsible for the cytopathological effects observed during oxidative stress in vivo (9). HNE exhibits a wide range of biological activities, including inhibition of protein and DNA synthesis, inactivation of enzymes, stimulation of phospholipase C, reduction of gap junction communication, and stimulation of neutrophil migration. Many of these in vitro effects, which are observed at low micromolar or even submicromolar concentrations of HNE, have been attributed to the modification of cellular proteins by HNE. HNE protein adducts have been detected in various human tissue samples, including atherosclerotic lesions (10), nigral neurons in Parkinson's disease (11), renal cell carcinomas (12), amyloid deposits in systemic amyloidosis (13) and Alzheimer's disease (14, 15), and trophoblast cells of pre-eclamptic placentas (16).

An iron chelate, ferric nitrilotriacetate (Fe3+-NTA), induces acute renal proximal tubular necrosis, a consequence of free radical-mediated oxidative tissue damage, that eventually leads to a high incidence of renal adenocarcinoma in rodents (17-19). In the present study, we investigated the free radical-induced oxidative stress in this carcinogenesis model, focusing on the turnover of HNE-modified proteins, and we demonstrated that the removal of HNE-modified proteins is ubiquitin/proteasome-dependent.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Animals-- Male ddY mice (Shizuoka Laboratory Animal Center, Shizuoka, Japan), weighing 25-35 g (6 weeks of age) were used. They were kept in a plastic cage and given commercial chow (CE-2) as well as deionized water (Millipore Japan, Osaka) ad libitum. The animals were housed in a room with a temperature of 23 ± 2 °C and a 12/12-h light/dark cycle. Forty-two animals were divided into time course study groups. In the time course study, mice received a single intraperitoneal injection of Fe3+-NTA. They were sacrificed at 0, 1, 3, 6, 16, 24, and 48 h after the administration. Each subgroup contained 6 animals.

Materials-- Ferric nitrate enneahydrate and sodium carbonate were from Wako (Osaka); nitrilotriacetic acid disodium salt was from Nacalai Tesque, Inc. (Kyoto, Japan). The anti-ubiquitin polyclonal antibody was obtained from Biomeda Co. (Foster City, CA), and 20 S proteasome polyclonal antibody was generated by Matthews et al. (20). Horseradish peroxidase-linked anti-rabbit IgG or anti-mouse IgG and enhanced chemiluminescence (ECL) and Western blotting detection reagents were obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). Protein A-Sepharose 4 Fast Flow was obtained from Amersham Pharmacia Biotech. Protein concentration was measured using the BCA protein assay reagent obtained from Pierce. Succinyl-leucine-leucine-valine-tyrosine-MCA (s-LLVY-MCA) for the chymotrypsin activity and butoxycarbonyl-leucine-serine-threonine-arginine-MCA (Boc-LSTR-MCA) for the trypsin activity of proteasome and the proteasome inhibitor (benzyloxycarbonyl-leucine-leucine-leucinal) were obtained from the Peptide Institute, Inc. (Osaka, Japan). Benzyloxycarbonyl-leucine-leucine-glutamate-beta NA (Z-LLE-beta NA), a substrate of peptidylglutamyl peptide hydrolase activity of proteasome, leupeptin, pepstatin, and N-tosylphenylalanine chloromethyl ketone were obtained from Sigma. Glyceraldehyde-3-phosphate dehydrogenase was obtained from Roche Molecular Biochemicals.

Preparation and Injection of Fe3+-NTA-- The Fe3+-NTA solution was prepared immediately before use as described previously (21). Briefly, ferric nitrate enneahydrate and nitrilotriacetic acid disodium salt were each dissolved in deionized water to form 300 and 600 mM solutions. They were mixed at the volume ratio of 1:2 (molar ratio, 1:4), and the pH was adjusted with sodium hydrocarbonate to 7.4. The experimental group was given an intraperitoneal injection of Fe3+-NTA at a dose of 5 mg of Fe/kg body weight. The kidneys were homogenized in a Teflon homogenizer in 10 volumes of 50 mM sodium phosphate buffer (pH 7.2). The homogenate was centrifuged at 10,000 × g for 10 min, and supernatants were used for the enzyme and TBARS assays. The supernatant was centrifuged at 105,000 × g for 60 min to obtain microsome fractions, whereas the supernatant was taken as the cytosolic fractions.

2-Thiobarbituric Acid-reactive Substances-- The amount of 2-thiobarbituric acid-reactive substances (TBARS) was determined according to the method described by Masaki et al. (22). Renal subcellular fractions (0.1 ml) were treated with 0.5 ml of 2.8% (w/v) trichloroacetic acid and 0.5 ml of 1% 2-thiobarbituric acid in 0.05 N NaOH and then boiled for 20 min. After cooling, the sample was centrifuged (11,000 × g, 3 min), and the absorbance of the supernatant solution was measured at 534 nm. Malondialdehyde bis(dimethyl acetal) (Aldrich), which yields malondialdehyde after acid treatment, was used as a standard.

8-Hydroxy-2'-deoxyguanosine Assay-- DNA isolated from the kidney samples, as described previously (23), was digested to deoxyribonucleotide levels by treatment with nuclease P1 and alkaline phosphatase (Sigma). After proper dilution of the DNA, 8-OHdG levels were determined using a competitive ELISA kit (8-OHdG check, Japan Institute for the Control of Aging, Fukuroi, Shizuoka, Japan). The determination range was 0.64-2000 ng/ml. The specificity of the monoclonal antibody N45.1 used in the competitive ELISA kit has been established (23).

ELISA-- The HNE-modified protein was determined by a competitive ELISA. To coat the wells of the microtiter plate, 100 µl/well of the HNE-modified bovine serum albumin in 50 mM sodium phosphate buffer (pH 7.2) was used and incubated overnight at 4 °C. Following washing with TBS containing 1% Tween 20 (TBS/Tween), each well was filled with 200 µl of 4% Blockace solution for 30 min at 37 °C. The kidney homogenates incubated with the partially purified mouse monoclonal antibody against HNE-modified proteins (HNEJ-2) (24) for 20 h at 4 °C were added to each well and incubated for 1 h at 37 °C. After three washings with TBS/Tween, 100 µl/well of peroxidase-conjugated anti-mouse IgG (1:2000) was added and incubated for 1 h at 37 °C. After washing, 100 µl of 0.05 M citrate buffer (pH 5.0) containing 0.4 mg/ml o-phenylenediamine and 0.003% H2O2 was added and incubated for several minutes at room temperature. The reaction was terminated by adding 2 M sulfuric acid, and the absorbance at 492 nm was read on a micro-ELISA plate reader.

Western Blotting-- For detection of ubiquitin, proteasome, and HNE-modified protein, cytosolic proteins from kidney homogenates of mice treated with Fe3+-NTA were incubated with Laemmli sample buffer (25) for 5 min at 100 °C. The samples were separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). One gel was used for staining with Coomassie Brilliant Blue; the other was transblotted to Immobilon polyvinylidene difluoride membranes, incubated with Blockace for blocking, washed, and incubated with the antibody. This procedure was followed by the addition of horseradish peroxidase conjugated to either goat anti-rabbit IgG or rabbit anti-mouse IgG, and ECL reagents. The bands were visualized by exposing the membranes to autoradiography film.

Immunoprecipitation-- Renal cytosolic fractions (approximately 400 µg of protein/0.1 ml) of Fe3+-NTA-treated mice were incubated with 10 µl of primary antibody (10 µg of IgG) on ice for 3 h. The mixture was then treated with 50 µl of protein A-Sepharose 4 Fast Flow and incubated on ice for 1 h. The mixture was then centrifuged (10,000 × g, 3 min), rinsed three times with 0.1 M Hepes buffer (pH 8.0), and then treated with Laemmli sample buffer for SDS-PAGE/immunoblot.

Immunohistochemistry-- The avidin-biotin complex method was used. After deparaffinization, normal rabbit serum (diluted to 1:75; Dako, Kyoto, Japan) was used for the inhibition of nonspecific binding of secondary antibody. Primary antibody (HNEJ-2, 25 µg/ml), biotin-labeled rabbit anti-mouse IgG serum (diluted 1:300; Dako), and avidin-biotin complex (diluted 1:100; Vector Laboratories, Burlingame, CA) were sequentially used. The substrate for alkaline phosphatase (black) was obtained from Vector. Procedures using phosphate-buffered saline, normal rabbit serum, or the IgG fraction of normal rabbit serum instead of the antibody against HNE-modified proteins exhibited no signal or a negligible signal.

Proteasome Activity-- Peptidase activity of proteasomes was measured using three peptidase activities (chymotrypsin-like, trypsin-like and peptidylglutamyl peptide hydrolase) with fluorogenic peptides as substrates according to the method of previous reports (26, 27).

In Vitro Metabolism of HNE-modified Proteins in the Kidney Homogenates-- The kidney homogenates treated with HNE (50 µM) for 1 h were further incubated in the presence or absence of protease inhibitors. After the incubation, the reaction mixtures were heated for 10 min at 95 °C to inactivate the proteases, and then the HNE-modified proteins remaining in the kidney homogenates were measured by the competitive ELISA.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oxidative Stress in the Kidney of Mice Treated with Fe3+-NTA-- A single intraperitoneal Fe3+-NTA treatment (5 mg Fe/kg body weight) caused oxidative stress, as the earliest event, in the kidney of male ddY mice. As shown in Fig. 1A, Fe3+-NTA treatment resulted in the accumulation of lipid peroxidation products (TBARS) that reached the peak at 1 h after administration of Fe3+-NTA and gradually decreased thereafter. The amount of TBARS increased dose-dependently to the Fe3+-NTA administered (data not shown). Oxidative stress was also assessed by the formation of 8-OH-dG in the DNA of the mouse kidney after treatment with Fe3+-NTA. As shown in Fig. 1B, the level of 8-OH-dG significantly increased with the maximal amount of 12.7 ng/mg DNA at 3 h and returned to the untreated level at 16 h. We have also observed that, along with the formation of these oxidized products, the Fe3+-NTA treatment led to a decline in the activity of antioxidant enzymes, including glutathione S-transferase, glutathione peroxidase, and superoxide dismutase (data not shown).


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  		Fig. 1.   Fe3+-NTA-induced oxidative stress in the kidney of mice. Mice were intraperitoneally treated with Fe3+-NTA (5 mg of Fe per kg body weight), and oxidative stress was monitored by the formation of TBARS (A) and 8-OH-dG (B).

Transient Accumulation of HNE-modified Proteins-- Oxidative stress is known to generate proteins oxidatively modified by a variety of reactive species including free radicals and membrane lipid peroxidation-derived aldehydes (28). Among the aldehydes, HNE exhibits the highest reactivity with proteins and is believed to be largely responsible for cytopathological effects during oxidative stress. Hence, we examined the formation of HNE-modified proteins in the kidney of mice treated with Fe3+-NTA by the immunochemical procedures using a monoclonal antibody directed to the protein-bound HNE. As shown in Fig. 2, competitive ELISA analysis demonstrated that the amount of HNE-modified proteins reached a peak (approximately 1.14 nmol/mg of protein) at 6 h after administration of Fe3+-NTA and gradually decreased thereafter. The transient accumulation of HNE-modified proteins was also validated by an immunohistochemical analysis of the kidney (data not shown). Untreated control animals showed weak positivity in all the proximal tubules. Intense positivity was detected in the renal proximal tubular cells at 3 and 6 h after treatment with Fe3+-NTA. In particular, intense positivities were observed in the degenerating cells which were indicated by pyknosis. Some of the proteinaceous casts were also stained. Most of the positivity was diminished after 24 h, suggesting that HNE-modified proteins once generated by the Fe3+-NTA-induced lipid peroxidation were eliminated from the kidney. These results were in fair agreement with the observations (Fig. 1) that oxidative damages, monitored by the formation of TBARS and 8-oxo-dG, were transiently detected in the kidney of mice treated with Fe3+-NTA.


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  		Fig. 2.   Fe3+-NTA-induced transient accumulation of HNE-modified proteins in the kidney of mice. Mice were intraperitoneally treated with Fe3+-NTA (5 mg of Fe per kg body weight). A, chemical structure of HNE. B, competitive ELISA analysis of HNE-modified proteins.

Ubiquitination of HNE-modified Protein-- It is anticipated that the Fe3+-NTA-induced oxidative stress leading to the formation of oxidatively modified proteins provokes the misfolding of proteins, which may then be targeted for degradation by the ubiquitin/proteasome-dependent proteolytic pathway. To examine whether the ubiquitin/proteasome pathway is activated by the Fe3+-NTA-induced oxidative stress, ubiquitin-protein conjugates generated in the kidney were analyzed by an immunoblot analysis. As shown in Fig. 3A, ubiquitin-protein conjugates were detected from 1 to 6 h after administration of Fe3+-NTA and returned to the level of control at 16 h. We then performed immunoprecipitation with anti-ubiquitin monoclonal antibody followed by detection of ubiquitinated HNE-modified proteins by immunoblotting with anti-HNE monoclonal antibody. As shown in Fig. 3B, the level of ubiquitinated HNE-modified proteins in the kidney reached a maximum at 3 h and returned to the undetectable level at 16 h. These data suggest that recovery from oxidative stress is associated with the increased ubiquitination of modified proteins, including HNE-modified proteins, followed by degradation with proteasomes.


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  		Fig. 3.   Ubiquitination of proteins in the kidney of mice treated with Fe3+-NTA. Mice were intraperitoneally treated with Fe3+-NTA (5 mg of Fe per kg of body weight). A, detection of ubiquitin-protein conjugates by an immunoblot analysis. B, detection of ubiquitinated HNE-modified proteins by an immunoprecipitation/immunoblot analysis. Total renal cytoplasmic proteins were treated with anti-ubiquitin monoclonal antibody and precipitated with protein A-Sepharose 4 Fast Flow. Ubiquitinated HNE-modified proteins were then analyzed by immunoblotting with anti-HNE monoclonal antibody. The arrowhead and arrow represent nonspecific staining of IgG and ubiquitinated HNE-modified proteins, respectively.

Involvement of Proteasomes in the Metabolism of HNE-modified Proteins in Vitro-- Transient accumulation of HNE-modified proteins in the kidney during Fe3+-NTA-induced oxidative stress was reproduced with the in vitro incubation of the kidney homogenates with HNE. As shown in Fig. 4A, when the kidney homogenates treated with 50 µM HNE for 1 h were further incubated at 37 °C, time-dependent reduction of the level of HNE-modified proteins was observed and approximately 30% of the HNE-modified proteins diminished after 6 h of incubation. To test whether proteasome is involved in the metabolism of HNE-modified proteins generated in the kidney homogenates, the effect of inhibitors on the removal of HNE-modified proteins was tested. The partial disappearance of HNE-modified protein was significantly inhibited by the proteasome inhibitor, benzyloxycarbonyl-leucine-leucine-leucinal (ZLLLal), whereas lysosomal inhibitors including leupeptin (serine and cysteine protease inhibitor), N-tosylphenylalanine chloromethyl ketone (cathepsin B inhibitor), and pepstatin (asparagine protease inhibitor such as cathepsin D and cathepsin E) were all ineffective (Fig. 4B). Similar data was also obtained from the experiment using HNE-modified glyceraldehyde-3-phosphate dehydrogenase as the exogenous substrate, in which the disappearance of HNE-modified glyceraldehyde-3-phosphate dehydrogenase, upon incubation with kidney homogenates, was significantly suppressed by the proteasome inhibitor (data not shown).


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  		Fig. 4.   Involvement of proteasomes in the metabolism of HNE-modified proteins in the kidney homogenates. A, time-dependent changes in the level of HNE-modified proteins generated in the kidney homogenates treated with HNE. The kidney homogenates (10 mg of protein/ml) treated with HNE (50 µM) for 1 h were further incubated at 37 °C. After the incubation, the reaction mixtures were heated for 10 min at 95 °C to inactivate the proteases and then the HNE-modified proteins remaining in the kidney homogenates were measured by the competitive ELISA. B, effect of protease inhibitors. The kidney homogenates treated with HNE (50 µM) for 1 h were further incubated at 37 °C in the presence and absence of protease inhibitors.

Impairment of Proteasome Activities during Oxidative Stress-- The observations that HNE-modified proteins were ubiquitinated (Fig. 3) and the proteasome inhibitor significantly suppressed the metabolism of HNE-modified proteins in vitro (Fig. 4) indicated that proteasomes might contribute to the metabolism of HNE-modified proteins during oxidative stress in vivo. Hence, we examined the changes in the proteasome activities in the kidney of mice treated with Fe3+-NTA, using the fluoropeptides (s-LLVY-MCA for the chymotrypsin activity, Boc-LSTR-MCA for the trypsin activity, and Z-LLE-beta NA for the peptidylglutamyl peptide hydrolase activity) as proteolytic substrates. As shown in Fig. 5, although the chymotrypsin activity was scarcely altered, a significant decline in both trypsin and peptidylglutamyl peptide hydrolase activities of proteasome was observed after the 1st h of the Fe3+-NTA treatment. The trypsin activity decreased 31% after 16 h, and the peptidylglutamyl peptide hydrolase activity decreased 23% after 6 h of Fe3+-NTA treatment. By 48 h, both activities returned to the pretreatment level. These data led to the assumption that Fe3+-NTA-induced oxidative stress resulted in the loss of proteasome activities, leading to the transient accumulation of HNE-modified proteins in the kidney.


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  		Fig. 5.   Changes in proteasome activities in the kidney of mice treated with Fe3+-NTA. Mice were intraperitoneally treated with Fe3+-NTA (5 mg of Fe per kg body weight). The proteasome activities in the kidney of mice treated with Fe3+-NTA were measured using the fluoropeptides, s-LLVY-MCA for the chymotrypsin activity (open circle ), Boc-LSTR-MCA for the trypsin activity (), and Z-LLE-beta NA for the peptidylglutamyl peptide hydrolase activity (triangle ), as proteolytic substrates.

We then tested the effect of HNE treatment on the proteasome activity in the kidney homogenates. As shown in Fig. 6, when the kidney homogenates were incubated with 0-10 mM HNE, the HNE concentration up to 0.1 mM was without detectable effect on the proteasome activities, whereas treatment with high concentrations (>= 1 mM) of HNE resulted in significant reduction of proteasome activities; the trypsin and peptidylglutamyl peptide hydrolase activities of proteasome decreased to 74 and 84% of the control values of untreated kidney homogenates, respectively, as the concentration of HNE was increased to 1 mM. In accordance with the results of Fig. 5, the chymotrypsin activity of the proteasomes in the kidney homogenates was scarcely affected by the addition of HNE.


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  		Fig. 6.   Effect of HNE addition on the proteasome activities in the kidney homogenates. The kidney homogenates (10 mg of protein/ml) were incubated with HNE (0-10 mM) for 1 h at 37 °C, and the proteasome activities in the kidney of mice treated with Fe3+-NTA were measured using the fluoropeptides, s-LLVY-MCA for the chymotrypsin activity (open circle ), Boc-LSTR-MCA for the trypsin activity (), and Z-LLE-beta NA for the peptidylglutamyl peptide hydrolase activity (triangle ), as proteolytic substrates.

Direct Attachment of HNE to Proteasome-- Because the protein level of proteasomes (20 S proteasome) was nearly unchanged during oxidative stress (Fig. 7B), it was presumed that the partial loss of proteasome activity during oxidative stress (Fig. 5) could be ascribed to the direct interaction of HNE with proteasomes. To detect HNE-proteasome conjugates, immunoprecipitation with anti-protein-bound HNE monoclonal antibody followed by detection of proteasomes by immunoblotting with anti-proteasome antibody, which allowed the specific detection of HNE-modified proteasomes, was performed. As shown in Fig. 7A, the HNE-modified proteasomes were detected at 3 h after Fe3+-NTA treatment. The level of modified proteasomes in the kidney reached a maximum at 6 h and returned to the pretreatment level at 24 h. These data suggest that the loss of proteasome activities in the kidney during oxidative stress (Fig. 5) can be ascribed, at least in part, to the covalent binding of HNE to the protein.


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  		Fig. 7.   Detection of HNE-modified proteasome in the kidney of mice treated with Fe3+-NTA. Mice were intraperitoneally treated with Fe3+-NTA (5 mg of Fe per kg body weight). A, detection of HNE-modified proteasome by an immunoprecipitation/immunoblot analysis. Total renal cytoplasmic proteins were treated with anti-protein-bound HNE monoclonal antibody and precipitated with protein A-Sepharose 4 Fast Flow. The HNE-modified proteasome was then analyzed by immunoblotting with anti-proteasome antibody. B, immunoblot analysis of proteasome.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A single injection of Fe3+-NTA causes a number of time-dependent morphological alterations in the structure and the function of renal proximal tubular cells and their mitochondria (29). During the early stage of injury, typical cellular changes are loss of brush border, cytoplasmic vesicles, mitochondrial disorganization, and dense cytoplasmic deposits in proximal tubular cells. Most of the damaged epithelia show a typical appearance of necrotic cells, and more than half of the proximal tubular cells are removed. It has been suggested that oxidative stress is one of the basic mechanisms of Fe3+-NTA-induced acute renal injury and is closely associated with renal carcinogenesis (30). In this study, to evaluate an overall oxidative damage which occurred in the kidney of mice treated with Fe3+-NTA, we measured two distinct parameters of oxidative stress, lipid peroxidation and DNA damage. The data that the amount of free radical-mediated products, including TBARS (Fig. 1A) and 8-OH-dG (Fig. 1B), reached a maximum at an early time point within 3 h after an Fe3+-NTA treatment indicated that oxidative stress might be the earliest event occurring in the kidney.

A number of reactive lipid peroxidation-derived aldehydes have been characterized and shown to display a wide variety of damaging actions (9). Among the aldehydes that originate from the peroxidation of cellular membrane lipids, 4-hydroxy-2-alkenals such as HNE are particularly interesting as they are highly reactive electrophils that exhibit a variety of cytopathological effects because of their facile reactivities with biological molecules, particularly with proteins. Alkylating agents including HNE are versatile mutagens and/or carcinogens. Once formed, they induce diverse aspects of severe cellular stress, including chromosomal aberrations, sister chromatid exchanges, point mutations, and cell killing. It has been shown that HNE exogenously added to the cells or endogenously generated in the cells binds to different proteins and impairs their function; examples include the Na+,K+-ATPase (31), neuronal glucose transporter GLUT3 (32), the astrocyte glutamate transporter GLT-1 (33), and the GTP-binding protein Galpha q/11 (34). HNE is relatively stable and can pass among the subcellular compartments; therefore it has the potential to interact with many different cell proteins including tau (35). Upon reaction with proteins, HNE reacts with the imidazole moiety of histidine residues, the epsilon -amino group of lysine residues, and the sulfhydryl group of cysteine residues (36-41). With cysteine and histidine residues, HNE forms Michael addition-type products having a hemiacetal structure, whereas with lysine residues, HNE forms pyrroles (42) and, to a lesser extent, fluorescent cross-linking products (43). The primary products are simple Michael addition-type products, which further undergo cyclization between the aldehyde moiety and the C-4 position of HNE to form a hemiacetal structure. In this work, the formation of HNE-modified proteins in the kidney of mice following treatment with Fe3+-NTA was validated immunologically by the competitive ELISA and immunohistochemical procedures. The ELISA demonstrated that, in a manner similar to the formation of TBARS and 8-OH-dG, the HNE-modified proteins were detected transiently at an early stage of injury (Fig. 2). This was also attested to by the immunohistochemical experiment, in which the increase in antibody labelings of various patches in some of the renal proximal tubular cells was observed within 6 h of Fe3+-NTA treatment, whereas little or no immunoreactivity was detected with proteins from mice treated with Fe3+-NTA for 24 h (data not shown). Thus, the accumulation of HNE-modified proteins appeared to be dependent upon the period after Fe3+-NTA treatment. Furthermore, we noted that the elimination of lipid peroxidation products from the renal cytoplasms proceeded concomitantly with the recovery of renal proximal tubular epithelium. These observations led to the assumption that cytoprotectants such as intracellular proteases played a role in preventing aberrant proteins from precipitating in the cells and aid in the removal of these proteins.

Ubiquitination of proteins occurs post-translationally and is a complex ATP-dependent process in which ubiquitin is sequentially activated, transferred to ubiquitin-conjugating enzymes, and then ligated to protein substrates (44). Very often, more than one ubiquitin is attached to the target proteins, forming polyubiquitin chains (45). Covalent binding of ubiquitin to proteins in the cytosol and in the nucleus is frequently viewed as a means by which proteins are marked for subsequent degradation by the ubiquitin/ATP-dependent proteinase, commonly known as the 26 S proteasome (46, 47). Here we found that the Fe3+-NTA-induced oxidative stress transiently induced the ubiquitination of proteins (Fig. 3A). More significantly, we detected ubiquitinated HNE-modified proteins in the kidney (Fig. 3B), suggesting that the ubiquitin pathway was involved in the removal of HNE-modified proteins from the kidney of Fe3+-NTA-treated mice.

Proteasomes are large multisubunit protease complexes that selectively degrade intracellular proteins (48). Most of the proteins removed by these proteases are tagged for destruction by ubiquitination. Proteasomes have a role to play in controlling cellular processes, such as metabolism and the cell cycle, through signal-mediated proteolysis of key enzymes and regulatory proteins. They also operate in the stress response, by removing abnormal proteins, and in the immune response, by generating antigenic peptides. In mammalian cells, the proteasome complex exists in both an ATP-independent 20 S form and an ATP-dependent 26 S form (49). Some studies indicated that the ATP-independent 20 S proteasome is the form that recognizes and selectively degrades oxidatively modified protein substrates (50-52), whereas others (53-55) suggested the involvement of both ATP/ubiquitin-independent (20 S proteasome) and ATP/ubiquitin-stimulated (26 S proteasome) pathways in degrading oxidatively modified proteins. In either way, it is hypothesized that one role for this pathway during oxidative stress is to remove damaged and cytotoxic proteins such as HNE-modified protein.

Involvement of proteasomes in the removal of HNE-modified protein was suggested through in vitro assays using tissue homogenates. It was revealed that incubation of kidney homogenates that had been treated with HNE resulted in a time-dependent reduction of the level of HNE-modified proteins generated in the homogenates (Fig. 4A). This and the results that the proteasome inhibitors significantly suppressed the decay of the modified protein generated in the kidney homogenates (Fig. 4B) suggested a possible involvement of the proteasomes in the degradation of HNE-modified proteins in the kidney. This was also supported by the in vitro experiment where the degradation of the exogenous substrate (HNE-modified glyceraldehyde-3-phosphate dehydrogenase) with the kidney homogenates was significantly suppressed by the proteasome inhibitors.

On the other hand, proteasomes were identified to be major intracellular target molecules of HNE under the oxidative stress induced by Fe3+-NTA. We observed a transient decrease in both trypsin-like and peptidylglutamyl peptide hydrolase-like activities of proteasomes in the kidney of mice during oxidative stress (Fig. 5). In addition, in vitro incubation of kidney homogenates with HNE also resulted in significant loss of both trypsin and peptidylglutamyl peptide hydrolase activities of proteasomes (Fig. 6). The data (Fig. 7) that the HNE-modified proteasomes were detected concomitantly with the decrease in the proteasome activities suggested that the loss of proteasome activities could be explained, at least in part, by direct attachment of HNE to the proteasomes. Partial reduction of proteasome activity upon incubation of isolated 20 S proteasome with HNE has also been reported (56). Although the detailed mechanism of inactivation of proteasomes with HNE remains unclear, it is postulated that the active site of the enzymes (trypsin and peptidylglutamyl peptide hydrolase) preferentially reacts with HNE, leading to the inactivation. Alternatively, as reported by Friguet and Szweda (57), the inhibition of proteasomes by HNE-modified (cross-linked) proteins generated in the kidney may not be unlikely.

In the present study, we have found that the intracellular proteolytic systems play a role in the metabolism of HNE-modified proteins in vivo. The observation that the HNE-modified protein was significantly ubiquitinated and that the proteolytic activities of proteasomes in the kidney were diminished in accordance with the accumulation of HNE-modified proteins suggested the involvement of proteasomes in the removal of HNE-modified proteins. In addition, the loss of proteasome activities during oxidative stress was partially ascribed to the covalent binding of HNE to proteasomes. In vitro evaluation of the effects of proteasome inhibitors also indicated the proteasome-dependent removal of HNE-modified proteins. These results indicate that the ubiquitin/proteasome system plays a crucial role in the metabolism of HNE-modified proteins in vivo.

    FOOTNOTES

* This work was supported in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, Japan. Fax: 81-52-789-5296; E-mail: uchidak@agr.nagoya-u.ac.jp.

    ABBREVIATIONS

The abbreviations used are: HNE, 4-hydroxy-2-nonenal; Fe3+-NTA, ferric nitrilotriacetate; ELISA, enzyme-linked immunosorbent assay; 8-OH-dG, 8-hydroxy-2'-deoxyguanosine; TBARS, 2-thiobarbituric acid-reactive substances; s-LLVY-MCA, succinyl-leucine-leucine-valine-tyrosine-MCA; Boc-LSTR-MCA, butoxycarbonyl-leucine-serine-threonine-arginine-MCA; Z-LLE-beta NA, benzyloxycarbonyl-leucine-leucineglutamate-beta NA.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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