Ubiquitin Conjugation Is Not Required for the Degradation of Oxidized Proteins by Proteasome*

Reshma ShringarpureDagger §, Tilman GruneDagger , Jana Mehlhase, and Kelvin J. A. DaviesDagger ||

From the Dagger  Ethel Percy Andrus Gerontology Center and Division of Molecular and Computational Biology, University of Southern California, Los Angeles, California 90089-0191 and the  Neuroscience Research Center, Medical Faculty (Charité), Humboldt University, D-10098 Berlin, Germany

Received for publication, June 24, 2002, and in revised form, October 23, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oxidatively modified proteins that accumulate in aging and many diseases can form large aggregates because of covalent cross-linking or increased surface hydrophobicity. Unless repaired or removed from cells, these oxidized proteins are often toxic, and threaten cell viability. Most oxidatively damaged proteins appear to undergo selective proteolysis, primarily by the proteasome. Previous work from our laboratory has shown that purified 20 S proteasome degrades oxidized proteins without ATP or ubiquitin in vitro, but there have been no studies to test this mechanism in vivo. The aim of this study was to determine whether ubiquitin conjugation is necessary for the degradation of oxidized proteins in intact cells. We now show that cells with compromised ubiquitin-conjugating activity still preferentially degrade oxidized intracellular proteins, at near normal rates, and this degradation is still inhibited by proteasome inhibitors. We also show that progressive oxidation of proteins such as lysozyme and ferritin does not increase their ubiquitinylation, yet the oxidized forms of both proteins are preferentially degraded by proteasome. Furthermore, rates of oxidized protein degradation by cell lysates are not significantly altered by addition of ATP, excluding the possibility of an energy requirement for this pathway. Contrary to earlier popular belief that most proteasomal degradation is conducted by the 26 S proteasome with ubiquitinylated substrates, our work suggests that oxidized proteins are degraded without ubiquitin conjugation (or ATP hydrolysis) possibly by the 20 S proteasome, or the immunoproteasome, or both.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Accumulation of oxidatively damaged proteins is a characteristic feature of aging cells and a number of age-related pathologies (1-3). In young, healthy cells moderately oxidized soluble proteins are selectively recognized and rapidly degraded by the proteasome (4-8). We have previously demonstrated that exposure of mammalian cells in culture to moderate oxidative stress significantly increases the degradation of intracellular proteins (5, 9). Proteasome immunoprecipitation, treatment of cells with antisense oligonucleotides to essential proteasome subunits, and proteasome inhibitor profiles (4, 5, 9) all confirm that proteasome is primarily responsible for the degradation of oxidized proteins in mammalian cells. The 20 S proteasome constitutes the catalytic core, whereas the 26 S proteasome is formed by addition of two 19 S regulators, which have subunits for ATP hydrolysis and polyubiquitin recognition (10-12). We have extensive evidence that purified 20 S proteasome preferentially degrades oxidized proteins in vitro in an ATP- and ubiquitin-independent manner (4, 5, 9, 13, 14), however, it has not been clear if proteasome can degrade oxidized proteins without ubiquitin and ATP in vivo.

The 20 S proteasome now appears to be the predominant proteasome species in mammalian cells (15). Furthermore, a number of proteins have now been shown to undergo ubiquitin-independent degradation by the 26 S proteasome (16-19). Our studies on comparative resistance of the 20 S and 26 S forms of the proteasome in response to hydrogen peroxide treatment show that the 26 S proteasome is much more susceptible to oxidative stress than is the 20 S form (20, 21), suggesting that it is probably the 20 S proteasome, which is responsible for the degradation of oxidized proteins when cells are exposed to oxidative stress.

The current study was undertaken to determine whether ubiquitin conjugation is essential for degradation of oxidized proteins by the proteasome in intact cells. For this we utilized the Chinese hamster lung fibroblast cell line, CH E36 and a cell cycle mutant derived from this line, ts20, which harbors a thermolabile ubiquitin-activating enzyme (E1)1 (22-24). These cell lines have been previously used to study the role of ubiquitin conjugation in various processes (24-26). We also tested ubiquitin conjugation of progressively oxidized substrates, to determine whether oxidation actually increases the tendency of a protein to be ubiquitinylated.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- CH E36 and clone ts20 cells were a kind gift from Dr. Alan L. Schwartz, Washington University, St. Louis, MI. The parental cell line E36 is a hypoxanthine guanosine-phosphoribosyl transferase-negative mutant (HGPRT-) derived from the Chinese hamster lung fibroblast cell line V-79 (22). Clone ts20 is a cell cycle mutant derived from E36 cells by ethylmethane sulfonate mutagenesis (23), which contains a thermolabile ubiquitin-activating E1 enzyme (24). Both, E36 cells (wild-type for E1) and E1 mutant ts20 cells were grown as monolayers at 30.5 °C on minimal essential medium-alpha (Invitrogen) supplemented with 10% fetal bovine serum and 4.5 g/liter glucose under 5% CO2. Flasks of both E36 and ts20 cells were shifted to the restrictive temperature, 39.5 °C, for a minimum of 2 h to inactivate E1.

Preparation of Cell Extracts-- Cells were washed twice in phosphate-buffered saline, bathed in cold phosphate-buffered saline containing 1 mM EDTA, and scraped with a rubber policeman. About 107 cells were resuspended in 300 µl of 1 mM dithiothreitol and incubated at 4 °C for 1 h, with vigorous shaking for hypotonic lysis. The crude extracts were centrifuged at 13,000 × g in an Eppendorf centrifuge for 15 min at 4 °C, supernatants were collected and stored at -70 °C until used. Protein content was determined using the BCA assay (Pierce) or the DC assay (Bio-Rad).

SDS-PAGE and Immunoblotting-- Total soluble cell protein was separated according to Laemmli (27) by SDS-PAGE using a mini-PROTEAN II electrophoresis cell (Bio-Rad). Following transfer, the nitrocellulose membranes were probed with antibodies to E1 (Calbiochem) or ubiquitin (Chemicon) and visualized using the ECL chemiluminescent system (Amersham Biosciences). Antibodies to ubiquitin conjugation substrates were obtained from Roche Molecular Biochemicals (anti-ferritin) or Chemicon (anti-lysozyme).

Cell Counting and Cell Survival Analysis-- Live, attached cells were harvested by trypsinization and counted in a Z1S Coulter particle counter. Growth curves were performed by seeding equal numbers of cells in 24-well plates and then harvesting and counting cells at various time points. To test if H2O2 treatments were toxic, cell survival analysis was performed using neutral red (Sigma) uptake as described previously (28).

Determination of Activity of the 20 S and 26 S Proteasome-- 20 S Proteasome activity was measured at 37 °C as described previously (29) by monitoring the release of 7-amido-4-methylcoumaric acid during degradation of the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amido-4-methylcoumaric acid by extracts of CH E36 and ts20 cells. Activity of the 26 S proteasome was determined by monitoring ability of cell lysates to degrade pre-formed lysozyme-ubiquitin conjugates. Ubiquitin conjugates were prepared from heat-denatured lysozyme as described below. These conjugates were then incubated either with extracts from ts20 cells at the restrictive temperature or with buffer alone, for 20 min at 39.5 °C. The reaction mixtures were separated by 12.5% SDS-PAGE, transferred to nitrocellulose, and the membranes were probed with an anti-lysozyme antibody. The amount of total lysozyme present in each lane was then quantified using NIH Image software to determine the net loss of lysozyme.

Metabolic Labeling, Exposure of Cells to Oxidative Stress, and Proteolysis Measurements-- Cells were seeded at 2-5 × 105 cells/ml in 24-well plates and grown at 31 °C. On day 3, cells were metabolically labeled with a [35S]Cys/Met mixture for 2 h (to generate predominantly "short lived" proteins) or for 16 h (to generate mostly "long lived" proteins) as described (5, 9). Both, CH E36 and ts20 cells were then shifted to the restrictive temperature (39.5 °C) for 2 h to inactivate the E1 ubiquitin-activating enzyme in ts20 cells (cells were maintained at 39.5 °C for all further treatments). The monolayers were then exposed to oxidative stress by bolus addition of increasing concentrations (0.1 to 1 mM) of H2O2 in PBS, for 30 min. Following oxidative stress, cells were incubated in cold (nonradioactive) medium, with or without proteasome inhibitors, and percent protein degradation was monitored by measuring acid-soluble [35S] counts from previously acid-precipitable 35S-labeled proteins as described (5, 9).

Determination of Protein Carbonyls-- A sensitive ELISA method as described by Winterbourn and colleagues (30) with modifications described by Sitte et al. (31) was used for the quantitative determination of protein carbonyls.

Oxidative Modification and Heat Denaturation of Ferritin/Lysozyme-- Isolated ferritin and lysozyme (Sigma) were oxidized with various concentrations of hydrogen peroxide as described previously (29) with a few modifications. Proteins (1 mg) were exposed to various concentrations of hydrogen peroxide in an Eppendorf thermomixer for 2 h at 30 °C in a total volume of 1 ml of phosphate buffer. For heat denaturation, the proteins were incubated in a boiling water bath for 5 min, followed by quick chilling on ice. Untreated proteins, peroxide-treated proteins, and heat-denatured proteins were extensively dialyzed overnight at 4 °C against 200 volumes of buffer, with 2 changes, to remove any residual hydrogen peroxide or spontaneously generated fragments.

Ubiquitin Conjugation of Ferritin/Lysozyme in Vitro-- Ubiquitin conjugation of purified protein substrates was carried out as described by Finley et al. (32), except that in our assay, the P-enolpyruvate/pyruvate kinase system was used for ATP regeneration and glutathione S-transferase-tagged ubiquitin was used as the source of ubiquitin. Briefly, the assay mixture contained 20 µl of CH E36 cell lysate (4 mg/ml), 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol, 3.6 mM P-enolpyruvate, 0.5 units of pyruvate kinase, 5 mM ATP, 5 µg of glutathione S-transferase-ubiquitin, 0.09 units of inorganic pyrophosphatase (to reduce the build-up of PPi), 50 µM lactacystin (to inhibit proteasomal degradation of Ub conjugates), as well as 1 µM ubiquitin-aldehyde (to inhibit de-ubiquitinating enzymes). The reaction was initiated by adding 10 µg of oxidized or heat-denatured ferritin. For ubiquitin conjugation of lysozyme, the assay was similar except that the widely used creatine phosphate (10 mM)/creatine phosphokinase (100 µg/ml) system was used for ATP regeneration. In some reactions, ATP-depleted lysates were produced by incubation with 10 mM 2-deoxyglucose and 0.5 units of hexokinase. After 20 min at 30 °C, the reaction was stopped by addition of SDS-PAGE loading buffer and an aliquot of the mixture was loaded on 12.5% polyacrylamide gels after boiling. After transfer, the blots were probed with antibodies to the substrate proteins ferritin (Roche Molecular Biochemicals) or lysozyme (Chemicon).

Degradation of Ferritin and Lysozyme-- Degradation of ferritin by purified 20 S proteasome was monitored by measuring acid-soluble free amines using fluorescamine, as described (8, 29). To determine the role of ATP, degradation of oxidized [3H]ferritin by cell lysates (60 µl; 2.5 mg/ml) was measured both in the presence and absence of 10 mM ATP. Percent degradation was calculated by measuring acid soluble radioactivity as described previously (14, 29). Lysozyme was labeled with [3H] by reductive methylation (33) and percent degradation was measured by formation of acid-soluble counts, as described (14, 29).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

E1 Inactivation Results in Decreased Ubiquitinylation and Decreased Basal Proteolysis of Short Lived Proteins-- Incubation of temperature-sensitive ts20 cells for 2 h at the restrictive temperature (39.5 °C) resulted in loss of E1. Cells were initially shifted to the restrictive temperature for 2 h, followed by sham treatment with PBS (or treatment with 500 µM hydrogen peroxide) for 30 min. In extracts of E1-mutant ts20 cells incubated at the restrictive temperature for more than 2.5 h, the enzyme could not be detected immunologically with an anti-E1 antibody (Calbiochem) at the expected position of 110 kDa (Fig. 1A, lanes 4 and 6). There was, however, no difference in the levels of E1 in the wild-type CH E36 cells when incubated at the restrictive temperature (Fig. 1A, lanes 3 and 5). Both E36 cells and ts20 cells exhibited comparable amounts of enzyme at the permissive temperature of 30.5 °C (Fig. 1A, lanes 1 and 2). Similar results were obtained for cells treated with hydrogen peroxide (data not shown).


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Fig. 1.   E1 inactivation results in decreased ubiquitinylation and decreased basal proteolysis of short lived proteins in ts20 cells. Panel A, Western blot, anti-E1 antibody. The left panel shows levels of E1 in cells incubated at the permissive temperature of 30.5 °C. Right panel, levels of E1 in CH E36 cells (lanes 3 and 5) and E1 mutant ts20 cells (lanes 2 and 4) at 39.5 ° C. Panel B, Western blot, anti-ubiquitin antibody. Soluble cell proteins from cells incubated at the restrictive temperature of 39.5 °C for 3 h were separated on a 12.5% SDS-PAGE and probed with an anti-ubiquitin antibody. The blot was stripped and reprobed with an anti-actin antibody (Sigma) as a loading control (panel B, lower box). Panel C shows basal proteolysis of short lived proteins; mean ± S.E. of three independent determinations. Cells were metabolically labeled as described under "Experimental Procedures," incubated at the restrictive temperature for 2 h, and then sham treated with PBS or H2O2 (not shown) for 30 min. Percent degradation was measured by formation of acid-soluble [35S] counts from previously acid-precipitable 35S-labeled proteins as described (5, 9). Panel D, reports the quantification of an anti-lysozyme Western blot (inset) of pre-formed lysozyme-ubiquitin conjugates in the absence (100%; inset, lane 1) or presence (40%; inset, lane 2) of ts20 lysates. Pre-formed lysozyme-Ub conjugates were incubated with buffer alone, or with ts20 lysates, for 20 min at 39.5 °C followed by separation on a 12.5% SDS-PAGE. Following transfer, the membranes were probed with an anti-lysozyme antibody (inset) and the chemiluminescent signal was later quantified using NIH Image software.

To determine whether this was a truly functional inactivation of the enzyme, we looked at the ability of ts20 cells to form high molecular weight ubiquitin conjugates de novo. CH E36 cells and ts20 cells were incubated at 39.5 °C for 3 h, after which equal amounts of protein from cell extracts were separated by a 12.5% SDS-PAGE. Following transfer, the membranes were probed with an anti-ubiquitin antibody (Chemicon) to detect protein-ubiquitin conjugates. As seen in Fig. 1B, the steady-state levels of endogenous, high molecular weight ubiquitin conjugates were considerably lower in ts20 cells, than in wild type CH E36 cells, indicating markedly compromised ubiquitin-conjugating activity in ts20 cells at the restrictive temperature. Similar results were obtained by Kulka et al. (24) presumably because there is no de novo synthesis of high molecular weight Ub conjugates in ts20 cells at the restrictive temperature. Although this has been previously seen in ts20 cells (24), we reconfirmed these properties of the temperature-sensitive ts20 mutants in light of recent reports of residual ubiquitin-conjugation activity for a similar, yet distinct ts85 mutant cell line (34).

Most short lived proteins are degraded by the 26 S proteasome in an ATP and ubiquitin-dependent fashion (35). Baseline proteolysis of short lived proteins was reduced by almost 50% in ts20 cells, indicating that ubiquitin-dependent proteolysis was indeed compromised in temperature-sensitive E1 mutants (Fig. 1C). It should be noted that besides ubiquitin-independent proteolysis by the proteasome, proteolysis is also conducted by other proteases. Similar studies have been performed in ts20 cells and in another E1 mutant cell line, ts85 derived from FM3A mouse carcinoma cells (36).

We next ensured that the thermolability of ubiquitin-dependent proteolysis was solely because of a defect in ubiquitin conjugation and not inactivation of the 26 S proteasome at the restrictive temperature. For this we tested the degradation of pre-formed lysozyme-ubiquitin conjugates by extracts of ts20 cells incubated at the restrictive temperature. Ubiquitin conjugates of heat-denatured lysozyme were incubated with buffer alone (lane 1 in Fig. 1D, and lane 1 in the inset) or with E1-inactivated ts20 extracts (lane 2 in Fig. 1D, and lane 2 in the inset). After separation of the reaction mixtures by SDS-PAGE and transfer, the membranes were probed with an anti-lysozyme antibody and the resulting chemiluminescence was quantified using NIH Image software. Besides disappearance of individual high molecular weight lysozyme-Ub conjugates, densitometric quantification revealed a 60% loss of total lysozyme after incubation with ts20 lysates. Thus, ts20 cells incubated at the restrictive temperature for 3 h were capable of degrading lysozyme-ubiquitin conjugates, confirming that the 26 S proteasome was not affected in these cells. Similar results were obtained using extracts of wild-type CH E36 cell lysates (data not shown).

Growth Properties, H2O2 Toxicity, and Proteasome Activity Profiles-- Before testing a possible ubiquitin involvement in the degradation of oxidized proteins, we first compared growth properties at 30.5 °C (Fig. 2A), 39.5 °C (Fig. 2B), and with H2O2 in wild-type CH E36 cells and E1 mutant ts20 cells (Fig. 2C). Hydrogen peroxide caused a similar growth arrest in both cell lines (Fig. 2C) as expected from previous work (37, 38). Because the cells were maintained at the restrictive temperature for only 24-26 h, the two cell lines did not show a major difference in cell number. To test whether the ts20 mutants were more susceptible than CH E36 cells to hydrogen peroxide toxicity because of their thermolabile E1, we analyzed cell survival in response to H2O2 treatment in both cell lines by the neutral red uptake assay (Fig. 2, D and E). There were no significant differences in survival over a 24-h period following H2O2 treatment, further indicating that lack of ubiquitin-conjugating activity did not alter the short term ability of ts20 cells to survive an acute oxidative stress.


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Fig. 2.   Growth curves, hydrogen peroxide toxicity, and proteasome activity. Panel A, on the first day 1 × 105 cells were seeded in 24-well plates and cells were counted in an automated cell counter. Both wild-type CH E36 cells and E1 mutant ts20 cells grew at comparable rates at the permissive temperature of 30.5 ° C. Panels B and C, wild-type, CH E36, and E1 mutant ts20 cells were grown at the permissive temperature (30.5 °C) for 48 h, then shifted to the restrictive temperature (39.5 °C) to inactivate E1. At 50 h, the cells were either sham treated with PBS (B) or with 0.4 mM hydrogen peroxide (C) for 30 min at 39.5 ° C. Panels D and E, effect of hydrogen peroxide on cell survival was tested with the neutral red assay (Sigma). Effect of treatment with 0.6 mM peroxide immediately after treatment and 24 h later is shown for both wild-type CH E36 cells (D) and E1 mutant ts20 cells (E) at the permissive and restrictive temperatures. Panel F demonstrates that proteasome activity is not affected in ts20 cells at the restrictive temperature. Cells were grown at 30.5 °C, or were incubated for 3 h at 39.5 °C and then harvested. Proteasome activity in lysates was measured by degradation of Suc-LLVY-7-amido-4-methylcoumaric acid for 1 h at 37 °C. All data points are mean ± S.E. of at least three independent experiments.

Because most proteolysis experiments were performed at 39.5 °C, we measured the activity of the 20 S core proteasome in both cell lines, at both temperatures, to determine whether the proteasome was inactivated at the restrictive temperature. As seen in Fig. 2F, the proteasome was not inactivated at the restrictive temperature and loss of proteasome-dependent proteolysis may, therefore, be attributed to defects in ubiquitin conjugation.

E1 Mutants Degrade Oxidized Proteins Despite Compromised Ubiquitinylation-- To test whether ubiquitin conjugation is necessary for the degradation of oxidized proteins, we studied the turnover of short lived and long lived proteins after treatment with hydrogen peroxide in ts20 cells with compromised ubiquitin-conjugating activity. CH E36 and ts20 cells were metabolically labeled for 2 h (Fig. 3A) or 16 h (Fig. 3B) with [35S]Cys/Met to label pools of either short lived (2 h) or mostly long lived (16 h) proteins, respectively. As seen in Fig. 3, intracellular proteolysis was considerably higher in cells treated with 400 µM H2O2 than in untreated cells. We have previously reported a similar oxidation-induced increase in overall proteolysis for many different cell types (4, 5, 9, 39) but we can now show that E1 mutant ts20 cells still preferentially degrade oxidized proteins despite the lack of an E1. Because a number of short lived regulatory proteins are known to be degraded by the ubiquitin-proteasome pathway (35, 40), the total turnover of short lived proteins was considerably lower in ts20 cells, as expected. However, the relative increase in degradation because of oxidation was comparable for both cell lines, indicating that the pool of oxidized proteins can still be eliminated in ts20 cells. The degradation of long lived proteins (Fig. 3B) also showed a similar oxidation-induced increase in both CH E36 and E1 mutant ts20 cells as seen previously for other cell types. In fact, it may be noted that the absolute increase in proteolysis because of H2O2 is actually higher in the ts20 E1 mutant cells than in the parent CH E36 cells (Fig. 3B). In earlier studies we reported that moderate oxidation of proteins rapidly increases their degradation, whereas severe oxidation causes a gradual decline in intracellular proteolysis (4, 5, 9, 29). The E1 mutant ts20 cells exhibited exactly this pattern of response to hydrogen peroxide concentration (Fig. 3C), indicating that the E1 mutant ts20 cells utilize the same pathway for degradation of oxidized proteins described previously (4, 5, 9).


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Fig. 3.   E1 mutant ts20 cells degrade oxidized proteins despite compromised ubiquitinylation. CH E36 and E1 mutant ts20 cells were metabolically labeled with [35S]Cys/Met for 2 h (panels A and C) or 16 h (panel B) to label pools of short lived, or mostly long lived, proteins, respectively. After incubation of these cells for 2 h at the restrictive temperature (39.5 °C), the cells were treated with PBS (no H2O2) or increasing concentrations of hydrogen peroxide for 30 min at 39.5 °C, followed by incubation in regular, cold medium for a further 24 h at 39.5 °C. Percent degradation was measured by formation of acid-soluble [35S] counts from previously acid-precipitable 35S-labeled proteins as described (5, 9). All data points are mean ± S.E. of at least three independent experiments.

The Proteasome Is Largely Responsible for the Degradation of Oxidized Proteins in ts20 Cells-- To determine whether the proteasome is still responsible for oxidation-induced protein degradation in ts20 cells with compromised ubiquitin-conjugating activity, we examined the effects of proteasome inhibitors. The selective proteasome inhibitor NLVS (5 µM) strongly inhibited the increase in degradation because of oxidation in both CH E36 control cells and E1 mutant ts20 cells, indicating that the proteasome was still largely responsible for the degradation of oxidized proteins even without the synthesis of ubiquitin conjugates (Fig. 4). Similar results were also obtained with another well known proteasome inhibitor, lactacystin (not shown).


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Fig. 4.   The proteasome inhibitor NLVS inhibits H2O2-induced proteolysis. Proteolysis was measured exactly as described in the legend to Fig. 3A, except that the cells were treated either with 5 µM NLVS or Me2SO vehicle alone. Me2SO or NLVS was added 2 h prior to treatment with peroxide, and remained in the final incubation medium. All data points are mean ± S.E. of at least three independent experiments.

E1 Mutant ts20 Cells Can Eliminate Carbonyl-containing Proteins without Ubiquitin Conjugation-- Because the above experiments measured overall proteolysis, we next studied protein carbonyls to address the turnover of oxidized proteins. The total carbonyl content of both wild type CH E36 cells and E1 mutant ts20 cells was increased by H2O2 but then dramatically decreased over the following 24 h, whether measured by ELISA or Western blot assays (Fig. 5, A and B), showing that the ts20 cells could still degrade oxidized, carbonyl-containing proteins without the presence of an active ubiquitin-conjugating system.


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Fig. 5.   E1 mutants can still degrade carbonyl-containing proteins. Panel A, ELISA for the quantitative determination of protein carbonyls using an anti-DNP antibody (31, 32). Cells were incubated at the restrictive temperature for 2 h to inactivate E1, followed by treatment with hydrogen peroxide (or PBS in controls). Protein carbonyls were measured in untreated cells and hydrogen peroxide-treated cells for both the CH E36 and the E1 mutant ts20 cell lines, either immediately after treatment (0 h), or 24 h after treatment (24 h) with 0.5 mM hydrogen peroxide. Panel B, Western blot (anti-DNP antibody) to detect carbonyl content in ts20 cells sham treated with PBS (C), or in ts20 cells treated with hydrogen peroxide (P), at both 0 and 24 h. In both panels cells were treated with H2O2 or PBS for 30 min, and H2O2 was then replaced by dialysis with medium. Results of panel A are mean ± S.E. of at least three independent experiments and the Western blot of panel B was repeated several times with very similar results.

Progressive Oxidation of Ferritin Does Not Promote Its Ubiquitinylation in Vitro Yet the Protein Is Degraded by Proteasome without ATP Hydrolysis-- Fig. 6A shows an in vitro ubiquitin-conjugation assay for native, heat-denatured, and progressively oxidized ferritin. Native ferritin (lanes 1 and 2) was modified either by heat denaturation (lanes 3 and 4) or increasing concentrations of hydrogen peroxide (lanes 5-10). The protein was then incubated either by itself (lanes 1, 3, 5, 7, and 9) or in the presence of a complete ubiquitin-conjugation system (lanes 2, 4, 6, 8, and 10) for 30 min. at 30 °C. The high molecular weight ferritin-immunoreactive bands represent high molecular weight ferritin-ubiquitin conjugates with a progressively increasing number of ubiquitin moieties. Glutathione S-transferase-tagged ubiquitin (Mr 38,500) was used to obtain better separation of ubiquitin conjugates, because "naked" ubiquitin usually produces a smear-like pattern.


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Fig. 6.   In vitro ubiquitin conjugation and degradation of oxidized ferritin. Panel A, purified horse spleen ferritin was purchased from Sigma and used in its native form (lanes 1 and 2), modified by heat denaturation (lanes 3 and 4), or by progressive oxidation with 10 (lanes 5 and 6), 20 (lanes 7 and 8), and 30 mM (lanes 9 and 10) hydrogen peroxide. These model protein substrates were then incubated either alone (lanes 1, 3, 5, 7, and 9) or in the presence of a complete ubiquitin-conjugation system that included CH E36 cell lysates (lanes 2, 4, 6, 8, and 10) for 30 min at 30 °C. Loading buffer was added to stop the reaction and the reaction mixtures were separated by 12.5% SDS-PAGE. After transfer, membranes were probed with an anti-ferritin antibody (Roche Molecular Biochemicals). Panel B, oxidation of ferritin was assessed by an ELISA for detecting DNP-derivatized protein carbonyls with an anti-DNP antibody (31, 32). Panel C, the proteolytic susceptibility of oxidized ferritin to degradation by purified 20 S proteasome was monitored by measuring release of acid-soluble free amines using fluorescamine. Panel D, progressively oxidized [3H]ferritin was incubated with lysates of either wild-type CH E36 cells (white bars), or E1 mutant ts20 cells (gray bars), in the absence (solid bars) or presence (patterned bars) of 10 mM ATP for 2 h at 39.5 °C. Percent degradation was calculated by measuring the increase in acid soluble radioactivity as described under "Experimental Procedures."

Heat-denatured ferritin showed a marked increase in ubiquitin conjugation as seen by the both the intensity and the number of high molecular weight bands (Fig. 6A, lane 4) in comparison with native ferritin (lane 2). Progressive oxidation of ferritin by 10 (lanes 5 and 6), 20 (lanes 7 and 8), or 30 mM (lanes 9 and 10) hydrogen peroxide, however, did not increase ubiquitin conjugation: actually, H2O2 caused a progressive decrease in the number and intensity of high molecular weight ferritin-ubiquitin conjugates, in comparison with untreated ferritin (lane 2), showing that this oxidized protein is not preferentially ubiquitinylated.

Fig. 6B shows that ferritin was indeed progressively oxidized, as seen by the increase in carbonyl content. Protein carbonyls were assessed by an ELISA using an anti-DNP antibody as described under "Experimental Procedures." This modified ferritin was still preferentially degraded by purified 20 S proteasome in the absence of ATP (Fig. 6C). As described for several proteins in our earlier work (4, 5, 8, 9, 13, 14, 20, 21, 29, 41, 47, 48, 51), moderately oxidized ferritin was preferentially degraded by the 20 S proteasome whereas severely oxidized ferritin tended to be a poorer substrate.

Based on our results, as well as new evidence in the literature (21, 41), we speculated that this degradation of oxidized proteins is most likely conducted by the 20 S proteasome or the immunoproteasome. Because the 26 S proteasome requires ATP hydrolysis, we tested the effect of ATP on degradation of oxidized ferritin by both CH E36 and E1 mutant ts20 cell lysates (Fig. 6D). As seen in Fig. 6D, ATP did not enhance the degradation of oxidized ferritin by the wild-type, CH E36 cell lysates, or E1 mutant ts20 cell lysates, thus excluding any energy requirement for the degradation of oxidized protein substrates.

Progressive Oxidation of Lysozyme Does Not Promote Its Ubiquitinylation in Vitro and Oxidized Lysozyme Is Still Preferentially Degraded by Lysates of ts20 Cells-- A similar ubiquitin-conjugation assay was performed with the widely used proteasome substrate, lysozyme. As seen in Fig. 7A, heat-denatured lysozyme showed a large increase in ubiquitin conjugates as seen by the dark smear (lane 12) as compared with native lysozyme (lanes 1 and 2). Neither heat-denatured lysozyme by itself (lane 11), nor heat-denatured lysozyme when incubated with an ATP-depleted extract (lane 13), nor a ts20 extract incubated at 39.5 °C with inactive E1 (lane 14) showed a similar increase in high molecular weight lysozyme-ubiquitin conjugates ruling out nonspecific ubiquitinylation. Progressive oxidation of lysozyme (lanes 3-10), however, did not increase its tendency to be ubiquitinylated (as also seen for ferritin in Fig. 6A), further confirming that ubiquitin conjugation may not be involved in targeting oxidized proteins to the proteasome.


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Fig. 7.   In vitro ubiquitin conjugation and degradation of oxidized lysozyme. Panel A, an in vitro ubiquitin-conjugation assay using lysozyme (Sigma) as the substrate. Native lysozyme (lanes 1 and 2), lysozyme modified by 10 (lanes 3 and 4), 20 (lanes 5 and 6), 30 (lanes 7 and 8), or 40 mM (lanes 9 and 10) hydrogen peroxide, or heat-denatured lysozyme (lanes 11-14) was used as a substrate for ubiquitin conjugation. Lysozyme was incubated either by itself (lanes 1, 3, 5, 7, 9, and 11) or in the presence of a complete ubiquitin-conjugating system that included lysates from CH E36 cells (lanes 2, 4, 6, 8, 10, and 12). When heat-denatured lysozyme was incubated by itself (lane 11), with an ATP-depleted extract (lane 13) or with a heat-inactivated ts20 extract containing an inactive E1 (lane 14), there was no formation of high molecular weight lysozyme-ubiquitin conjugates as expected. Panel B, quantification of the lysine oxidation product 2-aminoadipic semialdeheyde upon progressive oxidation of lysozyme with hydrogen peroxide. At 40 mM hydrogen peroxide, the lysine oxidation product 2-aminoadipic semialdehyde itself appears to undergo oxidative destruction. Panel C, in vitro degradation of oxidized [3H]lysozyme by lysates of wild-type CH E36 cells and E1 mutant ts20 cells at 39.5 °C. Panel D, in vitro degradation of heat-denatured [3H]lysozyme by lysates of wild-type CH E36 cells and E1 mutant ts20 cells at 39.5 °C. In panels C and D degradation of [3H]lysozyme was measured by increasing production of acid-soluble [3H] counts from previously precipitable [3H]lysozyme (13, 30, 42). Results are mean ± S.E. of at least three independent experiments.

Fig. 7B reveals a progressive oxidation of lysine residues (some of which are required for forming polyubiquitin conjugates) with H2O2 exposures up to 30 mM. At 40 mM H2O2 the lysine oxidation product 2-aminoadipic semialdehyde itself underwent oxidative destruction.

Degradation of [3H]lysozyme by extracts of E36 and (E1-inactivated) ts20 cells at 39.5 °C was monitored as described previously (13, 29, 42). The E1 mutant ts20 cells degraded lysozyme as well as the wild-type CH E36 cells, confirming that even in wild-type cells, ubiquitin conjugation is not required for the degradation of oxidized proteins (Fig. 7C). Interestingly, the wild-type cells degraded heat-denatured lysozyme much more efficiently than did the ts20 mutants (Fig. 7D). Because heat-denatured lysozyme is degraded by the ubiquitin 26 S proteasome system this experiment clearly shows that oxidized proteins must be degraded by another proteasome pathway.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have shown that E1 mutant ts20 cells can eliminate oxidized proteins despite their lack of ubiquitin-conjugating activity, and that this degradation is still catalyzed by the multicatalytic proteinase complex, proteasome. The proteasome exists in multiple forms including free 20 S, 26 S, immunoproteasome, and hybrid proteasomes (10, 11, 15). The 26 S proteasome requires ATP hydrolysis and mostly degrades ubiquitin-conjugated proteins (10-12), with a few known exceptions (17, 43-45). The 19 S regulator is required for recognition and binding of ubiquitin-conjugated proteins, followed by their deubiquitinylation and unfolding to allow entry into the catalytic core (20 S) of the proteasome. Pickart and co-authors (46, 47) have shown that it is primarily the hydrophobic effect that contributes to polyubiquitin chain recognition by the 26 S proteasome. Many substrates of the 26 S proteasome are short lived regulatory proteins that are not necessarily damaged or denatured and, therefore, have to be tagged with an external hydrophobic patch in the form of a polyubiquitin chain. ATP hydrolysis is required for unfolding such substrates so that they can enter the catalytic core of the proteasome (43). Oxidative damage to a protein, however, directly results in partial unfolding and exposure of otherwise buried hydrophobic residues (48-51). Therefore, an oxidized protein does not need to be further modified by ubiquitin conjugation to confer a hydrophobic patch, nor does it require energy from ATP hydrolysis as it is already unfolded. We (14, 42, 52) and others (53, 54) have shown that the 20 S proteasome has a distinct preference for hydrophobic and bulky (aromatic) residues. We therefore suggest that the 20 S proteasome selectively recognizes oxidatively modified, partially denatured proteins because of their exposed hydrophobic moieties.

Contrary to earlier belief that the 26 S proteasome is the major intracellular proteasome species, recent literature on proteasome stoichiometry suggests that there is a significant excess of free (and enzymatically active) 20 S proteasome form (15, 55-57) in mammalian cells. Several examples of ubiquitin-independent degradation of proteins by the proteasome have now been reported (16-19, 44, 58). We have demonstrated that the 20 S proteasome is resistant to oxidative stress, whereas the 26 S proteasome is quite susceptible. Taylor and co-authors (41, 59, 60) have shown that activity of ubiquitin-activating and -conjugating enzymes (E1 and E2s) is reversibly depressed during oxidative stress, possibly because of glutathiolation of the active site cysteine residues in both E1 and the family of E2 enzymes. Because both the 26 S proteasome and the ubiquitin-conjugation system are inhibited/inactivated by oxidative stress, it is unlikely that the ubiquitin-26 S proteasome pathway is involved in the degradation of oxidized proteins in cells undergoing oxidative stress.

Our studies on ubiquitin conjugation of oxidized substrates in vitro show that progressive oxidation of a protein does not increase its propensity to be ubiquitinylated (Figs. 6 and 7). Conjugation of the polyubiquitin chain occurs via internal lysine residues of the substrate protein and progressive oxidation of a protein actually modifies an increasing number of lysine residues as seen by formation of the oxidation product, 2-aminoadipic semialdehyde. Despite the lack of ubiquitinylation, and loss of lysine residues, oxidized proteins such as ferritin and lysozyme are still preferentially degraded in wild-type cells and E1 mutant ts20 cells, and by purified 20 S proteasome. Besides, addition of ATP did not enhance the degradation of oxidized proteins either by wild-type CH E36 or E1 mutant ts20 lysates, suggesting that the 26 S proteasome, which requires ATP hydrolysis, may not be involved in oxidized protein degradation. Our current data, especially when combined with previous reports by us (4, 5, 8, 9, 13, 14, 20, 21, 29, 31, 39, 42, 52), and others (41, 59, 60), appear to rule out any significant involvement of the ubiquitin-26 S proteasome system in the degradation of oxidatively modified proteins. Both this study and previous reports, however, clearly demonstrate that some form of the proteasome is essential for selective intracellular clearance of oxidized proteins.

Careful quantification has now revealed that the free 20 S proteasome is the predominant form of the enzyme in mammalian cells (15, 55, 57). In vitro studies by our group have repeatedly shown that the free 20 S proteasome efficiently and aggressively degrades a wide variety of oxidized proteins (4, 5, 9, 13, 14, 29, 31, 39, 42, 52) and our preliminary studies indicate that the immunoproteasome can also degrade oxidized proteins (data not shown). Although our data does not conclusively rule out involvement of the 26 S proteasome in the degradation of oxidized proteins, it now appears to be less likely, because our results reveal that oxidized proteins are degraded with no requirement for either ATP hydrolysis or ubiquitin conjugation. It will now be important to directly test this hypothesis in intact cells, and to determine the relative contributions of the free 20 S proteasome and the immunoproteasome to the degradation of oxidized proteins.

    ACKNOWLEDGEMENT

We thank Dr. Alan Schwartz for providing the Chinese hamster lung fibroblast cell lines, CH E36 and ts20.

    FOOTNOTES

* This work was supported by NIEHS National Institutes of Health Grant ES03598 (to K. J. A. D.).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.

§ Current address: Dana-Farber Cancer Inst., 44 Binney St., Mayer 551, Boston, MA 02115. E-mail: Reshma_Shringarpure@dfci.harvard.edu.

|| To whom correspondence should be addressed: Andrus Gerontology Center, GER 306C, University of Southern California, 3715 McClintock Ave., Los Angeles, CA 90089-0191. Tel.: 213-740-8959; Fax: 213-740-6462; E-mail: kelvin@usc.edu.

Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M206279200

    ABBREVIATIONS

The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; ELISA, enzyme-linked immunosorbent assay; DNP, 2,4-dinitrophenol.

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