Differential Impairment of 20S and 26S Proteasome Activities in Human Hematopoietic K562 Cells during Oxidative Stress

Archives of Biochemistry and Biophysics Vol. 377, No. 1, May 1, pp. 65– 68, 2000 doi:10.1006/abbi.2000.1717, available online at http://www.idealibrary.com on

Differential Impairment of 20S and 26S Proteasome Activities in Human Hematopoietic K562 Cells during Oxidative Stress 1 Thomas Reinheckel,* ,† Oliver Ullrich,* Nicolle Sitte,* and Tilman Grune* ,2 *Clinics of Physical Medicine and Rehabilitation, Medical Faculty (Charite´), Humboldt University Berlin, Schumannstrasse 20/21, D-10098 Berlin, Germany; and †Department of Experimental Surgery, Medical Faculty, University of Magdeburg, D-39120 Magdeburg, Germany

Received September 3, 1999, and in revised form November 22, 1999

The 20S proteasome and the 26S proteasome are major components of the cytosolic and nuclear proteasomal proteolytic systems. Since proteins are known to be highly susceptible targets for reactive oxygen species, the effect of H 2 O 2 treatment of K562 human hematopoietic cells toward the activities of 20S and 26S proteasomes was investigated. While the ATP-independent degradation of the fluorogenic peptide suc-LLVY-MCA was not affected by H 2 O 2 concentrations of up to 5 mM, the ATP-stimulated degradation of suc-LLVY-MCA by the 26S proteasome began to decline at 400 ␮M and was completely abolished at 1 mM oxidant treatment. A combination of nondenaturing electrophoresis and Western blotting let us believe that the high oxidant susceptibility of the 26S proteasome is due to oxidation of essential amino acids in the proteasome activator PA 700 which mediates the ATP-dependent proteolysis of the 26S-proteasome. The activity of the 26S-proteasome could be recovered within 24 h after exposure of cells to 1 mM H 2 O 2 but not after 2 mM H 2 O 2 . In view of the specific functions of the 26S proteasome in cell cycle control and other important physiological functions, the consequences of the higher susceptibility of this protease toward oxidative stress needs to be considered. © 2000 Academic Press Key Words: oxidative stress; protease regulation; proteolysis; protein oxidation.

Proteins are increasingly recognized as important targets for the action of free oxygen radicals that are formed under physiological and pathological conditions (1, 2). The accumulation of oxidatively damaged proteins which lack their functional properties could lead to a pool of useless cellular debris. Thus the removal of oxidized proteins or protein fragments by proteases and the subsequent reuse of the produced amino acids in metabolism seem to make “sense” in order to keep cellular integrity. For this concept the term “proteolysis as secondary antioxidant defense” was established (3). It was shown by several groups that oxidized proteins are preferentially degraded by several proteases as compared to their nonoxidized forms. For instance, the oxidation of 6 of the 16 methionine residues of glutamine synthetase results in increased degradation of the enzyme by isolated 20S proteasome (4). The oxidation of substrate proteins leads to increased susceptibility of proteins toward degradation. These in vitro findings could be confirmed in viable and dividing cell cultures, where an increased intracellular protein turnover was found in response to mild oxidative stress (5). In all instances the proteasome was identified as the protease responsible for most of the breakdown of cytosolic and nuclear oxidatively modified proteins (5– 9). A number of regulators of the proteasomal activity have been described (10). The association of the cylinder-shaped 20S “core” proteasome with the proteasome activator PA 3 700 is well characterized. This results in the formation of the ATP-stimulated and ubiquitindependent 26S proteasome. Both forms of the protea-

1

Parts of this publication were presented at the SFRR (Europe) Meeting in Dresden, Germany, July 2– 6, 1999. 2 To whom correspondence and reprint requests should be addressed. Fax: (49-30)-2093-7204. E-mail: tilman.grune@charite. de. 0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

3 Abbreviations used: PA, proteasome activator; DMSO, dimethylsulfoxide; MCA, monochloroacetic acid; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; PARP, poly-ADP ribose polymerase.

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somal system, i.e., the 20S proteasome and the 26S proteasome, are apparently involved in the degradation of various abnormally folded proteins. However, the main function of the 26S proteasome appears to be the highly specific and effective degradation of proteins during various events in regulated cellular processes like transcription and cell cycle (10). Thus it was interesting to investigate the effects of oxidative stress on the 20S and 26S proteasomes in a cell culture model. K562 human hematopoietic cells previously used to demonstrate the increased proteolysis after oxidative stress (7), were explored to demonstrate the regulatory effect of oxidative stress on the proteasomal system. MATERIALS AND METHODS Cell culture. K562 cells (chronic myelogenous leukemia, human) were obtained from American Tissue and Cell Culture (ATCC CCL 243). The cells were cultured in 90% RPMI 1640 medium, supplemented with 10% fetal bovine serum. Cells were seeded initially at a density of 0.4 ⫻ 10 6 cells/ml. Cells at third day of growth were used for the experiments. Hydrogen peroxide treatment. Cells were washed with phosphate buffer and afterward exposed to the indicated concentrations of hydrogen peroxide in phosphate-buffered saline (pH 7.4) for 30 min at 37°C. In the case of the 24-h experiments the cells were washed again after hydrogen peroxide treatment and recultivated under the conditions described above. Lysis of cells. For the experiments the cells were washed twice with phosphate buffer and then lysed by repeated freeze–thaw cycles in 0.25 M sucrose, 25 mM Hepes (pH 7.8), 10 mM MgCl 2, 1 mM EDTA, and 1 mM dithiothreitol. Lysates were centrifuged at 14,000g for 30 min. Protein was determined using a Bio-Rad reagent. Proteolysis measurement. Cell lysates were diluted with proteolysis assay buffer (50 mM Hepes (pH 7.8), 20 mM KCl, 5 mM MgCl 2, and 1 mM dithiothreitol) to a protein concentration of 10 ␮g/ml. The peptidase activity was measured by addition of 170 ␮l proteolysis assay buffer and 30 ␮l suc-LLVY-MCA (2 mM stock solution in DMSO) to 100 ␮l of the diluted cell lysate. Where indicated, ATP was added to 5 mM final concentration. To determine the specific degradation of suc-LLVY-MCA by proteasome 5 ␮M of lactacystin, a proteasome inhibitor, was added. The mixture was incubated at 37°C for 1 h. The reaction was stopped by addition of an equal volume of ice-cold ethanol and 10 vol 0.125 M sodium borate (pH 9.0). The fluorescence determination was performed at 380 nm excitation and 440 nm emission using free MCA as a standard. Nondenaturing electrophoresis and protease activity stain. Analysis of the 20S proteasome was performed by a nondenaturing PAGE using a 3% stacking and a 6% separating gel according to Hough et al. (11) with modifications of Reinheckel et al. (12). The separation was performed for 650 Vh at 4°C. Directly after the run the gel was either stained with Coomassie or analyzed for proteasome activity by incubation in 50 mM Tris (pH 7.8), 25 mM KCl, 10 mM NaCl, 1 mM MgCl 2, 1 mM dithiothreitol, 0.1 mM EDTA, and 10% glycerol for 15 min at 37°C. After incubation, the gel was mounted on a light box (emitting light: 366 nm) and overlaid with a 200 ␮M suc-LLVY-MCA solution solved in 50 mM Tris (pH 7.8), 25 mM KCl, 10 mM NaCl, 5 mM MgCl 2, 1 mM dithiothreitol, 0.1 mM EDTA, and 5 mM ATP. The fluorescence was photographed within 15 to 45 min of incubation. SDS–PAGE and immunoblotting. After equalizing the protein content of cell lysates an SDS–PAGE under reducing conditions according to Laemmli et al. (13) was performed. After transferring the samples, the blot membranes were incubated with either an

TABLE I

ATP-Independent and ATP-Stimulated Degradation of the Fluorogenic Peptide suc-LLVY-MCA in Lysates of K562 Cells after Exposure of Cells to H 2O 2 suc-LLVY-MCA degradation (nmol MCA ⫻ min ⫺1 ⫻ mg protein ⫺1) H 2O 2 (mM)

no ATP

5 mM ATP

Ratio (ATP-stimulated/ ATP-independent)

0 0.4 1.0 2.0 5.0

1.50 ⫾ 0.11 1.41 ⫾ 0.10 1.44 ⫾ 0.13 1.47 ⫾ 0.07 1.39 ⫾ 0.15

2.81 ⫾ 0.19 2.20 ⫾ 0.10 1.68 ⫾ 0.24 1.55 ⫾ 0.15 1.42 ⫾ 0.14

1.87 1.56 1.16 1.05 1.05

Note. As the degradation of the fluorogenic peptide by the 26S proteasome is stimulated by ATP, the ratio of ATP-stimulated and ATP-independent lactacystine sensitive proteolysis is indicative for the activity of the 26S proteasome, whereas the ATP-independent degradation represents the activity of the 20S proteasome. Values are the means ⫾ SE for five independent experiments.

anti-proteasome antibody (Prof. K. Tanaka, Japan) or an antiMSS-1- antibody (Affinity, Exeter, UK) and analyzed using a chemiluminescence detection kit. Statistical evaluation. Statistical significance of the data was tested using the Student t test for unpaired samples. Differences were considered to be significant if P ⱕ 0.05 (*) or P ⱕ 0.01 (**).

RESULTS AND DISCUSSION

In the present study K562 cells were treated with H 2O 2 and proteasome activities were measured in cell lysates as ATP-independent and ATP-stimulated degradation of the fluorogenic peptide suc-LLVY-MCA for 20S proteasome and 26S proteasome, respectively. Lactacystin, a proteasome inhibitor, was used to ensure the specificity of the assays. The incubation of K562 cells with H 2O 2 concentrations of up to 5 mM for 30 min did not affect the activity of the 20S proteasome significantly (Table I). In contrast, the ATP-stimulated activity of the 26S-proteasome was significantly reduced by 0.4 mM H 2O 2. At 1 mM H 2O 2 the ATP-stimulated activity and thus the specific activity of the 26S proteasome was completely inhibited (Table I). To give further evidence for the higher sensitivity of the 26S proteasome we used a nondenaturing gel electrophoresis combined with a protease activity stain. This technique allows the physical separation of the two proteasomal forms and is probably more accurate, therefore, in terms of differentiating between the activities of both forms, rather than the presence or absence of ATP. As demonstrated in Fig. 1A a treatment of cells with 1 mM H 2O 2 leads to a complete loss of the activity of the 26S proteasomal form. This result supports therefore the results from the proteolysis measurement in the presence or absence of ATP (see Table I).

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PROTEASOMAL ACTIVITIES DURING OXIDATIVE STRESS TABLE II

Recovery of ATP-Stimulated Proteolysis in Lysates of K562 Cells during 24 h after Exposure of Cells to H 2O 2 Lactacystin-sensitive and ATP-stimulated degradation of suc-LLVY-MCA (nmol MCA ⫻ min ⫺1 ⫻ mg protein ⫺1) H 2O 2 (mM)

0 h after H 2O 2 treatment

24 h after H 2O 2 treatment

Increase during recovery (%)

0 1.0 2.0

1.31 ⫾ 0.17 0.24 ⫾ 0.11 0.07 ⫾ 0.06

1.40 ⫾ 0.15 0.86 ⫾ 0.13 0.08 ⫾ 0.06

106, ns 358** 114, ns

Note. Only the ATP-stimulated activity (difference of the 26S proteasomal activity and the 20S proteasomal activity) is shown. The ATP-independent activity remained unchanged in all instances (see Table I). Values are the Means ⫾ SE for five independent experiments. ** P ⬍ 0.01; ns, not significant. FIG. 1. Analysis of 20S and 26S proteasomes of hydrogen peroxidetreated K562 cells. The experimental conditions of cell culture, hydrogen peroxide treatment, and cell lysis are described under Materials and Methods. (A) The proteolytic activity of proteasomes after electrophoresis under nondenaturing conditions as described by Hough et al. (11). After separation the degradation of the fluorogenic peptide suc-LLVY-MCA was detected by overlaying the gel with 200 ␮M suc-LLVY-MCA. 20S and 26S proteasome bands were identified by comigration of purified proteasomes. (B) The immunoblot of an SDS–PAGE of the samples as presented in A. The 20S proteasome consists of 14 distinct subunits ranging in size from 20 to 35 kDa. The immunoblot was developed with a preparation of IgG purified from an anti-serum raised against isolated 20S proteasome. Therefore a number of subunits of the 20S proteasome were detected. The antibody was generously provided by Prof. K. Tanaka, University of Tokushima, Japan. (C) An immunoblot of the same samples using an anti-MSS-1 antibody (Affinity, UK). Electrophoretic analysis represent one of three experiments.

Remarkably, there was no increase in activity of the 20S proteasome band, as expected if there would be a simple dissociation of the 26S proteasome in the 20S “core” and PA 700. Further analysis of the subunits of the 20S proteasome (Fig. 1B) as well as of a PA 700 subunit MSS-1 (Fig. 1C) revealed no change in the band intensity of these polypeptides. Therefore, a fragmentation or rapid proteolytic degradation of subunits does not seem to occur. Thus, the sensitivity of the 26S proteasome toward oxidative stress is possibly due to the oxidation of amino acids essential for the function of the proteasome activator PA 700. On the other hand, heat shock protein 90 has been implicated in protection against oxidative inactivation of the 20S proteasome (14, 15). This could, at least in part, explain the comparatively high oxidant resistance of these protease during oxidative stress in K562 cells. The fate of cells after oxidative injury depends largely on their ability to recover from the stress. Since the 26S-proteasome is essential for numerous physiological processes (10) it is essential for cellular vitality to reconstitute the activity

of the 26S proteasome. Therefore, we tested for the reconstitution of the proteasomal system 24 h after oxidative stress. As demonstrated in Table II in our model the ATP-stimulated 26S proteasome activity increased during the 24 h after treatment of cells with 1 mM H 2O 2. However, after an incubation with 2 mM H 2O 2 the activity of the 26S proteasome remained completely abolished for 24 h (Table II). These results could be also demonstrated by using the nondenaturing gel electrophoresis (Fig. 2) where no reconstitution of the 26S proteasome could be demonstrated 24 h after treatment with 2 mM H 2O 2 and at least a partial

FIG. 2. Recovery of the 26S proteasome in K562 cells during 24 h after exposure to H 2O 2. Nondenaturing electrophoresis and the protease activity stain using suc-LLVY-MCA were performed as described under Materials and Methods. (A) The activities of 20S and proteasomes immediately after exposure of the cells to H 2O 2 for 30 min. (B) The activities of 20S and proteasomes 24 h after exposure of the cells to H 2O 2. The results represent one typical of three experiments.

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reconstitution of the 26S proteasomal form was found 24 hours after treatment with 1 mM H 2O 2 (Fig. 2). It should be noted that after treatment of K562 cells with 0.5 mM and 1 mM H 2O 2 the viability of cells, as judged by trypan blue exclusion, was unchanged as compared to the control (viability: control 96 ⫾ 5%, 0.5 mM H 2O 2 93 ⫾ 7%, 1.0 mM H 2O 2 92 ⫾ 6%). At 2 mM H 2O 2 treatment the percentage of intact cells started to decline, reaching 51 ⫾ 11% at 5 mM H 2O 2. Thus, the proteasomal system appears to play a central role in the maintenance of cellular integrity after oxidative stress. The 20S proteasome is capable of degrading unfolded (oxidized) proteins and short peptides even after extensive oxidative stress in vitro (12). Only the aggregation of cellular proteins due to free radical induced protein cross-linking can limit the accessibility of substrates to the proteasome and, therefore, compromise the 20S proteasome-mediated proteolysis as part of the secondary antioxidant defense system (6). While the role of the 20S proteasome in response to oxidative stress is relatively well established, the high susceptibility of the 26S proteasome to free radical attack raises a multitude of questions regarding the consequences of oxidant induced 26S proteasome inactivation for cellular function and integrity. Here we could demonstrate for the first time, that the inhibition of the 26S proteasome due to oxidative stress, which was previously reported (12), is not due to a dissociation of the 20S proteasome (“core” proteasome) and the PA 700 activator. On the other hand, no decline of the amount of the MSS-1 subunit of the PA 700 activator could be demonstrated and consequently no selective degradation occurs. In cells the 20S proteasome appears to be relatively resistant to oxidative inactivation. Furthermore, there probably exist mechanisms involved in the activation of the 20S proteasome during oxidative stress, as shown for the nuclear 20S proteasome which can be activated by poly-ADP ribose polymerase (PARP) in order to degrade oxidatively damaged histones (9) or under certain oxidative conditions as shown by Strack et al. (16). However, in any case the protease complex seems to be able to fulfill the proposed role in the removal of oxidatively modified cellular proteins. On the other hand, the here presented results suggest that the specific ATP-dependent activity of the 26S proteasome is highly susceptible toward oxidative stress. As the 26S proteasome degrades polyubiquitinated substrate proteins oxidative inactivation of this protease would result in the accumulation of ubiquitin and ubi-

quitinated proteins in stressed or aged tissues (17). From the point of view of the functions of the 26Sproteasome in the cell cycle progression the impairment of this protease could contribute to the growth arrest frequently observed in response to oxidative stress (18). The ability of a cell to reestablish the 26S proteasome seems to depend on the degree of oxidative stress. Failure to reconstitute the functional 26S proteasome might ultimately lead to some form of cell death or permanent growth arrest. ACKNOWLEDGMENT This work was in parts supported by the Deutsche Forschungsgemeinschaft.

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