Proteasome-Dependent Turnover of Protein Disulfide Isomerase in Oxidatively Stressed Cells

Archives of Biochemistry and Biophysics Vol. 397, No. 2, January 15, pp. 407– 413, 2002 doi:10.1006/abbi.2001.2719, available online at http://www.idealibrary.com on

Proteasome-Dependent Turnover of Protein Disulfide Isomerase in Oxidatively Stressed Cells Tilman Grune,* ,† Thomas Reinheckel,* ,‡ Rui Li,* James A. North, and Kelvin J. A. Davies* ,1 *Ethel Percy Andrus Gerontology Center and the Division of Molecular Biology, the University of Southern California, Los Angeles, California 90089-0191; †Clinics of Physical Medicine and Rehabilitation, Medical Faculty (Charite), Humboldt University, Berlin, Germany; and ‡Center for Surgery and Institute of Clinical Chemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

Received October 22, 2001, and in revised form November 20, 2001

Generalized increases in protein oxidation and protein degradation in response to mild oxidative stress have been widely reported, but only a few individual proteins have actually been shown to undergo selective, oxidation-induced proteolysis. Our goal was to find such proteins in Clone 9 liver cells exposed to hydrogen peroxide. Using metabolic radiolabeling of intracellular proteins with [ 35S]cysteine/methionine, and analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), we found at least three labeled proteins (“A,” “B,” and “C”) whose levels were decreased significantly more than the generalized protein loss after mild oxidative stress. “Protein C” was excised from 2-D PAGE and subjected to N-terminal amino acid microsequencing. “Protein C” was identified as Protein Disulfide Isomerase or PDI (E.C. 5.3.4.1), and this identity was reconfirmed by Western blotting with a C-terminal antiPDI monoclonal antibody. A combination of quantitative radiometry and Western blotting in 2-D PAGE revealed that PDI was selectively degraded and then new PDI was synthesized, following H 2O 2 exposure. PDI degradation was blocked by inhibitors of the proteasome, and by cell treatment with proteasome C2 subunit antisense oligonucleotides, indicating that the proteasome was largely responsible for oxidation-induced PDI degradation. © 2002 Elsevier Science Key Words: protein disulfide isomerase; proteasome; protein oxidation; protein degradation; protein turnover; oxidative stress; free radicals; aging.

Living organisms posses multiple layers of defense, damage removal, and repair systems to deal with the 1 To whom correspondence and reprint requests should be addressed. Fax: (213) 740-6462. E-mail: [email protected].

0003-9861/02 $35.00 © 2002 Elsevier Science All rights reserved.

ever-present threat of oxidative stress (1–3). It has been proposed that the multicatalytic proteinase complex, “Proteasome” constitutes one such damage removal/repair system by recognizing and selectively degrading oxidatively damaged proteins in the cytoplasm, nucleus, and endoplasmic reticulum of eukaryotic cells (3–34). Despite numerous reports of overall increases in proteasome-dependent proteolysis following oxidative stress; however, there are very few indications as to whether certain specific proteins might be preferentially degraded, or if most or even all cell proteins are equal targets of oxidation and degradation. Some of the most important work in this area is that of Stadtman, Levine, Rivett, and colleagues, who have provided vital data on the selective oxidative damage and subsequent degradation of glutamine synthetase (18 –34). Taylor and colleagues have studied lens crystallins (35–37) and Guo et al. have studied iron regulatory protein 2 (38). We have reported on the increased proteolytic susceptibility of oxidatively modified hemoglobin (5, 7, 9 –12, 15) superoxide dismutase (8, 10, 15), aconitase (6), and histone proteins (17). As important as these studies are in indicating possible targets of oxidative damage to proteins; however, most of the data so far collected (including our own) has tended to highlight relatively mild oxidative damage to purified proteins in vitro which can then be shown to exhibit increased proteolytic susceptibility when incubated (again in vitro) with proteolytic enzymes. It is also well established that very severely oxidized proteins, which form large aggregates of hydrophobic bonds, ionic bonds, and various covalent cross-links, become progressively more resistant to proteolysis (4 –17, 39, 40). The present report represents one of a series of studies in which we have tried to determine if certain proteins are actually preferential targets of oxidative 407

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damage and degradation in intact mammalian cells. To this end we have exposed Clone 9 liver cells (a normal rat liver, permanent epithelial line) containing metabolically radiolabeled cell proteins, to oxidative stress in the form of hydrogen peroxide (H 2O 2), established that overall proteolysis was significantly increased, and then compared the actual intracellular levels of individual proteins using two-dimensional gel electrophoresis. Candidate electrophoresis “spots” were excised from the gels and microsequenced. As we now report, one candidate protein to be identified in this way was protein disulfide isomerase (PDI), 2 and we further studied the selective degradation of this oxidized protein, and its poststress resynthesis, using monoclonal antibodies, proteasome inhibitors, and proteasome antisense oligonucleotides. MATERIALS AND METHODS Cells and cell culture. Clone 9 liver cells (normal rat liver epithelia) obtained from American Type Culture Collection (ATCC CRL 1439) were maintained in 90% Ham’s F-12K medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 Units/ml penicillin, and 10 ␮g/ml streptomycin. Cells were typically replated at a density of 2 ⫻ 10 4 cells/cm 2 every 2 to 3 days to maintain logarithmic growth. After experimental treatment, both control cultures and treated samples were harvested, using a tissue scraper and small amounts of cold phosphate-buffered saline (PBS) containing 1 mM EDTA. The cells were washed and resuspended in 300 ␮l PBS-EDTA in microcentrifuge tubes, disrupted by a 10 s burst of sonication (20 mW power), and the supernatant collected after centrifugation for 15 min at 10,000g. For electrophoresis and antibody studies the homogenizing buffer contained a protease inhibitor cocktail (Sigma catalog number P-8340) to decrease protein degradation during sample preparation. Metabolic radiolabeling of cellular proteins. Cell cultures were washed with PBS and incubated for 2 h in Eagle’s minimal essential medium without methionine or cysteine but containing 0.01 mCi/ml [ 35S]methionine/cysteine mixture (NEN catalog number NEG772). After cell proteins were metabolically radiolabeled, the cultures were washed twice with PBS and incubated with Ham’s complete medium for 2 h as a cold “chase.” These exact procedures have previously been used extensively in this laboratory for studies of protein turnover in various mammalian cells (13–17). Exposure to oxidative stress. Subconfluent monolayers of clone 9 liver epithelial cells were exposed to bolus additions of hydrogen peroxide. The amount of hydrogen peroxide used was corrected for the number of cells present in the culture so that, in every experiment, the concentration was normalized as ␮M H 2O 2 per 3 ⫻ 10 5 cells in a final exposure volume of 10 ml. After incubation at 37°C for 30 min, the cells were washed twice with PBS and harvested immediately (0 time point) or incubated with Ham’s complete medium before washing and harvesting at the appropriate time points. 2-D gel electrophoresis, Western blotting, and protein micrrosequencing. Two dimensional polyacrylamide gel electrophoresis (2-D PAGE), consisting of sodium dodecyl sulfate PAGE in the first di2

Abbreviations used: PBS, phosphate-buffered saline; 2-D PAGE, two-dimensional polyacrylamide gel electrophoresis; NLVS, trileucine vinyl sulfone; N-t-Boc-GAR-MCA, N-t-Boc-Gln-Ala-Arg 7-amido-4-methylcoumarin; H 2O 2, hydrogen peroxide; PDI, protein disulfide isomerase.

FIG. 1. Hydrogen peroxide causes proteolysis in clone 9 liver cells. Clone 9 liver cells containing metabolically 35S-radiolabeled proteins were exposed (using 3 ⫻ 10 5 cells in a 10 ml volume) to various concentrations of H 2O 2 for 30 min, at 37°C, as described under Materials and Methods. Overall protein degradation was measured by increases in acid-soluble radioactivity, following cell disruption and trichloroacetic acid precipitation of remaining intact proteins, as previously described (13–17). Results shown are means and SEs of three independent observations.

mension and isoelectric focusing in the second dimension, as described by O’Farrell (41), was performed as previously detailed (42). Western blots were visualized using a commercially available monoclonal antibody directed against the carboxyl terminal end of PDI and an alkaline phosphatase-tagged secondary antibody (Accurate Chemical & Scientific Corp., Product No. YSGSPA891, Westbury, NY). Amino-terminal protein micro sequencing was performed using a 12% SDS–PAGE separating gel (16 ⫻ 20 cm) by electroblotting proteins onto PVDF membranes. Spots of particular interest were sent to Immuno-Dynamics, Inc. (La Jolla, Ca) for the actual protein sequencing.

RESULTS

Exposure of Clone 9 liver cells to mild oxidative stress resulted in a progressive increase in proteolysis, as measured by the release of acid-soluble counts from (previously) metabolically 35S-radiolabeled cell proteins, which peaked at an H 2O 2 concentration of 400 ␮M and declined sharply at higher concentrations (Fig. 1). These results are consistent with previous reports indicating that mild oxidative stress increases proteolytic susceptibility whereas severely oxidized proteins become progressively poorer substrates (4 –17). The cell-permeant proteasome inhibitors lactacystin, NLVS, and epoxomicin, all caused 66 –76% inhibition of this oxidant-induced proteolysis, whereas the lysosomal proteolysis inhibitor leupeptin had little effect (Fig. 2). A 10-day preincubation of cells with an antisense oligonucleotide directed against the proteasome C2 subunit inhibited oxidant-induced proteolysis by

PDI TURNOVER IN OXIDATIVE STRESS

FIG. 2. Inhibitors of hydrogen peroxide-induced proteolysis in clone 9 liver cells. Metabolically radiolabeled cells (using 3 ⫻ 10 5 cells in a 10-ml vol) were exposed to 400 ␮M H 2O 2 for 30 min, as described in the legend of Fig. 1. In various experiments the selective proteasome inhibitors lactacystin (10 ␮M), NLVS (tri-leucine vinyl sulfone; -t-Boc-GAR-MCA, N-t-Boc-Gln-Ala-Arg 7-amido-4-methylcoumarin at a concentration of 10 ␮M), or epoxomicin (20 ␮M), or the lysosomal proteolysis inhibitor leupeptin (100 ␮M), were included in the incubation. In some experiments cells were preincubated for 7 days with daily 0.4 nmol/ml treatments of a proteasome C2 antisense oligonucleotide (5⬘-AGCTATGTTTCGCAA-3⬘), or a control oligonucleotide, in order to decrease intracellular proteasome levels and activity, as previously described (13, 14). Percentage inhibition values shown above are expressed relative to the maximal proteolysis observed at 400 ␮M H 2O 2 as described in the legend of Fig. 1. All values are means and standard errors of four independent observations.

85%, whereas a control oligonucleotide had no effect (Fig. 2). As we have previously demonstrated antisense oligonucleotides are highly effective in lowering the intracellular levels of both proteasome subunits, and overall proteasome activity in clone 9 cells (13, 15–17) and other cell types (14, 15). We next incubated clone 9 cells, containing metabolically radiolabeled 35S-proteins, with 400 ␮M H 2O 2 then disrupted the cells and separated proteins by 2-D PAGE. After repeating this experiment several times it became clear that the levels of at least three proteins were particularly diminished following H 2O 2 exposure; these three proteins have been labeled, “A,” “B,” and “C” in the partial 2-D PAGE shown in the left panel of Fig. 3. The N-terminus of Protein “A” was blocked, and the analysis of Protein “B” will be reported elsewhere, but protein “C” forms the subject of this paper. We followed the fate of the radiolabeled “spot” labeled protein “C” (at approximately 56,000 daltons and approx-

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imately 5.5 PI) for 24 h after H 2O 2 exposure and, as shown in Fig. 3, found a clear and repeatable loss. By reversing the order of oxidant treatment and metabolic radiolabeling used in Figs. 1–3 we were able to ask whether the unidentified protein “C” underwent resynthesis after H 2O 2 treatment. As shown in Fig. 4, cells treated with only 100 ␮M H 2O 2 and then metabolically radiolabeled with [ 35S]cysteine/methionine, exhibited an increase in the synthesis of the 56,000 Da, 5.5 PI protein “C,” especially after 24 h. Exposure to 400 ␮M H 2O 2 (which caused maximal proteolysis in Fig. 1) caused an even stronger induction of unknown protein “C” (Fig. 4). We next wanted to identify protein “C,” which, from analysis of our 2-D PAGE, had a molecular weight of 56,000 and a PI of 5.5. Protein “C” was excised from a 2-D gel and the first 19 N-terminal amino acids were micro-sequenced. A search of the SwissProt data base by the BLASTP program revealed a 95% match with an internal region close to the N-terminus (residues 26 – 43) of rat Protein Disulfide Isomerase (E.C. 5.3.4.1) or “PDI” (Table 1). As reported in the legend to Fig. 3, unknown “Protein C” had an apparent molecular weight of 56,000 and an apparent PI of 5.5. Literature reports indicate a PI of approximately 5.5 but a molec-

FIG. 3. Unidentified “protein C” is preferentially degraded after hydrogen peroxide treatment of clone 9 liver cells. Intracellular proteins were metabolically radiolabeled with [ 35S]cysteine/methionine, cells were exposed to 400 ␮M H 2O 2 and then disrupted and analyzed by 2-D electrophoresis, as described under Materials and Methods, using 3 ⫻ 10 5 cells in a 10-ml volume. The box to the left of the figure shows an expanded region of a 2-D electrophoresis gel showing three metabolically 35S-radiolabeled protein “spots” (A, B, and C) whose concentrations were significantly reduced by exposure to hydrogen peroxide. The pH gradient becomes more basic from left to right and the apparent molecular weight decreases going from the top to the bottom of the gel. Protein C had an apparent molecular weight of approximately 56,000 and an apparent PI of approximately 5.5 in comparison with standards. The main panel of the figure shows the effects of H 2O 2 exposure over a 24-h period on the quantity of the radioactive 35S-protein “C” spot at 56,000 daltons and 5.5 PI, remaining in cells. The experiment was repeated five times with similar results.

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otide all caused 70 –95% inhibition of oxidant-induced PDI degradation, whereas the lysosomal proteolysis inhibitor leupeptin and control oligonucleotides had little or no effect. DISCUSSION

FIG. 4. Induction of the unidentified “protein C” in hydrogen peroxide-treated clone 9 liver cells. This is a modification of the experiments described in the legend of Fig. 3, in which unlabeled cells (using 3 ⫻ 10 5 cells in a 10 ml volume) were first exposed to 400 ␮M H 2O 2 or used as controls, and all cells were then incubated with [ 35S]cysteine/methionine for metabolic radiolabeling of intracellular proteins, as described under Materials and Methods. Each panel shown the 35S-radioactive protein “C” spot at 56,000 Da and 5.5 PI, which increases with time and H 2O 2 exposure. The gels shown are representative of results that were repeated four times.

ular weight of 58,000 for PDI (42, 43). It seems likely that a region of 25 amino acids from the N-terminus of PDI is proteolytically removed under the conditions of our studies in Clone 9 liver cells. To confirm the identity of protein “C” as PDI, and to more carefully follow PDI degradation and resynthesis, we used a monoclonal antibody (see Materials and Methods) directed against the C-terminus of the enzyme. Western blots revealed an exact match between the radioactive “spot” initially identified as protein “C,” the excised “spot” subsequently identified by amino acid microsequence analysis as PDI, and the protein to which anti-PDI antibodies bound. Thus, we feel justified in positively identifying protein “C” as PDI. We next performed a series of detailed proteolysis and protein synthesis experiments of a “dual-label” type, in which loss of PDI radioactive counts or gain of radioactive counts was confirmed by immunoblotting. In these studies we found that PDI underwent extensive proteolysis following exposure to H 2O 2 (Fig. 5, left panel), and cells rapidly synthesized replacement PDI during a 24-h period after the oxidative stress (Fig. 5, right panel). Despite these quite dramatic increases in both PDI degradation and synthesis, however, no change in the steady-state cellular levels of PDI protein could be detected by immunoblotting analysis (data not shown). Since the results of Fig. 2 indicated that proteasome was mostly responsible for overall proteolysis following H 2O 2 exposure, we next wanted to test if proteasome might catalyze PDI degradation under the same conditions. As shown in Fig. 6, the selective proteasome inhibitors lactacystin, NLVS, epoxomicin, and the proteasome C2 antisense oligonucle-

We now report that PDI undergoes extensive proteolytic degradation in clone 9 liver cells exposed to the oxidative stress generated by hydrogen peroxide. This selective proteolysis is largely catalyzed by the proteasome multicatalytic proteinase complex. Although oxidatively stressed cells exhibit a significant overall increase in proteolysis, the extent of PDI degradation is far greater; reaching approximately 90% (as judged by tracer studies) within 24 h. While cells are rapidly degrading PDI they also begin extensive synthesis of new PDI. The net result of these rather dramatic increases in PDI degradation and synthesis is an almost constant steady-state intracellular level of PDI protein.

TABLE I

Comparison of Amino Acid Sequences of Protein “C” and Protein Disulfide Isomerase “Protein C”

Rat protein disulfide isomerase

Residue

Amino acid

Residue

Amino acid

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

? Aspartic acid Valine Leucine Glutamic acid Leucine Threonine Aspartic acid Glutamic acid Asparagine Phenylalanine Serine Threonine Arginine Valine Aspartic acid Isoleucine Threonine Glycine

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Serine Aspartic acid Valine Leucine Glutamic acid Leucine Threonine Aspartic acid Glutamic acid Asparagine Phenylalanine Serine Serine Arginine Valine Aspartic acid Isoleucine Threonine Glycine

Note. A 19 amino acid N-terminal stretch of unknown “Protein C” from Clone 9 rat liver cells was subjected to N-terminal sequencing as described under Materials and Methods. The first residue could not be resolved but the following 18 residues were successfully sequenced. Protein C is compared with an internal stretch of 19 amino acids in the sequence of rat liver Protein Disulfide Isomerase (E.C. 5.3.4.1). The PDI sequence was taken for Accession No. P11598 from the Swiss Protein Database, updated August 20, 2001. The 18 sequenced Protein C amino acids exhibited a 17/18 match with a sequence of PDI extending from residue 24 through 43 (95% sequence identity).

PDI TURNOVER IN OXIDATIVE STRESS

FIG. 5. Increased protein disulfide isomerase turnover induced by hydrogen peroxide in clone 9 liver cells. To measure PDI degradation (Right Panel) cell proteins were first metabolically radiolabeled with [ 35S]cysteine/methionine and cells were then exposed to 400 ␮M H 2O 2 for 30 min, at 37°C as per the legend of Fig. 3. To measure PDI protein synthesis (right panel) cells were first exposed to 400 ␮M H 2O 2 and then cell proteins were metabolically radiolabeled with [ 35S]cysteine/methionine, as described in the legend of Fig. 4. PDI degradation or synthesis were then measured by quantifying the radioactivity in 2-D gel electrophoresis spots whose identity was confirmed by immunoblotting with a monoclonal antibody directed against the PDI C-terminus (see Materials and Methods). All values shown are means and standard erors of at least three independent observations, using 3 ⫻ 10 5 cells in a 10-ml volume.

The most plausible explanation for this phenomenon may be that the enzyme is preferentially damaged or modified during the initial H 2O 2 exposure, is then recognized and selectively degraded by the proteasome, and must then be synthesized de novo to maintain cellular homeostasis. It is not yet known if PDI is especially sensitive to oxidative damage, modification, or “proteolytic marking.” Alternatively, since PDI interacts with proteins carrying oxidized sulfhydryl groups (43, 44), and since such proteins also frequently carry other oxidized residues (1– 40), it is entirely possible that increased selective interaction with protein oxidation products (during oxidative stress) may cause extensive PDI inactivation and increased proteolytic susceptibility. Other possibilities, such as a programmed reorganization of signaling enzymes, within complex oxidative stress signal transduction pathways, may also be possible. PDI is found at a concentration of approximately 10 mg/ml (approaching mM levels) in the lumen of the endoplasmic reticulum, where it has a very important role in the refolding of misfolded proteins, via isomerization of inappropriate protein disulfide bonds (43, 44). The enzyme is a member of the thioredoxin superfamily and is dependent on reducing equivalents from glutathione for its enzymatic activity (43, 44). A good deal of confusion once surrounded PDI, which, at various times, has been thought to be a phosphilipase C

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alpha (45) or even a cysteine protease (46). It now seems clear, however, that PDI is indeed a disulfide isomerase (43, 44), which may well also be a glucoseregulated chaperone (47, 48) and which has been shown to be induced during hypoxia/reperfusion injury where it seems to protect against apoptosis (49). Finally, PDI is also a subunit component of the microsomal triglyceride transfer protein complex (50, 51). The high intracellular levels of PDI, its interactions with oxidized protein substrates and glutathione, and its responsiveness to stress, may all contribute to its increased turnover during oxidative stress. It is not yet clear exactly how many proteins undergo selective proteolytic breakdown following oxidative stress, but it does appear that many proteins undergo a generalized increase in proteolysis while some, such as proteins A, B, and C in this paper, and others discussed below, are more extensively degraded. PDI now joins a relatively small group of proteins (at least so far) whose turnover is dramatically increased by oxidative stress. Other members of this “family” appear to include glutamine synthetase (18 –34), hemoglobin (5, 7, 9 –12, 15), aconitase (6), superoxide dismutase (8, 10, 15), lens crystallins (35–37), iron regulatory protein (38), histone proteins (17), and ezrin (52, 53). Whether these proteins all share particularly oxidation-sensitive motifs, geographic susceptibility,

FIG. 6. Inhibitors of hydrogen peroxide-induced protein disulfide isomerase degradation in clone 9 liver cells. PDI degradation was measured as described in the legend of Fig. 5, left panel. The effects of various protease inhibitors and proteasome C2 subunit antisense oligonucleotides were tested exactly as described in the legend of Fig. 2. Values shown are the means and standard errors of three independent observations, using 3 ⫻ 10 5 cells in a 10-ml volume.

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stress-involvement, or a mixture of such properties is not yet known. It is intriguing that all eight of these proteins seem to be degraded by the proteasome. The proteasome exists in several different forms in all mammalian cells (54) and is probably responsible for the turnover of most aberrant soluble proteins (53). Importantly, declining proteasome activity during aging may predispose cells to accumulating damaged proteins (55– 62) and is associated with degenerative problems such as Alzheimer disease (58, 61). Whether PDI is one of the proteins that accumulates in aging tissues has yet to be tested. ACKNOWLEDGMENTS This research was supported by NIH/NIEHS Grant ES-03589 to K.J.A.D. This paper is dedicated to Dr. Earl R. Stadtman on the occasion of his retirement as an Executive Editor of the journal Archives of Biochemistry and Biophysics. His energy, enthusiasm, and integrity are an inspiration to one and all.

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