Redox Control of 20S Proteasome

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redox sensor] Figure 7 shows Po graphs of two separate channels before and after labeling with CPM. Both channels respond strongly to symmetric - 1 8 0 mV transmembrane redox potential and are negatively regulated by a - 2 2 0 / - 1 8 0 mV cis/trans gradient [compare Fig. 7, bars 1-3 (left) and Po graphs (right)]. After exposing the channels to 20 nM CPM for 2 min [Fig. 7, bar 4 (marked with an asterisk), CPM removed from the bath by extensive perfusion of the chamber with 25 volumes of buffer], neither channel responds to a - 180/- 180 mV symmetric cis/trans redox gradient (compare bars 4 and 5 in Fig. 7). Acknowledgments This work was supported by NIH Grants 2RO 1AR43140 and 1POAR17605.

[23] R e d o x C o n t r o l By BERTRAND

of 20S

Proteasome

FRIGUET, A N N E - L A U R E BULTEAU, MARIANGELA CONCONI,

and ISABELLE

PETROPOULOS

Introduction Oxidative modifications of proteins have been implicated in age- and diseaserelated impairment of cellular functions and are known to affect protein turnover.l-3 Because the 20S proteasome has been shown to be the major actor in the degradation of oxidized protein4 and consequently to be important in the regulation of the steady state level of altered proteins in the cell, the fate of proteasome subjected to oxidative processes has deserved specific attention. 5-9 On oxidative stress, an increase in intracellular proteolysis of oxidized protein is well documented in different cell systems although no upregulation of proteasome subunits synthesis has 1 B. S. Berlett and E. R. Stadtman, J. Biol. Chem. 272, 20313 (1997). 2 T. Grune, T. Reinheckel, and K. J. Davies, FASEB J. 11, 526 (1997). 3 B. Friguet, A. L. Bulteau, N. Chondrogianni, M. Conconi, and I. Petropoulos, Ann. N. Y Acad. Sci. 908, 143 (2000). 4 T. Grune, T. Reinheckel, M. Joshi, and K. J. Davies, J. Biol. Chem. 270, 2344 (1995). 5 M. Conconi, L. I. Szweda, R. L. Levine, E. R. Stadtman, and B. Friguet, Arch. Biochem. Biophys. 331, 232 (1996). 6 p. R. Strack, L. Waxman, and J. M. Fagan, Biochemistry 35, 7142 (1996). 7 M. Conconi and B. Friguet, Mol. Biol. Rep. 24, 45 (1997). 8 M. Conconi, I. Petropoulos, I. Emod, E. Turlin, E Biville, and B. Friguet, Biochem. J. 333, 407 (1998). 9 T. Reinheckel, N. Sitte, O. Ullrich, U. Kuckelkorn, K. J. Davies, and T. Grune, Biochem. J. 335, 637 (1998).

METHODSIN ENZYMOLOGY,VOL.353

Copyright2002,ElsevierScience(USA). All rightsreserved. 0076-6879/02$35.00

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[23]

been reported. 2 This increase in proteasome-dependent proteolysis is likely the result of an increased proteolytic susceptibility of oxidized proteins, although a transient activation of the proteasomal system cannot be excluded. 6'8 The proteasomal system is made of a catalytic core, the 20S proteasome (EC 3.4.25.1), and several regulatory components that affect its specificity and activity.l°,11 The 20S proteasome is a high molecular weight (700,000) multicatalytic proteinase found both in the cytosol and nucleus of eukaryotic cells. The eukaryotic proteasome is made up of 28 (2 × 14) different subunits arranged as 4 stacked rings, each containing 7 subunits. The t~-type subunits form the outer tings and the 13-type subunits form the inner rings; three of the fl-type subunits have an N-terminal threonine that is critical for proteolytic activity.12' 13 Indeed, the hydroxyl group acts as a nucleophile in the active site and defines a new type of protease as compared with serine and cysteine proteases. The 20S proteasome is characterized by three main proteolytic activities with distinct specificities against short synthetic peptides: the trypsin-like activity (which cleaves after basic residues like arginine or lysine) expressed by the Z and MECL1 subunits, the chymotrypsin-like activity (which cleaves after large hydrophobic residues such as tyrosine or phenylalanine) expressed by the X, LMP2, and LMP7 subunits, and the peptidylglutamyl peptide-hydrolyzing activity (which cleaves after acid residues such as glutamic acid) expressed by the Y subunits. 14 Exposure to metal-catalyzed oxidation of purified 20S proteasome in vitro or of rat hepatoma cells results in the inactivation of certain peptidase activities. 5'8 However, the alterations in peptidase activities observed in vitro depend on whether the purified proteasome is in its latent or active form before treatment with the reactive oxygen species-generating system. 8 Appropriate protocols aimed at isolating the 20S proteasome from different sources, as well as methods for promoting the conversion from latent to active proteasome, are described. Different ways of monitoring oxidation-mediated functional and structural changes of the 20S proteasome, either purified or within tissue and cell homogenates, are then presented. 20S Proteasome Isolation When assayed in crude homogenates, it is not always simple to distinguish between the proteasome peptidase activities and other intracellular protease activities. Even by using specific proteasome inhibitors that permit selectively measurement of its peptidase activities, these activities may still be affected by the presence of endogenous inhibitors, activators, and competing denatured protein substrates. In addition, the analysis of structural modifications of the 20S l00. Coux, K. Tanaka,and A. L. Goldberg,Annu. Rev. Biochem. 65, 801 (1996). II G, N. DeMartinoand C. A. Slaughter,J. Biol. Chem. 274, 22123 (1999). 12j. Lowe, D. Stock,B. Jap, P. Zwickl,W. Baumeister,and R. Huber, Science 268, 533 (1995). 13M. Groll,L. Ditzel,J. Lowe,D. Stock, M. Bochtler, H. D. Bartunik, and R. Huber,Nature (London) 386, 463 (1997). 14M. Orlowskiand S. Wilk,Arch. Biochem. Biophys. 383, 1 (2000).

[23]

REDOX CONTROLOF 20S PROTEASOME

255

proteasome can be conducted only after its isolation. Depending on the source and the amount of starting biological material, different purification protocols must be set up in order to obtain both good yields and purification factors. Accordingly, ammonium sulfate precipitation followed by conventional ion-exchange and gelfiltration chromatographic methods is recommended with such organs as human placenta or rat liver. Ultracentrifugation followed by ion-exchange or affinity chromatography is advisable when dealing with smaller organs or tissues such as rat heart, human epidermis biopsies, or cell cultures (e.g., fibroblasts or keratinocytes). Addition of EDTA to the homogenization buffer and working at a low temperature (4 ° ) are required to prevent inactivation and oxidative modification of the proteasome during the sample preparation procedure.

Purification by Precipitation and Conventional Chromatographic Methods Organs or tissues that have been kept frozen at - 7 0 ° are thawed and homogenized with either a Potter-Elvehjem glass homogenizer with a Teflon pestle (for rat liver) or a Waring Blender (for human placenta) in 20 mM HEPES, pH 7.8, supplemented with 0.1 mM EDTA and 1 mM 2-mercaptoethanol. The homogenate is centrifuged at 15,000g for 1 hr at 4 ° and the supernatant is subjected to a first precipitation with 35% (w/v) saturated ammonium sulfate followed by a second precipitation of the resulting supernatant with 60% (w/v) saturated ammonium sulfate. The pellet obtained after centrifugation at 10,000g for 30 min at 4 ° is then resuspended in 10 mM Tris-HC1, pH 7.2, supplemented with 100 mM KC1, 0.1 mM EDTA, and 1 mM 2-mercaptoethanol, and dialyzed overnight at 4 ° against the same buffer. A first ion exchange is performed on a DEAE-5PW column (TosoHaas, Stuttgart, Germany) in 10 mM Tris-HCl, pH 7.2, with a linear gradient of KC1 from 0.1 to 0.5 M on a Beckman Gold liquid chromatograph (BeckmanCoulter, Fullerton, CA). For the different chromatographic steps, the fractions are assayed for 20S proteasome activity with the synthetic substrate LLVY-MCA (see below). The pooled fractions containing the 20S proteasome are then subjected to a second ion exchange on a Mono Q HR 5/5 column (Amersham Pharmacia Biotech, Uppsala, Sweden) in 20 mM Tris-HC1, pH 7.2, with a linear gradient of KC1 from 0.1 to 0.5 M. Finally, the pooled fraction containing the 20S proteasome is chromatographed on a Superose 6 HR column (Amersham Pharmacia Biotech) in 50 mM potassium phosphate (pH 7), 100 mM KC1. Purified 20S proteasome is then dialyzed against a buffer such as 20 mM HEPES, pH 7.8, and stored at - 7 0 °. For long storage periods, the addition of glycerol up to 20% (v/v) is recommended.

Purification by Ultracentrifugation and Ion-Exchange or Affinity Chromatography The handling of smaller amounts of starting biological material prevents the use of the conventional purification procedure described above. Therefore, a two-step procedure consisting of ultracentrifugation followed by chromatography, either

256

PROTEIN STRUCTURE AND FUNCTION

[23]

ion-exchange chromatography on a Mono Q HR 5/5 column (Amersham Pharmacia Biotech) or affinity chromatography with human proteasome monoclonal antibody (MCP 21) coupled to a HiTrap NHS-activated column according to the manufacturer instructions (Amersham Pharmacia Biotech), has proved to be useful for purifying 20S proteasome from rat heart and human epidermis, respectively. After homogenization of the tissue and/or lysis of the cells, the homogenate is first centrifuged at 15,000g for 1 hr at 4 ° to remove cellular debris. The supernatant is then centrifuged at 100,000g for 16 hr at 4 ° to pellet the 20S proteasome. The pellet is dissolved in 20 mM Tris-HC1, pH 7.2, supplemented with 100 mM KC1, 0.1 mM EDTA, and 1 mM 2-mercaptoethanol before being loaded on a Mono Q HR 5/5 column (Amersham Pharmacia Biotech) as decribed above. Alternatively, the pellet is dissolved in 25 mM Tris-HC1, pH 7.5, before being loaded on the MCP 21 affinity column. After washing with 25 mM Tris-HC1, pH 7.5, the 20S proteasome is eluted from the affinity column with 2 M NaC1 in 25 mM Tris-HCl, pH 8, and then dialyzed against 25 mM Tris-HC1, pH 7.5. The original affinity purification of the human 20S proteasome using MCP 21 has been described by Hendil and Uerkvitz, 15 and MCP 21 is now available as purified antibody from Affiniti (Exeter, UK) or as a hybridoma cell line from either the European collection of cell cultures (ECACC, Salisbury, UK) or the American Type Culture Collection (ATCC, Manassas, VA). C o n v e r s i o n of 2 0 S P r o t e a s o m e f r o m L a t e n t to Active F o r m After purification, the proteasome is generally obtained in a latent form that can be further activated in vitro by various treatments such as incubation with poly-Llysine or fatty acids, heating, freezing and thawing, long storage in the absence of glycerol, addition of a low concentration of sodium dodecyl sulfate (SDS), or dialysis against water. 16-19 Depending on the treatment, 20S proteasome activation is characterized by increased proteolytic activity against both synthetic peptide substrates and protein substrates (e.g., oxidized protein and casein). This activation presumably results from conformational rearrangements of the proteasome complex.17,18,20.21 Activation of the 20S proteasome is also achieved by physiological activators of the proteasome, such as PA 28 (or 11S regulator) and PA 700 (or 19S regulator). 1°'11'22 The precise way these effectors regulate proteasome 15 K. B. Hendil and W. Uerkvitz, Z Biochem. Biophys. Methods' 22, 159 (1991). 16 M. J. McGuire, M. L. McCullough, D. E. Croall, and G. N. DeMartino, Biochim. Biophys. Acta 995, 181 (1989). L7p. E. Falkenburg and P. M. Kloetzel, J. Biol. Chem. 264, 6660 (1989). L8y. Saitoh, H. Yokosawa, and S. Ishii, Biochem. Biophys. Res. Commun. 162, 334 (1989). 19 T. Tokumoto and K. Ishikawa, Biochem. Biophys. Res. Commun. 192, 1106 (1993). 20 H. Djaballah, A. J. Rowe, S. E. Harding, and A. J. Rivett, Biochem. J. 292, 857 (1993). 21 M. E. Figueiredo-Pereira, W. E. Chen, H. M. Yuan, and S. Wilk, Arch. Biochem. Biophys. 317, 69 (1995). 22 D. Voges, P. Zwickl, and W. Baumeister, Annu. Rev. Biochem. 68, 1015 (1999).

[23]

REDOX CONTROLOF 20S PROTEASOME

257

activity is still unclear, but it has been suggested that binding of these activators promotes a different conformation of the proteasome. Addition of SDS at concentrations varying from 0.01 to 0.05% (w/v) has shown that full activation of the three proteasome peptidase activities can be achieved at about 0.03% (w/v) for 20S proteasome purified from rat liver or human placenta. On overnight dialysis of 20S proteasome from rat liver or human placenta against water, the proteasome peptidase activities become activated, as does the oxidized glutamine synthetase or casein degradation activity. In the following sections dealing with the effects of different oxidative treatment on 20S proteasome peptidase activity, the proteasome active form that has been used is that obtained after dialysis against water because, in this case, the proteasome preparation is devoid of any additives, such as SDS, that may interfere with the treatment.

20S Proteasome Functional Changes on Oxidative Treatments

Proteolytic Assays Using Peptide and Protein Substrates Proteasome peptidase activities can be monitored by using fluorogenic peptide substrates (available from Sigma, St. Louis, MO): Ala-Ala-Phe-amidomethylcoumarin (AAF-MCA) or succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (Suc-LLVY-MCA) at 20 # M for the chymotrypsin-like activity, N-tert-butyloxycarbonyl-Leu-Ser-Thr-Arg-amidomethylcoumarin (Boc-LSTR-MCA) at 20/zM for the trypsin-like activity, and N-benzyloxycarbonyl-Leu-Leu-Glu-/%naphthylamide (Cbz-LLE-NA) at 100/zM for the peptidylglutamyl-peptide hydrolase activity. In cell or tissue homogenates, the use of the AAF-MCA fluorogenic peptide should be avoided because it has an unprotected N terminus and is also a good substrate for aminopeptidases. Incubation is achieved with 1 to 5/zg of 20S proteasome in 200 #1 of 0.1 M HEPES, pH 7.8, or 25 mM Tris-HC1, pH 7.5, at 37 ° for 20 min, at which point the reaction is stopped with 300/zl of acid (30 mM sodium acetate, 70 mM acetic acid, 100 mM sodium chloroacetate, pH 4.3) or ethanol. After addition of 2 ml of distilled water, the fluorescence is monitored with a spectrofluorimeter at excitation and emission wavelengths of 350/440 and 333/410 nm for aminomethylcoumarin (MCA) and/%naphthylamine (NA) products, respectively. A calibration curve is obtained by using different concentrations, from 0.1 to 1 #M, of the product, MCA or NA. For a large number of samples (up to 96) the assay can be conducted with a temperature-controlled microplate fluorimetric reader (e.g., FLUOstar Galaxy; B & L Systems, Maarssen, The Netherlands), which has the additional advantage of allowing the kinetics with which the fluorescent products appear to be monitored. Oxidized proteins and casein are considered good protein substrates for the 20S proteasome. Proteolysis of these substrates is monitored by measuring the resulting small peptides as a function of incubation time of the protein with the proteasome. As opposed to the protein substrate and the proteasome, such small peptides are

258

PROTEINSTRUCTUREAND FUNCTION

[23]

soluble in 10% (w/v) trichloroacetic acid (TCA), and several methods can be used to quantify the concentration of the released peptides. A substrate of choice is radiolabeled [14C]casein (DuPont-NEN, Zaventem, Belgium) or oxidized proteins (glutamine synthetase). 23 Otherwise, the TCA supernatant is first neutralized with an equal volume of 2 M sodium borate, pH 10. When the substrate is casein-fluorescein isothiocyanate (Sigma), the concentration of small peptides is monitored directly with a spectrofluorimeter (excitation at 495 nm, emission at 515 nm). Alternatively, when protein substrates are not labeled by radioactivity or fluorescence, the concentration of small peptides may also be determined after reaction of their free NH2 groups with fluorescamine (0.06-mg/ml final concentration; stock solution, 0.3 mg/ml in acetone) by spectrofluorimetry (excitation at 375 nm, emission at 475 nm). Oxidation-Mediated Changes in Activity of Purified 20S Proteasome

20S proteasome from rat liver (at 0.1 mg/ml) has been exposed to metalcatalyzed oxidation on incubation in 0.1 M HEPES, pH 7.8, in the presence of 0.1 mM FeC13 and 25 mM ascorbate. At the indicated times, 50-#1 aliquots are diluted in 200/zl of 0.1 M HEPES, pH 7.8, containing the fluorogenic peptide of interest, with the intention of monitoring each specific peptidase activity. The results shown in Fig. 1A indicate that all three peptidase activities are increased when the 20S proteasome is in its latent form before oxidation, whereas the peptidase activities are decreased when carried by the 20S proteasome active form (Fig. 1B). The trypsin-like and peptidylglutamyl-peptide hydrolase activities are much more sensitive to oxidation than the chymotrypsin-like activity, which is only slightly inactivated. It is interesting to note that the trypsin-like activity is known to be the most sensitive to thiol-blocking agents (e.g., N-ethylmaleimide),24 which indicates that there is an important thiol for the proteolytic activity that may be a good target for reactive oxygen species. Of additional interest is the fact that the chaperone proteins Hsp90 (heat shock protein 90) and ot-crystallin (a member of the Hsp27 family) were found to specifically protect in vitro the trypsin-like activity and also a nonconventional N-benzyloxycarbonyl-Leu-Leu-Leu-amidomethylcoumarin(Cbz-LLLMCA) hydrolyzing activity of the 20S proteasome active form. Indeed, a 4-fold molar excess of either Hsp90 or a-crystallin was enough to prevent the oxidative inactivation of the 20S proteasome, whereas no protection was observed with thyroglobulin and glucose-6-phosphate dehydrogenase, used as controls. 8 This finding suggests that these chaperone proteins can bind to the proteasome and may serve as specific protectors against inactivation by reactive oxygen species.

23B. Friguet,E. R. Stadtman,and L. I. Szweda,J. Biol. Chem.269, 21639 (1994). 24p. j. Savoryand A. J. Rivett,Biochem. J. 289, 45 (1993).

[23] A

REDOX CONTROL OF 2 0 S PROTEASOME

2 5 0~

/ /

200

B

/11

100

80

~-

>,

259

/'~ > 60 "6 ,< ¢-

Le 4o

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i,

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2.5

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o

i ....

i ....

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i ....

i ....

1 1.5 2 Time (hours)

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FIG. 1. Peptidase activities after metal-catalyzed oxidation of 20S proteasome in latent and active forms. Latent and active forms of proteasome at 0.1 mg/ml in 0.1 M HEPES, pH 7.8, were incubated with 0.1 mM FeCI3 and 25 mM ascorbate at 37 °. At the indicated times peptidase activities were assayed as described. For each peptidase, 100% activity was defined as the peptidase activity at oxidation time 0. (A) Activities of the latent form: chymotrypsin-like activity (O); trypsin-like activity (11); peptidylglutamyl-peptide hydrolase activity (0). (B) Activities of the active form: chymotrypsin-like activity (O); trypsin-like activity ([~); peptidylglutamyl-peptide hydrolase activity (<>). [Reproduced with permission from M. Conconi, I. Petropoulos, I. Emod, E. Turlin, E Biville, and B. Friguet, Biochem. J. 333, 407 (1998), copyright © 1998 the Biochemical Society.]

In addition to the production of hydroxyl radicals by metal-catalyzed oxidation, the UV-A-mediated production of a variety of reactive oxygen species such as hydrogen peroxide and singlet oxygen is able to induce functional changes in the 20S proteasome. Indeed, on irradiation by UV-A (10 J/cm2) of the 20S proteasome (at 0.2 mg/ml in 25 mM Tris-HC1, pH 7.5) in its latent form, the trypsin-like activity undergoes a 2-fold increase whereas the chymotrypsin-like activity is only slightly increased and the peptidylglutamyl-peptide hydrolase activity remains unaffected. The same UV-A irradiation on the proteasome in its active form results in only a 30% decrease of the peptidylglutamyl-peptide hydrolase activity. As observed for metal-catalyzed oxidation, the peptidylglutamyl-peptide hydrolase activity is also a target for UV-A-mediated oxidative damage. Protein modifications may also originate from reactions with small aldehydes such as malonaldehyde or 4hydroxy-2-nonenal (HNE), which are the main products of lipid peroxidation. 4-Hydroxy-2-nonenal readily reacts at physiological pH with cystein, lysine, and histidine residues to form Michael adducts. 25-27 Not surprisingly, HNE was found 25 H. Esterbauer, R. J. Schaur, and H. Zollner, Free Radic. Biol. Med. 11, 81 (1991). 26 K. Uchida and E. R. Stadtman, Prec. Natl. Acad. Sci. U.S.A. 89, 5611 (1992). 27 L. I. Szweda, K. Uchida, L. Tsai, and E. R. Stadtman, J. Biol. Chem. 268, 3342 (1993).

260

[23]

PROTEIN STRUCTURE AND FUNCTION

to inactivate the trypsin-like activity of the 20S proteasome active form, 7 which corroborates the fact that this activity is the most sensitive to thiol-blocking agents, z4

20S Proteasome Activity in Cells Exposed to Metal-Catalyzed Oxidation Subconfluent FAO rat hepatoma cells are exposed to metal-catalyzed oxidation on addition of 25 mM ascorbate and 0.1 mM FeC13 in 0.5 mM ADP to the culture medium. After various durations of treatment, cells are harvested and disrupted by five sonications (5 sec each) in 0.25 M Tris-HC1, pH 7.8. After centrifugation at 20,000g for 20 min, the supernatant containing total soluble cellular protein is collected and the trypsin-like and peptidylglutamyl-peptide hydrolase activities are measured with 50 #g of total proteins in 0.1 M HEPES, pH 7.8, and the appropriate flurogenic peptide at the usual concentration in a final volume of 200 #I. 20S proteasome proteolytic activity is determined as the difference between the total activity and the remaining activity of the crude extract in the presence of 20 #Mproteasome inhibitor MG- 132 [N-carbobenzoxy-L-leucyl-L-leucyl-leucinal;available from Calbiochem (Meudon, France) or Affiniti (Mamhead, Exeter, UK)]. The results shown in Fig. 2A indicate that both trypsin-like and peptidylglutamyl-peptide B

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100

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FIG. 2. Peptidase activities of proteasome after metal-catalyzed oxidation in FAO cells pretreated or not pretreated with FeC13. FAO cells were subjected to pretreatment with 0.1 mM FeC13 for 16 hr. At the indicated times, 0.1 mM FeCl3 and 0.5 mM ascorbate were added to the cells. Crude extracts were prepared and peptidase activities of the proteasome were assayed as described. For each peptidase activity, 100% was defined as the value obtained at treatment time 0. (A) Trypsin-like activity; (B) peptidylglutamyl-peptide hydrolase activity. Open symbols, naive cells; solid symbols, FeCI3preincubated cells. [Reproduced with permission from M. Conconi, I. Petropoulos, I. Emod, E. Turlin, E Biville, and B. Friguet, Biochem. J. 333, 407 (1998), copyright © 1998 the Biochemical Society.]

6

[231

REDOX CONTROL OF 2 0 S PROTEASOME

261

hydrolase activities of the proteasome are inactivated on exposure of FAO cells to metal-catalyzed oxidation. Induction of Hsp90 by challenging the cells with 0.1 mM FeC13 before exposure to metal-catalyzed oxidation results in the protection of the 20S proteasome trypsin-like and Cbz-LLL-MCA-hydrolyzingactivities (Fig. 2B). 8 2 0 S P r o t e a s o m e S t r u c t u r a l C h a n g e s o n Oxidative T r e a t m e n t s A method of choice for investigating structural changes of the 20S proteasome is to analyze the two-dimensional (2D) gel electrophoresis pattern of subunits before and after oxidative treatment. The Multiphor system from Amersham Pharmacia Biotech has been used with Immobilines Drystrips (pH 3-10; length, 13 cm) for the first dimension. Purified proteasome (15 #g) is diluted in sample buffer [9 M urea, 2% (w/v) 3-[(3-cholamidopropyl)-dimethyl-ammonio]-l-propanesulfonate (CHAPS), 2% (v/v) Pharmalytes (pH 3-10), 20 mM dithiothreitol, and bromphenol blue]. The Drystrips are rehydrated in this solution in a reswelling tray (Amersham Pharrnacia Biotech) overnight at room temperature and then focused for 50,000 V. hr for 23 hr. After focusing, the Immobilines Drystrips are equilibrated for 10 min in equilibration buffer [50 mM Tris-HC1 (pH 6.8), 6 M urea, 30% (v/v) glycerol, 1% (w/v) SDS] supplemented with 1% (w/v) dithiothreitol and for 10 min in equilibration buffer containing 2.5 % (w/v) iodoacetamide and 0.01% (w/v) hromphenol blue. The second dimension is performed by the Laemmli method of SDS-polyacrylamide gel electrophoresis (PAGE) 28 on a 12% (w/v) polyacrylamide gel, using the Protean II system (Bio-Rad, Hercules, CA). Proteins are stained with silver nitrate29 and the gel is digitized on a JX-330 scanner (Sharp, Hamburg, Germany). Protein spot detection and quantification are performed with Imagemaster 2D Elite software (Amersham Pharmacia Biotech). Mobility shift and/or differences of staining intensity were observed for certain subunits of 20S proteasome that were exposed to different oxidative treatments (metal-catalyzed oxidation and UV-A irradiation), reflecting the occurrence of structural modifications for these subunits. Specific differences of staining of four subunits were also observed when we compared proteasome isolated from epidermis of young and old donors, suggesting an oxidative modification of these particular subunits during aging. 3° The appearance of oxidative modifications on proteasome subunits can be checked by detecting the presence of carbonyl groups, 31'32 using the Oxyblot 28 U. K. Laemmli, Nature (London) 227, 680 (1970). 29 C. R. Merril, D. Goldman, S. Sedman, and H. Ebert, Science 211, 1437 (1981). 3o A. Bultean, I. Petropoulos, and B. Friguet, Exp. Gerontol. 35, 767 (2000). 31 R. L. Levine, D. Garland, C. N. Oliver, A. Amici, I. Climent, A. G. Lenz, B. W. Alan, S. Shaltiel, and E. R. Stadtman, Methods Enzymol. 186, 464 (1990). 32 R. L. Levine, J. A. Williams, E. R. Stadtman, and E. Shacter, Methods Enzymol. 233, 346 (1994).

262

PROTEIN STRUCTURE AND FUNCTION

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technique after 1D (Quantum-Appligene, Illkirch, France) or 2D gel33 electrophoresis of purified 20S proteasome. Alternatively, the presence of specific modifications can be detected by Western blotting after 1D or 2D gel electrophoresis of purified 20S proteasome, using an antibody specific for the modification of interest. As an example, the proteasome isolated from kidney of mice treated with a renal carcinogen (ferric nitrilotriacetate) was shown to be modified by HNE adduct in Western blot experiments, using a monoclonal antibody specifically raised against this modification.34 A polyclonal antibody against HNE adducts can also be used 35 and is available from Calbiochem. Identification of modified 20S proteasome subunits can be achieved by 2D Western blot with monoclonal antibodies specific for the different subunits and available from Affiniti. Conclusion Growing evidence argues for the 20S proteasome being a target for oxidative modification on different in vitro and physiological oxidative stresses. Alteration of 20S proteasome peptidase activities as observed on aging and during several types of oxidative stress is likely to impair proteasome function and to have important physiological consequences. Indeed, the proteasomal system is pivotal not only for oxidized protein degradation and general protein turnover but also for specific cellular processes including activation of transcription factors such as NF-xB, antigen processing, progression of the cell cycle, and apoptosis through the activation of caspases. Activation of the proteasomal system by specific stimuli, including those affecting the redox status, remains a yet to be solved but interesting possibility.6'8 Such activation processes are likely to involve transient interactions with other components of the proteasomal system (e.g., PA 28 and PA 700) that may not be observed when analyzing only the 20S proteasome catalytic core. However, studying the pattern of 20S proteasome modifications and their consequences on 20S proteolytic activity has already yielded valuable information about the structurefunction relationship of this key enzyme for intracellular proteolysis. Acknowledgments Our laboratory is supported by funds from the MENRT (Institut Universitaire de France and Universit6 Denis Diderot-Paris 7) and by a European Union QLRT "Protage" Grant (QLK6-CT1999-02193).

33 j. M. Talent, Y. Kong, and R. W. Gracy, Anal Biochem. 263, 31 (1998). 34 K. Okada, C. Wangpoengtrakul, T. Osawa, S. Toyokuni, K. Tanaka, and K. Uchida, J. BioL Chem. 274, 23787 (1999). 35 L. I. Szweda, P. A. Szweda, and A. Holian, Methods Enzymol. 319, 562 (2000).