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FlG. 2. Peptidase activities of proteasome after metal-catalyzed oxidation in FAO cells pretreated or not pretreated with FeC^. FAO cells were subjected to pretreatment with 0.1 mM FeCl3 for 16 hr. At the indicated times, 0.1 mM FeC^ and 0.5 m M 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, FeClj-preincubated cells. [Reproduced with permission from M. Conconi, I. Petropoulos, I. Emod, E. Turlin, F. Biville, and B. Friguet, Biochem. J. 333,407 (1998), copyright © 1998 the Biochemical Society.]

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 FeCl3 before exposure to metal-catalyzed oxidation results in the protection of the 20S proteasome trypsin-like and Cbz-LLL-MC A-hydrolyzing activities (Fig. 2B).8

20S Proteasome Structural Changes on Oxidative Treatments

A method of choice for investigating structural changes of the 20S proteasome is to analyze the two-dimensional (2D) gel electrophoresis pattern of sub-units 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 fig) is diluted in sample buffer [9 M urea, 2% (w/v) 3-[(3-cholamidopropyl)-dimethyl-ammonio]-l-pro-panesulfonate (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 Pharmacia 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-HCl (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) bromphenol 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.30

The appearance of oxidative modifications on proteasome subunits can be checked by detecting the presence of carbonyl groups,31'32 using the Oxyblot

29 C. R. Merril, D. Goldman, S. Sedman, and H. Ebert, Science 211, 1437 (1981).

30 A. Bulteau, 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. Ahn, 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).

technique after ID (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 ID 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 used35 and is available from Calbiochem. Identification of modified 20S proteasome sub-units 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-kB, 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 structure-function relationship of this key enzyme for intracellular proteolysis.

Acknowledgments

Our laboratory is supported by funds from the MENRT (Institut Universitaire de France and Université 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).

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