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J. Biol. Chem., Vol. 280, Issue 12, 11840-11850, March 25, 2005

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Overexpression of Proteasome ₅ Assembled Subunit Increases the Amount of Proteasome and Confers Ameliorated Response to Oxidative Stress and Higher Survival Rates^*

Niki Chondrogianni, Christos Tzavelas¶, Alexander J. Pemberton||**, Ioannis P. Nezis, A. Jennifer Rivett||, and Efstathios S. Gonos

From the National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, 48 Vasileos Constantinou Avenue, Athens 116 35, Greece and ^||Department of Biochemistry, University of Bristol, School of Medical Sciences, Bristol BS8 1TD, United Kingdom

Received for publication, November 17, 2004 , and in revised form, January 10, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Proteasomes play a fundamental role in retaining cellular homeostasis. Alterations of proteasome function have been recorded in various biological phenomena including aging. We have recently shown that the decrease in proteasome activity in senescent human fibroblasts relates to the down-regulation of -type subunits. In this study we have followed our preliminary observation by developing and further characterizing a number of different human cell lines overexpressing the ₅ subunit. Stable overexpression of the ₅ subunit in WI38/T and HL60 cells resulted in elevated levels of other -type subunits and increased levels of all three proteasome activities. Immunoprecipitation experiments have shown increased levels of assembled proteasomes in stable clones. Analysis by gel filtration has revealed that the recorded higher level of proteasome assembly is directly linked to the efficient integration of "free" (not integrated) -type subunits identified to accumulate in vector-transfected cells. In support we have also found low proteasome maturation protein levels in ₅ transfectants, thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. Functional studies have shown that ₅-overexpressing cell lines confer enhanced survival following treatment with various oxidants. Moreover, we demonstrate that this increased rate of survival is due to higher degradation rates following oxidative stress. Finally, because oxidation is considered to be a major factor that contributes to aging and senescence, we have overexpressed the ₅ subunit in primary IMR90 human fibroblasts and observed a delay of senescence by 4-5 population doublings. In summary, these data demonstrate the phenotypic effects following genetic up-regulation of the proteasome and provide insights toward a better understanding of proteasome regulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Protein degradation is a major intracellular function, which is responsible not only for housekeeping but also for the regulation of important cellular functions, such as homeostasis and survival. The proteasome is a major cellular non-lysosomal threonine protease. Through its catabolic functions, it is implicated in many cellular processes including removal of abnormal, misfolded, denatured, or otherwise damaged proteins as well as normal proteins (reviewed in Refs. 1-3). The 20S proteasome, a 700-kDa multisubunit enzyme complex, is a stack of four heptameric rings with the two outer -type subunits rings embracing two central head-to-head oriented rings containing -type subunits. The inside rings give rise to the central cavity of the particle, where the catalytic sites of the complex are confined. Three of the seven -type subunits are proteolytically active in the mature 20S proteasome, namely, the ₁, ₂, and ₅ subunits. The proteasome has a broad specificity hydrolyzing peptide bonds on the carboxyl site of hydrophobic (CT-L),¹ acidic (PGPH), and basic (T-L) amino acids (reviewed in Refs. 4-6). The 20S proteasome is also central to the ATP/ubiquitin-dependent intracellular protein degradation pathway, where it represents the proteolytic core of the 26S complex (20S core capped on each side by 19S regulatory complexes; reviewed in Refs. 7 and 8).

Alterations in proteasome function have been found in many biological processes including aging (reviewed in Refs. 3, 9, and 10). We (11, 12) and others (13-19) have reported loss of proteasome function upon aging of several human tissues as well as in senescent primary cultures. Our work in human embryonic fibroblast cultures undergoing replicative senescence has shown that the reduced levels of proteasomal activities during the process are accompanied by lower proteasome content and protein expression levels of some, but interestingly not all, proteasome subunits. Specifically, we have found that loss of proteasome function is due to lower levels of -type subunits (the "rate-limiting" subunits), whereas -type subunits are in excess as "free" subunits in senescent cells (12). Finally, the fundamental link between cellular senescence and proteasome function is further supported by our recent study (20), in which we demonstrate that when the proteasome is partially inhibited in young primary fibroblast cultures, a senescence-like phenotype is triggered.

Data in the literature are very limited regarding the activation/up-regulation of the proteasome (reviewed in Refs. 7 and 8). Gaczynska et al. (21) have succeeded in the enhancement of hydrophobic (CT-L) and basic (T-L) proteasome activities following transfection of lymphoblasts and HeLa cells with the _5i subunit, along with an increase of basic activity (T-L) following transfection of the same cell lines with the _1i subunit. The same group has also shown the stimulation of acidic activity (PGPH) after overexpression of ₁ subunit in HeLa cells (22). Our preliminary efforts to "activate" the proteasome revealed that stable overexpression of ₁ or ₅ subunits results in increased proteasome activities and enhanced cellular survival following treatment with proteasome inhibitors or H₂O₂ (12). Furthermore, and in accordance with the earlier results of Rock and co-workers (22), we have observed a co-regulation of ₁ and ₅ subunits (12). Apart from these pilot proteasome subunit transfection studies, few additional data exist regarding proteasome activation. Davies and co-workers (23) have shown that poly(ADP-ribose) polymerase binds to nuclear proteasomes during oxidative stress by H₂O₂, leading to increased proteasomal activities. More recently, Ustrell et al. (24) have identified a novel nuclear protein, PA200, that activates the nuclear proteasome following -irradiation, thus indirectly involving the proteasome in DNA repair. Finally, Friguet and co-workers have described the ability of a lipid algae extract (Phaeodactylum tricornutum) to stimulate 20S proteasome peptidase activities following keratinocyte UVA and UVB irradiation.² In summary, although these preliminary observations indicate that the proteasome can be activated through other pathways than the standard 19S and 11S complexes, there is still limited knowledge regarding the molecular mechanisms of such activation.

Data have been emerging recently with regard to the regulation of proteasome assembly in mammals (reviewed in Ref. 25). Proteasome biogenesis is a precisely ordered multistep event involving the biosynthesis of all subunits, their assembly, and maturation processes. POMP (or human/mouse Ump1 or proteassemblin; Refs. 26-28) is a factor that has been characterized as a human homologue of the yeast proteasome maturation factor Ump1 (29). Ump1 has been identified as an accessory protein, a short-lived chaperone, that is required and essential for correct maturation and normal proteasome assembly in yeast (29). POMP is respectively responsible for the proteasome maturation and biogenesis of mammalian 20S proteasome; it is a constituent of a mammalian proteasome assembly intermediate that is detected only in precursor, inactive fractions (26) and also becomes the first substrate of the mature proteasome (27). It has been reported to interact with _1i, ₁, ₅, ₆, and ₇ subunits but not with -type subunits (30).

In this study, we have investigated proteasome up-regulation by means of stably overexpressing the ₅ subunit in different cell lines. We show that this overexpression leads to increased levels of assembled and functional proteasome. This regulation is linked with the efficient integration of "free" (not integrated) -type subunits identified to accumulate in vector-transfected cells. Additionally, we clearly demonstrate that proteasome up-regulated cell lines confer enhanced survival against various oxidants. Finally, we provide evidence that overexpression of the ₅ subunit delays senescence in IMR90 normal fibroblasts.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reagents and Antibodies—LLVY-AMC, LLE-NA, LSTR-AMC, and MG132 as well as primary proteasomal antibodies against ₄ (XAPC7, C6; PW8120), ₆ (C2; PW8100), ₇ (C8; PW8110), ₁ (Y, delta; PW8140), ₂ (Z; PW8145), and ₅ (X, MB1, ; PW8895) subunits were purchased from Affiniti Research Products Ltd. Primary antibody against POMP was a generous gift from Dr. E. Kruger (26). Primary antibody against -actin (sc1616) and secondary antibodies were purchased from Santa Cruz Biotechnology Inc. Oxidized proteins were detected with anti-dinitrophenol antibody from the OxyBlot^TM Protein Oxidation Detection Kit (Qbiogene).

Cell Lines and Culture Conditions—Human embryonic fibroblasts IMR90 and WI38/T (SV40 T Ag WI38 VA 13 cell line) were obtained from the European Collection of Cell Cultures and maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (Invitrogen), 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 1% nonessential amino acids (complete medium). HL60 cells were a kind gift of Dr. D. Rickwood and cultured at concentrations between 0.5 and 1 x 10⁶ cells/ml in RPMI 1640 medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum and BASH (7.36 mg/ml sodium pyruvate, 0.454 mg/ml hypoxanthine, 4.18 mg/ml penicillin, 4.5 mg/ml streptomycin, and 4.53 µg/l vitamin B₁₂). Cells were fed 16 h prior to the assay, and cell number was determined in duplicates using a Coulter Z2 counter (Coulter Corp.). Normal fibroblasts were subcultured when cells reached confluence at a split ratio of 1:2 until they entered senescence.

Stable Transfections—An expression vector encoding for the full-length ₅ subunit cDNA (plasmid pBJ1-neo.₅) was the generous gift of Drs. K. Tanaka and K. Rock (22, 31). WI38/T cells were transfected with either empty vector or ₅ plasmid by using the electroporation method. In brief, 10⁷ cells were mixed with 50 µg of plasmid DNA (plasmid pBJ1-neo.₅ or empty vector) and electroporated at 260 V, 960 microfarads (Gene Pulser^TM; Bio-Rad). 2.1 x 10⁵ IMR90 cells were transfected with either plasmid or empty vector by using the Effectene^TM transfection reagent (Qiagen) according to the manufacturer's instructions. Transfected cells (IMR90 or WI38/T cells) were split 48 h after transfection and maintained in complete medium containing 400 µg/ml G418. Colonies of stable transfectants were isolated following 3-4 weeks of selection and propagated into cell lines.

HL60 cells were transfected as originally described by Bildirici et al. (32). In brief, 4 x 10⁵ cells were washed twice with immunoporation washing medium (IPBiosciences) and then resuspended in immunoporation medium (Immunoporation Ltd.) containing antibody-coated Immunofect beads (IPBiosciences) in a ratio of 20 beads/cell. 0.2 µg of plasmid DNA (plasmid pBJ1-neo.₅ or empty vector) was added, and then cells were incubated in an end-over-end mixer at 40 rpm for 6 h at room temperature. Cells were separated from the beads using a magnetic separator (IPBiosciences) (32, 33), centrifuged, and suspended in 1 ml of complete medium. Three days after transfection, G418 was added to the culture medium at a concentration of 800 µg/ml. Stable transfectants were clonally selected by serially diluting 10³ cells in 96-well dishes covered with a small layer of 1% high purity agarose (Invitrogen) containing 800 µg/ml G418.

Survival Assays—10⁴ WI38/T or 2 x 10⁶ HL60 cells stably transfected with empty vector or ₅ plasmid were seeded in 6-well plates in duplicates. Cells were then treated immediately (HL60) or allowed to recover for 24 h prior to treatment (WI38/T) with 10 or 20 µM tBHP, 100 or 300 µM H₂O₂, 1% or 2% EtOH or being subjected to metal catalyzed oxidation (treatment with 0.1 mM FeCl₃/0.5 mM ADP in the presence of 25 mM L-ascorbic acid) for 2.5 h at 37 °C in fresh medium. Treated cultures were washed thoroughly in PBS, maintained in complete medium for 7 days, and counted. Each experiment was performed at least three times. For immunoblot detection of carbonyl groups into proteins, 10⁵ cells were seeded in 6-well plates in normal medium and treated with 300 µM H₂O₂ for 30 min. Proteins were extracted immediately after treatment and 24 h after treatment.

Immunofluorescence Antigen Staining and Confocal Laser Scanning Microscope Analysis—For immunofluorescence labeling, cells grown on coverslips were washed in ice-cold PBS and subsequently fixed with 4% freshly prepared paraformaldehyde in PBS followed by cell permeabilization with 0.2% Triton X-100 in PBS. Immunolabeling of proteasomes was carried out using antibodies against the ₇ and ₅ subunits. The antibodies were diluted in PBS containing 0.1% Tween 20 and 3% bovine serum albumin (blocking buffer). The secondary anti-mouse IgG/fluorescein isothiocyanate-conjugated antibody and anti-rabbit IgG/TRITC-conjugated antibody were diluted 1:250 in blocking buffer. Images of the mounted coverslips were taken by using a Nikon PCM 2000 confocal laser scanning microscope. Routine procedures, applied as controls to demonstrate the specificity of the antibody used, were as follows: (a) the usage of normal serum instead of the reactive antibody, and (b) omission of the first antibody. All controls appeared free of any immunofluorescence background.

Proteasome Peptidase Assays and Protein Determination—CT-L, PGPH, and T-L activities of the proteasome in crude extracts were assayed with hydrolysis of the fluorogenic peptides LLVY-AMC, LLE-NA, and LSTR-AMC, respectively, for 30 min at 37 °C, as described previously (11). Proteasome activity was determined as the difference between the total activity of crude extracts or fractions and the remaining activity in the presence of 20 µM MG132. Assays of 26S proteasomes were carried out in 25 mM Tris/HCl buffer, pH 7.5, containing 5 mM ATP (34). Fluorescence was measured using a PerkinElmer Life Sciences 650-40 fluorescence spectrophotometer. Protein concentrations were determined using the Bradford method with bovine serum albumin as a standard.

Immunoblot Analysis—Cells were harvested (for IMR90 and WI38/T cell lines) or collected (for HL60 cell lines) at the indicated time points, lysed in non-reducing Laemmli buffer, and fractionated by SDS-PAGE (12% separating gel) according to standard procedures (35). After electrophoresis, proteins were transferred to nitrocellulose membranes for blotting with appropriate antibodies. Secondary antibodies conjugated with horseradish peroxidase and enhanced chemiluminescence were used to detect the bound primary antibodies. Immunoblot detection of carbonyl groups into proteins was performed with OxyBlot^TM Protein Oxidation Detection Kit (Qbiogene) according to the manufacturer's instructions. Protein loading was tested by stripping each membrane and reprobing it with a -actin antibody.

Preparation of Cell Extracts and Separation of Proteasome Complexes by Gel Filtration—WI38/T and HL60 cells (vector- and ₅-transfected cells) were lysed in 20 mM Tris/HCl buffer, pH 7.5, containing 5 mM ATP and 0.2% Nonidet P-40. Extracts were centrifuged at 13,000 rpm for 10 min at 4 °C, and then an equal amount of protein (1.5 mg) from empty vector or ₅ subunit transfectants was fractionated by gel filtration using an Amersham Biosciences Superose 6 HR10/30 fast protein liquid chromatography column equilibrated in 20 mM Tris/HCl buffer, pH 7.5, containing 10% glycerol, 5 mM ATP, and 100 mM NaCl (36). Fractions were collected, and samples were analyzed by SDS-PAGE and immunoblot analysis. Proteasome fractions were identified by quantifying the CT-L activity in duplicate experiments.

Immunoprecipitation of Proteasomes—Immunoprecipitated proteasomes were prepared as follows: cell monolayers of 80% confluent WI38/T vector- and ₅-transfected cultures were washed and scraped into ice-cold PBS containing 10 mM phenylmethylsulfonyl fluoride and 10 µg/ml aprotinin. 10⁷ HL60 vector- and ₅-transfected cells were collected accordingly. For radioactive labeled immunoprecipitation, prior to washing and scraping/collection, cells were starved in methionine-deficient medium for 1 h, followed by pulse labeling with 100 µCi/ml [³⁵S]methionine for 4 h in methionine-deficient medium. Collected cells were diluted directly in 20 mM Tris/HCl buffer, pH 7.5, containing 5 mM ATP, 10% glycerol, 0.2% Nonidet P-40, 10 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin (lysis buffer). Cell extracts (equilibrated in lysis buffer) were then cleared by adding normal mouse serum and protein A-agarose beads for 3 h at 4 °C. Meanwhile, protein A-agarose beads equilibrated in lysis buffer were coupled with 1-2 µg of antibody against the ₆ subunit for 3 h at 4 °C with constant rocking. The target antigen was then immunoprecipitated by adding the pre-cleared extracts in the pre-coupled antibody against ₆ subunit-protein A. Binding reactions were performed with constant rocking at 4 °C overnight. Immunoprecipitated protein complexes were collected; washed four times in 50 mM Tris/HCl buffer, pH 7.5, containing 5 mM ATP, 75 mM NaCl, 10% glycerol, and 0.2% Triton X-100 (washing buffer); and eluted from the agarose beads by boiling for 5 min in non-reducing Laemmli buffer. Controls were used to demonstrate the specificity of the observed immunoprecipitations. Following immunoprecipitation of proteasomes, the number of counts due to incorporated radioactive [³⁵S]methionine in the immunoprecipitated proteasomes was determined using a Wallac 1409 DSA liquid scintillation counter. Nonradioactive samples were processed for one-dimensional SDS-PAGE as described.

Analysis of Protein Turnover—Quantification of degradation of metabolically radiolabeled proteins in cell cultures was performed as follows: cells were starved in methionine-deficient medium for 1 h, incubated with [³⁵S]methionine in methionine-deficient medium for 4 h at 37 °C, washed twice in PBS, and treated with oxidants (metal catalyzed oxidation and tBHP) for 30 min as described. Following treatment, cells were washed thoroughly in PBS and maintained in normal medium for the indicated time points up to 72 h. The degradation of metabolically labeled proteins was quantified by adding an equal volume of 20% trichloroacetic acid on 1 ml of culture supernatant for 30 min on ice. Scintillation counting of the acid-soluble counts in the supernatant was performed using a Wallac 1409 DSA liquid scintillation counter after centrifugation for 10 min at 13,000 rpm.

Statistical Analysis and Densitometry—Statistical calculations and graphs were performed with Microsoft Excel software. Data were analyzed by single-factor analysis of variance, and p value was used to determine the level of significance (p < 0.05). All values were reported as mean (the average of three independent experiments) ± S.E., unless otherwise indicated. Densitometric analysis was performed with Gel Analyzer Version 1.0.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Overexpression of the ₅ Catalytic Subunit in WI38/T and HL60 Cells Leads to Increased Proteasomal Activities and Increased Protein Expression Levels of Proteasome Subunits—We aimed to reveal changes of the overall proteasome status and function in WI38/T and HL60 ₅-transfected cells. Several stable WI38/T and HL60 clones were selected and propagated into cell lines. These cell lines exhibited similar growth rates and morphological characteristics as compared with the parental cell lines, even after several months of cultivation. Two representative cell lines, WI38/T/₅.8 and HL60/₅.3, were chosen for additional studies. As shown in Fig. 1, all three major proteasomal activities (CT-L, PGPH, and T-L) were found to be significantly increased in both WI38/T and HL60 clones (p < 0.05) as compared with their control counterparts. The activities were increased from 1.3- to 2.1-fold, with the CT-L and PGPH activities being the most affected. Transfection with vector alone (pBJ1-neo) had no effect on proteasome activities.

View larger version (24K): [in this window] [in a new window]   FIG. 1. Specific proteasomal activities in 5-overexpressing cell lines. Percentage of CT-L, PGPH, and T-L activities in vector (CON)- and 5-transfected (A) WI38/T/5.8 and (B) HL60/5 .3 cells. Use of a proteasome inhibitor (MG132) in control reactions ensured the specificity of the enzymatic reactions. Mean value of activities was set at 100% in vector-transfected cells. Each column shows the average of three independent experiments, and error bars denote S.E. All three proteasome activities were found increased in 5-overexpressing cells.

View larger version (53K): [in this window] [in a new window]   FIG. 2. Protein levels of proteasome subunits in 5-overexpressing cell lines. Immunoblot analysis of representative subunits of 20S complex, -type subunits (1, 25.3 kDa; 2, 30 kDa; and 5, 22.9 kDa), and -type subunits (6, 29.5 kDa; and 7, 28.4 kDa) in vector (CON)- and 5-transfected (A) WI38/T/5.8 and (B) HL60/5 .3 cells. Equal protein loading was verified by stripping the membranes and reprobing them with -actin (43 kDa) antibody (bottom panels). Protein expression levels of catalytic subunits (-type subunits) were elevated in 5-overexpressing cells.

View larger version (46K): [in this window] [in a new window]   FIG. 3. Proteasome subcellular distribution in WI38/T/5 cells. Confocal micrographs demonstrating in situ localization of proteasomes in vector (CON; left panels)- and 5-transfected WI38/T/5.8 (right panels) cells. A, labeling with antibody against the 5 proteasome subunit; B, labeling with antibody against the 7 proteasome subunit; and C, 5 and 7 labeling overlapping localization signals. Nuclei (Nu) and cytoplasm (Cyt) are indicated by arrows. 5-Overexpressing WI38/T cells reveal an enhanced immunolocalization signal of the examined subunits.

View larger version (59K): [in this window] [in a new window]   FIG. 4. Proteasome immunoprecipitation followed by immunoblot analysis of proteasome subunits in 5-overexpressing cell lines. Immunoblot analysis of whole extracts used for the immunoprecipitation (left panels) and immunoprecipitated proteasomes (right panels) of representative subunits of 20S complex (2, 30 kDa; 4 kDa; 27.9 6, 29.5 kDa; and 7, 28.4 kDa) in vector (CON)- and 5-transfected (A) WI38/T/5.8 and (B) HL60/5.3 cells. Immunoprecipitations were initiated by using the same amount of total crude extracts. All subunits tested were found to be increased in 5-overexpressing cells in the immunoprecipitated proteasomes as compared with the whole extracts.

View larger version (45K): [in this window] [in a new window]   FIG. 6. Gel filtration analysis of proteasome in WI38/T/5.8 cell line. Cell extracts from vector (CON)- and 5-transfected WI38/T/5.8 cells were chromatographed on a Superose 6 HR10/30 fast protein liquid chromatography column equilibrated in 20 mM Tris/HCl buffer, pH 7.5, containing 10% glycerol, 5 mM ATP, and 100 mM NaCl. A, assays of 26S proteasomes for CT-L activity. Values represent the average of two independent experiments. B, late gel filtration fractions (fractions 32-46) were immunoblotted with antibodies against -type subunits (4, 27.9 kDa; 6, 29.5 kDa; and 7, 28.4 kDa) as indicated. C, immunoblot analysis of POMP in fractions 24-31. Because proteasome activities are detected in fractions 17-24 (A), precursor proteasomes are expected after fraction 25. "Free" (not integrated) -type subunits and precursor proteasome complexes were more abundant in extracts from vector-transfected WI38/T cells.

View larger version (33K): [in this window] [in a new window]   FIG. 5. Immunoprecipitation of labeled proteasomes from whole extracts of 5-overexpressing cell lines. Cells were labeled with [35S]methionine for 4 h, and proteasomes were immunoprecipitated at the indicated time points (0, 2, 4, 16, 24, and 40 h after labeling) by using a mouse monoclonal antibody against the 6 subunit (conditions for immunoprecipitation of 26S complex) as described under "Experimental Procedures." Immunoprecipitations were initiated by using the same amount of total crude extracts from vector (CON)- and -transfected (A) WI38/T/5.8 and (B) HL60/5.3 cells. Mean value of recorded counts of vector- and 5-transfected cells at 0 h post-labeling was set at 100%. Values represent the average of two independent experiments for each time point. Newly synthesized labeled subunits are used directly in the 5-overexpressing cell lines.

View larger version (41K): [in this window] [in a new window]   FIG. 7. Gel filtration analysis of proteasome in the HL60/5.3 cell line. Cell extracts from vector (CON)- and 5-transfected HL60/5.3 cells were processed as described in Fig. 6. A, assays of 26S proteasomes for CT-L activity. Values represent the average of two independent experiments. B, late gel filtration fractions (fractions 32-46) were immunoblotted with antibodies against -type subunits (4, 27.9 kDa; 6, 29.5 kDa; and 7, 28.4 kDa) as indicated. C, immunoblot analysis of POMP in fractions 24-31. Because proteasome activities are detected in fractions 19-26 (A), precursor proteasomes are expected in later fractions. "Free" (not integrated) -type subunits and precursor proteasome complexes were more abundant in extracts from vector-transfected HL60 cells.

Overexpression of the ₅ Catalytic Subunit in WI38/T and HL60 Cells Leads to Better Response to Oxidative Stress and Ameliorated Survival—Having established that overexpression of the ₅ catalytic subunit results in higher proteasome content, next we addressed whether this "proteasome up-regulation" results in functional differences. For these studies, we treated both WI38/T/₅.8 and HL60/₅.3 cell lines and controls with several oxidants, and we determined cell survival. Specifically, cell lines were exposed to 10 or 20 µM tBHP, 100 or 300 µM H₂O₂, 1% or 2% EtOH or were subjected to metal catalyzed oxidation for 2.5 h, and their survival capacity was recorded following a recovery period of 7 days (Fig. 8). Both ₅ cell lines exhibited higher survival rates as compared with the corresponding control cells. Specifically, WI38/T/₅.8 cells exhibited higher survival rates of 1.2-1.8-fold for EtOH, 1.8-2.0-fold for H₂O₂, 1.8-1.9-fold for tBHP, and 2.1-fold for metal-catalyzed oxidation as compared with control cells. Similarly, HL60/₅.3 cells exhibited higher survival rates of 1.6-1.7-fold for EtOH, 1.4-1.5-fold for H₂O₂, 1.8-1.9-fold for tBHP, and 2.3-fold for metal-catalyzed oxidation as compared with control cells. Analysis of oxidized proteins in HL60 cells following treatment with 300 µM H₂O₂ revealed lower levels of oxidized proteins in ₅ transfectants both right after treatment and following a recovery period of 24 h (Fig. 9). Similar results have been obtained in WI38/T cells (12). Thus, we conclude that the differences in the proteolytic activities of ₅-overexpressing cell lines can be translated into functional differences of the proteasome because transfectants exhibit an increased capacity to cope better with various oxidants by decreasing their oxidative load.

View larger version (36K): [in this window] [in a new window]   FIG. 8. Survival rates of 5-overexpressing cell lines following treatments with oxidants. Number of vector (CON)- and 5-transfected (A) WI38/T/5.8 and (B) HL60/5.3 cells following a single treatment with 1% or 2% EtOH, 100 or 300 µM H2O2, 10 or 20 µM tBHP or after being subjected to metal-catalyzed oxidation after a recovery period of 1 week. Each column shows the average of three independent experiments, and error bars denote S.E. 5-Overexpressing cell lines proliferate significantly (p < 0.05) better than the vector-transfected cells.

View larger version (24K): [in this window] [in a new window]   FIG. 9. Levels of oxidized proteins in HL60/5.3 cells. Analysis of oxidized proteins by oxyblot (for conditions, see "Experimental Procedures") in vector (CON)- and 5-transfected treatment HL60/5.3 cells following a single with 300 µM H2O2, either immediately after the treatment (no recovery) or after a recovery period of 24 h (24 recovery). Equal protein loading was verified by the use of a -actin (43 kDa) antibody (bottom panel). Molecular mass (in kDa) is shown to the left of the blot. Levels of oxidized proteins were decreased in 5-overexpressing cells.

View larger version (26K): [in this window] [in a new window]   FIG. 10. Rate of overall proteolysis following oxidative stress in 5-overexpressing cell lines. Rate of overall proteolysis following metal catalyzed oxidation (A and B) and tBHP treatment (C and D) in vector (CON)- and 5-transfected WI38/T/5.8 (A and C) and HL60/5.3 (B and D) cells for the indicated post-labeling time points. Degradation (i.e. recorded scintillation counts) for vector-transfected cells at 0.5 h was set to 100%. Values represent the average of two independent experiments for each time point. Overall proteolysis was increased in 5-overexpressing cells following treatment with oxidants.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this article, we have shown that overexpression of the ₅ proteasome subunit in different cell lines leads to enhanced proteasome activities, increased protein expression levels of the proteasome subunits, and efficiently assembled proteasome. We have found that this increased amount of assembled proteasome is translated to more functional proteasome that confers enhanced survival following treatment with oxidants, primarily through a higher rate of degradation. Finally, we provide evidence that proteasome up-regulation is due, in part, to the recruitment of "free" (not integrated) -type subunits.

In a previous work (12), pilot analysis has indicated enhanced proteasome function (i.e. elevated levels of CT-L and PGPH activities) after stable transfections of either the ₁ or ₅ catalytic subunit in WI38/T cells. In this article and after screening a large number of stable clones, we show that all three major proteolytic activities (CT-L, PGPH, and T-L) are enhanced not only in WI38/T immortalized fibroblasts but also in two other unrelated cell lines, namely, HL60 (a leukemic cell line) and IMR90 (normal fibroblasts), following overexpression of the ₅ subunit. This finding further strengthens the suggestion that proteasome can be up-regulated through additional molecular pathways, apart from its association with internal protein activators, such as 19S or 11S complexes (reviewed in Refs. 7 and 8), poly(ADP-ribose) polymerase (23), or PA200 (24).

To understand the mechanism(s) of proteasome up-regulation, we have taken a detailed molecular and biochemical approach to WI38/T and HL60 ₅-overexpressing clones. Our analysis indicates that there is a common regulation between the -type subunits because overexpression of the ₅ subunit has resulted in the overexpression of other -type subunits. In support of our assumption, previous studies have shown that overexpression of either the ₁ or ₅ subunit resulted in overexpression of the other subunit (12, 22). Furthermore, our preliminary analysis and our previously reported data demonstrate that stable overexpression of the ₁ subunit in WI38/T cells also results in elevated levels of all three proteasomal activities, increased amounts of other subunits, and, finally, enhanced survival of the stable clones following treatment with either proteasome inhibitors or oxidants (12).³ Based on these data, we speculate that overexpression of just one -type subunit (₅ or ₁, according to our results) is sufficient to increase proteasome assembly and function. A potential auto-regulatory mechanism regarding proteasome has also been suggested by other studies. Davies and co-workers (reviewed in Ref. 2) indirectly implied that few proteasomal subunits may be regulated in the same way because daily treatment with an ₆ antisense oligonucleotide severely depressed the intracellular levels of several, but not all, proteasome subunits in both cultured liver epithelial cells and K562 human hemopoietic cells. In addition, a common transcriptional regulation of the proteasomal subunits has been reported in yeast (42-44), in which 26 of the 32 proteasomal subunit genes have been found to be preceded in their promoters by proteasome-associated control element (PACE). RPN4 has been found to be the factor that binds on this element and transcriptionally activates these genes (42). However, no human homologue of RPN4 has been identified thus far. Additional support regarding auto-regulation of proteasome subunits comes from the work of Wojcik and DeMartino (45) in S2 cells derived from Drosophila. Silencing of different proteasomal genes, by the use of small interfering RNA technology, resulted in reduction of the mRNA level of the respective targeted subunit but, moreover, changes in the mRNA levels of several other (but not all) non-targeted subunits. More recently, Meiners et al. (46) have also suggested an auto-regulatory feedback mechanism that allows the compensation of reduced proteasome activity in mammalian cells after exposure to proteasome inhibitors. Their results clearly show that although a concerted regulation of proteasome genes takes place after proteasome inhibition, all subunits are not up-regulated to the same extent. These results are in agreement with our suggestion regarding a different regulation between different proteasome subunits.

Because the -type subunits are co-regulated and consequently overexpressed following transfection of the ₅ subunit, where does the cell find additional -type subunits to produce more assembled proteasome? The gel filtration results clearly show that there are "free" (not integrated in active proteasomes) -type subunits in the control cells as opposed to the ₅ clones (see also below). The reported immunoprecipitation experiments show that when ₅ subunit is overexpressed, the whole proteasome is up-regulated and more efficiently assembled. This is in accordance with our previously reported data regarding the assembly of the proteasome in early- and late-passage human embryonic fibroblasts (12), in which the -type subunits appear to be "rate-limiting" subunits in late-passage cells, whereas -type subunits were also found in excess, being "free" (not integrated). Radiolabeled immunoprecipitation experiments in this study demonstrate a lag phase of 16-24 h regarding the use and integration of labeled, newly synthesized subunits into assembled proteasomes in control cells but not in the ₅ clones. We suggest that in clones in which the -type subunits are in excess, radiolabeled subunits can be used at once because they can find their -type partners for the assembly process. Thus, we hypothesize that the intracellular levels of -type subunits may determine the amount of assembled proteasome. This hypothesis is further strengthened by the fact that we have never detected any free -type subunits in all cell lines studied.

The gel filtration analysis also shows that there are more inactive proteasome precursor fractions in the control cells as opposed to the ₅ clones. It is known that there are assembly intermediates (13S and 16S proteasome precursor complexes) in the formation of mammalian 20S proteasomes (Refs. 47-50; reviewed in Ref. 51) that are inactive. During proteasome fractionation, late fractions of control cells (in which no activity was detected) reveal higher amounts of these complexes as compared with the corresponding late fractions of the ₅ clones. Furthermore, our data concerning POMP expression analysis strengthen our assumption that in control cells there are more precursor and thus inactive complexes. In accordance with our findings, Witt et al. (26) have reported that in cell lines that accumulate precursor proteasome complexes, POMP is stabilized and accumulated in the inactive proteasome precursor fractions. Finally, we have found more abundant -type subunits (₄, ₆, and ₇) in late fractions derived from control cells than in ones from ₅ clones. Although little is known about the early steps in the assembly of subunits in eukaryotes, human subunit ₇, when overexpressed in Escherichia coli, has been shown to spontaneously form double ring-like structures (52). The ₆ and ₁ subunits are unable to form ring-like structures when expressed by themselves; however, they are incorporated in such assemblies when they are co-expressed with ₇ (53). Similar observations have also been reported for the ₅ subunit (54). Moreover, Seemuller and co-workers (55) have characterized the proteasome as a self-compartmentalizing protease, where proteasomal -type subunits spontaneously self-assemble into seven-membered rings that serve as templates into which -type subunits are incorporated. Mayer and co-workers (56) have recently shown that several -type subunits are found in low-density fractions, giving the molecular basis for the formation of 13S and 16S assembly intermediates. They suggest that in addition to the 13S precursor complex, there are smaller complexes that are composed of at least the ₄ and ₇ subunits (subunits that we have also identified in the late fractions). Our results are in accordance with the existence of such complexes. It is shown here that an excess of -type subunits in the control cells could possibly give rise to such early complexes (because we have detected ₄, ₆, and ₇ subunits in different late fractions), whereas these complexes are not detected in the ₅ clones, possibly because they are quickly used to produce the higher amounts of assembled proteasomes. Finally, it is also feasible that these subunits act as free monomers. Jorgensen and Hendil (57) have reported that the ₅ subunit seems to exhibit an RNase activity even as a free monomer. In addition, the S5a/Rpn10 yeast subunit of the 19S complex has been identified to exist in two forms, as either a proteasome subunit or a free form (58).

A major point of this study is that the "proteasome up-regulated" cell lines have increased capacity to cope with various stresses. Accumulation of abnormal proteins is determined by their rates of formation, but their rates of hydrolysis and elimination are of equal importance. Because the proteasomal system is responsible for the degradation of non-functional proteins as well as for the cellular response to oxidative stress (reviewed in Ref. 59), it is expected that cells possessing elevated proteasome activities, like our ₅ clones, exhibit enhanced survival to the different oxidants used in this study. In addition we demonstrate that ₅ clones exhibit higher degradation rates following oxidative stress. Thus the increased capacity to respond to oxidative stress of the clones relates to the elevated protein turnover.

In a previous work, we have studied proteasome activity in fibroblasts derived from healthy donors of different ages (18-80 years) including centenarians, because they represent the best model of successful aging (reviewed in Ref. 60). We have found that healthy centenarians possess an active proteasome (11). Davies and co-workers (2) have hypothesized that the ability of the proteasome to degrade oxidized proteins serves as a secondary cellular antioxidant defense system. In the current study, we show that overexpression of the ₅ subunit in primary IMR90 fibroblasts delays senescence. Because oxidation is a major factor that contributes to aging and replicative senescence (reviewed in Ref. 38), the retardation of the senescent phenotype observed in IMR90 clones could be considered an ameliorated response against oxidative stress.

Proteasome function appears to be dependent on both genetic and environmental factors. This study highlights the observation that the proteasome can be genetically "up-regulated," thus resulting in enhanced cellular capacity against oxidants. Because the proteasome is down-regulated in several biological processes, including aging and diseases, anti-aging/therapeutic strategies should be aimed at proteasome activation and identification of the rules that govern proteasome assembly and regulation. Furthermore, the search for compounds that may activate the proteasome is expected to be of great interest.

	FOOTNOTES

 
^* This work was supported in part by European Union FOOD/FP-6 "Zincage" Grant FOOD-CT-2003-506850 (to E. S. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Fig. 1.

Recipient of a Ph.D. fellowship from the Bodosaki Foundation.

^¶ Recipient of a postdoctoral fellowship from Greek State's Scholarship Foundation (IKY).

^** Funded by a Cancer Research-UK studentship.

To whom correspondence should be addressed. Tel.: 30-210-7273756; Fax: 30-210-7273677; E-mail: sgonos{at}eie.gr.

¹ The abbreviations used are: CT-L, chymotrypsin-like; EtOH, ethanol; LLE-NA, N-Cbz-Leu-Leu-Glu--naphthylamine; LLVY-AMC, Suc-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin; LSTR-AMC, N-t-Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin; PBS, phosphate-buffered saline; PGPH, peptidylglutamylpeptide hydrolyzing; POMP, proteasome maturation protein; tBHP, tert-butyl hydroperoxide; T-L, trypsin-like; TRITC, tetramethylrhodamine isothiocyanate.

² C. Nizaed, B. Friguet, M. Moreau, A. L. Bulteau, and A. Saunois, patent PCT Application WO 02/080876.

³ N. Chondrogianni and E. S. Gonos, unpublished observations.

	ACKNOWLEDGMENTS

 
We thank Dr. B. Friguet for critical reading of the manuscript and Dr. I. P. Trougakos for helpful discussions during the course of this work. We are grateful to Drs. E. Kruger, D. Rickwood, K. Rock, and K. Tanaka for cell lines, antibodies, and plasmids. Drs. H. Moutsopoulos and D. Liakos are acknowledged for the use of microscopy facilities.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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