Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts NICOLLE SITTE,*,† MICHAEL HUBER,* TILMAN GRUNE,† AXEL LADHOFF,* WOLF-DIETRICH DOECKE,‡ THOMAS VON ZGLINICKI,* AND KELVIN J. A. DAVIES§,1 *Institute of Pathology, †Clinics of Physical Medicine and Rehabilitation, and ‡Institute of Medical Immunology, Charite´, Humboldt University, Berlin, Germany; and §Ethel Percy Andrus Gerontology Center and Division of Molecular Biology, the University of Southern California, Los Angeles, California 90089-0191, USA ABSTRACT We have studied the effects of hyperoxia and of cell loading with artificial lipofuscin or ceroid pigment on the postmitotic aging of human lung fibroblast cell cultures. Normobaric hyperoxia (40% oxygen) caused an irreversible senescence-like growth arrest after about 4 wk and shortened postmitotic life span from 1–1/2 years down to 3 months. During the first 8 wk of hyperoxia-induced ‘aging’, overall protein degradation (breakdown of [35S]methionine metabolically radiolabeled cell proteins) increased somewhat, but by 12 wk and thereafter overall proteolysis was significantly depressed. In contrast, protein synthesis rates were unaffected by 12 wk of hyperoxia. Lysosomal cathepsin-specific activity (using the fluorogenic substrate z-FR-MCA) and cytoplasmic proteasome-specific activity (measured with suc-LLVY-MCA) both declined by 80% or more over 12 wk. Hyperoxia also caused a remarkable increase in lipofuscin/ceroid formation and accumulation over 12 wk, as judged by both fluorescence measurements and FACscan methods. To test whether the association between lipofuscin/ceroid accumulation and decreased proteolysis might be causal, we next exposed cells to lipofuscin/ceroid loading under normoxic conditions. Lipofuscin/ceroid-loaded cells indeed exhibited a gradual decrease in overall protein degradation over 4 wk of treatment, whereas protein synthesis was unaffected. Proteasome specific activity decreased by 25% over this period, which is important since proteasome is normally responsible for degrading oxidized cell proteins. In contrast, an apparent increase in lysosomal cathepsin activity was actually caused by a large increase in the number of lysosomes per cell. To test whether lipofuscin/ceroid could in fact directly inhibit proteasome activity, thus causing oxidized proteins to accumulate, we incubated purified proteasome with lipofuscin/ceroid preparations in vitro. We found that proteasome is directly inhibited by lipofuscin/ceroid. Our results indicate that an accumulation of oxidized proteins (and lipids) such as lipofuscin/ceroid may actually cause further increases in damage accumulation during aging by
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inhibiting the proteasome.—Sitte, N., Huber, M., Grune, T., Ladhoff, A., Doecke, W.-D., von Zglinicki, T., Davies, K. J. A. Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts. FASEB J. 14, 1490 –1498 (2000) Key Words: ceroid pigment 䡠 protein turnover 䡠 lysosome 䡠 oxidative stress 䡠 free radicals
Cellular aging can be studied at two different levels: either as proliferative senescence, i.e., the loss of reproductive ability of a nontransformed cell culture; or as aging, and finally death, of individual (postmitotic) cells. Major discoveries in the field of proliferative senescence have included the finding that telomere shortening may act as a mitotic clock (1) and that cell cycle checkpoint control may be effected by the tumor suppressors p53 and pRB (2). The finding that telomere shortening can be induced by oxidative stress (3–5) recently connected this area of study with the free radical theory of aging. Our understanding of the aging of individual postmitotic cells is much less advanced, although many theories have been proposed. One such postulate is that the accumulation of heavily damaged, oxidized, and cross-linked proteins may contribute to the aging process in postmitotic cells (6 – 8). Numerous studies have identified a major role for the 20S proteasome in the removal of oxidatively modified proteins in mammalian cells (9 –12), and proteasome depletion prevents cells from degrading oxidized proteins (13, 14). Oxidized protein aggregates can inhibit the proteolytic activity of the proteasome in vitro (15, 16). Therefore, the accumulation of heavily damaged, oxidized, and aggregated proteins during postmitotic aging may diminish the effectiveness of proteolytic enzymes. 1 Correspondence: Ethel Percy Andrus Gerontology Center, University of Southern California, 3715 McClintock Ave., Room 306, Los Angeles, CA 90089-0191, USA. E-mail:
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0892-6638/00/0014-1490/$02.25 © FASEB
Figure 1. Protein synthesis and degradation in WI-38 fibroblasts during hyperoxia-induced ‘aging’. WI-38 were cultivated under standard conditions except that the ambient oxygen concentration was raised to 40%. After the indicated time periods, the cells were exposed for 16 h to radiolabeled [35S] methionine as described in Materials and Methods. A) Protein synthesis was measured by the amount of radioactivity incorporated into cells after removing the unincorporated [35S] methionine. B) Protein degradation was measured after 0, 5, 8, and 12 wk of cell incubation under hyperoxic conditions. After removal of unincorporated label, the liberation of TCAsoluble counts was followed for up to 144 h. Percent degradation was calculated as: (TCA-soluble counts ⫺ blank)/(total incorporated counts ⫺ blank) ⫻ 100. The insert compares the percent degradation 144 h after labeling as a function of the time of cell incubation under hyperoxia. The data in both panels represent the means of three independent experiments, each with four measurements. A, B) Inset: sds are shown for each mean. In the main portion of panel B, sds were always less than 10%.
Lipofuscin and ceroid are fluorescent pigments of aggregated polymers derived from oxidation products of proteins and lipids, which are cross-linked by covalent and hydrophobic bonds (17). Lipofuscin and ceroid accumulate during aging, most obviously in postmitotic cells. A close correlation has been reported between lipofuscin/ceroid accumulation and aging rate in several mammalian species despite widely differing maximum life spans (18, 19). Lipofuscin/ceroid accumulation within aging cells might be due to increased production of reactive oxygen species (7, 20) or a decline in the efficiency of protein repair and/or degradation systems (21, 22). Experimentally, lipofuscin/ceroid accumulation can be accelerated by increased oxidative stress (19, 23) and by inhibition of lysosomal proteases and lipases (24), conditions that accelerate the aging process in general (24 –26). Accordingly, lipofuscin/ceroid accumulation is regarded as one of the best-known biomarkers of aging (27). Since lipofuscin/ceroid is a biological marker of aging and cross-linked proteins are able to inhibit proteases, we decided to study the influence of oxidative stress and artificial lipofuscin/ceroid (4, 28) on protein turnover and proteolytic enzymes in aging postmitotic fibroblasts.
MATERIALS AND METHODS Cell culture and treatments WI-38 and MRC-5 human lung fibroblast strains were both obtained at a population doubling level (PDL) of 20 from ATCC (Rockville, Md.). F9 and F12 human lung fibroblast LIPOFUSCIN/CEROID INHIBITS THE PROTEASOME
strains were established from human bronchial tumor samples at the Institute of Pathology, Charite´, and assigned a starting PDL of 10 after growth of the first 25 cm2 monolayer. All cells were grown in EMEM plus 10% fetal calf serum (Seromed, Berlin). Cells with a PDL of between 20 and 30 (WI-38, MRC-5) or between 10 and 20 (F9, F12) were used. For several experiments, fibroblast cultures were exposed to hyperoxia under a 40% normobaric oxygen partial pressure as described previously (3, 4). This treatment results in an irreversible senescence-like growth arrest after ⬃4 wk and shortens the normal postmitotic life span of 1–2 years down to only about 3 months (4). Parallel cultures were grown under normoxia and treated with synthetic lipofuscin/ceroid twice a week for up to 4 wk. Artificial lipofuscin/ceroid was prepared from liver mitochondria as described (4, 28). Briefly, a crude liver mitochondrial preparation (3.0 mg protein/ml) was UV-peroxidized to completion as judged by return to basal levels of the production of thiobarbituric acid-reactive materials within the preparation. The preparation was extensively homogenized to obtain particles of mitochondrial size and the equivalent of 0.3 mg protein was added to 106 cells. Additional controls were performed with WI-38 cells held under normoxia for up to 2 months as density-inhibited cultures. Some of these control cultures were treated with nonfluorescent latex spheres (1 m in diameter, PLANO, Marburg, Germany) instead of lipofuscin/ceroid. Measurement of lipofuscin/ceroid content For electron microscopy, 1 ⫻ 106 cells were centrifuged at 900 rpm and the pellets were fixed in glutaraldehyde/osmium tetroxide. Between 25 and 35 cells were photographed at a final magnification of 5000⫻ using an EM 10 electron microscope (Zeiss, Oberkochen, Germany) in a systematic random manner. The fraction of secondary lysosomes within the fibroblast cytoplasm (excluding the nuclei) was estimated by point counting using a grid with a 10 mm mesh size. Cellular autofluorescence in the yellow-green range of the spectrum (563– 607 nm), which stems mainly from lipofuscin/ceroid, 1491
dithiothreitol during vigorous shaking for 1 h at 4°C. The lysates were immediately used for determination of proteolytic activities. Proteasome activity The lysates were centrifuged for 30 min at 14,000 g. Supernatants were incubated in 50 mM Tris-HCl buffer (pH 7.8) containing 20 mM KCl, 0.5 mM MgOAc, and 1 mM dithiothreitol. The fluorogenic peptide suc-LLVY-MCA was used as a substrate at a final concentration of 200 M. After a 1 h incubation at 37°C, proteolysis was terminated by addition of an equal volume of ice-cold ethanol. Measurements of proteolysis (release of the MCA fluor) were performed at 380 nm excitation and 440 emission after addition of 0.125 M sodium borate (pH 9.0) using free MCA (a fluorogenic peptide used to measure proteasome activity) as standard for quantification. Activity of lysosomal proteases
Figure 2. Specific activity of proteolytic systems in WI-38 cells during hyperoxia-induced ‘aging’. WI-38 fibroblasts were cultivated for 0, 5, 8, and 12 wk, then harvested and lysed. The lysates were sonicated for determination of lysosomal cathepsin activity (z-FR-MCA-degrading activity). To determine the activity of the cytosolic proteasome (suc-LLVY-MCA-degrading activity), the lysates were centrifuged. For details of cell lysis, see Materials and Methods. For activity determinations, the lysates were incubated for 30 min (z-FR-MCA) or 60 min (suc-LLVY-MCA) in the presence of 200 M of the respective peptide substrate. After stopping the reaction, the fluorescence of the free MCA fluor was determined at 380 nm excitation and 440 nm emission. The data represent means ⫾ sd of four independent experiments, each with four measurements. was also separately measured by flow cytometry using a Becton-Dickinson FACScan as described previously (4). Measurement of protein turnover For measurements of overall protein synthesis and proteolytic activity, cells were washed twice with phosphate-buffered saline and incubated for 16 h with 35S-labeled methionine (35S-Met) in methionine-free medium (Sigma). After labeling, cells were again washed twice and cultured further in (methionine-rich) standard medium. Protein synthesis was determined by calculating the difference between the radioactivity in the medium before and after 16 h of incubation. Radioactivity was measured by scintillation counting. For proteolysis measurements, trichloroacetic acid (TCA) soluble counts were determined as described previously (13, 14). Protease activity determinations The maximal activity of proteolytic systems was analyzed according to Inubushi et al. (29) and Grune et al. (13, 14). Between 0.3 and 1 ⫻ 106 cells were lysed in 150 l of 1 mM 1492
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Lysates were sonicated for 2 min on ice in a SONOPLUS GM70. The activity assay was performed by incubation of lysates at 37°C for 30 min in the presence of 200 M z-FR-MCA (a fluorogenic peptide used to measure lysosomal cathepsin activity) as substrate. The incubation buffer consisted of 50 mM sodium acetate (pH 5.5), 8 mM cysteine hydrochloride, and 1 mM EDTA. The reaction was stopped and measurements of MCA release were performed, as described for the determination of proteasome activity.
RESULTS ‘Aging’ of WI-38 fibroblasts is accelerated in an atmosphere of 40% oxygen (19). Under these conditions, proliferation ceases completely within 4 wk; this phenomenon is referred to as proliferative senescence (3). Most of the postmitotic life span is completed within the next 2 to 3 months, and extensive cell death occurs after 12 to 14 wk of hyperoxia (4). Protein turnover was measured in intact WI-38 fibroblasts. Incorporation of [35S] methionine into newly synthesized proteins was taken as a measure of protein synthesis. Incorporation of [35S] into proteins did not change significantly during 12 wk of fibroblast incubation under normobaric hyperoxia (Fig. 1A). Under the same conditions, however, protein degradation as measured by the liberation of TCA-soluble counts after removal of unincorporated label underwent a series of important changes (Fig. 1B). Total protein degradation increased by as much as 30% after 5 wk of hyperoxia, but thereafter declined to a level 25% lower than original after 12 wk of incubation under hyperoxic conditions (Fig. 1B, inset). From the results of Fig. 1, we concluded that the main changes in protein turnover during postmitotic aging might be those in protein catabolism. Therefore, we decided to test the overall capacity of the main proteolytic systems in WI-38 cells using various fluorogenic peptide substrates. Degradation of z-FR-
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Figure 3. Lipofuscin/ceroid and lysosome accumulation in WI-38 cells during hyperoxia or lipofuscin/ ceroid treatment. A) Autofluorescence was measured using a FACScan. Fluorescence of 5 ⫻ 104 cells was counted in triplicate after cultivation of cells under 40% oxygen for the indicated time periods. Four independent experiments were performed. B) WI-38 fibroblasts were cultivated under normoxia, with twice weekly additions of artificial lipofuscin/ceroid for the indicated periods of time. Cells were extensively washed before harvesting and the autofluorescence of 5 ⫻ 104 cells was counted in triplicate in a FACScan. Biologically inert, nonfluorescent, latex particles of similar size were used in control experiments (B, insert). C) The volume fraction of secondary lysosomes in the cytoplasm (excluding nuclei, in %) was measured by electron microscopic morphometry by counting 25–30 cells per group in triplicate. All experiments were repeated four times independently and the data in all panels represent means ⫾ sd.
MCA under acidic conditions in the cell lysate gives a good approximation for the activity of lysosomal cathepsins (29), whereas in the centrifuged (membrane-free) lysate of cells, proteasome represents the main suc-LLVY-MCA-degrading activity (13, 14). As reported in Fig. 2, a decline in the activity of both lysosomal proteases and cytosolic proteasome occurs during postmitotic aging. Both proteolytic systems lose more than 70% of their initial activity, indicating a dramatic loss of the overall capacity of the cell to degrade proteins. Hyperoxia causes increased oxygen radical production (30) and an increased rate of formation of radical-damaged proteins. Accumulation of lipofuscin/ceroid pigment is a major consequence of oxidative damage to proteins, which can be followed by the measurement of cellular yellow-green autofluorescence. As shown in Fig. 3A, the autofluorescence of WI-38 cells increases continuously during ‘aging’ under hyperoxic conditions, becoming especially marked after permanent inhibition of proliferation at around wk 4 of the treatment. Increased production of damaged proteins, due to increased formation of reactive oxygen species, may overwhelm the capacity of proteolytic systems. In addition, the accumulated oxidized proteins may cause a further decrease in the activity of proteolytic systems themselves, as discovered in earlier in vitro experiments (15, 16). Therefore, we decided to directly test whether exogenous lipofuscin/ceroid-like material, if taken up by cells, can change protein turnover in WI-38 fibroblasts. Confluent WI-38 fibroblasts were cultivated under normoxic conditions and synthetic lipofuscin/ceroid was added to the cells twice a week. Uptake and incorporation of exogenous lipofuscin/ceroid was carefully monitored both by electron microscopic LIPOFUSCIN/CEROID INHIBITS THE PROTEASOME
morphometry and flow cytometry with 563– 607 nm autofluorescence. The amount of lipofuscin/ceroid phagocytosed over time is shown as autofluorescence in Fig. 3B. A continuous increase in cellular autofluorescence was found during incubation with lipofuscin/ceroid. After 4 wk of lipofuscin/ceroid treatment, the level of autofluorescence was comparable to that of WI-38 cells after 7–10 wk of normobaric hyperoxia (compare Fig. 3, panels A and B). Electron microscopic morphometry of secondary lysosomes confirmed this result (data not shown). Increases in secondary lysosomes after 2 wk of lipofuscin/ceroid treatment were similar to those seen after 7 wk of hyperoxia as measured by morphometry and autofluorescence (Fig. 3C). Control experiments using latex particles of a size similar to the lipofuscin/ceroid-like material showed only a slight increase in autofluorescence (Fig. 3B, insert), which was actually equal to the accumulation of lipofuscin/ceroid we observed in untreated confluent fibroblasts under normoxia (data not shown). To test the influence of lipofuscin/ceroid accumulation on protein turnover in WI-38 fibroblasts, we performed [35S] incorporation experiments with lipofuscin/ceroid-treated cells. As shown in Fig. 4, overall protein degradation declined in a time-dependent manner in cells treated with lipofuscin/ ceroid, so that after 4 wk of lipofuscin/ceroid treatment protein degradation had decreased by ⬃20%. As measured by [35S] incorporation, protein synthesis remained unchanged (Fig. 4, insert). Control incubations using inert latex particles revealed no influence on protein turnover. There was, however, a significant influence of lipofuscin/ceroid on the proteolytic activities of both the lysosomal and proteasomal systems. As demonstrated in Fig. 5, the activity of lysosomal cathepsins increased by ⬃30%, 1493
ceroid accumulated whereas proteasome activity and overall proteolysis declined. Lipofuscin/ceroid-treatment of WI-38 fibroblasts caused a gradual lipofuscin/ceroid accumulation and a similarly gradual decline in both proteasome activity and overall protein degradation over a 4 wk period (Fig. 7B). The data of Fig. 7 reveal a rather tight negative correlation between cellular accumulation of lipofuscin/ ceroid (whether added directly or induced by hyperoxia), proteasome inactivation, and loss of intracellular proteolysis. To test whether or not the inverse relationship between lipofuscin/ceroid accumulation and proteasomeactivity/overall proteolysis seen in Fig. 7 and the divergence of lysosomal and proteasomal activities during lipofuscin/ceroid treatment seen in Fig. 5 are limited to the WI-38 cell line, we next examined three other nontransformed fibroblast cell lines. In all these cell lines, lipofuscin/ceroid treatment caused lipofuscin/ceroid accumulation (Fig. 8D) and gradually and significantly decreased overall protein degradation over a 4 wk treatment period Figure 4. Protein degradation and synthesis in WI-38 fibroblasts after lipofuscin/ceroid-induced ‘aging’. Lipofuscin/ ceroid treatment was performed as per Fig. 3. After 0, 2, 3, and 4 wk of lipofuscin/ceroid treatment and 2 or 4 wk of treatment with latex particles, protein turnover rates were measured. Protein degradation was measured as described in Materials and Methods and in Fig. 1B. The data represent the means of three independent experiments, with four separate measurements, for which sds were always less than 10%. The insert shows protein synthesis rates (means ⫾ sd of four independent experiments with four separate measurements) measured according to Materials and Methods and Fig. 1A.
whereas the cytosolic activity of proteasome declined by 25%. Therefore, despite the overall decrease in proteolytic specific activities observed in ‘aging’ WI-38 fibroblasts (Fig. 2), an increase in the total activity of lysosomal cathepsins occurs. It will also be noted that both proteasome and lysosomal cathepsin-specific activities decreased in the normobaric hyperoxia studies of Fig. 2, whereas lysosomal cathepsins actually increased after external treatment with lipofuscin/ceroid. This apparent contradiction is, in fact, explained by an increase in secondary lysosomes induced by artificial lipofuscin/ceroid in WI-38 cells (see Fig. 6, and Fig. 3C). To determine whether the changes in proteasome activity, overall proteolysis, and lipofuscin/ceroid accumulation reported in Figs. 1– 6 were temporally related, we performed studies of hyperoxia-treatment and lipofuscin/ceroid treatment in WI-38 cells in which all data was collected on the same time scale. As shown in Fig. 7A, hyperoxia caused an initial increase in overall protein degradation until the point (5 wk) when lipofuscin/ceroid began to accumulate. From wk 5 through wk 12, lipofuscin/ 1494
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Figure 5. Proteasome and lysosomal cathepsin activities of WI-38 cells after lipofuscin/ceroid-induced ‘aging’. WI-38 fibroblasts were cultivated and treated with lipofuscin/ceroid or inert latex particles, as described in Materials and Methods and Fig. 4. The proteolytic capacities of both proteasome and lysosomal cathepsins were measured in cell lysates prepared as described in Materials and Methods. Lysosomal cathepsin activity (z-FR-MCA-degrading activity) and cytosolic proteasome activity (suc-LLVY-MCA-degrading activity) were measured as described in the legend to Fig. 2. All data represent the means ⫾ sd of four independent experiments, each with four separate measurements.
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Since our results indicated that lipofuscin/ceroid might inhibit the proteasome during aging, we decided to directly test the ability of lipofuscin/ceroid to inhibit proteasome in vitro. Using purified proteasome and our artificial lipofuscin/ceroid preparation, we indeed found a clear, dose-dependent inhibition of proteasome activity (Fig. 9). These data clearly demonstrate the strong, direct inhibitory effect of lipofuscin/ceroid on the proteasome.
DISCUSSION
Figure 6. Accumulation of lysosomes in WI-38 fibroblasts after lipofuscin/ceroid-induced ‘aging’. A) A representative electron micrograph of control WI-38 fibroblasts. B) WI-38 fibroblasts after 2 wk of treatment with synthetic lipofuscin/ceroid; C) cells after 2 wk of treatment with inert latex spheres. The ultrastructure of secondary lysosomes after 2 wk of treatment with synthetic lipofuscin/ceroid is shown at higher magnification in panel D. The bars indicate 1 m (A–C) or 500 nm (D).
(Fig. 8A). This was accompanied by a decline in the activity of proteasome (Fig. 8B), whereas lysosomal cathepsin activity actually increased significantly in all cell lines tested (Fig. 8C).
Since the free radical theory of aging was proposed by Harman (31), considerable evidence has been presented to support the involvement of free radicals in the aging process. One example is the demonstration of an accumulation of oxidized proteins during aging (32). The original free radical theory of aging concentrated on increased oxidant production and decreased primary antioxidant enzyme protection, but the discovery of damage removal and repair systems (or secondary antioxidant systems) has led to a reevaluation of the theory and new proposals for declining damage removal capacity (33). Indeed, age-associated accumulation of oxidized proteins may well be the result of a decline in the proteolytic activity of damage removal systems (16). Decreased proteolytic activities have been reported in aging
Figure 7. Time course of lipofuscin/ceroid accumulation, proteasome inactivation, and loss of overall proteolysis in hyperoxia-treated or lipofuscin/ceroid-treated WI-38 fibroblasts. WI-38 fibroblasts were cultivated and used as controls, exposed to up to 12 wk of hyperoxia (40% oxygen), or treated for up to 4 wk with lipofuscin/ceroid or inert latex particles, as described in Materials and Methods and the legends to Figs. 1–5. Measurements of proteasome activity (sLLVY-MCA degradation), overall protein degradation (turnover of metabolically radiolabeled 35S-cell proteins), and lipofuscin/ceroid accumulation (FACScan measurements of specific autofluorescence) were performed as described in Materials and Methods and in the legends to Figs. 1–5. LIPOFUSCIN/CEROID INHIBITS THE PROTEASOME
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Figure 8. Protein degradation in various fibroblast lines after lipofuscin/ceroid-induced ‘aging’. F9, F12, and MRC-5 fibroblast cell lines were cultivated under standard conditions. Lipofuscin/ceroid treatment was performed as described in Figs. 3– 6. A) Cellular proteins were metabolically labeled with [35S] methionine after 0 to 4 wk of cellular lipofuscin/ceroid treatment. After extensive washing, the liberation of TCA-soluble counts was taken as a measure of protein degradation as described in the legend to Fig. 1B. The percentage of labeled protein degraded was measured at 144 h after initial radiolabeling of cells. B, C) Cells were lysed as described in Materials and Methods and incubated with fluoropeptide proteolysis substrate. The experiments of panel D involved measurements of lipofuscin/ceroid accumulation, performed as described in the legend to Fig. 3B. Data are given for untreated controls (0 wk) and for cells exposed to 1, 2, 3, or 4 wk of lipofuscin/ceroid treatment. The assay for fluoropeptide degradation was performed in the presence of 200 M of the indicated fluoropeptide. Proteasome activity was measured with suc-LLVY-MCA (B) and lysosomal cathepsins were measured with z-FR-MCA (C), both by fluorometric measurement of MCA liberation. Values shown represent the means ⫾ sd of four independent experiments, each with four separate measurements.
brains (32). Additional studies using protease inhibitors like leupeptin or E-64 (24, 25) have indicated a key role for proteases in the aging process. Previous studies focused on the role of lysosomal proteases in aging, but it is known that the proteasome is mostly responsible for the degradation of oxidized cytosolic proteins (9 –12). Several groups have demonstrated a role of the proteasome in the selective recognition (34, 35) and degradation of oxidized proteins (9 –16, 21, 22). The proteasomal capacity of cells is high, and the removal of oxidized proteins depends largely on substrate supply (13, 14). The present data indicate an initial increase in overall protein degradation in cells exposed to hyperoxia, i.e., to an increased oxygen radical flux. Such an increase has previously been demonstrated using other radicalgenerating systems (13, 14). After 5 wk of hyperoxia, however, a progressive decline in protein degradation occurred in WI-38 cells, reflecting the decreased activity of proteolytic enzymes. In this study, lipofuscin/ceroid accumulation during hyperoxia-induced cellular ‘aging’ was corre1496
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lated with decreased overall protein degradation as well as with decreased proteasome activity and lysosomal cathepsin activity. In cells forced to accumulate artificial lipofuscin/ceroid under normoxic conditions, overall protein degradation again decreased, as did the activity of proteasome. An apparent increase in lysosomal cathepsin activity in cells treated with artificial lipofuscin/ceroid was actually caused by an increase in the number of secondary lysosomes. These results are in accord with a major role for the proteasome in normal protein turnover, as proposed previously (36). Inhibition of proteasome by cross-linked proteins was previously demonstrated only in in vitro systems, and now we present evidence that this process also occurs in living cells. Accumulation of lipofuscin/ceroid and cross-linked proteins during aging may be a self-accelerating process, leading to a gradual decline in cellular proteolytic activities. In retinal pigmented epithelial cells, it has been reported that lipofuscin/ceroid might contain lysosome-disrupting cationic detergents, which can inhibit proteolysis (37, 38).
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4. 5. 6. 7.
8. 9.
10. 11. 12.
Figure 9. Inhibition of the chymotrypsin-like activity of the proteasome by lipofuscin/ceroid. Purified 20S proteasome was isolated from red blood cells as described previously (9 –14). Proteasome was then incubated with artificial lipofuscin/ceroid for 30 min prior to addition of the fluorogenic peptide substrate suc-LLVY-MCA used to measure the chymotrypsin-like activity of the proteasome. Proteolysis was then measured by release of the MCA fluor, as described in Materials and Methods. Data shown are means ⫾ sd of four independent experiments, each with four separate measurements.
One should consider that the activity of proteolytic systems measured using only artificial substrates as performed by others (39, 40) provides only limited insight into the real protein turnover of living cells. From the present results, combining both artificial substrates and overall intracellular proteolysis, we conclude that oxidized cross-linked proteins and lipofuscin/ceroid are possible inhibitors of the proteasome during postmitotic aging. The proteasome seems to be an important proteolytic system underlying age-dependent changes.
13. 14. 15. 16.
17. 18. 19.
20. 21.
This work was supported by grants from the Swedish Medical Research Council, by the foundation Verhalten und Umwelt (Munich, Germany), and by NIH/NIEHS grant ES 03598 to K.J.A.D. The authors thank Professor Ulf Brunk, Linko¨ping, Sweden, for helpful suggestions.
22. 23.
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The FASEB Journal
Received for publication September 17, 1999. Accepted for publication December 7, 1999.
SITTE ET AL.
