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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 1, 2003 as doi:10.1096/fj.03-0177fje. |
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* Neuroscience Research Center,
Institute of Anatomy, and
Clinic of Gastroenterology and Hepatology, Medical Faculty (Charité), Humboldt University Berlin, D-10098 Berlin, Germany
2Correspondence: Neuroscience Research Center, Medical Faculty (Charité), Humboldt University Berlin, Schumannstr. 20/21, D-10117 Berlin, Germany. E-mail: tilman.grune{at}charite.de
SPECIFIC AIMS
Accumulation of oxidized proteins and a loss of activity of the proteolytic enzymes, including the proteasome, are highlights of age-related changes of cellular metabolism. We tested whether cytosolic and nuclear proteasomes are equally affected by age-related changes and whether the proteasomal system is able to remove oxidized proteins from both compartments.
PRINCIPAL FINDINGS
1. Protein turnover and proteasomal activity in the nucleus of MRC-5 fibroblasts is stable during senescence
In a first series of experiments we tested the overall turnover of proteins in the cytosol and the nucleus of young and senescent cells. As shown in Fig. 1
, overall protein degradation declines dramatically during proliferative senescence. Nuclear proteins turn over significantly slower than the total protein pool. But in disagreement with the total degradation rates, nuclear proteins seem to turn over much faster in senescent fibroblasts. Therefore, it can be concluded that the nuclear protein turnover is stabilized during senescence.
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We reported earlier that the decline of the overall protein turnover is accompanied by a decline in the cytosolic proteasomal activity, so we tested both compartments—the cytosol and the nuclei—for proteasomal activity. The activity of the cytosolic proteasomal system declined with increasing proliferation level, but for nuclear protein turnover, the activity of the proteasomal system is higher at higher PD levels.
2. Stable proteasome concentration in the nucleus of senescent cells
Next we tested both compartments for the concentration of the proteasome. Using immunofluorescence studies, we were able to compare directly the proteasomal concentration in the cytosolic and nuclear compartment within young and senescent cells. Quantification of these data allowed us to conclude that proteasomal concentration in the nucleus is not changing during the senescence process, whereas the cytosolic proteasome concentration is increasing somewhat. However, the proteasome concentration in the nuclear compartment is clearly not changing during senescence.
3. Low response of protein turnover to oxidative stress in cytosol and nucleus of senescent cells
We tested whether the protein turnover is changing during oxidative stress in the cytosol and the nucleus. The overall protein turnover in fibroblasts of young MRC-5 fibroblasts increases in a dose-related manner after hydrogen peroxide treatment. This result agrees with numerous investigations performed by our group and reflects the functionality of the proteasomal system in the younger fibroblasts. However, senescent fibroblasts are losing the ability to respond on oxidative stress with an increased proteolysis. The same seems to be true for the nuclear protein turnover. Whereas the nuclei of young fibroblasts are able to increase the protein turnover by almost 50%, senescent fibroblasts lack this ability. Protein turnover in the younger fibroblasts rises to approximately the same level as in the old fibroblasts before oxidative stress.
Therefore, the high protein turnover in senescent fibroblast nuclei might well be the result of a permanent oxidative stress exposure due to higher oxidant production rates or lower primary antioxidative defenses in senescent cells.
4. Oxidative stress-related activation of nuclear proteasome in young but not in senescent cells
We were interested in changes in activity of the proteasomal system in the cytosol and nuclei of young and senescent fibroblasts. The cytosolic proteasome is relatively resistant to oxidative stress. Some unexpected results were achieved by measuring the proteasomal activity in the nucleus. Hydrogen peroxide treatment in young fibroblasts causes a >15-fold increase in activity 24 h after treatment with 0.4 mM hydrogen peroxide. Senescent fibroblasts, however, double their activity directly only after oxidative stress; no further changes were observed. As tested by immunoblotting, this increase in proteasomal activity in the nucleus in young MRC-5 fibroblasts is not due to an increasing proteasome amount in the nucleus. Further studies revealed that this increase is transient and, after 48, h proteasomal activity is returning to the preoxidation level. No major change of the proteasomal activity in the nucleus of senescent fibroblasts could be observed in this time frame.
5. High levels of oxidized proteins in the nucleus and cytosol of senescent cells
To test this, we measured the amount of protein-bound carbonyl groups, an established marker of protein oxidation. We were able to demonstrate that cytosolic proteins as well as nuclear proteins have higher protein-bound carbonyl levels in senescent fibroblasts than the same compartment in young MRC-5 cells. Nondegraded oxidized proteins tend to cross-link and form highly polymerized protein aggregates. We measured the appearance of cellular autofluorescence by fluorescence microscopy and were able to show that the high level of autofluorescence in MRC-5 fibroblasts is exclusively located outside the nucleus.
6. Slow removal of oxidized proteins from cytosol and nucleus in senescent cells
The above-described increase in accumulation of protein oxidation products might be the result of a reduction of the efficiency of the removal of oxidized proteins or the result of an increased oxidant production. We therefore tested the ability of the proteasome in the cytosol and the nucleus to remove oxidized proteins after an exposure to a bolus concentration of hydrogen peroxide. Oxidation via hydrogen peroxide increases the protein carbonyl content, but in the cytosol this increase is much more pronounced in senescent fibroblasts than in the cytosol of young fibroblasts. This suggests the efficiency of the cytosolic primary antioxidative defense mechanism in young fibroblasts. The proteins of the nucleus of young as well as of old fibroblasts seem to be less protected; therefore, a high amount of protein carbonyls is formed in young and senescent cells. The removal of oxidized proteins was determined by measuring the protein carbonyl content 24 h after oxidative stress. Senescent fibroblasts were less able to cope with the removal of oxidized proteins, and there is only a partial reduction of protein carbonyls in the cytosol and nucleus of senescent fibroblasts 24 h after oxidative stress. Therefore, the functionality of the proteasomal system is not sufficient to remove all the oxidized protein moieties at late proliferation stages, a malfunction that also seems prominent in the nucleus.
CONCLUSIONS
It is known that a dramatic decline of protein turnover and proteasomal activity, but not enzyme content, occurs during proliferative senescence (Fig. 2
). Although it has been known for some time that the proteasome is distributed within the cytosol and the nucleus, nothing is known about the senescent-related changes in concentration and functionality of proteasomes in various cellular compartments.
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We found that the nuclear protein does not undergo a senescent-related decline in activity and therefore there is no decline in the turnover of nuclear proteins in senescent MRC-5 fibroblasts. The proteasomal concentration in the nucleus is higher than the cytosol in young or senescent cells. However, the proteasome content in the cytosol is rising with senescence while the nuclear proteasomal content is not. Since the accumulation of oxidized/cross-linked proteins during senescence happens outside the nucleus, we suggest that the proteasomal activity in the cytosol is inhibited by protein aggregates but the nuclear proteasome is not affected.
Oxidative stress induced a clear increase in the turnover of nuclear proteins in young cells, whereas senescent cells are unable to respond this way. Since the nuclei of senescent fibroblasts already have a high protein turnover per se, the proteasomal degradation ability seems to reach a limit. A pronounced activation of the nuclear proteasome after oxidative stress was described that is prominent only in young MRC-5 cells. This indicates some limitations of the adaptability of the nuclear proteasomal system toward oxidative stress in senescent cells.
Our results led us to conclude that the nuclear proteasome is relatively stable during senescence, although some malfunction of the proteasomal activation after oxidative stress takes place.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0177fje; doi: 10.1096/fj.03-0177fje 
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