Age-related changes in protein oxidation and proteolysis in mammalian cells.

Reactive oxygen species generated as by-products of oxidative metabolism, or from environmental sources, frequently damage cellular macromolecules. Proteins are recognized as major targets of oxidative modification, and the accumulation of oxidized proteins is a characteristic feature of aging cells. An increase in the amount of oxidized proteins has been reported in many experimental aging models, as measured by the level of intracellular protein carbonyls or dityrosine, or by the accumulation of protein-containing pigments such as lipofuscin and ceroid bodies. In younger individuals, moderately oxidized soluble cell proteins appear to be selectively recognized and rapidly degraded by the proteasome. An age-related accumulation of oxidized proteins could, therefore, be a result of declining activity of the proteasome. Previous research to investigate the notion of an age-related decline in the content and/or activity of the proteasome has generated contradictory results. The latest evidence, including our own recent findings, indicates that proteasome activity does, indeed, decline during aging as the enzyme complex is progressively inhibited by oxidized and cross-linked protein aggregates. We propose that cellular aging involves both an increase in (mitochondrial) oxidant production and a progressive decline in proteasome activity. Eventually so much proteasome is inactivated that oxidized proteins begin to accumulate rapidly and contribute to cellular dysfunction and senescence.

BJ fibroblasts display high antioxidant capacity and slow telomere shortening independent of hTERT transfection.

Human foreskin BJ fibroblasts are well protected against oxidative stress as shown by their low intracellular peroxide content, low levels of protein carbonyls, and low steady-state lipofuscin content as compared to other primary human fibroblasts. This correlates with a long replicative life span of the parental cells of about 90 population doublings and a telomere-shortening rate of only 15-20 bp/PD. This value might define the upper limit of a telomere-shortening rate that can still be explained by the end replication problem alone. In BJ clones immortalized by transfection with hTERT, the catalytic subunit of telomerase, the same telomere-shortening rate as in parental cells is observed over a long time despite strong telomerase activity. Hyperoxia, which induces oxidative stress and accelerates telomere shortening in a variety of human fibroblast strains, does not do so in BJ cells. It is possible that the high antioxidative capacity of BJ cells, by minimizing the accumulation of genomic damage, is instrumental in the successful immortalization of these cells by telomerase.

Protein oxidation and proteolysis in RAW264.7 macrophages: effects of PMA activation.

Macrophages are stimulable cells able to increase the production of reactive oxygen and nitrogen species dramatically for a short period of time. Free radicals and other oxidants are able to oxidize the intracellular protein pool. These oxidized proteins are selectively recognized and degraded by the intracellular proteasomal system. We used the mouse macrophage-like cell line RAW264.7 to test whether macrophagial cells are able to increase their protein turnover after oxidative stress and whether this is accompanied by an increased protein oxidation. Macrophagial cells are particularly susceptible to bolus additions of hydrogen peroxide and peroxynitrite. In further experiments we activated RAW264.7 cells with PMA to test whether the production of endogenous oxidants has analogous effects. A clear dependence of the protein turnover and protein oxidation on the oxidative burst could be measured. In further experiments the role of the proteasomal system in the selective removal of oxidized proteins could be revealed exploring the proteasome specific inhibitor lactacystin. Therefore, although oxidants are able to attack the intracellular protein pool in macrophages, these cells are able to remove oxidized proteins selectively and protect the intracellular protein pool from oxidation.

beta-Endorphin-containing memory-cells and mu-opioid receptors undergo transport to peripheral inflamed tissue.

Immunocyte-derived beta-endorphin can activate peripheral opioid receptors on sensory neurons to inhibit pain within inflamed tissue. This study examined mu-opioid receptors (MOR) on sensory nerves and beta-endorphin (END) in activated/memory CD4(+) cells (the predominant population homing to inflamed tissue). We found an upregulation of MOR in dorsal root ganglia, an increased axonal transport of MOR in the sciatic nerve and an accumulation of MOR in peripheral nerve terminals in Freund's adjuvant-induced hindpaw inflammation. A large number of CD4(+) cells containing beta-endorphin, but very few naive cells (CD45RC(+)), were observed in inflamed tissue, suggesting that this opioid is mainly present in activated/memory cells (CD4(+)/CD45RC(-)). Taken together, our results indicate an enhanced transport of both MOR and of the endogenous ligand beta-endorphin to injured tissue. This unique simultaneous upregulation of both receptors and ligands may serve to prevent excessive and/or chronic inflammatory pain.

Lipofuscin accumulation in proliferating fibroblasts in vitro: an indicator of oxidative stress.

The amount of the ageing pigment, lipofuscin, found in replicating cells depends both on its rate of formation as well as its rate of dissolution by cell division. We present a model which allows the calculation of the lipofuscin accumulation rate from measurements of its amount and of the cell cycle duration. In two human fibroblast strains, the accumulation rate correlates well with differences in oxidative stress/antioxidative defence as measured by intracellular peroxide generation, protein carbonyl content, telomere shortening rate and replicative life span. The lipofuscin content increases with replicative age in both cultures. The rather steep increase in presenescent fibroblasts is not solely due to a slowing down of the cell turnover, but is partially caused by an increased rate of lipofuscin formation/ accumulation. This might indicate an increased level of oxidative stress in presenescent fibroblasts, or a decreased efficiency of proteolytic systems, or both. The results are in accordance with data demonstrating an adverse effect of lipofuscin accumulation on cellular protein turnover and suggest an active role for lipofuscin accumulation in cellular senescence.

4-Hydroxynonenal-modified amyloid-beta peptide inhibits the proteasome: possible importance in Alzheimer's disease.

The amyloid beta-peptide (Abeta) is a 4-kDa species derived from the amyloid precursor protein, which accumulates in the brains of patients with Alzheimer's disease. Although we lack full understanding of the etiology and pathogenesis of selective neuron death, considerable data do imply roles for both the toxic Abeta and increased oxidative stress. Another significant observation is the accumulation of abnormal, ubiquitin-conjugated proteins in affected neurons, suggesting dysfunction of the proteasome proteolytic system in these cells. Recent reports have indicated that Abeta can bind and inhibit the proteasome, the major cytoslic protease for degrading damaged and ubiquitin-conjugated proteins. Earlier results from our laboratory showed that moderately oxidized proteins are preferentially recognized and degraded by the proteasome; however, severely oxidized proteins cannot be easily degraded and, instead, inhibit the proteasome. We hypothesized that oxidatively modified Abeta might have a stronger (or weaker) inhibitory effect on the proteasome than does native Abeta. We therefore also investigated the proteasome inhibitory action of Abeta1-40 (a peptide comprising the first 40 residues of Abeta) modified by the intracellular oxidant hydrogen peroxide, and by the lipid peroxidation product 4-hydroxynonenal (HNE). H2O2 modification of Abeta1-40 generates a progressively poorer inhibitor of the purified human 20S proteasome. In contrast, HNE modification of Abeta1-40 generates a progressively more selective and efficient inhibitor of the degradation of fluorogenic peptides and oxidized protein substrates by human 20S proteasome. This interaction may contribute to certain pathological manifestations of Alzheimer's disease.

Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part I--effects of proliferative senescence.

Oxidized and cross-linked proteins tend to accumulate in aging cells. Declining activity of proteolytic enzymes, particularly the proteasome, has been proposed as a possible explanation for this phenomenon, and direct inhibition of the proteasome by oxidized and cross-linked proteins has been demonstrated in vitro. We have further examined this hypothesis during both proliferative senescence (this paper) and postmitotic senescence (see the accompanying paper, ref 1 ) of human BJ fibroblasts. During proliferative senescence, we found a marked decline in all proteasome activities (trypsin-like activity, chymotrypsin-like activity, and peptidyl-glutamyl-hydrolyzing activity) and in lysosomal cathepsin activity. Despite the loss of proteasome activity, there was no concomitant change in cellular levels of actual proteasome protein (immunoassays) or in the steady-state levels of mRNAs for essential proteasome subunits. The decline in proteasome activities and lysosomal cathepsin activities was accompanied by dramatic increases in the accumulation of oxidized and cross-linked proteins. Furthermore, as proliferation stage increased, cells exhibited a decreasing ability to degrade the oxidatively damaged proteins generated by an acute, experimentally applied oxidative stress. Thus, oxidized and cross-linked proteins accumulated rapidly in cells of higher proliferation stages. Our data are consistent with the hypothesis that proteasome is progressively inhibited by small accumulations of oxidized and cross-linked proteins during proliferative senescence until late proliferation stages, when so much proteasome activity has been lost that oxidized proteins accumulate at ever-increasing rates. Lysosomes attempt to deal with the accumulating oxidized and cross-linked proteins, but declining lysosomal cathepsin activity apparently limits their effectiveness. This hypothesis, which may explain the progressive intracellular accumulation of oxidized and cross-linked proteins in aging, is further explored during postmitotic senescence in the accompanying paper (1).

Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part II--aging of nondividing cells.

Oxidized/cross-linked intracellular protein materials, known as ceroid pigment, age pigment, or lipofuscin, accumulate in postmitotic tissues. It is unclear, however, whether diminishing proteolytic capacities play a role in the accumulation of such oxidized intracellular proteins. Previous studies revealed that the proteasome is responsible for the degradation of most oxidized soluble cytoplasmic and nuclear proteins and, we propose, for the prevention of such damage accumulations. The present investigation was undertaken to test the changes in protein turnover, proteasome activity, lysosome activity, and protein oxidation status during the aging of nondividing cells. Since the companion paper shows that both proteasome activity and the overall protein turnover decline during proliferative senescence whereas the accumulation of oxidized proteins increases significantly, we decided to use the same human BJ fibroblasts, this time at confluency, at different PD levels (including those that are essentially postmitotic) to investigate the same parameters under conditions where cells do not divide. We find that the activity of the cytosolic proteasome declines dramatically during senescence of nondividing BJ fibroblasts. The peptidyl-glutamyl-hydrolyzing activity was particularly affected. This decline in proteasome activity was accompanied by a decrease in the overall turnover of short-lived (radiolabeled) proteins in the nondividing BJ fibroblasts. On the other hand, no decrease in the actual cellular proteasome content, as judged by immunoblots, was found. The decline in the activity of the proteasome was also accompanied by an increased accumulation of oxidized proteins, especially of oxidized and cross-linked material. Unlike the loss of lysosomal function seen in our accompanying studies of proliferative senescence (1), however, the present study of hyperoxic senescence in nondividing cells actually revealed marked increases in lysosomal cathepsin activity in all but the very 'oldest' postmitotic cells. Our comparative studies of proliferating (1) and nonproliferating (this paper) human BJ fibroblasts reveal a good correlation between the accumulation of oxidized/cross-linked proteins and the decline in proteasome activity and overall cellular protein turnover during in vitro senescence, which may predict a causal relationship during actual cellular aging.

Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts.

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 [(35)S]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 inhibiting the proteasome.

Ferritin oxidation in vitro: implication of iron release and degradation by the 20S proteasome.

Ferritin, the major iron storage protein in mammalian cells, was treated with various concentrations of different oxidants: xanthine/xanthine oxidase, Sin-1 (3-morpholinosydnonimine, purchased from Alexis, Grunberg, Germany), DEA-NO (Diethylamine NONOate, purchased from Calblochem-Novabiochem, Schwalbach, Germany), and hydrogen peroxide. The proteolytic susceptibility towards the isolated 20S proteasome of untreated ferritin and oxidized ferritin was measured in parallel with the iron liberated by these oxidants. With increasing hydrogen peroxide, Sin-1, and xanthine oxidase concentrations, the measured proteasomal degradation of ferritin also increased. At higher oxidant concentrations, however, the proteolytic susceptibility began to decrease. The oxidation of ferritin by DEA-NO was accompanied by a lesser increase of proteolytic susceptibility in comparison with the effects of the other oxidants. Addition of DEA-NO to Sin-1 suppressed the increase in proteolytic susceptibility of ferritin, whereas adding xanthine/xanthine oxidase had no additional effect. Iron was liberated readily from ferritin as a result of the oxidation process, although the increase in proteolytic susceptibility was not always correlated to the iron release. In fact, the degradation of oxidatively damaged ferritin was not accompanied by a further increase of free iron. Therefore, we conclude that the proteasome is a secondary antioxidative defense system that degrades only nonfunctional ferritin.

Differential impairment of 20S and 26S proteasome activities in human hematopoietic K562 cells during oxidative stress.

The 20S proteasome and the 26S proteasome are major components of the cytosolic and nuclear proteasomal proteolytic systems. Since proteins are known to be highly susceptible targets for reactive oxygen species, the effect of H(2)O(2) treatment of K562 human hematopoietic cells toward the activities of 20S and 26S proteasomes was investigated. While the ATP-independent degradation of the fluorogenic peptide suc-LLVY-MCA was not affected by H(2)O(2) concentrations of up to 5 mM, the ATP-stimulated degradation of suc-LLVY-MCA by the 26S proteasome began to decline at 400 microM and was completely abolished at 1 mM oxidant treatment. A combination of nondenaturing electrophoresis and Western blotting let us believe that the high oxidant susceptibility of the 26S proteasome is due to oxidation of essential amino acids in the proteasome activator PA 700 which mediates the ATP-dependent proteolysis of the 26S-proteasome. The activity of the 26S-proteasome could be recovered within 24 h after exposure of cells to 1 mM H(2)O(2) but not after 2 mM H(2)O(2). In view of the specific functions of the 26S proteasome in cell cycle control and other important physiological functions, the consequences of the higher susceptibility of this protease toward oxidative stress needs to be considered.

Protein oxidation and degradation during proliferative senescence of human MRC-5 fibroblasts.

One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The aging process on a cellular level can be treated either as the ongoing proliferation until a certain number of cell divisions is reached (the Hayflick limit) or as the aging of nondividing cells, that is, the age-related changes in cells without proliferation. The present investigation was undertaken to reveal the changes in protein turnover, proteasome activity, and protein oxidation status during proliferative senescence. We were able to demonstrate that the activity of the cytosolic proteasomal system declines dramatically during the proliferative senescence of human MRC-5 fibroblasts. Regardless of the loss in activity, it could be demonstrated that there are no changes in the transcription and translation of proteasomal subunits. This decline in proteasome activity was accompanied by an increased concentration of oxidized proteins. Cells at higher proliferation stages were no longer able to respond with increased degradation of endogenous [(35)S]-Met-radiolabeled proteins after hydrogen peroxide- or quinone-induced oxidative stress. It could be demonstrated that oxidized proteins in senescent human MRC-5 fibroblasts are not as quickly removed as they are in young cells. Therefore, our study demonstrates that the accumulation of oxidized proteins and decline in protein turnover and activity of the proteasomal system are not only a process of postmitotic aging but also occur during proliferative senescence and result in an increased half-life of oxidized proteins.

Hydrogen peroxide-mediated protein oxidation in young and old human MRC-5 fibroblasts.

It is suggested that the aging process is dependent on the action of free radicals. One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The present investigation was undertaken to reveal the proliferation-related changes in the protein oxidation and proteasome activity during and after an acute oxidative stress. It could be demonstrated that the activity of the cytosolic proteasomal system declines during proliferative senescence of human MRC-5 fibroblasts and is not able to remove oxidized proteins in old cells efficiently. Whereas in young cells removal of oxidized proteins was accompanied by an increase in the overall protein turnover, this increase in protein turnover could not be seen in old MRC-5 fibroblasts. Therefore, our studies demonstrate that old fibroblasts are much more vulnerable to the accumulation of oxidized proteins after oxidative stress and are not able to remove these oxidized proteins as efficiently as young fibroblasts.

Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts.

Telomere shortening triggers replicative senescence in human fibroblasts. The inability of DNA polymerases to replicate a linear DNA molecule completely (the end replication problem) is one cause of telomere shortening. Other possible causes are the formation of single-stranded overhangs at the end of telomeres and the preferential vulnerability of telomeres to oxidative stress. To elucidate the relative importance of these possibilities, amount and distribution of telomeric single-strand breaks, length of the G-rich overhang, and telomere shortening rate in human MRC-5 fibroblasts were measured. Treatment of nonproliferating cells with hydrogen peroxide increases the sensitivity to S1 nuclease in telomeres preferentially and accelerates their shortening by a corresponding amount as soon as the cells proliferate. A reduction of the activity of intracellular peroxides using the spin trap alpha-phenyl-t-butyl-nitrone reduces the telomere shortening rate and increases the replicative life span. The length of the telomeric single-stranded overhang is independent of DNA damaging stresses, but single-strand breaks accumulate randomly all along the telomere after alkylation. The telomere shortening rate and the rate of replicative aging can be either accelerated or decelerated by a modification of the amount of oxidative stress. Quantitatively, stress-mediated telomere damage contributes most to telomere shortening under standard conditions.

LPS-induced protein oxidation and proteolysis in BV-2 microglial cells.

Exposure of proteins to oxidants leads to increased oxidation followed by preferential degradation by the proteasomal system. The role of the biological oxidant production in microglial BV-2 cells in the oxidation and turnover of endogenous proteins was measured. It could be demonstrated, that BV-2 cells are relatively resistant to fluxes of oxidants, but nevertheless protein oxidation occurs due to activation by LPS. This protein oxidation is followed by an enhanced degradation of endogenous proteins. Using PBN, a free radical scavenger and antioxidant, we could demonstrate the involvement of free radicals in the increased proteolysis in BV-2 cells after LPS-treatment. A slight but significant up-regulation of the proteasomal system after LPS activation takes place, indicating the importance of his proteolytic system in the maintenance of the protein pool of microglial cells.

Oxidized proteins as a marker of oxidative stress during coronary heart surgery.

The measurement of the degree of oxidative stress in patients often causes problems because of the lack of useful parameters. Therefore, we used an ELISA technique to evaluate serum protein carbonyls as a parameter of oxidative stress in patients during coronary heart surgery. Protein carbonyls were detected in serum samples of 14 patients undergoing coronary surgery and cardiopulmonary artery bypass grafting. A clear 2- to 3-fold increase in protein carbonyls in serum samples taken from human venous coronary sinus could be detected in the reperfusion period of the heart. We compared these data with markers of oxidative stress previously used, such as the glutathione status and the lipid peroxidation product malondialdehyde (MDA). Strong correlations of the protein carbonyl formation with MDA (r2 = 0.86) and oxidized glutathione (r2 = 0.81) were found in the early reperfusion stage. Increased levels of oxidized glutathione and MDA were detected only in the early reperfusion period. In contrast, the serum protein carbonyl content remained elevated for several hours, indicating a considerably slower serum clearance of oxidized proteins compared with that of lipid peroxidation products and the normalization of the glutathione status. We therefore concluded that the measurement of serum carbonyls by this ELISA technique is suitable to detect oxidative stress in serum samples of patients. The relative stability of the parameter makes the protein carbonyl detection even more valuable for clinical purposes.

Degradation of hypochlorite-damaged glucose-6-phosphate dehydrogenase by the 20S proteasome.

Glucose-6-phosphate dehydrogenase (G6PD) was treated with various concentrations of hypochlorite, which is produced by myeloperoxidase and is one of the most important oxidants during inflammatory processes. Inhibition of enzymatic activity, protein fragmentation, and proteolytic susceptibility toward the isolated 20S proteasome of G6PD were investigated. With rising hypochlorite concentrations, an increased proteasomal degradation of G6PD was measured. This occurred at higher hypochlorite concentrations than G6PD inactivation and at lower levels than G6PD fragmentation. The proteolytic activities of the 20S proteasome itself was determined by degradation of oxidized model proteins and cleavage of the synthetic proteasome substrate suc-LLVY-MCA. Proteasome activities remained intact at hypochlorite concentrations in which G6PD is maximally susceptible to proteasomal degradation. Only higher hypochlorite concentrations could decrease the proteolytic activities of the proteasome, which was accompanied by disintegration and fragmentation of the proteasome and proteasome subunits. Therefore, we conclude that the 20S proteasome can degrade proteins moderately damaged by hypochlorite and could contribute to an increased protein turnover in cells exposed to inflammatory stress.

Telomere shortening triggers a p53-dependent cell cycle arrest via accumulation of G-rich single stranded DNA fragments.

It has been repeatedly suspected that telomere shortening might be one possible trigger of the p53-dependent cell cycle arrest, although the mechanism of this arrest remained unclear. Telomeres in human cells under mild oxidative stress accumulate single-strand damage faster than interstitial repetitive sequences. In MRC-5 fibroblasts and U87 glioblastoma cells, which both express wild-type p53, oxidative stress-mediated production of single-strand damage in telomeres is concomitant to the accumulation of p53 and p21 and to cell cycle arrest. This response can be modeled by treatment of cells with short single stranded telomeric G-rich DNA fragments. The arrest is transient in U87 cells. Recovery from it is accompanied by up-regulation of telomerase activity and elongation of telomeres. Overexpression of mutated p53 is sufficient to reverse the phenotype of inhibition as well as the delayed activation of telomerase. These data suggest that the production of G-rich single stranded fragments during the course of telomere shortening is sufficient to trigger a p53 dependent cell cycle arrest.

Poly-ADP ribose polymerase activates nuclear proteasome to degrade oxidatively damaged histones.

The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified proteins in the cytoplasm. Nuclear proteins are also subject to oxidation, and the nucleus of mammalian cells contains proteasome. In human beings, tumor cells frequently are subjected to oxidation as a consequence of antitumor chemotherapy, and K562 human myelogenous leukemia cells have a higher nuclear proteasome activity than do nonmalignant cells. Adaptation to oxidative stress appears to be one element in the development of long-term resistance to many chemotherapeutic drugs and the mechanisms of inducible tumor resistance to oxidation are of obvious importance. After hydrogen peroxide treatment of K562 cells, degradation of the model proteasome peptide substrate suc-LLVY-MCA and degradation of oxidized histones in nuclei increases significantly within minutes. Both increased proteolytic susceptibility of the histone substrates (caused by modification by oxidation) and activation of the proteasome enzyme complex occur independently during oxidative stress. This rapid up-regulation of 20S proteasome activity is accompanied by, and depends on, poly-ADP ribosylation of the proteasome, as shown by inhibitor experiments, 14C-ADP ribose incorporation assays, immunoblotting, in vitro reconstitution experiments, and immunoprecipitation of (activated) proteasome with anti-poly-ADP ribose polymerase antibodies. The poly-ADP ribosylation-mediated activated nuclear 20S proteasome is able to remove oxidatively damaged histones more efficiently and therefore is proposed as an oxidant-stimulatable defense or repair system of the nucleus in K562 leukemia cells.