Oxidized proteins: intracellular distribution and recognition by the proteasome

The formation of oxidized proteins is one of the highlights of oxidative stress. In order not to accumulate such proteins have to be degraded. The major proteolytic system responsible for the removal of oxidized proteins is the proteasome. The proteasome is distributed throughout the cytosolic and nuclear compartment of mammalian cells, with high concentrations in the nucleus. On the other hand a major part of protein oxidation is taking place in the cytosol. The present review highlights the current knowledge on the intracellular distribution of oxidized proteins and put it into contrast with the concentration and distribution of the proteasome.     

Oxidative stress-mediated regulation of proteasome complexes

Oxidative stress has been implicated in aging and many human diseases, notably neurodegenerative disorders and various cancers. The reactive oxygen species that are generated by aerobic metabolism and environmental stressors can chemically modify proteins and alter their biological functions. Cells possess protein repair pathways to rescue oxidized proteins and restore their functions. If these repair processes fail, oxidized proteins may become cytotoxic. Cell homeostasis and viability are therefore dependent on the removal of oxidatively damaged proteins. Numerous studies have demonstrated that the proteasome plays a pivotal role in the selective recognition and degradation of oxidized proteins. Despite extensive research, oxidative stress-triggered regulation of proteasome complexes remains poorly defined. Better understanding of molecular mechanisms underlying proteasome function in response to oxidative stress will provide a basis for developing new strategies aimed at improving cell viability and recovery as well as attenuating oxidation-induced cytotoxicity associated with aging and disease. Here we highlight recent advances in the understanding of proteasome structure and function during oxidative stress and describe how cells cope with oxidative stress through proteasome-dependent degradation pathways.     

Degradation of oxidized proteins by the 20S proteasome

Oxidatively modified proteins are continuously produced in cells by reactive oxygen and nitrogen species generated as a consequence of aerobic metabolism. During periods of oxidative stress, protein oxidation is significantly increased and may become a threat to cell survival. In eucaryotic cells the proteasome has been shown (by purification of enzymatic activity, by immunoprecipitation, and by antisense oligonucleotide studies) to selectively recognize and degrade mildly oxidized proteins in the cytosol, nucleus, and endoplasmic reticulum, thus minimizing their cytotoxicity. From in vitro studies it is evident that the 20S proteasome complex actively recognizes and degrades oxidized proteins, but the 26S proteasome, even in the presence of ATP and a reconstituted functional ubiquitinylating system, is not very effective. Furthermore, relatively mild oxidative stress rapidly (but reversibly) inactivates both the ubiquitin activating/conjugating system and 26S proteasome activity in intact cells, but does not affect 20S proteasome activity. Since mild oxidative stress actually increases proteasome-dependent proteolysis (of oxidized protein substrates) the 20S 'core' proteasome complex would appear to be responsible. Finally, new experiments indicate that conditional mutational inactivation of the E1 ubiquitin-activating enzyme does not affect the degradation of oxidized proteins, further strengthening the hypothesis that oxidatively modified proteins are degraded in an ATP-independent, and ubiquitin-independent, manner by the 20S proteasome. More severe oxidative stress causes extensive protein oxidation, directly generating protein fragments, and cross-linked and aggregated proteins, that become progressively resistant to proteolytic digestion. In fact these aggregated, cross-linked, oxidized proteins actually bind to the 20S proteasome and act as irreversible inhibitors. It is proposed that aging, and various degenerative diseases, involve increased oxidative stress (largely from damaged and electron 'leaky' mitochondria), and elevated levels of protein oxidation, cross-linking, and aggregation. Since these products of severe oxidative stress inhibit the 20S proteasome, they cause a vicious cycle of progressively worsening accumulation of cytotoxic protein oxidation products.     

Oxidized protein degradation and repair in ageing and oxidative stress

Cellular ageing is characterized by the accumulation of oxidatively modified proteins which may be due to increased protein damage and/or decreased elimination of oxidized protein. Since the proteasome is in charge of protein turnover and removal of oxidized protein, its fate during ageing and upon oxidative stress has received special attention, and evidence has been provided for an age-related impairment of proteasome function. However, proteins when oxidized at the level of sulfur-containing amino acids can also be repaired. Therefore, the fate of the methionine sulfoxide reductase system during ageing has also been addressed as well as its role in protection against oxidative stress.     

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).     

Ump1 extends yeast lifespan and enhances viability during oxidative stress: Central role for the proteasome?

Increasing evidence suggests that the proteasome may play an important role in both oxidative stress response and cellular aging, although considerable controversy exists as to the exact role the proteasome plays in each of these paradigms. In the present study we examined the contribution of impaired proteasome function to the regulation of oxidative damage (oxidized protein levels) following the administration of oxidative stressors, and to the cytotoxicity observed in aging and oxidatively challenged cells. In these studies the preservation of proteasome-mediated protein degradation was achieved via increased expression of the proteasome assembly protein Ump1. We observed that Saccharomyces cerevisiae transformed to express increased levels of Ump1 exhibited increased viability in response to a variety of oxidative stressors (menadione, hydrogen peroxide, 4-hydroxynonenal). The increased viability observed in each of these paradigms was associated with an enhanced preservation of proteasome-mediated protein degradation, consistent with the preservation of proteasome function being sufficient to ameliorate oxidative stress-induced cytotoxicity. Interestingly, cells expressing Ump1 were observed to initially have robust elevations in oxidized protein levels following the addition of oxidative stressors, but exhibited a significantly reduced level of oxidized proteins following the removal of oxidative stressors. Cells expressing elevated levels of Ump1 also exhibited an enhanced preservation of proteasome-mediated protein degradation, and enhanced viability during stationary-phase aging. Taken together these data strongly support a role for the proteasome serving as a central regulator of cellular viability during oxidative stress and during aging.     

Proteasome modulates mitochondrial function during cellular senescence

Proteasome plays fundamental roles in the removal of oxidized proteins and in the normal degradation of short-lived proteins. Previously we have provided evidence that the impairment in proteasome observed during the replicative senescence of human fibroblasts has significant effects on MAPK signaling, proliferation, life span, senescent phenotype, and protein oxidative status. These studies have demonstrated that proteasome inhibition and replicative senescence caused accumulation of intracellular protein carbonyl content. In this study, we have investigated the mechanisms by which proteasome dysfunction modulates protein oxidation during cellular senescence. The results indicate that proteasome inhibition during replicative senescence has significant effects on intra- and extracellular ROS production in vitro. The data also show that ROS impaired the proteasome function, which is partially reversible by antioxidants. Increases in ROS after proteasome inhibition correlated with a significant negative effect on the activity of most mitochondrial electron transporters. We propose that failures in proteasome during cellular senescence lead to mitochondrial dysfunction, ROS production, and oxidative stress. Furthermore, it is likely that changes in proteasome dynamics could generate a prooxidative condition at the immediate extracellular microenvironment that could cause tissue injury during aging, in vivo.     

Regulation of proteasome-mediated protein degradation during oxidative stress and aging

Protein degradation is a physiological process required to maintain cellular functions. There are distinct proteolytic systems for different physiological tasks under changing environmental and pathophysiological conditions. The proteasome is responsible for the removal of oxidatively damaged proteins in the cytosol and nucleus. It has been demonstrated that proteasomal degradation increases due to mild oxidation, whereas at higher oxidant levels proteasomal degradation decreases. Moreover, the proteasome itself is affected by oxidative stress to varying degrees. The ATP-stimulated 26S proteasome is sensitive to oxidative stress, whereas the 20S form seems to be resistant. Non-degradable protein aggregates and cross-linked proteins are able to bind to the proteasome, which makes the degradation of other misfolded and damaged proteins less efficient. Consequently, inhibition of the proteasome has dramatic effects on cellular aging processes and cell viability. It seems likely that during oxidative stress cells are able to keep the nuclear protein pool free of damage, while cytosolic proteins may accumulate. This is because of the high proteasome content in the nucleus, which protects the nucleus from the formation and accumulation of non-degradable proteins. In this review we highlight the regulation of the proteasome during oxidative stress and aging.     

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.     

Ubiquitin-proteasome pathway and cellular responses to oxidative stress

The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Substrate proteins of the canonical UPP are first tagged by multiple ubiquitin molecules and then degraded by the 26S proteasome. However, in noncanonical UPP, proteins can be degraded by the 26S or the 20S proteasome without being ubiquitinated. It is clear that a proteasome is responsible for selective degradation of oxidized proteins, but the extent to which ubiquitination is involved in this process remains a subject of debate. Whereas many publications suggest that the 20S proteasome degrades oxidized proteins independent of ubiquitin, there is also solid evidence indicating that ubiquitin and ubiquitination are involved in degradation of some forms of oxidized proteins. A fully functional UPP is required for cells to cope with oxidative stress and the activity of the UPP is also modulated by cellular redox status. Mild or transient oxidative stress up-regulates the ubiquitination system and proteasome activity in cells and tissues and transiently enhances intracellular proteolysis. Severe or sustained oxidative stress impairs the function of the UPP and decreases intracellular proteolysis. Both the ubiquitin-conjugating enzymes and the proteasome can be inactivated by sustained oxidative stress, especially the 26S proteasome. Differential susceptibilities of the ubiquitin-conjugating enzymes and the 26S proteasome to oxidative damage lead to an accumulation of ubiquitin conjugates in cells in response to mild oxidative stress. Thus, increased levels of ubiquitin conjugates in cells seem to be an indicator of mild oxidative stress.     

Coordination of oxidized protein hydrolase and the proteasome in the clearance of cytotoxic denatured proteins

Intracellular accumulation of denatured proteins impairs cellular function. The proteasome is recognized as an enzyme responsible for the effective clearance of those cytotoxic denatured proteins. As another enzyme that participates in the destruction of damaged proteins, we have identified oxidized protein hydrolase (OPH) and found that OPH confers cellular resistance to various kinds of oxidative stress. In this study, we demonstrate the roles of the proteasome and OPH in the clearance of denatured proteins. The inhibition of proteasome activity results in the elevation of protein carbonyls in cells under oxidative stress. On the other hand, cells overexpressing OPH retain higher resistance to oxidative stress, even though the proteasome activity is inhibited. Furthermore, upon inhibition of the proteasome activity, OPH is recruited to a novel organelle termed the aggresome where misfolded or denatured proteins are processed. Thus, OPH and the proteasome coordinately contribute to the clearance of cytotoxic denatured proteins.     

Protein oxidation and proteolysis

One of the hallmarks of chronic or severe oxidative stress is the accumulation of oxidized proteins, which tend to form high-molecular-weight aggregates. The major proteolytic system responsible for the removal of oxidized cytosolic and nuclear proteins is the proteasome. This complicated proteolytic system contains a core proteasomal form (20S proteasome) and several regulators. All of these components are affected by oxidative stress to various degrees. The ATP-stimulated 26S proteasome is sensitive to oxidative stress, whereas the 20S form seems to be more resistant. The nuclear proteasome selectively degrades oxidatively damaged histones in the nuclei of mammalian cells, where it is activated and regulated by automodified PARP-1 after oxidative challenge. In this brief review we highlight the proteolysis and its regulatory effects during oxidative stress.     

Hsp27: novel regulator of intracellular redox state

Small stress proteins are molecular chaperones that modulate the ability of cells to respond to several types of injuries. In this regard, a protection generated by the expression of mammalian small stress proteins against the cell death induced by oxidative stress has been described. This review summarizes the current knowledge of the protective mechanism generated by the expression of the major mammalian small stress protein Hsp27 in cells exposed to oxidative stress. A possible role of this chaperone protein in the presentation of oxidized proteins to the proteasome degradation machinery is proposed.     

Chaperones, but not oxidized proteins, are ubiquitinated after oxidative stress

After oxidative stress, proteins that are oxidatively modified are degraded by the 20S proteasome. However, several studies have documented an enhanced ubiquitination of yet unknown proteins. Because ubiquitination is a prerequisite for degradation by the 26S proteasome in an ATP-dependent manner this raises the question whether these proteins are also oxidized and, if not, what proteins need to be ubiquitinated and degraded after oxidative conditions. By determination of oxidized and ubiquitinated proteins we demonstrate here that most oxidized proteins are not preferentially ubiquitinated. However, we were able to confirm an increase in ubiquitinated proteins 16 h after oxidative stress. Therefore, we isolated ubiquitinated proteins from hydrogen peroxide-treated cells, as well as from control cells and cells treated with lactacystin, an irreversible proteasome inhibitor, and identified some of these proteins by MALDI tandem mass spectrometry. As a result we obtained 24 different proteins that can be categorized into the following groups: chaperones, energy metabolism, cytoskeleton/intermediate filaments, and protein translation/ribosome biogenesis. The special set of identified, ubiquitinated proteins confirms the thesis that ubiquitination upon oxidative stress is not a random process to degrade the mass of oxidized proteins, but concerns a special group of functional proteins.     

Metabolism-induced oxidative stress is a mediator of glucose toxicity in HT22 neuronal cells

Oxidative stress has been widely considered as a key player in the adverse effects of hyperglycaemia to various tissues, including neuronal cells. This study examined the participation of oxidative stress in injurious effects of high glucose on HT22 cells along with the activity of proteasome, a proteolytic system responsible for degradation of oxidized proteins. Although 10-fold glucose concentration caused non-significant viability changes, a significant reduction of cell proliferation was found. Moreover, the cell morphology was also altered. These changes were followed by an enhancement of intracellular ROS generation, however without any significant boost of the carbonyl group concentration in proteins. Correspondingly, only a slight decline in the 20S proteasome activity was found in high-glucose-treated cells. On the other hand, substances affecting glucose metabolism or antioxidants partially preserved the oxidative stress in high glucose treated cells. In summary, these results highlight the role of metabolic oxidative stress in hyperglycaemia affecting neurons.     

Hyperhomocysteinemia-induced oxidative stress differentially alters proteasome composition and activities in heart and aorta

Background and aims

Hyperhomocysteinemia (HHcy) is associated with cardiovascular diseases and is thought to induce endogenous oxidative stress and causes many cellular damages. Proteasome that degrades oxidized and ubiquitinated proteins can regulate the cellular response to oxidative stress. We aimed to investigate whether hyperhomocysteinemia induces oxidative stress and alters proteasome function and composition in heart and aorta tissues of rat.

Methods and results

To create hyperhomocysteinemia, male Wistar rats (Pasteur Institute-Algiers) were received daily intraperitoneal injections of dl-homocysteine (0.6–1.2 μM/g body weight) for 3 weeks. Biomarkers of oxidative stress (malondialdehyde (MDA), protein carbonyl (PC), superoxide dismutase (SOD) and catalase (CAT)) were first measured by biochemical methods and tissue damages by histological sections. Proteasome activities were quantitated using fluorogenic synthetic peptides; ubiquitinated proteins and proteasome subunits expression were then evaluated by SDS PAGE and Western blot analysis. We showed increased MDA and PC but decreased SOD and CAT levels both in plasma, heart and aorta accompanied by histological changes. A significant decrease of proteasome activities was observed in heart, whereas proteasome activity was not affected in aorta. However proteasome composition was altered in both tissues, as the accumulation of ubiquitinated proteins.

Conclusion

Data demonstrated an alteration of the ubiquitin–proteasome system in hyperhomocysteinemia as a result of accumulating oxidized and ubiquitinated proteins in response to oxidative stress. Further studies must be conducted to better understanding mechanisms responsible of proteasome alterations in hyperhomocysteinemia.     

Analysis of proteasomal proteolysis during the in vitro metacyclogenesis of Trypanosoma cruzi

Proteasomes are large protein complexes, whose main function is to degrade unnecessary or damaged proteins. The inhibition of proteasome activity in Trypanosoma cruzi blocks parasite replication and cellular differentiation. We demonstrate that proteasome-dependent proteolysis occurs during the cellular differentiation of T. cruzi from replicative non-infectious epimastigotes to non-replicative and infectious trypomastigotes (metacyclogenesis). No peaks of ubiquitin-mediated degradation were observed and the profile of ubiquitinated conjugates was similar at all stages of differentiation. However, an analysis of carbonylated proteins showed significant variation in oxidized protein levels at the various stages of differentiation and the proteasome inhibition also increased oxidized protein levels. Our data suggest that different proteasome complexes coexist during metacyclogenesis. The 20S proteasome may be free or linked to regulatory particles (PA700, PA26 and PA200), at specific cell sites and the coordinated action of these complexes would make it possible for proteolysis of ubiquitin-tagged proteins and oxidized proteins, to coexist in the cell.     

Age-related differences in oxidative protein-damage in young and senescent fibroblasts

Aging is accompanied by an accumulation of oxidized proteins and cross-linked modified protein material. The intracellular formation and accumulation of highly oxidized and cross-linked proteins, the so-called lipofuscin, is a typical sign of senescence. However, little is known whether the lipofuscin accumulation during aging is related to environmental conditions, as oxidative stress, and whether the accumulation of oxidized proteins and lipofuscin is preferentially taking place in the cytosol or the nucleus and finally, what is the role of lysosomes in this process.Therefore, we investigated human skin fibroblasts in an early stage of proliferation (“young cells”) and in a late stage (“senescent cells”). Such cells were compared for the amount of protein carbonyls and lipofuscin and their distribution within the cytosol and the nucleus. Furthermore, cells were exposed to single and repeated doses of hydrogen peroxide and paraquat, measuring the same set of parameters. In addition to that the role of the proteasome to degrade oxidized proteins in young and senescent cells was tested. Furthermore, detailed microscopic analysis was performed testing the intracellular distribution of lipofuscin. The results clearly demonstrated that repeated/chronic oxidative stress induces a senescence-like phenotype of the distribution of oxidized proteins as well as of lipofuscin. It could be demonstrated that most of the lipofuscin is located in lysosomes and that senescent cells contain less lysosomes not lipofuscin-laden in comparison to young cells.     

Protein turnover by the proteasome in aging and disease

A significant body of evidence supports a key role for free radicals in causing cumulative damage to cellular macromolecules, thereby contributing to senescence/aging, and a number of age-related disorders. Proteins are recognized as major targets for oxidative damage (in addition to DNA and lipids) and the accumulation of oxidized proteins has been reported for many experimental aging models, as measured by several markers for protein oxidation. In young and healthy individuals, moderately oxidized soluble cell proteins are selectively and rapidly degraded by the proteasome. However, severely oxidized, cross-linked proteins are poor substrates for degradation and actually inhibit the proteasome. Considerable evidence now indicates that proteasome activity declines during aging, as the protease is progressively inhibited by binding to ever increasing levels of oxidized and cross-linked protein aggregates. Cellular aging probably involves both an increase in the generation of reactive oxygen species and a progressive decline in proteasome activity, resulting in the progressive accumulation of oxidatively damaged protein aggregates that eventually contribute to cellular dysfunction and senescence.