Chromatin repair after oxidative stress: Role of PARP-mediated proteasome activation

Oxidative stress is an inevitable process in the nucleus, especially in antitumor chemotherapy, and adaptation by defense mechanisms seems to be one element in the development of long-term resistance to many chemotherapeutic drugs. In this study, a potential chromatin repair mechanism during oxidative stress was investigated in HT22 cells. The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified histone proteins in the nucleus. Poly(ADP-ribosyl)ation reactions also play an important role in DNA repair as a consequence of oxidative damage and single-strand breaks. Such a reaction may occur also with the 20S proteasome—with a known increase in enzymatic activity—and also with histones—reducing their proteolytic susceptibility as shown for the first time here. After hydrogen peroxide treatment of HT22 cells, degradation of the model peptide substrate suc-LLVY-MCA and degradation of oxidized histones by nuclear proteasome increased. During the removal of protein carbonyls, single-strand breaks and 8-hydroxy-2′-deoxyguanosine, proteasome, and poly(ADP-ribose) polymerase-1 enzymes were shown to play tightly interacting roles. Our results following the repair of oxidative damage show the proteolytic activation of proteasome concerning poly(ADP-ribosyl)ation together with a decline in poly(ADP-ribosyl)ation of oxidized histones, leading to a selective recognition of oxidatively modified histones.     

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.     

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.     

Limited degradation of oxidized calmodulin by proteasome: formation of peptides

Oxidized proteins are recognized and degraded preferentially by the proteasome. This is true for numerous proteins including calmodulin (CaM). The degradation of CaM was investigated in a human fibroblast cell line under conditions of oxidative stress. Low molecular CaM fragments or peptides were found under such conditions. In in vitro experiments it was investigated whether this CaM breakdown product formation is induced by protein oxidation or is due to a limited proteolysis-derived degradation by the 20S proteasome. Native unoxidized CaM was not degraded by 20S proteasome, oxidized CaM was degraded in a time- and H₂O₂ concentration-dependent manner. Peptides of similar molecular weight were detected in isolated calmodulin as in oxidatively stressed fibroblasts. The peptides were identified using isolated calmodulin. Therefore, in oxidatively stressed fibroblasts and in vitro CaM is forming oxidation-driven fragments and proteasomal cleavage peptides of approximately 30 amino acids which undergo a slow or no degradation.     

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.     

Intracellular distribution of oxidized proteins and proteasome in HT22 cells during oxidative stress

The production of free radicals and the resulting oxidative damage of cellular structures are always connected with the formation of oxidized proteins. The 20S proteasome is responsible for recognition and degradation of oxidatively damaged proteins. No detailed studies on the intracellular distribution of oxidized proteins during oxidative stress and on the distribution of the proteasome have been performed until now. Therefore, we used immunocytochemical methods to measure protein carbonyls, a form of protein oxidation products, and proteasome distribution within cells. Both immunocytochemical methods of measurement are semiquantitative and the load of oxidized proteins is increased after various oxidative stresses explored, with the highest increase in the perinuclear region of the cell. Distribution of the proteasome and the total protein content revealed the highest concentration of both in the nucleus. No redistribution of the proteasome during oxidative stress occurs. The normalized ratio of protein carbonyls to protein content was formed, indicating the highest concentration of oxidized proteins in the cytosolic region near the cell membrane. By forming the protein oxidation-to-proteasome ratio it was concluded that the highest load of oxidized proteins to the proteasome takes place in the cytosol, independent of the oxidant explored.     

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.     

Ubiquitin conjugation is not required for the degradation of oxidized proteins by proteasome

Oxidatively modified proteins that accumulate in aging and many diseases can form large aggregates because of covalent cross-linking or increased surface hydrophobicity. Unless repaired or removed from cells, these oxidized proteins are often toxic, and threaten cell viability. Most oxidatively damaged proteins appear to undergo selective proteolysis, primarily by the proteasome. Previous work from our laboratory has shown that purified 20 S proteasome degrades oxidized proteins without ATP or ubiquitin in vitro, but there have been no studies to test this mechanism in vivo. The aim of this study was to determine whether ubiquitin conjugation is necessary for the degradation of oxidized proteins in intact cells. We now show that cells with compromised ubiquitin-conjugating activity still preferentially degrade oxidized intracellular proteins, at near normal rates, and this degradation is still inhibited by proteasome inhibitors. We also show that progressive oxidation of proteins such as lysozyme and ferritin does not increase their ubiquitinylation, yet the oxidized forms of both proteins are preferentially degraded by proteasome. Furthermore, rates of oxidized protein degradation by cell lysates are not significantly altered by addition of ATP, excluding the possibility of an energy requirement for this pathway. Contrary to earlier popular belief that most proteasomal degradation is conducted by the 26 S proteasome with ubiquitinylated substrates, our work suggests that oxidized proteins are degraded without ubiquitin conjugation (or ATP hydrolysis) possibly by the 20 S proteasome, or the immunoproteasome, or both.     

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.     

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.     

The proteasomal system and HNE-modified proteins

Metabolic processes and environmental conditions cause the constant formation of oxidizing species over the lifetime of cells and organisms. This leads to a continuous oxidation of intracellular components, including lipids, DNA and proteins. During the extensively studied process of lipid peroxidation, several reactive low-molecular weight products are formed, including reactive aldehydes as 4-hydroxynonenal (HNE). These aldehydic lipid peroxidation products in turn are able to modify proteins. The degradation of oxidized and oxidatively modified proteins is an essential part of the oxidant defenses of cells. The major proteolytic system responsible for the removal of oxidized cytosolic and nuclear proteins is the proteasomal system. The proteasomal system by itself is a multicomponent system responsible for the degradation of the majority of intracellular proteins. It has been shown that some, mildly cross-linked, HNE-modified proteins are preferentially degraded by the proteasome, but extensive modification with this cross-linking aldehyde leads to the formation of protein aggregates, that can actually inhibit the proteasome. This review summarizes our knowledge of the interactions between lipid peroxidation products, proteins, and the proteasomal system.     

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.     

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.     

Nuclear proteasome activation and degradation of carboxymethylated histones in human keratinocytes following glyoxal treatment

Nuclear DNA damage has been studied in detail, but much less is known concerning the occurrence and fate of nuclear protein damage. Glycoxidation, protein damage that results from a combination of protein glycation and oxidation, leads to the formation of protein-advanced glycation end products (AGE) of which N(epsilon)-carboxymethyllysine (CML) is a major AGE. We have used glyoxal, a product of environmental exposures that readily leads to the formation of CML, to study nuclear protein glycoxidation in HaCaT human keratinocytes. Glyoxal treatment that did not affect cell viability but inhibited cell proliferation in a dose-dependent manner that led to accumulation of CML-modified histones. Modified histones were slowly degraded but persisted for more than 3 days following treatment. Preincubation of cells with a proteasome inhibitor following glyoxal treatment led to an increase in CML-modified histones. While glyoxal treatment resulted in a slight decrease in total cellular proteasome activity, a dose dependent increase of up to 4-fold in nuclear proteasome activity was observed. The increase in nuclear proteasome activity was due to both increased nuclear proteasome protein content and increased activity, neither of which were affected by cyclohexamide. The increase also was unaffected by inhibitors of poly(ADP-ribose) polymerases, which have been previously implicated in nuclear proteasome activation by oxidizing agents. Accumulation of CML-modified histones over time may lead to epigenetic changes that contribute to various pathologies including aging and cancer, and upregulation of nuclear proteasome activity under conditions of glyoxidative stress may function to limit such damage.     

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.     

The immunoproteasome, the 20S proteasome and the PA28αβ proteasome regulator are oxidative-stress-adaptive proteolytic complexes

Oxidized cytoplasmic and nuclear proteins are normally degraded by the proteasome, but accumulate with age and disease. We demonstrate the importance of various forms of the proteasome during transient (reversible) adaptation (hormesis), to oxidative stress in murine embryonic fibroblasts. Adaptation was achieved by 'pre-treatment' with very low concentrations of H2O2, and tested by measuring inducible resistance to a subsequent much higher 'challenge' dose of H2O2. Following an initial direct physical activation of pre-existing proteasomes, the 20S proteasome, immunoproteasome and PA28αβ regulator all exhibited substantially increased de novo synthesis during adaptation over 24?h. Cellular capacity to degrade oxidatively damaged proteins increased with 20S proteasome, immunoproteasome and PA28αβ synthesis, and was mostly blocked by the 20S proteasome, immunoproteasome and PA28 siRNA (short interfering RNA) knockdown treatments. Additionally, PA28αβ-knockout mutants achieved only half of the H2O2-induced adaptive increase in proteolytic capacity of wild-type controls. Direct comparison of purified 20S proteasome and immunoproteasome demonstrated that the immunoproteasome can selectively degrade oxidized proteins. Cell proliferation and DNA replication both decreased, and oxidized proteins accumulated, during high H2O2 challenge, but prior H2O2 adaptation was protective. Importantly, siRNA knockdown of the 20S proteasome, immunoproteasome or PA28αβ regulator blocked 50-100% of these adaptive increases in cell division and DNA replication, and immunoproteasome knockdown largely abolished protection against protein oxidation.     

The nuclear proteasome and the degradation of oxidatively damaged proteins

Summary. The accumulation of oxidized proteins is known to be linked to some severe neurodegenerative diseases like Alzheimer’s, Parkinson’s and Huntington’s disease. Furthermore, the aging process is also accompanied by an ongoing aggregation of misfolded and damaged proteins. Therefore, mammalian cells have developed potent degradation systems, which selectively degrade damaged and misfolded proteins. The proteasomal system is largely responsible for the removal of oxidatively damaged proteins form the cellular environment. Not only cytosolic proteins are prone to oxidative stress, also nuclear proteins are readily oxidized. The nuclear proteasomal system is responsible for the degradation of these proteins. This review is focused on the specific degradation of oxidized nuclear proteins, the role of the proteasome in this process and the regulation of the nuclear proteasomal system under oxidative conditions.     

Proteasome inactivation upon aging and on oxidation-effect of HSP 90

Increases of oxidatively modified protein in the cell have been associated with the aging process. Such an accumulation of damaged protein may be the result of increase in the rate of protein oxidation and/or decrease in the rate of degradation of oxidized protein. The multicatalytic proteinase or proteasome is known to be the major proteolytic system involved in the removal of oxidized protein. We have reported that, after isolation of the 20S proteasome from the liver of young and old male Fischer 344 rat, out of the three peptidase activities (chymotrypsin-like, trypsin-like and peptidyl-glutamyl peptide hydrolase) we assayed with fluorogenic peptides, the peptidyl-glutamyl peptide hydrolase activity was declining with age to a value approximately 50% of that observed for protease purified from young rats. The proteasome was subjected to metal catalyzed oxidation to determine the susceptibility of the different peptidase activities to oxidative inactivation. Both trypsin-like and peptidyl-glutamyl peptide hydrolase activities were found sensitive to oxidation. Treatment of the proteasome with 4-hydroxy-2-nonenal, a major lipid peroxidation product, was also found to inactivate the trypsin-like activity. However, the trypsin-like activity was protected from inactivation by metal catalyzed oxidation in proteasome preparations contaminated with HSP 90, a protein that often copurifies with the proteasome. Upon addition of HSP 90 to pure 20S active proteasome, the trypsin-like activity was protected from inactivation by metal catalyzed oxidation and from inactivation by treatment with 4-hydroxy-2-nonenal. These results suggest a possible intervention of HSP 90 in response to oxidative stress in preventing the inactivation of the proteasome by oxidative damage. Abbreviations: AAF-amc – Ala-Ala-Phe-7-amido-4-methylcoumarin; LSTR-amc – N-t-Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin; LLE-na – Leu-Leu-Glu-b-naphthylamide; HSP 90: heat shock protein 90, MCP – multicatalytic proteinase or 20S proteasome.     

Proteasome activation by poly-ADP-ribose-polymerase in human myelomonocytic cells after oxidative stress

Cytotoxic action of a variety of antitumor drugs generate oxidatively modified proteins that are predominantly metabolized via the proteasome. In the present study, a differentiation-retrodifferentiation cell system was exposed to oxidative stress by hydrogen peroxide treatment. Thus, the activity of the nuclear proteasome in proliferating human U937 leukemic cells increased by 2.5-fold after hydrogen peroxide treatment. In contrast, growth-arrested differentiated U937 cells demonstrated 40% less constitutive proteasomal activity, which was not inducible after hydrogen peroxide exposure. After a retrodifferentiation process, however, in which differentiated U937 cells resume autonomous growth again, the proteasomal activity was indistinguishable from that in U937 control cells, both constitutively and after induction of oxidative stress. Moreover, cells of TUR, a differentiation-resistant U937 subclone, expressed an elevated constitutive proteasomal activity that increased by 2.5-fold after oxidative stress. Immunoblot analysis revealed that these differences in proteasomal activities did not correlate with proteasome protein expression but with protein levels of the nuclear enzyme poly-ADP-ribose-polymerase (PARP). Further studies using specific PARP inhibitors revealed that the noninducible proteasome activity in differentiated U937 cells was PARP independent, whereas the increased activity level in oxidatively stressed TUR cells was downregulated upon PARP inhibition. Immunoprecipitation experiments demonstrated a protein-protein interaction of the functional active PARP with the proteasome in correlation with the proteasome activity. Similar results were obtained by analyzing protein carbonyls after oxidative stress. Taken together, these data suggest that proliferating, rather than growth-arrested, cells metabolize oxidatively damaged nuclear proteins via the proteasome by expressing high levels of PARP.     

Degradation of oxidized extracellular proteins by microglia

In living organisms a permanent oxidation of protein oxidation occurs. The degradation of intracellular oxidized proteins is intensively studied, but knowledge about the fate of oxidatively modified extracellular proteins is still limited. We studied the fate of exogenously added oxidized proteins in microglial cells. Both primary microglial cells and RAW cells are able to remove added oxidized laminin and myelin basic protein from the extracellular environment. Moderately oxidized proteins are degraded most efficiently, whereas strongly oxidized proteins are taken up by the microglial cells without an efficient degradation. Activation of microglial cells enhances the selective recognition and degradation of moderately oxidized protein substrates by proteases. Inhibitor studies also revealed an involvement of the lysosomal and the proteasomal system in the degradation of extracellular proteins. These studies let us conclude that microglial cells are able to remove oxidized proteins from the extracellular environment in the brain.