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Age-Related Changes in Protein Oxidation and Proteolysis in Mammalian Cells

^a Clinics of Physical Medicine and Rehabilitation, Medical Faculty (Charité), Berlin, Germany
^b Ethel Percy Andrus Gerontology Center, Division of Molecular Biology, the University of Southern California, Los Angeles

Kelvin Davies, Ethel Percy Andrus Gerontology Center, University of Southern California, 3715 McClintock Avenue, Room 306, Los Angeles, CA 90089-0191 E-mail: kelvin{at}usc.edu.

Decision Editor: John Faulkner, PhD

	Abstract

Top Abstract Protein Oxidation Products Protein Degradation After... Aging Models of Relevance... Markers of Protein Oxidation... Protein Turnover During Aging... Prevention of Accumulation of... Protein Oxidation and Aging:... References

 
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.

AEROBIC organisms are continuously exposed to free radicals and other reactive oxygen and nitrogen species. To deal with this constant threat, aerobic organisms have developed defense mechanisms for the detoxification of reactive (oxidizing) species, and the repair of oxidant-induced damage. Preserving cell function and viability during oxidative stress depends on both antioxidant capacity and the efficiency of damage removal and repair systems. Paradoxically, though oxygen is required for cellular metabolism, oxidative stress is an inevitable consequence of aerobic life.

In 1956, Denam Harman proposed the "free radical theory of aging," which suggested that oxygen radicals are one of the major factors responsible for the aging of cells ⁽¹⁾. With an increasing acceptance of the role of reactive oxygen species in the pathogenesis of many diseases, the free radical theory of aging has matured further ^(2)(3)(4). It is now well established that protein oxidation is a significant consequence of oxidative stress, in addition to free-radical-mediated damage of DNA and lipids. Oxidative modification to proteins (by reactive oxygen and nitrogen species) is often accompanied by unfolding of the protein and loss of enzymatic function. If such protein oxidation products are not removed rapidly, they tend to accumulate in cells and may contribute to the formation of large, cross-linked aggregates, which can threaten cell function and viability. Cells have therefore developed efficient enzymatic systems for the removal of oxidatively damaged proteins. Such rapid removal of oxidized proteins by degradation constitutes an important antioxidant defense strategy.

	Protein Oxidation Products

Top Abstract Protein Oxidation Products Protein Degradation After... Aging Models of Relevance... Markers of Protein Oxidation... Protein Turnover During Aging... Prevention of Accumulation of... Protein Oxidation and Aging:... References

 
The oxidation of free amino acids as well as the oxidation of peptides and proteins has been studied by many laboratories ^{(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)}. The degree of protein damage caused by a given oxidative stress depends on many factors, including the nature and relative location of the oxidant or free radical source, the proximity of the radical–oxidant to a protein target, and the nature and concentrations of available antioxidant enzymes and compounds. A number of derivatized amino acid side chains have been described in proteins ^{(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)}. Some of the most commonly measured protein oxidation products are carbonyl groups. Carbonyl groups have the advantage of being abundant, but they have the disadvantage of being nonspecific oxidation markers. Some modifications include the oxidation of leucine resulting in the formation of various hydroxyleucines, tryptophan oxidation to form N-formylkynurenine, histidine oxidation to form aspartate or asparagine, tyrosine oxidation to form 3,4-dihydroxyphenylalanine (DOPA), and methionine oxidation to form methionine sulfoxide ⁽⁶⁾. In addition to the modification of amino acid side chains, oxidation reactions can also mediate fragmentation of polypeptide chains, and both intramolecular and intermolecular cross-linking of peptides and proteins ^{(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)}.

Some free radical reactions result in the formation of protein aggregates ^(5)(6)(8), many of which are formed largely as a result of hydrophobic and electrostatic interactions ^{(6)(9)(10)(11)(12)(13)(14)}. In addition, a number of covalent cross-links are involved in protein aggregation. One of the most thoroughly investigated cross-links is the 2,2'-biphenyl cross-link (called bityrosine or dityrosine) formed by the reaction of two tyrosyl radicals ^{(5)(6)(7)(15)(16)(17)}. Dityrosine is a very specific, but not a particularly abundant, protein oxidation product. In contrast, the formation of S-S bonds as a result of the oxidation of sulfhydryls is quite widespread. In addition to the cross-links directly involving amino acids, several "natural" cross-linking reagents are formed as a result of oxidative stress. A number of lipid peroxidation products act as protein cross-linkers. For the most abundant lipid peroxidation products—malondialdehyde and 4-hydroxynonenal—protein cross-linking effects have been demonstrated ^(9)(18)(19). Another group of potential protein cross-linking compounds are carbohydrates or oxidized carbohydrates, although these compounds have not been investigated as thoroughly. It is believed that the cross-linked proteins ultimately form insoluble, fluorescent materials that accumulate within cells (Fig. 1). This material is referred to as lipofuscin (if it becomes encapsulated by lysosomes), ceroid (in the cytoplasm), or AGE (advanced glycation end products) pigment-like fluorophore by various authors, indicating the involvement of carbohydrates in final fluorophore formation ^(20)(21). Several investigators believe that all these pigments have the same principal origin ⁽²⁰⁾, although there may be tissue-specific, age-related, and disease-specific differences ^{(20)(21)(22)(23)(24)(25)(26)}. Many groups have observed that lipofuscin does indeed accumulate in postmitotic cells with age ^{(27)(28)(29)(30)}. The involvement of free radicals was hypothesized as one of the initial steps in the formation of fluorescent oxidized–cross-linked aggregates ^{(28)(31)(32)(33)}, with further emphasis on the role of catalytic iron, which may be released from autophagocytosed mitochondria ^(27)(32). Lipid peroxidation products are also evidently involved in the cross-linking reactions of undegraded material ^(20)(21)(34). Because large amounts of the fluorescent oxidized–cross-linked aggregates are found in lysosomes, there has been considerable speculation about dysfunction of the lysosomal proteases ^{(27)(29)(30)(35)(36)}.

View larger version (129K): [in this window] [in a new window]   Figure 1. Accumulation of fluorescent material in fibroblasts. An example of young and old BJ fibroblasts is shown; they were cultivated in Dulbecco–Vogt modified Eagle's medium supplemented with 10% fetal calf serum under normoxia (air plus 5% CO2), and subcultivated at confluency by using a seeding density of 0.3 x 104 cells/cm2. The medium was changed once a week. A, young fibroblasts after 42 population doublings. B, postmitotic cells (this cell line reaches the postmitotic stage after 72 population doublings) aged for 20 weeks under hyperoxic conditions (40% oxygen, 5% carbon dioxide, 55% nitrogen). In both cases the cells were fixed and stained with the nuclear stain DAPI (4',6-diamidino-2 phenylindole), and they were analyzed on a fluorescence microscope. The blue fluorescence demonstrates the nuclei of cells, whereas the yellow-brown pigment is the cell's autofluorescence caused by an accumulation of oxidized/cross-linked material. This picture was taken with the support of Dr. T. von Zglinicki (Institute of Pathology, Humboldt-University Berlin), who supplied the BJ fibroblasts.

	Protein Degradation After Oxidative Stress

Top Abstract Protein Oxidation Products Protein Degradation After... Aging Models of Relevance... Markers of Protein Oxidation... Protein Turnover During Aging... Prevention of Accumulation of... Protein Oxidation and Aging:... References

View larger version (23K): [in this window] [in a new window]   Figure 2. Regulation of the cytosolic 20S proteasome. The 20S proteasome is the catalytic core of the proteasome. This particle consists of two sets of 14 different subunits, which are located in four rings of seven subunits each. The two outer rings are made up of -type subunits and may serve as binding sites for the 19S (and 11S) regulators; the two inner rings are made up of ß-type subunits and contain catalytically active residues. The 19S regulator (also called PA 700) can bind to each end of the 20S proteasome to form the larger 26S proteasome, which is responsible for the adensosine triphosphate-dependent degradation of ubiquitinylated substrates. An alternative form of the proteasome, commonly referred to as the immunoproteasome, comprises the 20S core bound on either side by the 11S (or PA 28) regulator. Although this structure (11S-20S-11S) is not as thoroughly investigated, it is believed to play a role in antigen presentation and has high peptidase activity. Hybrid complex formation (11S–20S–19S) has also been demonstrated, though its functional significance remains unclear. Proteasome activity can also be modulated by phosphorylation of certain subunits or by -interferon-induced subunit exchange.

Although degradation of a protein substrate by the proteasome initially increases with increasing oxidant exposure of the protein, strongly oxidized proteins tend to aggregate and form covalent cross-links. Such protein aggregates are, in fact, poor proteasome substrates ^{(9)(18)(19)(42)(46)(47)(48)(49)(50)}. To prevent the formation of such protein aggregates, cells tend to degrade moderately oxidized proteins rapidly. We have demonstrated an enhanced protein turnover after oxidative stress in various cell lines ^(37)(38)(51). This increased protein turnover is accompanied by a decline in the cellular content of oxidized proteins as demonstrated in MRC-5 fibroblasts ⁽⁵¹⁾. The ability of cells to preferentially degrade oxidized proteins is lost in "proteasome-deficient" cells, as demonstrated by using antisense oligonucleotides directed against one of the essential proteasome subunits ^(37)(38)(42). The proteasome evidently plays a significant role in the elimination of oxidized proteins. Not much is known about the role of other proteases in the degradation of oxidized soluble proteins, outside of the lysosomal compartment.

	Aging Models of Relevance for Investigating the Turnover of Oxidized Proteins

Top Abstract Protein Oxidation Products Protein Degradation After... Aging Models of Relevance... Markers of Protein Oxidation... Protein Turnover During Aging... Prevention of Accumulation of... Protein Oxidation and Aging:... References

 
It is difficult to study such a complex process as aging in humans. The availability of human material is often limited, and an easily accessible postmortem tissue or organ may not always be appropriate to answer a specific question. Additionally, because of variability between individuals, it is difficult to conclude whether the changes are primarily a result of aging or are a result of other parameters such as environment, disease, and living conditions. Overall, it is difficult and expensive to follow protein oxidation and proteolysis in different organs during the entire life span of most complex organisms. A number of simpler models have been developed instead to measure parameters of protein turnover and oxidation associated with aging. The preferred animal models are organisms with short life spans, such as the fruit fly, Drosophila melanogaster ⁽⁵²⁾, and the nematode, Caenorhabditis elegans ⁽⁵³⁾. Nevertheless, studies have been performed in rodents ^(54)(55), and limited investigations on aging have been initiated in monkeys ⁽⁵⁶⁾, to enable better application of these data to humans.

Another widely used model for studies on aging is the tissue culture model. Cells in culture lack interaction with other tissues, hormone stimulation, or regulation, and therefore they do not "age" in a normal environment. Nevertheless, they provide a useful working model for the investigation of biochemical changes in metabolism. In this regard, a number of models have been developed using different cell types and tissue culture protocols ^{(29)(30)(50)(57)(58)(59)}. In vitro aging of cells entails growing primary, nonimmortalized cells in culture and allowing them to age with increasing population doublings, whereas in vivo aging allows the cells to age within the organism before isolating them to study age-related changes. Some protocols involve a combination of both techniques, allowing in vivo aging of cells followed by their investigation under in vitro conditions, that is, isolation of cells from young and old animals or humans ^(60)(61). However, this experimental model has its own limitations, as it is possible that very old cells are more susceptible to disruption during the isolation procedure. Additionally, because of changes in connective tissue, the identity of fractions isolated from young and old cells can sometimes be questionable.

Thus, as mentioned above, there are several models to study aging, many of which have been appropriately used to study the course of protein oxidation and proteolysis during aging. Although the use of different systems makes it more difficult to compare the results, it also reveals some of the common principles underlying the mechanism of aging in general.

	Markers of Protein Oxidation During Aging

Top Abstract Protein Oxidation Products Protein Degradation After... Aging Models of Relevance... Markers of Protein Oxidation... Protein Turnover During Aging... Prevention of Accumulation of... Protein Oxidation and Aging:... References

 
There is a large body of literature documenting studies on protein oxidation during aging in different models ^{(29)(30)(48)(50)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76)(77)(78)(79)(80)(81)(82)(83)(84)(85)(86)(87)(88)(89)(90)(91)(92)(93)(94)(95)(96)(97)(98)(99)(100)(101)(102)(103)(104)(105)(106)(107)(108)(109)(110)(111)(112)(113)}. A majority of these studies used protein-bound carbonyls as a marker for protein oxidation ^{(48)(52)(54)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76)(77)(78)(79)(80)}. Although this method has been seriously questioned ⁽⁸³⁾, the criticism has also been strongly rebutted ⁽⁷¹⁾. Other methods included detecting the products of specific amino acid modifications, such as dityrosine ^{(7)(15)(16)(17)}, O-tyrosine or nitrotyrosine ^{(84)(85)(86)(87)(88)}, 5-hydroxyl-2-amino valeric acid ⁽⁸⁹⁾, or methionine sulfoxide ⁽⁸⁶⁾, to name a few. Changes in overall protein thiol content are also considered a good indication of the age-related changes in protein structure and oxidant status ⁽⁹⁰⁾. Alterations in protein structure, as seen by increased surface hydrophobicity and aggregation, have also been used as markers of an age-related increase in protein oxidation ^{(20)(28)(48)(91)}.

Several studies using animal models have verified that cellular protein carbonyl content increases with age, as reported for the brain of gerbils ^(62)(71), humans ⁽⁶³⁾, rats ^(70)(73)(80), and mice ^(69)(75)(76). Among the different tissues examined, an increase in the amount of oxidized proteins has been reported for the liver, lens, lymphocytes, heart, skin, and skeletal muscle of various species ^{(40)(48)(54)(64)(65)(69)(72)(81)(82)(85)(86)(88)}. An inverse correlation between life expectancy and protein oxidation has been established in a number of rodent and fly species ^(52)(54). Because of its short life span, and simple and inexpensive laboratory requirements, the fruit fly D. melanogaster has been extensively used to investigate age-related changes in protein oxidation ^{(66)(67)(68)(90)}. As an extension of the free radical theory of aging, the central role of mitochondria in the process of generating reactive oxygen intermediates has been emphasized, and age-related changes in mitochondrial protein oxidation have received significant attention. Sohal and coauthors ^(68)(92) have reported an age-related increase in the levels of oxidized proteins in fly muscle mitochondria, whereas Martinez and coworkers ^(74)(77) have made similar observations for mice synaptic mitochondria.

It is interesting to note that although the oxidation of many proteins is a random process, certain proteins do seem to accumulate at higher rates in oxidized forms, as demonstrated for high-molecular-weight mitochondrial proteins ⁽⁹²⁾, mitochondrial aconitase ⁽⁹³⁾, and the cytosolic carbonic anhydrase III ⁽⁹⁴⁾. Whether this accumulation is a result of increased susceptibility or specific targeting of these proteins for oxidation, or a result of diminished degradation of these particular oxidized proteins, remains unclear. Ross and colleagues ^(114)(115) have suggested that protein oxidation in bacteria and yeast is a very specific process, and this view is also supported by the aconitase studies of Sohal and colleagues ⁽⁹³⁾. In contrast, from our earliest studies in bacteria ^(116)(117) and mammalian cells ^(118)(119) it was clear that the extent of both protein damage and proteolysis, in response to oxidants, is too great to be explained by only one or two proteins. This apparent discrepancy is probably only a question of degree of damage and degradation. A careful analysis of data from literally hundreds of experiments reveals that as much as 10–30% of newly synthesized (metabolically radiolabeled) intracellular proteins can undergo proteolytic degradation in both eukaryotic and prokaryotic cell lines following exposure to oxidative stress ^{(9)(15)(16)(29)(30)(37)(38)(46)(47)(49)(50)(51)}. This is simply too much protein turnover to be explained by a process that is specific to one or two proteins. In contrast, some proteins are clearly more sensitive to oxidative damage than others ^(92)(93)(94). If one imagines that most proteins experience 5–10% damage when cells are exposed to oxidative stress whereas particularly sensitive proteins experience 50–60% damage, we can reconcile all the available data in the literature. Techniques such as gel electrophoresis, Western blots, and enzyme activity assays will not reliably detect a 5–10% change, causing some investigators (we suggest) to mistakenly assume that only a few proteins are sensitive to oxidative damage.

A decline in the activity of certain enzymes has also been observed during aging in some experimental models. This is true for glutamine synthetase ^{(62)(63)(64)(79)(80)}, glucose-6-phosphate dehydrogenase ^(64)(67), tyrosine hydroxylase ⁽⁷³⁾, and some enzymes of the antioxidant defense system ^(95)(96)(97). Rikans and coauthors report an age-related increase in ferritin content and discuss its role in preventing the accumulation of free iron ⁽⁹⁸⁾. However, none of these studies clarify whether the observed loss of protein function is a result of changes that occur before or during translation, or is a result of posttranslational modifications, suggesting that we exercise caution in interpreting the conclusions.

Nonetheless, one conclusion emerges from all the above-mentioned studies, performed in different models using different methods of detection, confirming that oxidized proteins do tend to accumulate in most organs and tissues during aging. Whether this is the result of an increased oxidation of these proteins or a decline in the efficiency of the repair and removal system requires further clarification. It is noteworthy that the accumulation of some oxidized proteins appears to be a random process, whereas other proteins appear to undergo selective oxidation and accumulation.

	Protein Turnover During Aging and the Significance of Degrading Oxidized Proteins

Top Abstract Protein Oxidation Products Protein Degradation After... Aging Models of Relevance... Markers of Protein Oxidation... Protein Turnover During Aging... Prevention of Accumulation of... Protein Oxidation and Aging:... References

 
The age-related accumulation of oxidized proteins may result from an increase in protein oxidation or a decline in the degradation of oxidized proteins. We suggest that both processes actually contribute to an age-related accumulation of oxidized proteins. It is therefore important to investigate age-related changes in the activity of intracellular proteolytic systems. We recently investigated oxidized protein turnover during cellular senescence of BJ fibroblasts in proliferating ⁽²⁹⁾ as well as nondividing ^(30)(50) cells. Our comparative studies reveal a good correlation between the accumulation of oxidized or cross-linked proteins and the decline in proteasome activity and overall cellular protein turnover during in vitro senescence. We found a marked decline in all three proteasome activities (trypsin-like, chymotrypsin-like, and peptidyl-glutamyl-hydrolyzing activities). This decline in overall protein turnover was accompanied by a dramatic increase in the levels of oxidized and cross-linked proteins. In both of our models of cellular senescence, the proteasome content and the transcription of proteasome subunits, as determined by immunoblots and Northern blots, was unchanged, despite significantly decreased intracellular proteasome activities. The clear conclusion was that proteasome was being inhibited by the accumulated oxidized or cross-linked protein aggregates, leading to a progressively diminishing cellular ability to degrade oxidized proteins.

Carney and colleagues ⁽⁶²⁾, Stadtman and colleagues ⁽⁶⁴⁾, and Starke-Reed and Oliver ⁽⁹⁹⁾ have shown that the neutral alkaline protease activity in rodent brain and liver declines with age. Although other authors have also reported a decline in the function of the major cytosolic proteolytic system ^(9)(100), results of some investigations found no changes in the activity of the proteasome during aging in houseflies ⁽¹⁰¹⁾ or in rat liver ⁽¹⁰²⁾. Because these experiments were performed under diverse conditions, it is difficult to determine the reason(s) for these different findings. The enzyme activity assays for the different proteases using potentially natural substrates (in this case oxidized proteins; 102) seem to be more reliable than the artificial fluorogenic peptide substrates, which are very common in proteolysis research today. Although the fluorogenic peptide substrates give more insight into the changes of different catalytic properties of the proteasome, some groups have been skeptical about the reliability of these substrates ⁽¹⁰³⁾. By using fluorogenic peptides with different amino acids at the P1 position, investigators can distinguish between the three known catalytic activities of the proteasome. Based on studies using substrates specific for certain catalytic activities, it was shown that only the peptidylglutamyl hydrolyzing activity of the proteasome is severely affected with age ^{(104)(105)(106)}. Although the trypsin-like activity is also oxidation-dependent, it is apparently protected during aging as a result of the interaction of the proteasome with HSP 90 ⁽¹⁰⁵⁾. All these investigations analyzed the activity of the 20S "core" proteasome, because it is believed that this proteasomal form is responsible for the degradation of oxidized proteins. Very little is known about changes in the content or activity of the ATP-stimulated 26S proteasome during aging with respect to the accumulation of oxidized proteins. However, some groups have studied several components of the ubiquitin–proteasome pathway in response to oxidative stress and aging. Taylor and coworkers ^(55)(107) have reported an elevation of ubiquitin-mRNA, high-molecular-weight ubiquitin aggregates, and an increase in the activities of the E1 and E2 ubiquitinylating enzymes in the liver of aged Emory mice. The same group has described a response of the ubiquitin system to oxidative stress in cultured lens cells ^{(40)(108)(109)}. Because the proteasome is known to be responsible for the degradation of the bulk of intracellular proteins ⁽³⁹⁾ and oxidatively modified proteins ^{(9)(37)(38)(48)(49)}, little is known about the oxidation- and age-related changes of lysosomal proteases, which may remain unchanged ^(91)(110). In our studies, lysosomal cathepsin activity declined during proliferative senescence, but it increased markedly during aging of nondividing cells, except in the very "oldest" postmitotic cells, demonstrating a clear dissociation between lysosomal proteolytic capacity and the accumulation of oxidized proteins ^(29)(30)(50).

	Prevention of Accumulation of Oxidized Proteins

Top Abstract Protein Oxidation Products Protein Degradation After... Aging Models of Relevance... Markers of Protein Oxidation... Protein Turnover During Aging... Prevention of Accumulation of... Protein Oxidation and Aging:... References

 
The age-related accumulation of oxidized material, including protein oxidation products, could be prevented either by blocking the damaging action of free radicals with various antioxidants, or better still, by preventing the actual formation of free radicals. The only proposed method to date to accomplish a reduced rate of free radical generation in organisms is the restriction of dietary input of calories and/or proteins.

Among the different antioxidants, the spin-trap N-tert-butyl–phenylnitrone (PBN) has been used as an antioxidant in a number of animal studies. This compound was found to be effective in preventing or reducing the formation of protein carbonyls during aging ^{(62)(64)(71)(111)} as well as in preventing the age-related decline of glutamine synthase activity ^(52)(65). However, Cao and Cutler found no influence of PBN on protein carbonyl content or protease activity ⁽¹⁰³⁾, whereas Stadtman and colleagues ⁽⁶⁴⁾ demonstrated a restoration of protease activity after PBN treatment. PBN also appears to be effective in protecting synaptosomal membrane proteins from oxidation in senescence-accelerated mice ⁽⁷⁹⁾.

More studies have been conducted on animals subjected to caloric restriction. Lower levels of protein carbonyls were found in rat liver, brain, and lymphocytes and in heart, brain, and kidneys of mice under dietary restriction in comparison with controls ^{(65)(69)(72)(76)(80)}. Besides, a reduced accumulation of N-(carboxymethyl)lysine and pentosidine was demonstrated in skin collagen of calorie-restricted rats ⁽¹¹²⁾, and lower levels of dityrosine were measured in the heart and skeletal muscle of calorie-restricted rodents ⁽⁸⁰⁾. Apparently, dietary restriction prevents activation of the ubiquitin-conjugating system in old mice ^(55)(107) as well as the decline of the peptidylglutamyl hydrolyzing activity of the proteasome in rats ⁽¹⁰⁶⁾.

	Protein Oxidation and Aging: Summary

Top Abstract Protein Oxidation Products Protein Degradation After... Aging Models of Relevance... Markers of Protein Oxidation... Protein Turnover During Aging... Prevention of Accumulation of... Protein Oxidation and Aging:... References

 
In summary, the majority of studies on protein oxidation confirm that protein oxidation products, such as protein carbonyls and oxidized–cross-linked material, increase with age (Fig. 3). There is also a strong indication that protein turnover in cells and tissues tends to decline with age, and we now have good evidence demonstrating an actual decline in the activity of the major cytosolic protease—the proteasome ^(29)(30)(50). Not much is known about the changes of proteolytic systems in other cellular compartments, including nuclei and mitochondria, during aging. It may be important to investigate proteolytic activity in a given cellular compartment relative to the amount of substrate located in the same compartment. Little is known about the regulation of the proteasome and the ubiquitin-conjugating machinery during oxidative stress or other stress conditions. Similarly, our understanding of how proteasome is regulated during aging is still in its infancy.

View larger version (30K): [in this window] [in a new window]   Figure 3. Possible changes in cellular antioxidative defenses leading to enhanced protein oxidation in old cells. Whereas proteolytic enzymes can rapidly degrade oxidized or damaged proteins in younger cells, some unfolded, oxidized, and cross-linked proteins accumulate in older cells, forming lipofuscin or lipofuscin-like fluorophores. This figure suggests possible reasons for the accumulation of oxidized proteins in older cells, ranging from an increased generation of free radicals to age-related changes in proteolytic systems.

Besides regulation of the proteasome and protein oxidation status, one has to consider other nonoxidative protein modifications such as glycation, glycoxidation, and deamidation, among others, because these processes may also influence the susceptibility of proteins to proteolysis by various proteases. Furthermore, processes such as glycation and oxidation are probably interactive and mutually exacerbating in vivo.

The malfunction of the proteolytic machinery of cells may also be combined with the increased free radical formation in older cells, or a diminished response of the primary antioxidant defenses, or both. Thus, age-related changes in protein oxidation status appear to be a combination of complex biochemical changes at the level of oxidant production, primary antioxidant defense, and repair of oxidant-induced damage (Fig. 3).

	Acknowledgments

 
T. Grune was supported by Stiftung Verhalten und Umwelt and the Deutsche Forschungsgemeinschaft (SFB 507). K.J.A. Davies was supported by the National Institutes of Health/National Institute of Environmental Health Sciences under Grant ES 03598, and by the National Institutes of Health/National Institute on Aging under Grant AG 16256.

Received January 18, 2001

Accepted April 5, 2001

	References

Top Abstract Protein Oxidation Products Protein Degradation After... Aging Models of Relevance... Markers of Protein Oxidation... Protein Turnover During Aging... Prevention of Accumulation of... Protein Oxidation and Aging:... References

 

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