Full text of DOI:10.1093/gerona/56.11.B459

REVIEW ARTICLE
Copyright 2001 by The Gerontological Society of America
Journal of Gerontology: BIOLOGICAL SCIENCES
2001, Vol. 56A, No. 11, B459–B467
Age-Related Changes in Protein Oxidation and
Proteolysis in Mammalian Cells
Tilman Grune,1,2 Reshma Shringarpure,2 Nicolle Sitte,1 and Kelvin Davies2
1Clinics of Physical Medicine and Rehabilitation, Medical Faculty (Charité), Berlin, Germany.
2Ethel Percy Andrus Gerontology Center, and Division of Molecular Biology, the University of Southern California,
Los Angeles.
Reactive oxygen species generated as by-products of oxidative metabolism, or from environ-
mental 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 dity-
rosine, or by the accumulation of protein-containing pigments such as lipofuscin and ceroid bod-
ies. 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, indi-
cates that proteasome activity does, indeed, decline during aging as the enzyme complex is pro-
gressively 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 be-
gin to accumulate rapidly and contribute to cellular dysfunction and senescence.
EROBIC organisms are continuously exposed to free
radicals and other reactive oxygen and nitrogen spe-
moval of oxidized proteins by degradation constitutes an
important antioxidant defense strategy.
A
cies. 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. Paradoxi-
cally, though oxygen is required for cellular metabolism, ox-
idative stress is an inevitable consequence of aerobic life.
In 1956, Denam Harman proposed the “free radical the-
ory 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 oxy-
gen species in the pathogenesis of many diseases, the free
radical theory of aging has matured further (2–4). It is now
well established that protein oxidation is a significant conse-
quence of oxidative stress, in addition to free-radical-medi-
ated 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 enzy-
matic function. If such protein oxidation products are not re-
moved rapidly, they tend to accumulate in cells and may
contribute to the formation of large, cross-linked aggre-
gates, which can threaten cell function and viability. Cells
have therefore developed efficient enzymatic systems for
the removal of oxidatively damaged proteins. Such rapid re-
PROTEIN OXIDATION PRODUCTS
The oxidation of free amino acids as well as the oxidation
of peptides and proteins has been studied by many laborato-
ries (5–17). The degree of protein damage caused by a given
oxidative stress depends on many factors, including the na-
ture and relative location of the oxidant or free radical
source, the proximity of the radical–oxidant to a protein tar-
get, and the nature and concentrations of available antioxi-
dant enzymes and compounds. A number of derivatized
amino acid side chains have been described in proteins (5–
17). Some of the most commonly measured protein oxida-
tion products are carbonyl groups. Carbonyl groups have
the advantage of being abundant, but they have the disad-
vantage of being nonspecific oxidation markers. Some mod-
ifications include the oxidation of leucine resulting in the
formation of various hydroxyleucines, tryptophan oxidation
to form N-formylkynurenine, histidine oxidation to form as-
partate or asparagine, tyrosine oxidation to form 3,4-dihy-
droxyphenylalanine (DOPA), and methionine oxidation to
form methionine sulfoxide (6). In addition to the modifica-
tion of amino acid side chains, oxidation reactions can also
mediate fragmentation of polypeptide chains, and both in-
tramolecular and intermolecular cross-linking of peptides
and proteins (5–17).
B459

B460
GRUNE ET AL.
Some free radical reactions result in the formation of pro-
compounds have not been investigated as thoroughly. It is
believed that the cross-linked proteins ultimately form in-
soluble, fluorescent materials that accumulate within cells
(Figure 1). This material is referred to as lipofuscin (if it be-
comes encapsulated by lysosomes), ceroid (in the cyto-
plasm), or AGE (advanced glycation end products) pig-
ment-like fluorophore by various authors, indicating the
involvement of carbohydrates in final fluorophore forma-
tion (20,21). Several investigators believe that all these pig-
ments have the same principal origin (20), although there
may be tissue-specific, age-related, and disease-specific dif-
ferences (20–26). Many groups have observed that lipofus-
cin does indeed accumulate in postmitotic cells with age
(27–30). The involvement of free radicals was hypothesized
as one of the initial steps in the formation of fluorescent ox-
idized–cross-linked aggregates (28,31–33), with further em-
phasis on the role of catalytic iron, which may be released
from autophagocytosed mitochondria (27,32). Lipid peroxi-
dation products are also evidently involved in the cross-
tein aggregates (5,6,8), many of which are formed largely as
a result of hydrophobic and electrostatic interactions (6,9–
14). In addition, a number of covalent cross-links are in-
volved in protein aggregation. One of the most thoroughly
investigated cross-links is the 2,2ꢀ-biphenyl cross-link (called
bityrosine or dityrosine) formed by the reaction of two ty-
rosyl radicals (5–7,15–17). 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 oxida-
tion 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 pro-
tein cross-linkers. For the most abundant lipid peroxidation
products—malondialdehyde and 4-hydroxynonenal—pro-
tein cross-linking effects have been demonstrated (9,18,19).
Another group of potential protein cross-linking compounds
are carbohydrates or oxidized carbohydrates, although these
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 con-
fluency by using a seeding density of 0.3 ꢁ 104 cells/cm2. The medium was changed once a week. A, young fibroblasts after 42 population dou-
blings. B, postmitotic cells (this cell line reaches the postmitotic stage after 72 population doublings) aged for 20 weeks under hyperoxic condi-
tions (40% oxygen, 5% carbon dioxide, 55% nitrogen). In both cases the cells were fixed and stained with the nuclear stain DAPI (4ꢀ,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.

AGING, PROTEIN OXIDATION, AND PROTEOLYSIS
B461
linking reactions of undegraded material (20,21,34). Because
large amounts of the fluorescent oxidized–cross-linked ag-
gregates are found in lysosomes, there has been considerable
speculation about dysfunction of the lysosomal proteases
(27,29,30,35,36).
broblasts (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.
PROTEIN DEGRADATION AFTER OXIDATIVE STRESS
Mammalian cells contain major proteolytic systems such
as the lysosomal cathepsins, the cytoskeletal calpains, and
the 20S and 26S proteasomes located within the cytosol, nu-
clei, and endoplasmic reticulum. We have previously dem-
onstrated that the proteasome can recognize and selectively
degrade oxidized proteins (9–16,37,38). The proteasome is
also responsible for the degradation of a vast majority of in-
tracellular soluble proteins (39). Although in vitro experi-
ments have shown that the isolated 20S proteasome or the
catalytic “core” can recognize and degrade oxidatively
modified proteins, it has not previously been clear that the
core proteasome actually performs this function in vivo (9,
39,40). Indeed, many researchers have assumed that only
the 26S proteasome is catalytically active in vivo, and that
the 26S proteasome can only degrade ubiquitinylated pro-
teins. Recent (and ongoing) work with cells incapable of
ubiquitinylating protein substrates for the 26S proteasome
indicates that the 20S proteasome does, indeed, conduct the
recognition and degradation of oxidatively modified pro-
teins in vivo (41,42). It has also become apparent that even
the 26S proteasome can degrade certain protein substrates
without ubiquitin conjugation (42). It now seems quite clear
that the 20S proteasome is mostly responsible for the recog-
nition and degradation of oxidatively modified soluble pro-
teins in the cytoplasm, nucleus, and endoplasmic reticulum,
in a ubiquitin-independent and ATP (adenosine triphosphate)-
independent process. This subject has recently been exten-
sively reviewed (42). The activity of the proteasome in cells
may also be modulated by the action of multiple regulators
(39), phosphorylation status (43), and subunit composition
(44; see Figure 2).
AGING MODELS OF RELEVANCE FOR INVESTIGATING THE
TURNOVER OF OXIDIZED PROTEINS
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. Addi-
tionally, because of variability between individuals, it is dif-
ficult to conclude whether the changes are primarily a result
of aging or are a result of other parameters such as environ-
ment, 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 devel-
oped instead to measure parameters of protein turnover and
oxidation associated with aging. The preferred animal mod-
els are organisms with short life spans, such as the fruit fly,
Drosophila melanogaster (52), and the nematode, Cae-
norhabditis elegans (53). Nevertheless, studies have been
In 1987 we provided evidence that the oxidation of pro-
teins is a denaturing process similar in many ways to heat
denaturation that exposes hydrophobic amino acid residues
from the protein’s interior (5). We further proposed that
these exposed hydrophobic moieties were the actual “oxida-
tive modifications” recognized by proteolytic enzymes that
caused increased proteolytic susceptibility (5). Since that
time, we and others have shown that the increased surface
hydrophobicity of oxidized proteins is indeed the key factor
in the proteolytic recognition of these oxidized substrates
(42,45–49).
Although degradation of a protein substrate by the pro-
teasome 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 (9,18,19,42,46–49,50).
To prevent the formation of such protein aggregates, cells
tend to degrade moderately oxidized proteins rapidly. We
have demonstrated an enhanced protein turnover after oxi-
dative 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 fi-
Figure 2. Regulation of the cytosolic 20S proteasome. The 20S
proteasome is the catalytic core of the proteasome. This particle con-
sists 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
ꢂ-type subunits and may serve as binding sites for the 19S (and 11S)
regulators; the two inner rings are made up of ꢃ-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 triphos-
phate-dependent degradation of ubiquitinylated substrates. An alter-
native form of the proteasome, commonly referred to as the immuno-
proteasome, 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 presen-
tation and has high peptidase activity. Hybrid complex formation
(11S–20S–19S) has also been demonstrated, though its functional sig-
nificance remains unclear. Proteasome activity can also be modulated
by phosphorylation of certain subunits or by ꢄ-interferon-induced
subunit exchange.

B462
GRUNE ET AL.
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.
cause 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 (66–68,90). 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 mitochon-
dria, whereas Martinez and coworkers (74,77) have made
similar observations for mice synaptic mitochondria.
Another widely used model for studies on aging is the tis-
sue culture model. Cells in culture lack interaction with
other tissues, hormone stimulation, or regulation, and there-
fore they do not “age” in a normal environment. Neverthe-
less, they provide a useful working model for the investiga-
tion of biochemical changes in metabolism. In this regard, a
number of models have been developed using different cell
types and tissue culture protocols (29,30,50,57–59). 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, iso-
lation of cells from young and old animals or humans
(60,61). However, this experimental model has its own lim-
itations, as it is possible that very old cells are more suscep-
tible to disruption during the isolation procedure. Addition-
ally, because of changes in connective tissue, the identity of
fractions isolated from young and old cells can sometimes
be questionable.
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 demon-
strated for high-molecular-weight mitochondrial proteins
(92), mitochondrial aconitase (93), and the cytosolic car-
bonic anhydrase III (94). Whether this accumulation is a re-
sult 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 oxida-
tion 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 bac-
teria (116,117) and mammalian cells (118,119) it was clear
that the extent of both protein damage and proteolysis, in re-
sponse 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 re-
veals that as much as 10–30% of newly synthesized (meta-
bolically radiolabeled) intracellular proteins can undergo
proteolytic degradation in both eukaryotic and prokary-
otic cell lines following exposure to oxidative stress (9,15,
16,29,30,37,38,46,47,49–51). This is simply too much pro-
tein 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 (92–94). If
one imagines that most proteins experience 5–10% damage
when cells are exposed to oxidative stress whereas particu-
larly 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 ac-
tivity assays will not reliably detect a 5–10% change, caus-
ing 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 (62–64,79,80), glucose-
6-phosphate dehydrogenase (64,67), tyrosine hydroxylase
(73), and some enzymes of the antioxidant defense system
(95–97). Rikans and coauthors report an age-related in-
crease 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-
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
There is a large body of literature documenting studies on
protein oxidation during aging in different models (29,30,
48,50,52–113). A majority of these studies used protein-
bound carbonyls as a marker for protein oxidation (48,52,
54,60–80). Although this method has been seriously ques-
tioned (83), the criticism has also been strongly rebutted
(71). Other methods included detecting the products of spe-
cific amino acid modifications, such as dityrosine (7,15–17),
O-tyrosine or nitrotyrosine (84–88), 5-hydroxyl-2-amino
valeric acid (89), or methionine sulfoxide (86), to name a
few. Changes in overall protein thiol content are also con-
sidered a good indication of the age-related changes in pro-
tein structure and oxidant status (90). Alterations in protein
structure, as seen by increased surface hydrophobicity and ag-
gregation, 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 re-
ported for the brain of gerbils (62,71), humans (63), rats
(70,73,80), and mice (69,75,76). Among the different tis-
sues examined, an increase in the amount of oxidized pro-
teins 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 be-
tween life expectancy and protein oxidation has been estab-
lished in a number of rodent and fly species (52,54). Be-

AGING, PROTEIN OXIDATION, AND PROTEOLYSIS
B463
mentioned studies, performed in different models using dif-
ferent methods of detection, confirming that oxidized pro-
teins 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 notewor-
thy that the accumulation of some oxidized proteins appears
to be a random process, whereas other proteins appear to un-
dergo selective oxidation and accumulation.
that only the peptidylglutamyl hydrolyzing activity of the
proteasome is severely affected with age (104–106). Al-
though the trypsin-like activity is also oxidation-dependent,
it is apparently protected during aging as a result of the inter-
action of the proteasome with HSP 90 (105). All these inves-
tigations analyzed the activity of the 20S “core” proteasome,
because it is believed that this proteasomal form is responsi-
ble 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–protea-
some pathway in response to oxidative stress and aging.
Taylor and coworkers (55,107) have reported an elevation of
ubiquitin-mRNA, high-molecular-weight ubiquitin aggre-
gates, and an increase in the activities of the E1 and E2 ubiq-
uitinylating 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). Be-
cause the proteasome is known to be responsible for the deg-
radation of the bulk of intracellular proteins (39) and oxida-
tively 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 prolif-
erative 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 pro-
teins (29,30,50).
PROTEIN TURNOVER DURING AGING AND THE
SIGNIFICANCE OF DEGRADING OXIDIZED PROTEINS
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 pro-
teolytic systems. We recently investigated oxidized protein
turnover during cellular senescence of BJ fibroblasts in pro-
liferating (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, chy-
motrypsin-like, and peptidyl-glutamyl-hydrolyzing activi-
ties). This decline in overall protein turnover was accom-
panied by a dramatic increase in the levels of oxidized and
cross-linked proteins. In both of our models of cellular se-
nescence, the proteasome content and the transcription of
proteasome subunits, as determined by immunoblots and
Northern blots, was unchanged, despite significantly de-
creased intracellular proteasome activities. The clear con-
clusion was that proteasome was being inhibited by the
accumulated oxidized or cross-linked protein aggregates,
leading to a progressively diminishing cellular ability to de-
grade 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 de-
clines with age. Although other authors have also reported a
decline in the function of the major cytosolic proteolytic sys-
tem (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 deter-
mine 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, inves-
tigators can distinguish between the three known catalytic
activities of the proteasome. Based on studies using sub-
strates specific for certain catalytic activities, it was shown
PREVENTION OF ACCUMULATION OF OXIDIZED PROTEINS
The age-related accumulation of oxidized material, in-
cluding protein oxidation products, could be prevented ei-
ther 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 pro-
tein carbonyls during aging (62,64,71,111) as well as in pre-
venting the age-related decline of glutamine synthase activ-
ity (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 res-
toration of protease activity after PBN treatment. PBN also
appears to be effective in protecting synaptosomal mem-
brane 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 com-
parison 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

B464
GRUNE ET AL.
(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).
modifications such as glycation, glycoxidation, and deami-
dation, among others, because these processes may also in-
fluence the susceptibility of proteins to proteolysis by vari-
ous proteases. Furthermore, processes such as glycation and
oxidation are probably interactive and mutually exacerbat-
ing in vivo.
The malfunction of the proteolytic machinery of cells
may also be combined with the increased free radical for-
mation 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 combina-
tion of complex biochemical changes at the level of oxidant
production, primary antioxidant defense, and repair of oxi-
dant-induced damage (Figure 3).
PROTEIN OXIDATION AND AGING: SUMMARY
In summary, the majority of studies on protein oxidation
confirm that protein oxidation products, such as protein car-
bonyls and oxidized–cross-linked material, increase with
age (Figure 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 de-
cline 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, in-
cluding nuclei and mitochondria, during aging. It may be
important to investigate proteolytic activity in a given cellu-
lar compartment relative to the amount of substrate located
in the same compartment. Little is known about the regula-
tion of the proteasome and the ubiquitin-conjugating ma-
chinery during oxidative stress or other stress conditions.
Similarly, our understanding of how proteasome is regu-
lated during aging is still in its infancy.
Acknowledgments
T. Grune was supported by Stiftung Verhalten und Umwelt and the
Deutsche Forschungsgemeinschaft (SFB 507). K.J.A. Davies was sup-
ported by the National Institutes of Health/National Institute of Environ-
mental Health Sciences under Grant ES 03598, and by the National Insti-
tutes of Health/National Institute on Aging under Grant AG 16256.
Address correspondence to Prof. Kelvin J. A. Davies, Ethel Percy Andrus
Gerontology Center, University of Southern California, 3715 McClintock
Avenue, Room 306, Los Angeles, CA 90089-0191. E-mail: kelvin@usc.edu
An improved understanding of protein oxidation, protea-
some inhibitors, and proteasome regulation could be ex-
tremely helpful in a number of “age-related diseases.” For
example, reaction of the lipid oxidation product 4-hydroxy-
nonenal with the amyloid ꢃ-peptide generates a powerful
proteasome inhibitor that could be important in Alzheimer’s
disease (113). Much more work will be required to deter-
mine the actual pathological significance of oxidized and
cross-linked aggregates of various proteins.
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Received January 18, 2001
Accepted April 5, 2001
Decision Editor: John Faulkner, PhD