From the Ethel Percy Andrus Gerontology Center and
Division of Molecular and Computational Biology, University of
Southern California, Los Angeles, California 90089-0191 and the
¶ Neuroscience Research Center, Medical Faculty
(Charité), Humboldt University, D-10098
Berlin, Germany
Received for publication, June 24, 2002, and in revised form, October 23, 2002
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ABSTRACT |
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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.
Accumulation of oxidatively damaged proteins is a characteristic
feature of aging cells and a number of age-related pathologies (1-3).
In young, healthy cells moderately oxidized soluble proteins are
selectively recognized and rapidly degraded by the proteasome (4-8).
We have previously demonstrated that exposure of mammalian cells in
culture to moderate oxidative stress significantly increases the
degradation of intracellular proteins (5, 9). Proteasome immunoprecipitation, treatment of cells with antisense oligonucleotides to essential proteasome subunits, and proteasome inhibitor profiles (4,
5, 9) all confirm that proteasome is primarily responsible for the
degradation of oxidized proteins in mammalian cells. The 20 S
proteasome constitutes the catalytic core, whereas the 26 S proteasome
is formed by addition of two 19 S regulators, which have subunits for
ATP hydrolysis and polyubiquitin recognition (10-12). We have
extensive evidence that purified 20 S proteasome preferentially
degrades oxidized proteins in vitro in an ATP- and
ubiquitin-independent manner (4, 5, 9, 13, 14), however, it has not
been clear if proteasome can degrade oxidized proteins without
ubiquitin and ATP in vivo.
The 20 S proteasome now appears to be the predominant proteasome
species in mammalian cells (15). Furthermore, a number of proteins have
now been shown to undergo ubiquitin-independent degradation by the 26 S
proteasome (16-19). Our studies on comparative resistance of the 20 S
and 26 S forms of the proteasome in response to hydrogen peroxide
treatment show that the 26 S proteasome is much more susceptible to
oxidative stress than is the 20 S form (20, 21), suggesting that it is
probably the 20 S proteasome, which is responsible for the degradation
of oxidized proteins when cells are exposed to oxidative stress.
The current study was undertaken to determine whether ubiquitin
conjugation is essential for degradation of oxidized proteins by the
proteasome in intact cells. For this we utilized the Chinese hamster
lung fibroblast cell line, CH E36 and a cell cycle mutant derived from
this line, ts20, which harbors a thermolabile ubiquitin-activating enzyme (E1)1 (22-24). These
cell lines have been previously used to study the role of ubiquitin
conjugation in various processes (24-26). We also tested ubiquitin
conjugation of progressively oxidized substrates, to determine whether
oxidation actually increases the tendency of a protein to be ubiquitinylated.
Cell Culture--
CH E36 and clone ts20 cells were a kind gift
from Dr. Alan L. Schwartz, Washington University, St. Louis, MI. The
parental cell line E36 is a hypoxanthine guanosine-phosphoribosyl
transferase-negative mutant (HGPRT Preparation of Cell Extracts--
Cells were washed twice in
phosphate-buffered saline, bathed in cold phosphate-buffered saline
containing 1 mM EDTA, and scraped with a rubber policeman.
About 107 cells were resuspended in 300 µl of 1 mM dithiothreitol and incubated at 4 °C for 1 h,
with vigorous shaking for hypotonic lysis. The crude extracts were
centrifuged at 13,000 × g in an Eppendorf centrifuge
for 15 min at 4 °C, supernatants were collected and stored at
SDS-PAGE and Immunoblotting--
Total soluble cell protein was
separated according to Laemmli (27) by SDS-PAGE using a mini-PROTEAN II
electrophoresis cell (Bio-Rad). Following transfer, the nitrocellulose
membranes were probed with antibodies to E1 (Calbiochem) or ubiquitin
(Chemicon) and visualized using the ECL chemiluminescent system
(Amersham Biosciences). Antibodies to ubiquitin conjugation
substrates were obtained from Roche Molecular Biochemicals
(anti-ferritin) or Chemicon (anti-lysozyme).
Cell Counting and Cell Survival Analysis--
Live, attached
cells were harvested by trypsinization and counted in a Z1S Coulter
particle counter. Growth curves were performed by seeding equal numbers
of cells in 24-well plates and then harvesting and counting cells at
various time points. To test if H2O2 treatments were toxic, cell survival analysis was performed using neutral red
(Sigma) uptake as described previously (28).
Determination of Activity of the 20 S and 26 S
Proteasome--
20 S Proteasome activity was measured at 37 °C as
described previously (29) by monitoring the release of
7-amido-4-methylcoumaric acid during degradation of the
fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amido-4-methylcoumaric acid
by extracts of CH E36 and ts20 cells. Activity of the 26 S proteasome
was determined by monitoring ability of cell lysates to degrade
pre-formed lysozyme-ubiquitin conjugates. Ubiquitin conjugates were
prepared from heat-denatured lysozyme as described below. These
conjugates were then incubated either with extracts from ts20 cells at
the restrictive temperature or with buffer alone, for 20 min at
39.5 °C. The reaction mixtures were separated by 12.5% SDS-PAGE,
transferred to nitrocellulose, and the membranes were probed with an
anti-lysozyme antibody. The amount of total lysozyme present in each
lane was then quantified using NIH Image software to determine the net
loss of lysozyme.
Metabolic Labeling, Exposure of Cells to Oxidative Stress, and
Proteolysis Measurements--
Cells were seeded at 2-5 × 105 cells/ml in 24-well plates and grown at 31 °C. On
day 3, cells were metabolically labeled with a
[35S]Cys/Met mixture for 2 h (to generate
predominantly "short lived" proteins) or for 16 h (to generate
mostly "long lived" proteins) as described (5, 9). Both, CH E36 and
ts20 cells were then shifted to the restrictive temperature
(39.5 °C) for 2 h to inactivate the E1 ubiquitin-activating
enzyme in ts20 cells (cells were maintained at 39.5 °C for all
further treatments). The monolayers were then exposed to oxidative
stress by bolus addition of increasing concentrations (0.1 to 1 mM) of H2O2 in PBS, for 30 min.
Following oxidative stress, cells were incubated in cold
(nonradioactive) medium, with or without proteasome inhibitors, and
percent protein degradation was monitored by measuring acid-soluble
[35S] counts from previously acid-precipitable
35S-labeled proteins as described (5, 9).
Determination of Protein Carbonyls--
A sensitive ELISA method
as described by Winterbourn and colleagues (30) with modifications
described by Sitte et al. (31) was used for the quantitative
determination of protein carbonyls.
Oxidative Modification and Heat Denaturation of
Ferritin/Lysozyme--
Isolated ferritin and lysozyme (Sigma) were
oxidized with various concentrations of hydrogen peroxide as described
previously (29) with a few modifications. Proteins (1 mg) were exposed to various concentrations of hydrogen peroxide in an Eppendorf thermomixer for 2 h at 30 °C in a total volume of 1 ml of
phosphate buffer. For heat denaturation, the proteins were incubated in a boiling water bath for 5 min, followed by quick chilling on ice.
Untreated proteins, peroxide-treated proteins, and heat-denatured proteins were extensively dialyzed overnight at 4 °C against 200 volumes of buffer, with 2 changes, to remove any residual hydrogen peroxide or spontaneously generated fragments.
Ubiquitin Conjugation of Ferritin/Lysozyme in
Vitro--
Ubiquitin conjugation of purified protein substrates was
carried out as described by Finley et al. (32), except that
in our assay, the P-enolpyruvate/pyruvate kinase system was used for
ATP regeneration and glutathione S-transferase-tagged
ubiquitin was used as the source of ubiquitin. Briefly, the assay
mixture contained 20 µl of CH E36 cell lysate (4 mg/ml), 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol, 3.6 mM P-enolpyruvate, 0.5 units of pyruvate kinase, 5 mM ATP, 5 µg of glutathione
S-transferase-ubiquitin, 0.09 units of inorganic
pyrophosphatase (to reduce the build-up of PPi), 50 µM
lactacystin (to inhibit proteasomal degradation of Ub conjugates), as
well as 1 µM ubiquitin-aldehyde (to inhibit de-ubiquitinating enzymes). The reaction was initiated by adding 10 µg of oxidized or heat-denatured ferritin. For ubiquitin conjugation of lysozyme, the assay was similar except that the widely used creatine
phosphate (10 mM)/creatine phosphokinase (100 µg/ml) system was used for ATP regeneration. In some reactions, ATP-depleted lysates were produced by incubation with 10 mM
2-deoxyglucose and 0.5 units of hexokinase. After 20 min at 30 °C,
the reaction was stopped by addition of SDS-PAGE loading buffer and an
aliquot of the mixture was loaded on 12.5% polyacrylamide gels after
boiling. After transfer, the blots were probed with antibodies to the
substrate proteins ferritin (Roche Molecular Biochemicals) or lysozyme (Chemicon).
Degradation of Ferritin and Lysozyme--
Degradation of
ferritin by purified 20 S proteasome was monitored by measuring
acid-soluble free amines using fluorescamine, as described (8, 29). To
determine the role of ATP, degradation of oxidized
[3H]ferritin by cell lysates (60 µl; 2.5 mg/ml) was
measured both in the presence and absence of 10 mM ATP.
Percent degradation was calculated by measuring acid soluble
radioactivity as described previously (14, 29). Lysozyme was labeled
with [3H] by reductive methylation (33) and percent
degradation was measured by formation of acid-soluble counts, as
described (14, 29).
E1 Inactivation Results in Decreased Ubiquitinylation and Decreased
Basal Proteolysis of Short Lived Proteins--
Incubation of
temperature-sensitive ts20 cells for 2 h at the restrictive
temperature (39.5 °C) resulted in loss of E1. Cells were
initially shifted to the restrictive temperature for 2 h, followed
by sham treatment with PBS (or treatment with 500 µM hydrogen peroxide) for 30 min. In extracts of E1-mutant ts20 cells incubated at the restrictive temperature for more than 2.5 h, the
enzyme could not be detected immunologically with an anti-E1 antibody
(Calbiochem) at the expected position of 110 kDa (Fig. 1A, lanes 4 and
6). There was, however, no difference in the levels of E1 in
the wild-type CH E36 cells when incubated at the restrictive temperature (Fig. 1A, lanes 3 and 5). Both E36
cells and ts20 cells exhibited comparable amounts of enzyme at the
permissive temperature of 30.5 °C (Fig. 1A, lanes
1 and 2). Similar results were obtained for cells
treated with hydrogen peroxide (data not shown).
To determine whether this was a truly functional inactivation of the
enzyme, we looked at the ability of ts20 cells to form high molecular
weight ubiquitin conjugates de novo. CH E36 cells and ts20
cells were incubated at 39.5 °C for 3 h, after which equal
amounts of protein from cell extracts were separated by a 12.5%
SDS-PAGE. Following transfer, the membranes were probed with an
anti-ubiquitin antibody (Chemicon) to detect protein-ubiquitin conjugates. As seen in Fig. 1B, the steady-state levels of
endogenous, high molecular weight ubiquitin conjugates were
considerably lower in ts20 cells, than in wild type CH E36 cells,
indicating markedly compromised ubiquitin-conjugating activity in ts20
cells at the restrictive temperature. Similar results were obtained by
Kulka et al. (24) presumably because there is no de
novo synthesis of high molecular weight Ub conjugates in ts20
cells at the restrictive temperature. Although this has been previously
seen in ts20 cells (24), we reconfirmed these properties of the
temperature-sensitive ts20 mutants in light of recent reports of
residual ubiquitin-conjugation activity for a similar, yet distinct
ts85 mutant cell line (34).
Most short lived proteins are degraded by the 26 S proteasome in an ATP
and ubiquitin-dependent fashion (35). Baseline proteolysis of short lived proteins was reduced by almost 50% in ts20 cells, indicating that ubiquitin-dependent proteolysis was indeed
compromised in temperature-sensitive E1 mutants (Fig. 1C).
It should be noted that besides ubiquitin-independent proteolysis by
the proteasome, proteolysis is also conducted by other proteases.
Similar studies have been performed in ts20 cells and in another E1
mutant cell line, ts85 derived from FM3A mouse carcinoma cells
(36).
We next ensured that the thermolability of
ubiquitin-dependent proteolysis was solely because of a
defect in ubiquitin conjugation and not inactivation of the 26 S
proteasome at the restrictive temperature. For this we tested the
degradation of pre-formed lysozyme-ubiquitin conjugates by extracts of
ts20 cells incubated at the restrictive temperature. Ubiquitin
conjugates of heat-denatured lysozyme were incubated with buffer alone
(lane 1 in Fig. 1D, and lane 1 in the
inset) or with E1-inactivated ts20 extracts (lane
2 in Fig. 1D, and lane 2 in the
inset). After separation of the reaction mixtures by
SDS-PAGE and transfer, the membranes were probed with an anti-lysozyme
antibody and the resulting chemiluminescence was quantified using NIH
Image software. Besides disappearance of individual high molecular
weight lysozyme-Ub conjugates, densitometric quantification revealed a
60% loss of total lysozyme after incubation with ts20 lysates. Thus,
ts20 cells incubated at the restrictive temperature for 3 h were
capable of degrading lysozyme-ubiquitin conjugates, confirming that the
26 S proteasome was not affected in these cells. Similar results were
obtained using extracts of wild-type CH E36 cell lysates (data not shown).
Growth Properties, H2O2 Toxicity, and
Proteasome Activity Profiles--
Before testing a possible ubiquitin
involvement in the degradation of oxidized proteins, we first compared
growth properties at 30.5 °C (Fig.
2A), 39.5 °C (Fig.
2B), and with H2O2 in wild-type CH
E36 cells and E1 mutant ts20 cells (Fig. 2C). Hydrogen
peroxide caused a similar growth arrest in both cell lines (Fig.
2C) as expected from previous work (37, 38). Because the
cells were maintained at the restrictive temperature for only 24-26 h,
the two cell lines did not show a major difference in cell number. To
test whether the ts20 mutants were more susceptible than CH E36 cells
to hydrogen peroxide toxicity because of their thermolabile E1, we
analyzed cell survival in response to H2O2
treatment in both cell lines by the neutral red uptake assay (Fig. 2,
D and E). There were no significant differences
in survival over a 24-h period following H2O2
treatment, further indicating that lack of ubiquitin-conjugating
activity did not alter the short term ability of ts20 cells to survive
an acute oxidative stress.
Because most proteolysis experiments were performed at 39.5 °C, we
measured the activity of the 20 S core proteasome in both cell lines,
at both temperatures, to determine whether the proteasome was
inactivated at the restrictive temperature. As seen in Fig. 2F, the proteasome was not inactivated at the restrictive
temperature and loss of proteasome-dependent proteolysis
may, therefore, be attributed to defects in ubiquitin conjugation.
E1 Mutants Degrade Oxidized Proteins Despite Compromised
Ubiquitinylation--
To test whether ubiquitin conjugation is
necessary for the degradation of oxidized proteins, we studied the
turnover of short lived and long lived proteins after treatment with
hydrogen peroxide in ts20 cells with compromised ubiquitin-conjugating
activity. CH E36 and ts20 cells were metabolically labeled for 2 h
(Fig. 3A) or 16 h (Fig.
3B) with [35S]Cys/Met to label pools of either
short lived (2 h) or mostly long lived (16 h) proteins, respectively.
As seen in Fig. 3, intracellular proteolysis was considerably higher in
cells treated with 400 µM H2O2
than in untreated cells. We have previously reported a similar
oxidation-induced increase in overall proteolysis for many different
cell types (4, 5, 9, 39) but we can now show that E1 mutant ts20 cells
still preferentially degrade oxidized proteins despite the lack of an
E1. Because a number of short lived regulatory proteins are
known to be degraded by the ubiquitin-proteasome pathway (35, 40), the
total turnover of short lived proteins was considerably lower in ts20
cells, as expected. However, the relative increase in degradation
because of oxidation was comparable for both cell lines, indicating
that the pool of oxidized proteins can still be eliminated in ts20 cells. The degradation of long lived proteins (Fig. 3B) also
showed a similar oxidation-induced increase in both CH E36 and E1
mutant ts20 cells as seen previously for other cell types. In fact, it may be noted that the absolute increase in proteolysis because of
H2O2 is actually higher in the ts20 E1 mutant
cells than in the parent CH E36 cells (Fig. 3B). In
earlier studies we reported that moderate oxidation of proteins rapidly
increases their degradation, whereas severe oxidation causes a gradual
decline in intracellular proteolysis (4, 5, 9, 29). The E1 mutant ts20
cells exhibited exactly this pattern of response to hydrogen peroxide concentration (Fig. 3C), indicating that the E1 mutant ts20
cells utilize the same pathway for degradation of oxidized proteins described previously (4, 5, 9).
The Proteasome Is Largely Responsible for the Degradation of
Oxidized Proteins in ts20 Cells--
To determine whether the
proteasome is still responsible for oxidation-induced protein
degradation in ts20 cells with compromised ubiquitin-conjugating
activity, we examined the effects of proteasome inhibitors. The
selective proteasome inhibitor NLVS (5 µM) strongly inhibited the increase in degradation because of oxidation in both CH
E36 control cells and E1 mutant ts20 cells, indicating that the
proteasome was still largely responsible for the degradation of
oxidized proteins even without the synthesis of ubiquitin conjugates (Fig. 4). Similar results were also
obtained with another well known proteasome inhibitor, lactacystin (not
shown).
E1 Mutant ts20 Cells Can Eliminate Carbonyl-containing Proteins
without Ubiquitin Conjugation--
Because the above experiments
measured overall proteolysis, we next studied protein carbonyls to
address the turnover of oxidized proteins. The total carbonyl content
of both wild type CH E36 cells and E1 mutant ts20 cells was increased
by H2O2 but then dramatically decreased over
the following 24 h, whether measured by ELISA or Western blot
assays (Fig. 5, A and
B), showing that the ts20 cells could still degrade
oxidized, carbonyl-containing proteins without the presence of an
active ubiquitin-conjugating system.
Progressive Oxidation of Ferritin Does Not Promote Its
Ubiquitinylation in Vitro Yet the Protein Is Degraded by Proteasome
without ATP Hydrolysis--
Fig.
6A shows an in
vitro ubiquitin-conjugation assay for native, heat-denatured, and
progressively oxidized ferritin. Native ferritin (lanes 1 and 2) was modified either by heat denaturation (lanes
3 and 4) or increasing concentrations of hydrogen
peroxide (lanes 5-10). The protein was then incubated
either by itself (lanes 1, 3, 5,
7, and 9) or in the presence of a complete
ubiquitin-conjugation system (lanes 2, 4,
6, 8, and 10) for 30 min. at
30 °C. The high molecular weight ferritin-immunoreactive bands
represent high molecular weight ferritin-ubiquitin conjugates with a
progressively increasing number of ubiquitin moieties. Glutathione
S-transferase-tagged ubiquitin (Mr
38,500) was used to obtain better separation of ubiquitin
conjugates, because "naked" ubiquitin usually produces a smear-like
pattern.
Heat-denatured ferritin showed a marked increase in ubiquitin
conjugation as seen by the both the intensity and the number of high
molecular weight bands (Fig. 6A, lane 4) in
comparison with native ferritin (lane 2). Progressive
oxidation of ferritin by 10 (lanes 5 and 6), 20 (lanes 7 and 8), or 30 mM
(lanes 9 and 10) hydrogen peroxide, however, did
not increase ubiquitin conjugation: actually,
H2O2 caused a progressive decrease in the
number and intensity of high molecular weight ferritin-ubiquitin
conjugates, in comparison with untreated ferritin (lane 2),
showing that this oxidized protein is not preferentially ubiquitinylated.
Fig. 6B shows that ferritin was indeed progressively
oxidized, as seen by the increase in carbonyl content. Protein
carbonyls were assessed by an ELISA using an anti-DNP antibody as
described under "Experimental Procedures." This modified ferritin
was still preferentially degraded by purified 20 S proteasome in the
absence of ATP (Fig. 6C). As described for several proteins
in our earlier work (4, 5, 8, 9, 13, 14, 20, 21, 29, 41, 47, 48, 51),
moderately oxidized ferritin was preferentially degraded by the 20 S
proteasome whereas severely oxidized ferritin tended to be a poorer substrate.
Based on our results, as well as new evidence in the literature (21,
41), we speculated that this degradation of oxidized proteins is most
likely conducted by the 20 S proteasome or the immunoproteasome.
Because the 26 S proteasome requires ATP hydrolysis, we tested the
effect of ATP on degradation of oxidized ferritin by both CH E36 and E1
mutant ts20 cell lysates (Fig. 6D). As seen in Fig.
6D, ATP did not enhance the degradation of oxidized ferritin by the wild-type, CH E36 cell lysates, or E1 mutant ts20 cell lysates,
thus excluding any energy requirement for the degradation of oxidized
protein substrates.
Progressive Oxidation of Lysozyme Does Not Promote Its
Ubiquitinylation in Vitro and Oxidized Lysozyme Is Still Preferentially
Degraded by Lysates of ts20 Cells--
A similar ubiquitin-conjugation
assay was performed with the widely used proteasome substrate,
lysozyme. As seen in Fig. 7A, heat-denatured lysozyme showed a large increase in ubiquitin conjugates as seen by the dark smear (lane 12) as compared with native
lysozyme (lanes 1 and 2). Neither heat-denatured
lysozyme by itself (lane 11), nor heat-denatured lysozyme
when incubated with an ATP-depleted extract (lane 13), nor a
ts20 extract incubated at 39.5 °C with inactive E1 (lane
14) showed a similar increase in high molecular weight
lysozyme-ubiquitin conjugates ruling out nonspecific ubiquitinylation. Progressive oxidation of lysozyme (lanes 3-10), however,
did not increase its tendency to be ubiquitinylated (as also seen for ferritin in Fig. 6A), further confirming that ubiquitin
conjugation may not be involved in targeting oxidized proteins to the
proteasome.
Fig. 7B reveals a progressive oxidation of lysine residues
(some of which are required for forming polyubiquitin conjugates) with
H2O2 exposures up to 30 mM. At 40 mM H2O2 the lysine oxidation product 2-aminoadipic semialdehyde itself underwent oxidative destruction.
Degradation of [3H]lysozyme by extracts of E36 and
(E1-inactivated) ts20 cells at 39.5 °C was monitored as described
previously (13, 29, 42). The E1 mutant ts20 cells degraded lysozyme as
well as the wild-type CH E36 cells, confirming that even in wild-type
cells, ubiquitin conjugation is not required for the degradation of
oxidized proteins (Fig. 7C). Interestingly, the wild-type
cells degraded heat-denatured lysozyme much more efficiently than did
the ts20 mutants (Fig. 7D). Because heat-denatured lysozyme is degraded by the ubiquitin 26 S proteasome system this experiment clearly shows that oxidized proteins must be degraded by another proteasome pathway.
We have shown that E1 mutant ts20 cells can eliminate oxidized
proteins despite their lack of ubiquitin-conjugating activity, and that
this degradation is still catalyzed by the multicatalytic proteinase
complex, proteasome. The proteasome exists in multiple forms including
free 20 S, 26 S, immunoproteasome, and hybrid proteasomes (10, 11, 15).
The 26 S proteasome requires ATP hydrolysis and mostly degrades
ubiquitin-conjugated proteins (10-12), with a few known exceptions
(17, 43-45). The 19 S regulator is required for recognition and
binding of ubiquitin-conjugated proteins, followed by their
deubiquitinylation and unfolding to allow entry into the catalytic core
(20 S) of the proteasome. Pickart and co-authors (46, 47) have shown
that it is primarily the hydrophobic effect that contributes to
polyubiquitin chain recognition by the 26 S proteasome. Many substrates
of the 26 S proteasome are short lived regulatory proteins that are not
necessarily damaged or denatured and, therefore, have to be tagged with
an external hydrophobic patch in the form of a polyubiquitin chain. ATP
hydrolysis is required for unfolding such substrates so that they can
enter the catalytic core of the proteasome (43). Oxidative damage to a
protein, however, directly results in partial unfolding and exposure of
otherwise buried hydrophobic residues (48-51). Therefore, an oxidized
protein does not need to be further modified by ubiquitin conjugation
to confer a hydrophobic patch, nor does it require energy from ATP
hydrolysis as it is already unfolded. We (14, 42, 52) and others (53,
54) have shown that the 20 S proteasome has a distinct preference for
hydrophobic and bulky (aromatic) residues. We therefore suggest that
the 20 S proteasome selectively recognizes oxidatively modified,
partially denatured proteins because of their exposed hydrophobic moieties.
Contrary to earlier belief that the 26 S proteasome is the major
intracellular proteasome species, recent literature on proteasome stoichiometry suggests that there is a significant excess of free (and
enzymatically active) 20 S proteasome form (15, 55-57) in mammalian
cells. Several examples of ubiquitin-independent degradation of
proteins by the proteasome have now been reported (16-19, 44, 58). We
have demonstrated that the 20 S proteasome is resistant to oxidative
stress, whereas the 26 S proteasome is quite susceptible. Taylor and
co-authors (41, 59, 60) have shown that activity of
ubiquitin-activating and -conjugating enzymes (E1 and E2s) is
reversibly depressed during oxidative stress, possibly because of
glutathiolation of the active site cysteine residues in both E1 and the
family of E2 enzymes. Because both the 26 S proteasome and the
ubiquitin-conjugation system are inhibited/inactivated by oxidative
stress, it is unlikely that the ubiquitin-26 S proteasome pathway is
involved in the degradation of oxidized proteins in cells undergoing
oxidative stress.
Our studies on ubiquitin conjugation of oxidized substrates in
vitro show that progressive oxidation of a protein does not increase its propensity to be ubiquitinylated (Figs. 6 and 7). Conjugation of the polyubiquitin chain occurs via internal lysine residues of the substrate protein and progressive oxidation of a
protein actually modifies an increasing number of lysine residues as
seen by formation of the oxidation product, 2-aminoadipic semialdehyde. Despite the lack of ubiquitinylation, and loss of lysine residues, oxidized proteins such as ferritin and lysozyme are still
preferentially degraded in wild-type cells and E1 mutant ts20 cells,
and by purified 20 S proteasome. Besides, addition of ATP did not
enhance the degradation of oxidized proteins either by wild-type CH E36
or E1 mutant ts20 lysates, suggesting that the 26 S proteasome, which requires ATP hydrolysis, may not be involved in oxidized protein degradation. Our current data, especially when combined with previous reports by us (4, 5, 8, 9, 13, 14, 20, 21, 29, 31, 39, 42, 52), and
others (41, 59, 60), appear to rule out any significant involvement of
the ubiquitin-26 S proteasome system in the degradation of oxidatively
modified proteins. Both this study and previous reports, however,
clearly demonstrate that some form of the proteasome is essential for selective intracellular clearance of oxidized proteins.
Careful quantification has now revealed that the free 20 S proteasome
is the predominant form of the enzyme in mammalian cells (15, 55, 57).
In vitro studies by our group have repeatedly shown that the
free 20 S proteasome efficiently and aggressively degrades a wide
variety of oxidized proteins (4, 5, 9, 13, 14, 29, 31, 39, 42, 52) and
our preliminary studies indicate that the immunoproteasome can also
degrade oxidized proteins (data not shown). Although our data does not
conclusively rule out involvement of the 26 S proteasome in the
degradation of oxidized proteins, it now appears to be less likely,
because our results reveal that oxidized proteins are degraded with no requirement for either ATP hydrolysis or ubiquitin conjugation. It will
now be important to directly test this hypothesis in intact cells, and
to determine the relative contributions of the free 20 S proteasome and
the immunoproteasome to the degradation of oxidized proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) derived from the
Chinese hamster lung fibroblast cell line V-79 (22). Clone ts20 is a
cell cycle mutant derived from E36 cells by ethylmethane sulfonate
mutagenesis (23), which contains a thermolabile ubiquitin-activating E1
enzyme (24). Both, E36 cells (wild-type for E1) and E1 mutant ts20
cells were grown as monolayers at 30.5 °C on minimal essential
medium-
(Invitrogen) supplemented with 10% fetal bovine
serum and 4.5 g/liter glucose under 5% CO2. Flasks of both
E36 and ts20 cells were shifted to the restrictive temperature,
39.5 °C, for a minimum of 2 h to inactivate E1.
70 °C until used. Protein content was determined using the BCA
assay (Pierce) or the DC assay (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
E1 inactivation results in decreased
ubiquitinylation and decreased basal proteolysis of short lived
proteins in ts20 cells. Panel A, Western blot,
anti-E1 antibody. The left panel shows levels of E1 in cells
incubated at the permissive temperature of 30.5 °C. Right
panel, levels of E1 in CH E36 cells (lanes 3 and
5) and E1 mutant ts20 cells (lanes 2 and
4) at 39.5 ° C. Panel B, Western blot,
anti-ubiquitin antibody. Soluble cell proteins from cells incubated at
the restrictive temperature of 39.5 °C for 3 h were separated
on a 12.5% SDS-PAGE and probed with an anti-ubiquitin antibody. The
blot was stripped and reprobed with an anti-actin antibody (Sigma) as a
loading control (panel B, lower box). Panel
C shows basal proteolysis of short lived proteins; mean ± S.E. of three independent determinations. Cells were metabolically
labeled as described under "Experimental Procedures," incubated at
the restrictive temperature for 2 h, and then sham treated with
PBS or H2O2 (not shown) for 30 min. Percent
degradation was measured by formation of acid-soluble
[35S] counts from previously acid-precipitable
35S-labeled proteins as described (5, 9). Panel
D, reports the quantification of an anti-lysozyme Western
blot (inset) of pre-formed lysozyme-ubiquitin conjugates in
the absence (100%; inset, lane 1) or presence
(40%; inset, lane 2) of ts20 lysates. Pre-formed
lysozyme-Ub conjugates were incubated with buffer alone, or with ts20
lysates, for 20 min at 39.5 °C followed by separation on a 12.5%
SDS-PAGE. Following transfer, the membranes were probed with an
anti-lysozyme antibody (inset) and the chemiluminescent
signal was later quantified using NIH Image software.
View larger version (39K):
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Fig. 2.
Growth curves, hydrogen peroxide toxicity,
and proteasome activity. Panel A, on the first
day 1 × 105 cells were seeded in 24-well plates and
cells were counted in an automated cell counter. Both wild-type CH E36
cells and E1 mutant ts20 cells grew at comparable rates at the
permissive temperature of 30.5 ° C. Panels B and
C, wild-type, CH E36, and E1 mutant ts20 cells were
grown at the permissive temperature (30.5 °C) for 48 h, then
shifted to the restrictive temperature (39.5 °C) to inactivate E1.
At 50 h, the cells were either sham treated with PBS
(B) or with 0.4 mM hydrogen peroxide
(C) for 30 min at 39.5 ° C. Panels D and
E, effect of hydrogen peroxide on cell survival was tested
with the neutral red assay (Sigma). Effect of treatment with 0.6 mM peroxide immediately after treatment and 24 h later
is shown for both wild-type CH E36 cells (D) and E1 mutant
ts20 cells (E) at the permissive and restrictive
temperatures. Panel F demonstrates that proteasome activity
is not affected in ts20 cells at the restrictive temperature. Cells
were grown at 30.5 °C, or were incubated for 3 h at 39.5 °C
and then harvested. Proteasome activity in lysates was measured by
degradation of Suc-LLVY-7-amido-4-methylcoumaric acid for 1 h at
37 °C. All data points are mean ± S.E. of at least three
independent experiments.
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Fig. 3.
E1 mutant ts20 cells degrade oxidized
proteins despite compromised ubiquitinylation. CH E36 and E1
mutant ts20 cells were metabolically labeled with
[35S]Cys/Met for 2 h (panels A and
C) or 16 h (panel B) to label pools of short
lived, or mostly long lived, proteins, respectively. After incubation
of these cells for 2 h at the restrictive temperature
(39.5 °C), the cells were treated with PBS (no
H2O2) or increasing concentrations of hydrogen
peroxide for 30 min at 39.5 °C, followed by incubation in regular,
cold medium for a further 24 h at 39.5 °C. Percent degradation
was measured by formation of acid-soluble [35S] counts
from previously acid-precipitable 35S-labeled proteins as
described (5, 9). All data points are mean ± S.E. of at least
three independent experiments.
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Fig. 4.
The proteasome inhibitor NLVS inhibits
H2O2-induced proteolysis. Proteolysis was
measured exactly as described in the legend to Fig. 3A,
except that the cells were treated either with 5 µM NLVS
or Me2SO vehicle alone. Me2SO or NLVS was added
2 h prior to treatment with peroxide, and remained in the final
incubation medium. All data points are mean ± S.E. of at least
three independent experiments.
View larger version (42K):
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Fig. 5.
E1 mutants can still degrade
carbonyl-containing proteins. Panel A, ELISA for the
quantitative determination of protein carbonyls using an anti-DNP
antibody (31, 32). Cells were incubated at the restrictive temperature
for 2 h to inactivate E1, followed by treatment with hydrogen
peroxide (or PBS in controls). Protein carbonyls were measured in
untreated cells and hydrogen peroxide-treated cells for both the CH E36
and the E1 mutant ts20 cell lines, either immediately after treatment
(0 h), or 24 h after treatment (24 h) with 0.5 mM
hydrogen peroxide. Panel B, Western blot (anti-DNP antibody)
to detect carbonyl content in ts20 cells sham treated with PBS
(C), or in ts20 cells treated with hydrogen peroxide
(P), at both 0 and 24 h. In both panels cells were
treated with H2O2 or PBS for 30 min, and
H2O2 was then replaced by dialysis with medium.
Results of panel A are mean ± S.E. of at least three
independent experiments and the Western blot of panel B was
repeated several times with very similar results.
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Fig. 6.
In vitro ubiquitin conjugation and
degradation of oxidized ferritin. Panel A,
purified horse spleen ferritin was purchased from Sigma and used in its
native form (lanes 1 and 2), modified by heat
denaturation (lanes 3 and 4), or by progressive
oxidation with 10 (lanes 5 and 6), 20 (lanes 7 and 8), and 30 mM
(lanes 9 and 10) hydrogen peroxide. These model
protein substrates were then incubated either alone (lanes
1, 3, 5, 7, and
9) or in the presence of a complete ubiquitin-conjugation
system that included CH E36 cell lysates (lanes 2,
4, 6, 8, and 10) for
30 min at 30 °C. Loading buffer was added to stop the reaction and
the reaction mixtures were separated by 12.5% SDS-PAGE. After
transfer, membranes were probed with an anti-ferritin antibody (Roche
Molecular Biochemicals). Panel B, oxidation of ferritin
was assessed by an ELISA for detecting DNP-derivatized protein
carbonyls with an anti-DNP antibody (31, 32). Panel C,
the proteolytic susceptibility of oxidized ferritin to degradation by
purified 20 S proteasome was monitored by measuring release of
acid-soluble free amines using fluorescamine. Panel D,
progressively oxidized [3H]ferritin was incubated with
lysates of either wild-type CH E36 cells (white bars), or E1
mutant ts20 cells (gray bars), in the absence (solid
bars) or presence (patterned bars) of 10 mM
ATP for 2 h at 39.5 °C. Percent degradation was calculated by
measuring the increase in acid soluble radioactivity as described under
"Experimental Procedures."
View larger version (54K):
[in a new window]
Fig. 7.
In vitro ubiquitin conjugation and
degradation of oxidized lysozyme. Panel A, an
in vitro ubiquitin-conjugation assay using lysozyme (Sigma)
as the substrate. Native lysozyme (lanes 1 and
2), lysozyme modified by 10 (lanes 3 and
4), 20 (lanes 5 and 6), 30 (lanes 7 and 8), or 40 mM
(lanes 9 and 10) hydrogen peroxide, or
heat-denatured lysozyme (lanes 11-14) was used as a
substrate for ubiquitin conjugation. Lysozyme was incubated either by
itself (lanes 1, 3, 5, 7,
9, and 11) or in the presence of a complete
ubiquitin-conjugating system that included lysates from CH E36 cells
(lanes 2, 4, 6, 8,
10, and 12). When heat-denatured lysozyme
was incubated by itself (lane 11), with an ATP-depleted
extract (lane 13) or with a heat-inactivated ts20 extract
containing an inactive E1 (lane 14), there was no formation
of high molecular weight lysozyme-ubiquitin conjugates as expected.
Panel B, quantification of the lysine oxidation product
2-aminoadipic semialdeheyde upon progressive oxidation of lysozyme with
hydrogen peroxide. At 40 mM hydrogen peroxide, the lysine
oxidation product 2-aminoadipic semialdehyde itself appears to undergo
oxidative destruction. Panel C, in vitro
degradation of oxidized [3H]lysozyme by lysates of
wild-type CH E36 cells and E1 mutant ts20 cells at 39.5 °C.
Panel D, in vitro degradation of
heat-denatured [3H]lysozyme by lysates of wild-type CH
E36 cells and E1 mutant ts20 cells at 39.5 °C. In panels
C and D degradation of [3H]lysozyme was
measured by increasing production of acid-soluble [3H]
counts from previously precipitable [3H]lysozyme (13, 30,
42). Results are mean ± S.E. of at least three independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Alan Schwartz for providing the Chinese hamster lung fibroblast cell lines, CH E36 and ts20.
![]() |
FOOTNOTES |
---|
* This work was supported by NIEHS National Institutes of Health Grant ES03598 (to K. J. A. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: Dana-Farber Cancer Inst., 44 Binney St., Mayer 551, Boston, MA 02115. E-mail: Reshma_Shringarpure@dfci.harvard.edu.
To whom correspondence should be addressed: Andrus Gerontology
Center, GER 306C, University of Southern California, 3715 McClintock Ave., Los Angeles, CA 90089-0191. Tel.: 213-740-8959; Fax:
213-740-6462; E-mail: kelvin@usc.edu.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M206279200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; ELISA, enzyme-linked immunosorbent assay; DNP, 2,4-dinitrophenol.
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