(Received for publication, July 22, 1994; and in revised form, October 7, 1994)
From the
Exposure to various forms of mild oxidative stress significantly
increased the intracellular degradation of both
``short-lived'' and ``long-lived,'' metabolically
radiolabeled, cell proteins in cultures of Clone 9 liver cells (normal
liver epithelia). The oxidative stresses employed were bolus
HO
addition; continuous H
O
flux; the redox cycling quinones, menadione and paraquat; and the
aldehydic products of lipid peroxidation, 4-hydroxynonenal,
malonyldialdehyde, and hexenal. In general, exposure to more severe
oxidative stress produced a concentration-dependent decline in
intracellular proteolysis, in some cases to below baseline levels.
Oxidatively modified ``foreign'' proteins (superoxide
dismutase and hemoglobin) were also selectively degraded, in comparison
with untreated foreign proteins, when added to lysates of Clone 9 liver
cells. As with intracellular proteolysis, the degradation of foreign
proteins added to cell lysates was greatly increased by mild oxidative
modification, but depressed by more severe oxidative modification. The
proteinase activity was recovered in >300-kDa cell fractions, and
inhibitor profiles and immunoprecipitation studies indicated that the
multicatalytic proteinase complex, proteasome, was responsible for most
of the selective degradation observed with mild oxidative stress; up to
approximately 95% for intracellular proteolysis and 65-80% for
degradation of foreign modified proteins. Seven days of daily treatment
with an antisense oligodeoxynucleotide, directed against the initiation
codon region of the proteasome C2 subunit gene, severely depressed the
intracellular levels of several proteasome subunit polypeptides (by
Western blot analysis), and also depressed the H
O
induced increase in intracellular proteolysis by approximately
95%, without significantly affecting baseline proteolytic rates.
Extensive studies revealed only small or no increases in the overall
capacity of oxidatively stressed cells to degrade oxidatively modified
protein substrates; a finding supported by both Western blot and
Northern blot analyses which revealed no significant increase in the
levels of proteasome subunit polypeptides or mRNA transcripts. We
conclude that mild oxidative stress increases intracellular proteolysis
by modifying cellular proteins, thus increasing their proteolytic
susceptibility. In contrast, severe oxidative stress diminishes
intracellular proteolysis, probably by generating severely damaged cell
proteins that cannot be easily degraded (e.g. cross-linked/aggregated proteins), and by damaging proteolytic
enzymes. We further conclude that the multicatalytic proteinase complex
proteasome is responsible for most of the recognition and selective
degradation of oxidatively modified proteins in Clone 9 liver cells.
Over the past several years a wide series of publications from this laboratory(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) and other groups(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) have reported a relationship between protein oxidation and proteolysis. Such studies have been conducted with erythrocytes and reticulocytes from rabbits, cows, and human beings (1-7, 9-11, 13-17, 19, 21), with Escherichia coli(1, 9, 10, 14, 21) , with isolated mitochondria in vitro(12, 14) , with rat muscles in vitro(1, 14) , with primary cultures of rat hepatocytes(31, 32) , and with purified proteins and proteases in vitro(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) . These studies have all concluded that proteins are inherently susceptible to oxidative damage, and that oxidative damage alters proteolytic susceptibility. Our own studies have consistently demonstrated that relatively low-level oxidative damage alters proteolytic degradation, whereas extensive oxidative damage causes decreased proteolysis due to cross-linking, aggregation, and decreased solubility(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) .
Both our group(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) and that of Stadtman and his colleagues(25, 26, 27, 28, 29, 30, 31, 32) have suggested that increased degradation of mildly oxidized proteins is a normal function of intracellular proteolytic systems, whereas inability to degrade extensively oxidized proteins may contribute to certain disease states and aging.
In bacteria a series of proteolytic enzymes appear to conduct the degradation of oxidatively modified proteins(9, 10) . In primary hepatocytes(31, 32) , and erythrocytes and reticulocytes (1-7, 9-11, 13-17, 19-21) strong evidence has been presented for the role of the multicatalytic proteinase complex, proteasome; initially referred to as macroxyproteinase by this group(13, 14, 15) . Evidence has also been provided for a mechanism of proteolytic recognition and degradation by bacterial proteases (30) and erythrocyte/reticulocyte proteasome(1, 2, 3, 4, 5, 13, 16, 17, 20, 21) , based on partial unfolding and exposure of (previously shielded) hydrophobic amino acid R groups, as a result of protein oxidation.
A
recent publication by Dean et al.(33) , however, has
seriously questioned whether increased degradation of mildly oxidized
proteins is, indeed, a widespread property of eucaryotic cells and
organisms. Dean et al.(33) question whether
terminally differentiated red blood cells (RBC) ()are a
suitable model for other eucaryotic cell types, and note that the
hypothesis has not been thoroughly tested in other mammalian cells.
Additionally Matthews et al.(34) have questioned the
proposed role of proteasome in degrading oxidatively modified proteins.
We undertook the present investigation with two major goals: first, to test the hypothesis that low-level oxidative stress can result in increased intracellular proteolysis, in dividing mammalian cells: and second, to test the possible involvement/importance of proteasome in this process. Since liver cells are continuously exposed to oxidative stress through the metabolism of numerous endogenous and exogenous toxicants, at least two reports have suggested the degradation of oxidatively modified proteins by proteasome in primary cultures of hepatocytes(31, 32) , and mammalian proteasome has been most extensively studied in liver cells(35, 36) , we selected an immortalized liver cell culture line for our studies.
The degradation of radiolabeled foreign proteins, and of foreign
peptides was assessed following addition of these substrates to
unlabeled cell lysates. Cells were lysed by a 1-h incubation in water,
supplemented with 1.0 mM dithiothreitol. Membrane debris,
nuclei, organelles, and unlysed cells were then removed by
centrifugation at 14,000 g.
Figure 1:
Degradation of short-lived cellular
proteins in hydrogen peroxide-treated Clone 9 liver cells. Cell
proteins were metabolically labeled with
[S]Met/Cys for 2 h, and then exposed for 30 min
in phosphate-buffered saline plus 5.0 mM glucose to either
bolus H
O
addition (panels A and B), or to a continuous H
O
flux
generated by 0.14-2.77 µg/ml glucose oxidase (panel
C), as described under ``Materials and Methods.'' After
treatment, or ``sham-treatment'' for control cells, the
culture plates were washed twice with phosphate-buffered saline,
replenished with culture medium, and incubated for 0-24 h at 37
°C. Protein degradation was measured, at the indicated times,
following addition of 20% (w/v) trichloroacetic acid, to lyse the cells
and precipitate remaining intact proteins. Percent protein degradation
was measured by liquid scintillation in the trichloroacetic acid
supernatants as: (acid soluble counts - background counts)
(total counts initially incorporated - background
counts)
100. Panel A reports cumulative proteolysis
over a 24-h period following bolus H
O
treatment. Panel B reports actual rates of protein
degradation measured at 1-h intervals during the first 7 h after bolus
H
O
treatment. Panel C shows the
effects of a 30-min continuous H
O
flux
(generated by 0.14-2.77 µg/ml glucose oxidase and 5.0 mM glucose) on cumulative protein degradation over a 24-h
post-treatment period. All values are means of eight independent
experiments, for which S.E. (in panels A and C) were
always less than 10%.
Since
HO
exposures in vivo are more likely
to involve sources of continuous H
O
production,
rather than a bolus addition, we also tested the effects of continuous
H
O
production by the enzyme glucose oxidase. As
shown in Fig. 1C, continuous exposure to
H
O
production, over a 30-min glucose oxidase
treatment, caused a generalized increase in subsequent proteolysis at
all H
O
exposure rates tested. Proteolysis began
to increase at 50 nmol of
H
O
ml
30
min
, peaked at 100 nmol of
H
O
ml
30
min
, and gradually decreased again at the higher
exposures of 250, 400, and 1,000 nmol of
H
O
ml
30
min
. Since the cumulative H
O
exposures during the 30-min treatments of Fig. 1C were similar to the bolus H
O
additions of Fig. 1, A and B, it is interesting to note
that no depression of proteolysis below control rates was observed at
any of the continuous H
O
flux rates tested.
Importantly, no significant loss of cell viability, as judged by trypan
blue exclusion, occurred during any of the experiments shown in Fig. 1, A-C (data not shown).
We next tested for
effects of the superoxide (O )
generating agents paraquat and menadione on the degradation of
short-lived proteins in Clone 9 liver cells (Fig. 2). Protein
degradation was maximally increased by treatment of cells with 20
µM paraquat (Fig. 2A) or 20 µM menadione (Fig. 2B). Higher concentrations of
either agent again resulted in decreased proteolysis; in the case of
300 µM menadione, to below control levels.
Figure 2: Degradation of short-lived cellular proteins in cells exposed to redox cycling quinones. Clone 9 liver cells were exposed, or sham-exposed to paraquat (panel A) or to menadione (panel B) for 30 min. Protein degradation was measured at 24 h of incubation after exposure. All exposure conditions and proteolysis measurements were performed as described in the legend to Fig. 1. Values reported are means ± S.E. of at least six independent determinations.
Membrane lipid peroxidation is a well known outcome of cellular oxidative stress, and lipid oxidation products are able to react with other cellular components; including proteins. To model possible lipid peroxidation effects on protein integrity and stability we incubated Clone 9 liver cells with the aldehydic lipid peroxidation products 4-hydroxynonenal, malonyldialdehyde, and hexenal (Fig. 3). At the low concentrations of 1 or 10 µM, both 4-hydroxynonenal (Fig. 3A) and malonyldialdehyde (Fig. 3B) caused increased degradation of short-lived cellular proteins (by as much as 100%), but proteolysis returned toward baseline levels following 100 µM treatments with either agent. Hexenal caused only a small increase in proteolysis during the first 3 h after treatment and this trend actually reversed from 6 to 24 h post-treatment (Fig. 3C).
Figure 3: Degradation of short-lived cellular proteins in cells exposed to aldehydic products of lipid peroxidation. Clone 9 liver cells were exposed, or sham-exposed to 4-hydroxynonenal (panel A), to malonyldialdehyde (panel B), or to hexenal (panel C) for 30-min periods. All exposure conditions and proteolysis measurements were conducted as described in the legend to Fig. 1. The 4-hydroxynonenal was the kind gift of Prof. Hermann Esterbauer (University of Graz, Austria). Malonyldialdehyde was prepared as described previously (37) and trans2-hexenal was obtained from Sigma. All three lipid aldehydes were extensively purified prior to use. Values reported in panels A-C are means of at least eight independent determinations, for which S.E. were always less than 10%.
Figure 4:
Degradation of long-lived cellular
proteins in cells exposed to hydrogen peroxide or paraquat. Cell
proteins were metabolically labeled with
[S]Met/Cys for 16 h, and then chased with excess
unlabeled Met/Cys for 2 h. After this 18-h period, cells were either
exposed or sham-exposed, to H
O
(panel
A) or paraquat (panel B) for 30 min, washed, and then
incubated for a further 0-24 h in culture medium for measurements
of protein degradation. All H
O
(bolus) and
paraquat exposure conditions, and all proteolysis measurements, were
performed as described in the legends to Fig. 1and Fig. 2. Values in both panels are means of at least six
independent determinations, for which S.E. were always less than
10%.
Undamaged superoxide
dismutase and Hb were degraded at low rates during incubation with
untreated Clone 9 liver cell lysates, but prior oxidative modification
(by HO
) of these foreign proteins increased
their proteolytic susceptibility by up to 5-8-fold (Fig. 5A). When the Clone 9 cells were pretreated by
exposure to 0.25 or 0.4 mM H
O
, or to
20 or 300 µM paraquat (as per Fig. 1and Fig. 3) before lysis, essentially the same difference in
degradation of control and oxidatively modified superoxide dismutase
and Hb was again observed (Fig. 5, B, C, and D). It would, thus, appear that oxidative stress damages
cellular proteins, making them more (or less) susceptible to
proteolysis, rather than activating the proteolytic machinery of the
cell.
Figure 5:
Degradation of HO
modified foreign superoxide dismutase and hemoglobin added to
lysates of control and oxidatively stressed Clone 9 liver cells.
Tritium-labeled superoxide dismutase (SOD) and hemoglobin were
added to centrifuged lysates of unlabeled Clone 9 liver cells for
measurements of proteolysis over a 6-h period. A proteolysis buffer
consisting of 50 mM Tris-HCl (pH 7.8), 20 mM KCl, 5
mM MgOAc, and 0.5 mM dithiothreitol was used, as
previously(14) . Both foreign protein substrates were
radiolabeled by reductive methylation with
[
H]formaldehyde and sodium cyanoborohydride as
described by Jentoft and Deanborn(38) , and then extensively
dialyzed. The tritiated superoxide dismutase and Hb were either
undamaged, or were oxidatively modified immediately prior to the
experiment by exposure to 15 mM H
O
for
2 h, as described previously(13, 15) . Percent
superoxide dismutase or Hb degradation was determined, as described
previously(14) , by liquid scintillation in supernatants of
trichloroacetic acid precipitated lysates, by the formula:
(acid-soluble counts - background counts)
(initial acid
precipitable counts - background counts)
100. Panel A reports the selective degradation of
H
O
-modified superoxide dismutase and Hb (in
comparison with undamaged superoxide dismutase and Hb) during a 2-h
incubation of these protein substrates with centrifuged lysates from
untreated Clone 9 liver cells. Panels B-D report the
degradation of both H
O
modified and undamaged
superoxide dismutase and Hb during incubation with centrifuged lysates
from control, H
O
-treated, and paraquat-treated
Clone 9 liver cells. The 30-min H
O
or paraquat
cell treatments were conducted as described in the legends for Fig. 1and Fig. 2. In panels B and C the
H
O
cell pretreatments were as follows:
,
none;
, 0.25 mM;
, 1.0 mM. In panel
D the paraquat pretreatment of cells was as follows:
, none;
, 20 µM;
, 300 µM. Both the
bovine RBC superoxide dismutase and the bovine Hb were obtained from
Sigma. Values reported in all four panels are means of at least four
independent determinations for which S.E. (not shown in B, C, and D) were always less than
10%.
Our first experiment was to compare the inhibitor
profile of Clone 9 cell lysates, RBC lysates, and purified RBC
proteasome, conducting the degradation of oxidatively modified
superoxide dismutase and Hb. As shown in Table 1the degradation
of oxidized superoxide dismutase and Hb was similarly inhibited by
serine reagents (phenylmethanesulfonyl fluoride and diisopropyl
fluorophosphate), sulfhydryl reagents (Nethylmaleimide), and
transition metal chelators (EDTA and 8-hydroxyquinoline) in all three
test systems. The lysosomal proteolysis inhibitor leupeptin and
ATP-Mg both caused only minor inhibition in all
cases.
We next separated Clone 9 liver cell lysates into fractions
of less than 300 kDa and fractions of greater than 300 kDa (Fig. 6). Since the proteasome complex has a molecular size of
approximately 670 kDa (13, 16, 21) it should
be present only in the greater than 300-kDa cell fraction. The
0-300-kDa fraction was always prepared from radiolabeled cells
whose proteins had been metabolically labeled by incorporation of
[S]Met/Cys for either 2 h (short-lived proteins)
or for 16 h plus a 2-h chase (long-lived proteins). These radiolabeled
0-300-kDa fractions were prepared from both untreated cells and
from H
O
-treated cells, and were used as
``substrate fractions'' for proteolysis. The >300 kDa
``proteasome fraction'' was always prepared from non-labeled
cells, either with no treatment, or following H
O
exposure. When the proteasome fraction was prepared from control
cells (Fig. 6A) it selectively catalyzed significant
degradation of both short-lived and long-lived proteins in the
0-300-kDa substrate fractions from
H
O
-treated cells. When the proteasome fraction
was prepared from H
O
-treated cells (Fig. 6B) it again catalyzed the proteolysis of both
long-lived and short-lived proteins in the 0-300-kDa substrate
fractions from H
O
-treated cells, although the
degradation of long-lived proteins was somewhat depressed in comparison
with the results of Fig. 6A. The experiments of Fig. 6are certainly permissive of a role for proteasome in the
degradation of oxidatively modified cell proteins, and also indicate
that stress-induced proteolysis is primarily the result of substrate
modification or damage, rather than protease (proteasome) activation.
Figure 6:
Proteolysis in recombined 0-300 kDa
and >300 kDa Clone 9 liver cell fractions. One set of cells was
cultured (in the absence of radionucleotides), treated with 0.4 mM HO
or used as a control, lysed,
centrifuged, and filtered through a 300-kDa cut-off membrane
(Amicon
, Beverly, MA). The >300-kDa fraction retained
by the membrane was re-solubilized and used as the (unlabeled)
proteasome fraction for all experiments. Another set of cells was
cultured identically, and then either short-lived or long-lived
cellular proteins were metabolically labeled with
[
S]Met/Cys as per Fig. 1or 4. After
labeling the cells were either exposed to 0.4 mM H
O
, or used as controls. The labeled cells
were then lysed, centrifuged, and filtered through the 300-kDa cut-off
membrane. With these labeled cells, only the <300-kDa filtrate was
retained for use as the substrate fraction for proteolysis. The
unlabeled >300-kDa proteasome fraction and the <300-kDa
radiolabeled substrate fraction were then recombined for proteolysis
measurements, using the conditions of Fig. 5. Panel A shows experiments in which cells used for the proteasome fraction
were untreated. Panel B shows experiments in which cells used
for the proteasome fraction were pretreated by exposure to 0.4 mM H
O
prior to lysis and filtration. Values
reported are means ± S.E. of eight independent
experiments.
The fluoropeptide succinyl-leucine-leucine-valine-tyrosine-MCA
(s-LLVY-MCA) has been extensively studied as a proteolytic substrate
for proteasome(35, 36) . To further test the possible
importance of proteasome in degrading oxidatively modified Clone 9
liver cell proteins, we studied the effects of proteasome
immunoprecipitation on the degradation of both s-LLVY-MCA and
radiolabeled cell proteins, in lysates from control and
HO
-treated cells (Fig. 7). As shown in Fig. 7A, proteasome immunoprecipitation depressed
s-LLVY-MCA degradation by 75-85% in lysates of both control cells
and H
O
-treated cells. The inset to Fig. 7B is a proteasome Western blot which shows major
loss of several proteasome subunit bands following the
immunoprecipitation procedure. The main panel of Fig. 7B reveals a 95% decrease in the H
O
induced
degradation of long-lived cellular proteins following proteasome
immunoprecipitation.
Figure 7:
Proteolysis in lysates of
HO
-treated Clone 9 liver cells following
proteasome immunoprecipitation. Control and 0.4 mM H
O
-treated cells were lysed and
centrifuged at 3 h after (30 min) H
O
treatment
or sham-treatment. Proteasome in the cell lysates was then either
immunoprecipitated with a purified rabbit anti-rat IgG(39) ,
directed against the 20 S proteasome, designated +IP, or
sham-precipitated and designated -IP. Preliminary
studies revealed that the optimal IgG/lysate protein ratio was 70
µg of IgG/mg of lysate protein, and this ratio was used. Cell
lysates were incubated with IgG for 3 h at 4 °C, and then
centrifuged at 34,000
g for 30 min. Purified rabbit
anti-rat IgG (38) was the kind gift of Dr. Keiji Tanaka
(University of Tokushima, Tokushima, Japan) and immunoprecipitation was
conducted according to Orino et al.(39) . Panel A shows the effects of proteasome immunoprecipitation, in extracts
from control and H
O
-treated cells, on
degradation of the fluoropeptide s-LLVY-MCA. Degradation of s-LLVY-MCA
was measured, as previously(14) , in 34,000
g supernatants by fluorescence at 365-nm excitation/460-nm emission
(within the linear range for fluorescence response) in comparison with
Pronase-digested standards. The fluoropeptide was dissolved in 10%
Me
SO and used at a final concentration of 50 µM in an assay buffer consisting of 50 mM Tris-HCl (pH 7.8),
20 mM KCl, 0.5 mM MgOAc, and 0.5 mg of cell lysate
protein. After 1-h incubation at 37 °C proteolysis was terminated,
prior to fluorescence measurements, by addition of 2.0 ml of a solution
containing 0.1 M sodium borate (pH 9.0) and ethanol/water
(144:16), to 200 µl of reaction mixture. The main portion of panel B shows the effects of proteasome immunoprecipitation on
the H
O
induced degradation of long-lived
radiolabeled (16-h labeling plus a 2-h chase, as per Fig. 4)
cell proteins, in 34,000
g supernatants of proteasome
immunoprecipitated (+IP) and sham-immunoprecipitated
(-IP) cell lysates. It should be noted that the
experiments of panel B involved a 30-min H
O
treatment (or sham-treatment) of intact radiolabeled cells
followed by cell lysis and centrifugation, immunoprecipitation (or
sham-precipitation) for 3 h followed by centrifugation at 34,000
g for 30 min, and a 37 °C proteolysis incubation
for 6 h. The inset to panel B is a Western blot
showing the effects of proteasome immunoprecipitation on proteasome
subunit protein bands in the 34,000
g cell
supernatants. Western blots of control (-IP) and
immunoprecipitated (+IP) cell lysates were performed
according to Towbin et al.(40) . To achieve best
results a mixture of proteasome anitsera was used. The mixed proteasome
antisera was 50% rabbit anti-chick (41) which was the kind gift
of Dr. Chin Ha Chung (Seoul National University, Seoul, Korea), and 50%
rabbit anti-rat (39) which was the kind gift of Dr. Keiji
Tanaka (University of Tokushima, Tokushima, Japan). All values in both
panels are means ± S.E. of at least six independent
experiments.
We next repeated the immunoprecipitation
studies of Fig. 7, but this time used HO
modified superoxide dismutase as the substrate for proteolysis in
lysates of control and H
O
-treated cells. As
shown in Fig. 8, proteasome immunoprecipitation decreased the
degradation of H
O
-modified superoxide dismutase
by approximately 55% in lysates of both control cells and
H
O
-treated cells. When the immunoprecipitates
were re-solubilized and incubated with
H
O
-modified superoxide dismutase, more than
100% (actually 126-145%) of the initial activity was recovered (Fig. 8, bars labeled Pcpt.), suggesting a
small activation of proteasome by the immunoprecipitation procedure.
Figure 8:
Degradation of
HO
-modified superoxide dismutase in lysates of
Clone 9 liver cells following proteasome immunoprecipitation.
Centrifuged lysates of control or 0.4 mM H
O
-treated cells (with or without
immunoprecipitation) were incubated with control and
H
O
-modified [
H]superoxide
dismutase for 1 h, as per Fig. 5. The lysates were first either
immunoprecipitated (+IP) with proteasome antibody as per Fig. 7, or were sham-precipitated (-IP), prior to
centrifugation at 34,000
g. Proteolysis was measured
as described in the legend to Fig. 5by release of acid-soluble
counts, from [
H]superoxide dismutase added to the
34,000
g supernatants. Degradation of
[
H]superoxide dismutase was also measured by
addition of this substrate to re-solubilized immunoprecipitates;
designated Pcpt. Values for the difference between degradation
of H
O
modified and control
[
H]superoxide dismutase are means ± S.E.
of at least three independent experiments.
A more rigorous test of proteasome involvement in the intracellular degradation of oxidatively modified proteins would be to use knock-out mutants in which one of the essential proteasome subunit genes was deleted. Unfortunately, for our purposes, proteasome appears to be essential for cell division and deletion mutations are typically lethal (42, 43) . We, therefore, decided to use the more gentle approach of diminishing total cellular proteasome activity by prolonged exposure of cells to an antisense oligodeoxynucleotide for an essential proteasome subunit. For these studies we synthesized both sense and antisense oligodeoxynucleotides directed against the initiation codon region of the proteasome C2 subunit gene(44, 45) .
After 7 days of daily exposure to
the antisense message the cellular concentrations of several proteasome
subunit polypeptides were significantly depressed, as shown by Western
blot analysis (Fig. 9, inset). The degradation of
labeled proteins in untreated cells was not affected by the sense
oligodeoxynucleotide and the antisense message decreased basal
proteolysis in control cells by only approximately 12% (Fig. 9, main panel). In contrast, the increased degradation of
oxidized proteins normally seen in HO
-treated
cells was almost completely abolished in antisense
oligodeoxynucleotide-treated cells. Interestingly, the sense
oligodeoxynucleotide actually increased proteolysis in
H
O
-treated cells, although only by a modest
16%, in comparison with cells that received no oligodeoxynucleotide
treatment (Fig. 9, main panel).
Figure 9:
Degradation of long-lived proteins in
Clone 9 liver cells treated with antisense oligodeoxynucleotide to the
proteasome C2 subunit gene. Cells were either untreated (None)
or were exposed for 7 days to daily additions of 0.4 nmol/ml of either
sense or antisense oligodeoxynucleotides to the initiation codon region
of the proteasome C2 subunit gene(44) . The sense
oligonucleotide used was 5`-AGCTATGTTTCGCAA-3`, and the antisense
oligonucleotide was 5`-TTGCGAAACATAGCT-3`. Both oligonucleotides were
synthesized on an ABI391 DNA synthesizer (Applied Biosystems), and
extensively purified prior to use. Long-lived cell proteins were then
metabolically radiolabeled with [S]Met/Cys for
16 h (plus a 2-h chase) as described in the legend to Fig. 4.
The cells were next either exposed to 0.4 mM H
O
(as per Fig. 1) or used as
controls. Degradation of long-lived cellular proteins was measured
during a 6-h incubation by production of acid-soluble counts, as per Fig. 1. Values in the main portion of the figure are means
± S.E. of 12 independent experiments. The inset is a
Western blot showing the depletion of several proteasome subunit
proteins in cells treated with antisense oligonucleotide for 7 days, in
comparison with sense oligonucleotide-treated cells. Molecular weight
standard markers are included in the inset for convenient
identification of individual bands. The proteasome C2 subunit has an
approximate molecular size of 30-32
kDa(44, 45) .
It should be noted
that cellular growth rates, rates of protein synthesis, and the
incorporation of [S]Met/Cys into newly
synthesized proteins (whose degradation was actually measured in Fig. 9), were all unaffected by either the sense or the
antisense oligodeoxynucleotides (data not shown). The results of Fig. 9, thus, provide strong evidence for a major involvement of
proteasome in the degradation of oxidatively modified Clone 9 liver
cell proteins.
Most of the experimental data presented thus far
would suggest that oxidative stress modifies cellular substrate
proteins, making them more (or less) susceptible to proteolysis by
proteasome. The results of Fig. 7A (-IP)
and Fig. 8(comparing total activity in re-solubilized
immunoprecipitates), however, suggest that a small activation or
induction of proteasome may actually occur with exposure of cells to
HO
stress. To further investigate this
possibility we compared the degradation of the proteasome substrate
s-LLVY-MCA in control cells, and cells exposed to H
O
or paraquat. For the most part both H
O
and paraquat exposures caused no change in s-LLVY-MCA degrading
activity, except for small increases at very high oxidant exposures
which were actually very poor initiators of intracellular proteolysis
(data provided to the reviewers). Thus neither activation nor induction
of proteasome appears to explain our results.
To our knowledge, the present investigation is the first to conclusively demonstrate oxidative stress-induced intracellular proteolysis in a mammalian cell culture line. It would, thus, appear that the reasonable concern raised by Dean et al.(33) , that such phenomena might be limited to bacteria and RBC, has been answered. Our studies reveal that mild forms of oxidative stress can increase the intracellular degradation of both short-lived and long-lived proteins in Clone 9 liver cells. In contrast, more severe levels of oxidative stress are less effective in initiating intracellular proteolysis and, at the extreme, can even depress protein degradation to below baseline values. Similar trends have now been observed in bacteria, isolated mitochondria, chloroplasts, erythrocytes, and reticulocytes, rat muscles, and primary hepatocytes(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 26, 27, 28, 29, 30, 31, 32) . It, thus appears clear that oxidative stress-induced intracellular proteolysis is a general property of both procaryotes and eucaryotes.
Our inhibitor profiles, proteasome immunoprecipitation experiments, and proteasome C2 subunit oligodeoxynucleotide studies all indicate that the multicatalytic proteinase complex, proteasome is largely responsible for the selective degradation of oxidatively modified proteins in oxidatively stressed Clone 9 liver cells. The same conclusion was previously reached with red blood cells (1-7, 9-11, 13-17, 19-21). It should also be noted that the multicatalytic proteinase complex, proteasome was the enzyme isolated by Rivett (31, 32) from primary hepatocyte cultures, based on its ability to selectively degrade oxidatively modified protein substrates.
The proteasome complex exists in both an ATP-independent 19-20 S (670-700 kDa) form, and an ATP-stimulated 26 S (1,500 kDa) form in mammalian cells(35, 36, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59) . Our previous work with RBC(1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 19, 20, 21) , and the work of Rivett (31, 32) with primary hepatocytes, provided experimental evidence that the ATP-independent 19-20 S (670-700 kDa) ``core'' proteasome complex is the form that recognizes and selectively degrades oxidatively damaged protein substrates. In this regard it is important to note that reticulocytes and terminally differentiated erythrocytes exhibit similar capacities to degrade oxidatively modified proteins, and similar inhibition profiles(1, 2, 3, 4, 5, 6, 7, 8, 13, 14, 15, 16, 17, 19, 20, 21) , despite the fact that the 26 S ATP-stimulated proteasome complex is lost during maturation, and erythrocytes retain only the 19-20 S ATP-independent core proteasome complex(13) . The degradation of oxidized proteins in red cell lysates, and the degradation of oxidized protein substrates by the purified erythrocyte or reticulocyte 19-20 S proteasome complex, are inhibited by 15-20% upon addition of ATP(13, 14, 15, 16, 17, 19, 20, 21) . In the present investigation ATP was also slightly inhibitory (by approximately 15%) to the selective degradation of oxidatively modified proteins in Clone 9 cell lysates, providing at least initial evidence that the 19-20 S core proteasome complex is also responsible for degrading oxidized proteins in Clone 9 liver cells.
The multicatalytic proteinase complex, proteasome, can bind (or release) both inhibitory and stimulatory polypeptide subunits(35, 36) . Additionally, the entire proteasome complex is overexpressed under certain conditions, at discrete developmental stages, and in transformed cells(59) . In initiating the present investigation we considered the hypothesis that oxidative stress might either activate proteasome (by releasing an inhibitory subunit or promoting expression of a stimulatory subunit), or might induce proteasome synthesis. Our present studies, involving comparisons of control and oxidatively stressed cells, however, provide little or no evidence to support such an hypothesis. By Western blot analyses with polyclonal antiproteasome antibodies, Northern blot analyses with a cDNA probe directed against C2 subunit transcripts, and actual proteinase assays measuring total cellular capacity to degrade control and oxidatively modified protein substrates (superoxide dismutase and Hb added to cell lysates), no evidence for increased proteasome transcription, message stabilization, translation, or total activity was found. Small increases in peptidase activity with the proteasome fluoropeptide substrate s-LLVY-MCA were observed with severe oxidative stress, but such high stress levels actually depressed the degradation of oxidatively modified proteins. Thus, with the exception of a possible small activation or induction of peptidase activity, oxidative stress does not appear to either activate proteasome or induce proteasome synthesis in Clone 9 liver cells.
Since neither proteasome activation nor induction seem to explain the large increases in total intracellular protein degradation we observe following oxidative stress, and since oxidatively modified foreign proteins are selectively degraded in lysates of non-stressed Clone 9 liver cells, it seems clear that oxidative modification of substrate proteins must be the major cause of increased protein degradation following oxidative stress. This same conclusion was previously reported for erythrocytes and reticulocytes(1, 2, 3, 4, 5, 6, 7, 15, 16, 17, 19, 20, 21) , isolated mitochondria(12) , and bacteria(9, 10, 25, 26, 27, 28, 29, 30) , and now appears to be a fairly general biological phenomenon. Protein re-arrangement, with exposure of hydrophobic ``patches'' of amino acids, has been proposed as the substrate activation mechanism to explain increased degradation of oxidatively modified proteins by both RBC(13, 15, 16, 17, 19, 20, 21) and E. coli(9, 10, 30) . The model foreign substrates (superoxide dismutase and Hb) used to link substrate hydrophobicity and proteolytic susceptibility in RBC(13, 15, 16, 17, 19, 20, 21) were the same substrates used in our present investigation, and proteasome appears responsible for the degradation of oxidized proteins in both cases. It, thus, seems reasonable to propose that increased intracellular proteolysis in oxidatively stressed Clone 9 liver cells may also be explained by modification of cell proteins to expose, proteasome sensitive, hydrophobic patches.
Although we were able to
document oxidative stress-induced increases in intracellular
proteolysis of 150% or more, the model oxidized substrates superoxide
dismutase and Hb were degraded at severalfold higher rates (in
comparison with the untreated proteins) in cell lysates. It is also
abundantly clear from numerous studies that proteins vary widely in
their susceptibility to various forms of oxidative stress, and to
proteolysis(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) .
It, thus, seems probable that oxidative stress causes some generalized
increase in the oxidation and degradation of cellular proteins, but
that certain proteins are particularly susceptible. Further
studies have revealed the existence of at least five
proteins in Clone 9 liver cells whose turnover (from radiolabeling
experiments both before and after cellular oxidative stress) is
increased severalfold following exposure to H
O
.
The identities and mechanisms of oxidation/degradation of these, and
other, Clone 9 liver cell proteins are under active investigation in
this laboratory.