From the Ethel Percy Andrus Gerontology Center,
University of Southern California, Los
Angeles, California 90089-0191, the § Clinics of Physical
Medicine and Rehabilitation, Medical Faculty (Charité),
Humboldt-University Berlin, D-10098 Berlin, Germany, and the
¶ Institute of Molecular Pharmacology, D-10315
Berlin, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We report that exposure of aconitase to moderate concentrations of peroxynitrite, 3-morpholinosydnonimine (SIN-1; a superoxide- and nitric oxide-liberating substance), or hydrogen peroxide, inhibits the enzyme and enhances susceptibility to proteolytic digestion by the isolated 20 S proteasome. Exposure to more severe levels of oxidative stress, from these same agents, causes further inhibition of the enzymatic activity of aconitase but actually decreases its proteolytic breakdown by proteasome. It should be noted that the superoxide and nitric oxide liberated by SIN-1 decomposition react to form a steady flux of peroxynitrite. S-Nitroso-N-acetylpenicillamine, a compound that liberates nitric oxide alone, causes only a small loss of aconitase activity (25% or less) and has no effect on the proteolytic susceptibility of the enzyme. Proteasome also seems to be the main protease in cell lysates that can degrade aconitase after it has been oxidatively modified by exposure to peroxynitrite, SIN-1, or hydrogen peroxide. Using cell lysates isolated from K562 cells treated for several days with an antisense oligodeoxynucleotide to the initiation codon region of the C2 subunit of proteasome (a treatment which diminishes proteasome activity by 50-60%), the enhanced degradation of moderately damaged aconitase was essentially abolished. Other model proteins as well as complex mixtures of proteins, such as cell lysates, also exhibit enhanced proteolytic susceptibility after moderate SIN-1 treatment. Therefore we conclude that peroxynitrite reacts readily with proteins and that mild modification by peroxynitrite results in selective recognition and degradation by proteasome.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A close relationship between protein oxidation and susceptibility
to proteolysis has been well demonstrated in a variety of biological
systems (1-16). A series of publications have demonstrated the
enhanced degradation of proteins oxidized by exposure to the superoxide
anion radical (O2), to hydrogen peroxide
(H2O2), or to the hydroxyl radical
(·OH), in isolated systems as well as in various cell types
(1-16). These studies were performed in erythrocytes and reticulocytes of various species (3, 6), in Escherichia coli (7, 9), in
rat muscles in vitro (1, 4), in primary cultures of rat hepatocytes (13, 14), and recently in dividing cell lines (15, 16).
Most of these studies revealed that the degradation of mildly oxidized
proteins is a normal cellular function, whereas extensively oxidized
proteins are poor substrates for proteases and may accumulate and,
therefore, contribute to diseases or aging processes (1, 2, 6, 17). In
these studies using cells, cell extracts and isolated proteins strong
evidence has been presented for the key role of a single proteolytic
complex, the 20 S proteasome, in the selective recognition,
degradation, and removal of oxidized proteins (1, 15, 16). Evidence has
also been documented for the mechanism of recognition based on a
partial unfolding of the oxidized protein with consequent exposure of
hydrophobic moieties on the protein surface (18-21).
Over the past several years a number of publications have dealt with
the reactions of new oxidants with proteins: nitric oxide (NO)1 and peroxynitrite
(ONOO). Nitric oxide and other nitrogen-based oxidants
are produced by a variety of mammalian cells, including endothelial
cells, neutrophils, and macrophages (22-24). While nitric oxide acts
as a signal-transduction molecule, it exhibits, together with other nitrogen based oxidants, cytotoxic properties (25-35). In particular the reaction of nitric oxide with mitochondrial aconitase in various cells, and the reaction with cytosolic aconitase, also called the
iron-responsive element-binding protein, or iron-responsive protein-1,
have been intensively investigated (30, 31, 34). Although NO is a
relatively weak oxidant, it is able to react very rapidly with
superoxide anion radical to form the strongly oxidizing product
peroxynitrite (36). Peroxynitrite reacts with tyrosine residues of
proteins, with sugars, and with DNA, and can also initiate membrane
lipid peroxidation (25, 26, 37-40). In recent papers it has been
demonstrated that aconitase, mitochondrial electron transport
components, and mitochondrial ATPase are damaged by ONOO
(41). Whether NO also exhibits some effects on aconitase activity is
not completely clear, because Hausladen and Fridovich (30) and Castro
et al. (31) found aconitase to be stable toward NO, whereas
Drapier et al. (34) found an inactivation by NO. Stadler et al. (33) described an inactivation of mitochondrial
aconitase, whereas they found the cytosolic enzyme to be stable to NO
treatment.
We undertook the present investigation to test whether inhibition of aconitase by various nitrogen related oxidants is also accompanied by an increase in proteolytic degradation by proteasome. Therefore we exposed aconitase to various oxidants and investigated both the enzymatic activity and the proteolytic susceptibility of the oxidized protein. To test whether oxidation by nitrogen related oxidants, and the resulting enhanced proteolytic susceptibility of oxidized proteins is a more general process, we also used various other purified proteins as substrates for protein oxidation and proteolysis. Finally we exposed the numerous proteins in centrifuged cell lysates to nitrogen-based oxidants and measured the proteolytic susceptibility of the oxidatively modified protein mixture.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials-- Porcine heart aconitase (essentially mitochondrial), catalase, ovalbumin, myosin, myoglobin, ferritin, and carbonic anhydrase were obtained from Sigma. Collagenase and superoxide dismutase were from Boehringer Mannheim. Trinitrobenzenesulfonic acid was purchased from Fluka. S-Nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine (SIN-1) were obtained from Alexis. Peroxynitrite was synthesized according to Beckman et al. (25). Radiolabeled formaldehyde was a product of Amersham Pharmacia Biotech. All other chemicals were of highest purity available.
Proteasome Isolation--
Proteasome was isolated as previously
(42), using outdated blood from the Charité Hospital,
Humboldt-University, Berlin. After cell lysis the lysate was frozen to
allow the 26 S proteasome complex to dissociate. The final step was a
re-chromotography on a Mono Q-fast protein liquid chromatography
column, which gave a single peak. Fractions with the highest activities
were pooled and stored at 20 °C until needed.
Cells, Cell Culture, and Cell Lysates-- K562 cells (chronic myelogenous leukemia, human) were obtained from American Tissue and Cell Culture (ATCC CCL 243). The cells were cultured in 90% RPMI 1640 medium, supplemented with 10% fetal bovine serum. Cells were initially seeded at a density of 5 × 106 cells/ml. Cells were harvested and washed three times. Afterward the cells were lysed for 1 h in 1 mM dithiothreitol followed by a centrifugation at 14,000 × g of the lysate.
Exposure of Proteins to Oxidative Stress--
Isolated proteins
or cell lysates (all at a concentration of 0.33 mg/ml) were exposed to
various concentrations of different oxidants, in a rotating bath, for
2 h at 37 °C. Incubations were carried out at 50 rotations/min.
Afterward any remaining reactive oxygen or nitrogen species were
removed by addition of catalase in the case of hydrogen peroxide and by
addition of glutathione and superoxide dismutase in the case of SNAP,
SIN-1, and ONOO. In several control experiments both
(oxidant) treated and nontreated samples were next dialyzed extensively
for 4 h (at 4 °C) against 200 volumes of appropriate buffer,
with two changes of dialysis medium, in order to test the possible
effects of residual hydrogen peroxide, ONOO
, SNAP, SIN-1,
or their reaction products. In additional control experiments, the
possible influence of substances other than nitric oxide or
peroxynitrite originating from SIN-1 or SNAP was also investigated by
"reversed order addition experiments" (31) involving addition
of SIN-1 or SNAP after decomposition of purified NO or ONOO
.
Measurement of Aconitase Activity--
Aconitase activity was
measured according to Kennedy et al. (43). Briefly,
aconitase or oxidized aconitase was incubated at 25 °C in 100 mM Tris (pH 7.6) containing 0.5 mM
cis-aconitate. The reaction was monitored at 240 nm ( = 3.6 mM
1 cm
1) for 5 min.
Proteolysis Measurements-- Proteolysis studies involved either the degradation of radiolabeled aconitase, followed by liberation of acid-soluble radioactive counts; the degradation of unlabeled proteins, measured by the liberation of acid soluble primary amines; or the degradation of the fluorogenic peptide s-LLVY-MCA, measured by liberation of the free fluorescent MCA moiety.
Aconitase Degradation--
Aconitase was radiolabeled by
reductive methylation using [3H]formaldehyde as described
by Jentoft and Dearborn (44). The degradation of aconitase was assessed
following addition of isolated proteasome (30 µg of proteasome/mg of
protein) to the damaged or undamaged protein. A proteolysis buffer
consisting of 10 mM HEPES, 100 mM KCl, 5 mM MgCl2, and 1 mM dithiothreitol
(pH 7.2) was used. Degradation was followed over a 2-h incubation
period conducted in a rotating bath set at 37 °C and 50 rpm.
Afterward the proteolysis was stopped by the addition of ice-cold
trichloroacetic acid as described in Ref. 4. A 3% albumin solution was
used as carrier. The percentage of degradation was determined as
described previously (4), by liquid scintillation counting, using the formula: (acid soluble counts background counts)/(initial acid precipitable counts
background counts) × 100.
Degradation of Nonlabeled Proteins-- Proteasome addition and incubation was carried out as described above. Reactions were stopped by addition of an equal volume of trichloroacetic acid (20%). The samples were centrifuged for 15 min at 14,000 × g, and the supernatants were neutralized by addition of 1 M NaOH. After treatment with N-ethylmaleimide (0.5 mM final concentration), twice the volume of trinitrobenzenesulfonic acid (0.33 mg/ml in 0.1 M borate buffer (pH 9)) was added. The samples were next incubated for 20 min at 40 °C in a shaking water bath. Afterward the reaction was stopped by addition of 10 volumes of 1.0 N HCl. The formation of primary amines was measured spectrophotometrically at 334 nm, using leucine as a standard.
Degradation of Fluorogenic Peptide-- The fluoropeptide s-LLVY-MCA was used as a model substrate for the proteasome, as described previously (4, 15, 16). The s-LLVY-MCA was dissolved in 10% dimethyl sulfoxide and used in a final concentration of 200 µM. The peptide was incubated for 1 h with cell lysates at 37 °C in a shaking water bath using the proteolysis buffer. The reaction was terminated by addition of 0.1 M borate/ethanol (9:1). Fluorescence of the proteolytically released MCA fluor was measured at 365 nm excitation/460 nm emission. A pure MCA standard was used for quantification.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Inactivation of Aconitase by Oxidative Stress-- The enzymatic activity of purified aconitase was decreased by various oxidants (Fig. 1). Whereas hydrogen peroxide inhibited the enzyme almost completely at concentrations of 30 mM, SNAP (a liberator of NO) was not able to decrease the activity by more then 25%. On the other hand peroxynitrite, in a bolus addition, significantly inhibited aconitase at much lower concentrations than required for H2O2 and completely inhibited activity at a concentration of 1 mM.
|
Oxidative Stress Affects the Degradation of Aconitase by Purified
Proteasome--
The proteolytic susceptibility of tritiated aconitase
was strongly affected by exposure of the protein to various oxidants (Fig. 2). Proteolysis of the enzyme
gradually increased after treatment with hydrogen peroxide
concentrations up to 2 mM. At this concentration an almost
9-fold increase in the rate of proteolysis, in comparison with
controls, was observed. At higher concentrations of
H2O2 the protein became a poor substrate for
the proteasome, probably because it was too seriously damaged, as
previously reported for other proteins (1-6, 15, 16, 18, 19). Fig. 2
also demonstrates the effects of various oxides of nitrogen on the proteolytic susceptibility of aconitase. Whereas SNAP (the
NO-liberating substance) did not show any significant influence on the
proteolysis of aconitase at concentrations up to 30 mM,
bolus addition of peroxynitrite significantly increased the proteolytic
susceptibility, even at very low concentrations. An ONOO
concentration of 1 mM, for example, increased the
proteolysis rate up to 6-fold in comparison with untreated controls.
Higher ONOO
concentrations, from 2 to 5 mM,
decreased proteolysis rates to below control levels.
|
Proteasome Is Mainly Responsible for Aconitase Degradation in Cell Lysates-- To begin exploring whether the proteasome is also mainly responsible for degradation of aconitase exposed to oxidative stress in intact cells, we next replaced the purified proteasome used in the experiments of Fig. 2 with cell lysates from K562 cells, containing various levels of proteasome. In order to manipulate the proteasome content of K562 cells we used an antisense oligodeoxynucleotide directed against the initiation codon region of the proteasome C2 subunit gene, exactly as described previously (15, 16), to deplete the cells (and the lysates prepared from them) of proteasome. Fig. 3 reports the degradation of tritiated aconitase by cell lysates. As shown in the upper part of Fig. 3, moderate concentrations of H2O2 increased the degradation of aconitase by control or sense-oligonucleotide-treated K562 cell lysates up to 3-fold, whereas high concentrations failed to increase the proteolysis rates. With lysates prepared from cells treated with the antisense oligonucleotide, no H2O2 concentration tested was able to significantly stimulate proteolytic susceptibility, and high H2O2 concentrations were strongly inhibitory.
|
Peroxynitrite Induces Proteolytic Degradation of Many
Proteins--
Since the increased proteolytic susceptibility of
ONOO exposed aconitase to degradation by proteasome has
not been previously reported, we thought it important to test whether
the proteolytic susceptibility of other proteins is also increased
after ONOO
treatment. Therefore, we exposed a number of
different proteins to SIN-1, dialyzed them, and then incubated them
with purified proteasome in vitro. Although the stimulation
of proteolysis induced by ONOO
varied from 2- to 12-fold,
a significant increase in proteolysis was found for every protein
tested after treatment with 2 mM SIN-1 (Fig.
4).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A number of studies have reported the inactivation of aconitase by various forms of activated oxygen, such as the superoxide anion radical and hydrogen peroxide (31, 45-47). Our own data show an inhibition of aconitase by moderately high concentrations of hydrogen peroxide, which is in agreement with the results of Gardner et al. (47) and Verniquet et al. (48). These authors found an inhibition of mitochondrial aconitase at between 100 and 500 µM H2O2. We further find that oxidative modification is followed by an enhanced proteolytic susceptibility, which results in increased degradation of aconitase (as well as other proteins) by proteasome.
The role of proteasome in the degradation of oxidized proteins has been demonstrated in a large number of studies from our laboratory (1-7, 15, 16, 18, 19) and others (8-14). Recently we were able to demonstrate that dividing mammalian cells in culture are able to degrade oxidatively damaged proteins preferentially and that the proteasome is mainly responsible for this activity (15, 16). We, therefore, felt we were able to answer the doubts raised Dean et al. (49, 50) in this regard. The present results even further extend our examples of both oxidants and protein substrates that appear to be involved in proteasome-directed protein turnover (recently reviewed in Ref. 51).
Studies of various model proteins have demonstrated in isolated systems
that mildly oxidized proteins are preferentially degraded by the
isolated 20 S proteasome, also called macroxyproteinase earlier by our
group (4, 5). Because this multicatalytic proteinase complex exists in
both an ATP- and ubiquitin-stimulated 26 S form, and an ATP- and
ubiquitin-independent 20 S form, it is important to note that oxidized
proteins are preferentially degraded only by the ATP- and
ubiquitin-independent form. In fact ATP actually exhibits a 10-20%
inhibition of the degradation of oxidized proteins in cell extracts,
and direct comparisons reveal that the 26 S proteasome (in the presence
of ubiquitin and a fully competent conjugating system) appears unable
to recognize the oxidized forms of various protein substrates (4, 5,
15, 18). Our current studies reveal, for the first time, that both ONOO- and H2O2-oxidized aconitase
is also a preferred substrate for proteolytic degradation by the 20 S
proteasome.
As reported previously (51), we suggest that polyubiquitin serves (at least in part) as a hydrophobic "tag" to target many soluble proteins to the 26 S proteasome and that ATP serves partly to de-ubiquitinate substrates and partly to activate their unfolding prior to degradation. Since oxidative inactivation generally causes some rearrangement of secondary and tertiary structure (partial protein unfolding) with consequent exposure of normally buried, hydrophobic domains (18-21, 51), we suggest that oxidation essentially "replaces" both ubiquitin and ATP in activating substrates for degradation by the 20 S proteasome complex. As observed previously with other proteins exposed to other oxidants, aconitase is degraded most efficiently after treatment with moderate oxidative stress, whereas greater oxidative damage actually leads to decreased proteolytic susceptibility.
The effects of nitric oxide and peroxynitrite on aconitase activity have been investigated by others (30-35). More recently Yan et al. (52) reported the exciting new observation that oxidative inactivation of mitochondrial aconitase can contribute to age-related deterioration of mitochondrial function in houseflies. Furthermore, by experimentally inactivating or inhibiting mitochondrial aconitase, Yan et al. (52) were actually able to decrease housefly lifespans. These results indicate that oxidative inactivation of mitochondrial aconitase is an important biological process and suggest (to us) that efficient removal/degradation of the damaged enzyme may be an important first step in coping with the problem, assuming that de novo synthesis of replacement aconitase can then occur.
It has been reported that aconitase is one of the major intracellular targets of nitric oxide, and the loss of activity observed by some authors has been attributed to the reaction of nitric oxide with the iron-sulfur cluster of the enzyme. However, two recent independent publications by Hausladen and Fridovich (30) and by Castro et al. (31), as well as the data presented here, demonstrate that nitric oxide itself does not exert a major effect on the activity of aconitase. We now report that nitric oxide causes only a mild decrease in enzymatic activity and produces no significant increase in susceptibility to degradation by the purified proteasome.
Bouton et al. (53) recently demonstrated both disulfide bond
formation at Cys437 and iron-sulfur cluster disruption in
the peroxynitrite-mediated inactivation of cytosolic aconitase or
iron-responsive protein-1. Earlier, the work of Hausladen and Fridovich
(30) and that of Castro et al. (31) conclude that
peroxynitrite exerts a strong inactivatory effect on aconitase. These
results are in agreement with our own investigation of aconitase
activity and our observation that aconitase is degraded more rapidly
after SIN-1 or ONOO treatment than is the untreated
enzyme. Castro et al. (31) reported a 50% inhibition of
aconitase activity at 60 µM ONOO
(30),
which is in good agreement with our own results (I50 of 75 µM). Castro et al. (31) also found that
ONOO
attacks the 4iron-4sulfur complex of aconitase,
leading to cluster disruption, and that this process is completely
reversible due to dissociation of iron-sulfur and reincorporation of
iron. However, either the changes in the prosthetic group are a
relevant signal for a protease to recognize the protein, or further
changes happened in the protein structure, perhaps similar to the
disulfide formation observed by Bouton et al. (53) with
iron-responsive protein-1. Because of the increased proteolytic
susceptibility in the case of mild oxidative damage, and the decreased
proteolysis rates after severe damage, it is likely that the proteasome
recognizes damaged proteins due to the exposure of hydrophobic moieties
on the protein surface, as reported previously (18-21). Loss of
aconitase activity of 15% in the case of H2O2
or even 50% with ONOO
(SIN-1) is not accompanied by
increased proteolytic susceptibility, but after further oxidative
modification the proteolytic susceptibility rises. From the fact that
the proteolytic susceptibility of aconitase starts to increase at
different levels of remaining activity one can conclude that
H2O2 and ONOO
attack aconitase
differently. It further appears likely that the oxidized moieties
recognized by proteasome do not necessarily include the active center
of the enzyme. This phenomenon was discussed previously by our group
(1-6, 18, 19) and more recently in detail by the group of Stadtman
(20, 21), in reference to the recognition and degradation of glutamine
synthetase by proteasome.
All peroxynitrite-treated proteins studied in the current investigation exhibited an enhanced proteolytic susceptibility toward degradation by proteasome. This was independent of the presence of iron-containing prosthetic groups in a particular protein. That the proteolytic degradation of peroxynitrite damaged proteins by proteasome is a general phenomenon may be inferred from our results of exposure of a complex protein mixture, such as cell lysates, to peroxynitrite.
The production of superoxide anion radical and nitric oxide is a well
characterized process, especially in macrophages, neutrophils, and
endothelial cells (22-24). Because of the fast reaction of NO with
O2, it is very likely that ONOO
is formed
in vivo. Due to its high reactivity, ONOO
presumably reacts with numerous cellular proteins. Therefore we
postulate that the reaction of peroxynitrite with proteins enhances
their proteolytic susceptibility and reflects a general signal for
proteolytic degradation by the 20 S proteasome complex, an efficient
mechanism of oxidative protein turnover.
![]() |
FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft grants (to T. G. and I. E. B.) and by National Institutes of Health/NIEHS Grant ES-03598 (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.
Senior author. To whom correspondence and reprint requests
should be addressed: Ethel Percy Andrus Gerontology Center, University of Southern California, 3715 McClintock Ave., Rm. 306, Los Angeles, CA
90089-0191. Tel.: 213-740-8959; Fax: 213-740-6462; E-mail: kelvin{at}rcf.usc.edu.
1
The abbreviations used are: O2,
superoxide anion radical; ·OH, hydroxyl radical; NO, nitric
oxide; ONOO
, peroxynitrite; SNAP,
S-nitroso-N-acetylpenicillamine; SIN-1, 3-morpholinosydnonimine; s-LLVY-MCA,
succinyl-leucine-leucine-valine-tyrosine-MCA (a fluorogenic
peptide substrate for proteolysis).
2 T. Grune, I. E. Blasig, N. Sitte, B. Roloff, R. Haseloff, and K. J. A. Davies, data not shown.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|